WO2006063285A2 - Methods for identifying risk of breast cancer or prostate cancer and treatments thereof - Google Patents

Abstract

Provided herein are methods for identifying a subject at risk of breast cancer or prostate cancer, methods for providing a prognosis to a subject at risk of breast cancer or suffering from breast cancer, reagents and kits for carrying out the methods, methods for identifying candidate therapeutics for treating breast cancer or prostate cancer, and therapeutic methods for treating breast cancer or prostate cancer in a subject. These embodiments are based upon and analysis of polymorphic variations in nucleotide sequences within the human genome.

Description

METHODS FOR H)ENTIFYING RISK OF BREAST CANCER OR PROSTATE CANCER AND TREATMENTS THEREOF

Related Patent Applications

This patent application claims the benefit of U.S. Provisional Patent Application Nos. 60/634,839 and 60/683,257 filed on 10 December 2004 and 20 May 2005, respectively, entitled "METHODS FOR

IDENTIFYING RISK OF BREAST CANCER OR PROSTATE CANCER AND TREATMENTS THEREOF," naming Richard B. Roth et al. as inventors, and designated by attorney docket no. SEQ-4093-PV and SEQ-4093- PV2, respectively. The contents of each of these provisional patent applications is hereby incorporated by reference in its entirety, including all text and drawings, in jurisdictions providing for such an incorporation.

Field of the Invention

The invention relates to genetic methods for identifying subjects at risk of breast cancer or prostate cancer and treatments that specifically target the disease(s).

Background

Breast cancer is the third most common cancer, and the most common cancer in women, as well as a cause of disability, psychological trauma, and economic loss. Breast cancer is the second most common cause of cancer death in women in the United States, in particular for women between the ages of 15 and 54, and the leading cause of cancer-related death (Forbes, Seminars in Oncology, vol.24(l), Suppl 1 , 1997: pp.Sl-20-Sl-35). Indirect effects of the disease also contribute to the mortality from breast cancer including consequences of advanced disease, such as metastases to the bone or brain. Complications arising from bone marrow suppression, radiation fibrosis and neutropenic sepsis, collateral effects from therapeutic interventions, such as surgery, radiation, chemotherapy, or bone marrow transplantation-also contribute to the morbidity and mortality from this disease.

While the pathogenesis of breast cancer is unclear, transformation of normal breast epithelium to a malignant phenotype may be the result of genetic factors, especially in women under thirty (Miki, et al., Science, 266: 66-71 (1994)). However, it is likely that other, non-genetic factors also have a significant effect on the etiology of the disease. Regardless of its origin, breast cancer morbidity increases significantly if it is not detected early in its progression. Thus, considerable efforts have focused on the elucidation of early cellular events surrounding transformation in breast tissue. Such efforts have led to the identification of several potential breast cancer markers. For example, alleles of the BRCAl and BRCA2 genes have been linked to hereditary and early-onset breast cancer (Wooster, et al., Science, 265: 2088-2090 (1994)). However, BRCAl is limited as a cancer marker because BRCAl mutations fail to account for the majority of breast cancers (Ford, et al., British J. Cancer, 72: 805-812 (1995)). Similarly, the BRCA2 gene, which has been linked to forms of hereditary breast cancer, accounts for only a small portion of total breast cancer cases.

Worldwide, prostate cancer is the fourth most prevalent cancer in men. In North America and Northern Europe, it is by far the most common male cancer and is the second leading cause of cancer death in men. In the United States alone, there will be an estimated 220,990 cases in 2003, and an estimated 28,900 men will die from prostate cancer-second only to lung cancer. The age-adjusted incidence rate per 100,000 in the US (for the years of 1995-99) is 168.9, which accounts for 30% of all cancer incidence in males. The age-adjusted death rate per 100,000 in the US (for the years of 1995-99) is 33.9, which is 13% of all cancer deaths in males.

Prostate cancer is generally considered a late-onset cancer, with most cases being found in men over the age of sixty-five. The incidence rate of this cancer has leveled off in recent years, but continues to effect an extremely large number of men. One possible reason for the high incidence rate of prostate cancer is there are no early warning signs of developing the disease. Also there is only a minimal set of known risk factors, including age, ethnicity (African- American men are at a higher risk of developing prostate cancer than Caucasian men), and family predisposition. Family studies have suggested that 5%-10% of all prostate cancer cases might be attributable to genetic factors. Although survival rates following the diagnosis of prostate cancer continue to improve, especially if detected at the local or regional stage, finding genetic links to prostate cancer will certainly provide a means for earlier detection as well as provide novel starting points for therapeutic modalities, both of which will enhance long term survival.

Summary

It has been discovered that polymorphic variations of the ICAM \oci (chromosome 19pl3.2) in human genomic DNA are associated with occurrence of breast cancer and prostate cancer. High-density SNP mapping showed that the extent of association spans 20 kb and includes the intercellular adhesion molecule genes JCAMl, ICAM4 and JCAM5. Thus, featured herein are methods for identifying a subject at risk of breast cancer or prostate cancer, and/or determining risk of breast cancer or prostate cancer in a subject, which comprise detecting the presence or absence of one or more polymorphic variations associated with breast cancer or prostate cancer in a nucleic acid sample from the subject. The one or more polymorphic variations often are detected in or near the ICAM nucleotide sequence, such as the nucleotide sequence set forth as SEQ ID NOs: 1, 2, 3 or 4 or a substantially identical nucleotide sequence thereof.

It has also been discovered that polymorphic variations of the ICAM loci in human genomic DNA are associated with occurrence of organ metastases. Thus, featured herein are methods for prognosing an aggressive form of breast cancer (e.g., a cancer with an increased risk of metastasis to other organs) in a subject, which comprise detecting the presence or absence of one or more polymorphic variations associated with an aggressive form of breast cancer in a nucleic acid sample from the subject, wherein the presence or absence of one or more of such polymorphic variations associated is indicative of an aggressive breast cancer prognosis in the subject. Also, featured is a method for inhibiting metastasis of breast cancer, which comprises inhibiting an ICAMl nucleic acid or substantially identical nucleic acid thereof (e.g., reducing the amount of polypeptide expressed from mRNA encoded by the nucleotide sequence), or inhibiting an ICAMl polypeptide or substantially identical polypeptide thereof (e.g., inhibiting the function of the ICAMl polypeptide with an antibody). The inhibition can be effected by contacting a system with a molecule having the inhibitory activity, where the system sometimes is a group of cells in vitro, a tissue sample in vitro, or an animal such as a human, often a female. In an embodiment, the ICAMl nucleic acid or substantially identical nucleic acid thereof is inhibited by contacting cells overexpressing the ICAMl nucleotide sequence with an RNA molecule, and in certain embodiments, the RNA molecule is double stranded with one strand complementary to a subsequence of the ICAMl nucleotide sequence. In some embodiments, the ICAMl function is inhibited by contacting an ICAMl molecule with an appropriate antibody specific for ICAMl. Such methods sometimes are employed after a prognostic test determinative of the risk of breast cancer or an aggressive form of breast cancer is performed. Also featured are nucleic acids that encode an ICAM polypeptide, and include one or more polymorphic variations associated with breast cancer or prostate cancer, and oligonucleotides which hybridize to those nucleic acids. Also provided are polypeptides encoded by nucleic acids having an ICAM nucleotide sequence, which include the full-length polypeptide, isoforms and fragments thereof. In addition, featured are methods for identifying candidate therapeutic molecules for treating breast cancer or prostate cancer, and related disorders, as well as methods of treating breast cancer in a subject by administering a therapeutic molecule.

Also provided are compositions comprising a breast cancer cell or prostate cancer cell and/or an ICAM nucleic acid, or a fragment or substantially identical nucleic acid thereof, with a RNAi, siRNA, antisense DNA or RNA, or ribozyme nucleic acid designed from an ICAM nucleotide sequence. In an embodiment, the nucleic acid is designed from an ICAM nucleotide sequence that includes one or more breast cancer or prostate cancer associated polymorphic variations, and in some instances, specifically interacts with such a nucleotide sequence. Further, provided are arrays of nucleic acids bound to a solid surface, in which one or more nucleic acid molecules of the array are ICAM nucleic acids, or a fragment or substantially identical nucleic acid thereof, or a complementary nucleic acid of the foregoing. Featured also are compositions comprising a breast cancer cell and/or a protein, polypeptide or peptide encoded by an ICAM nucleic acid with an antibody that specifically binds to the protein, polypeptide or peptide. Featured also are compositions comprising a prostate cancer cell and/or a protein, polypeptide or peptide encoded by an ICAM nucleic acid with an antibody that specifically binds to the protein, polypeptide or peptide. In an embodiment, the antibody specifically binds to an epitope in an ICAM protein, polypeptide or peptide that includes a non-synonymous amino acid modification associated with breast cancer or prostate cancer, such as a valine at position 301 of a ICAM5 protein, polypeptide or peptide.

A genomic ICAM nucleotide sequence is set forth in SEQ ID NO:1. In certain embodiments, a polymorphic variation selected from the group consisting of rs2884487, rs2358580, rs2304236, rsl059840, rslO59843, rsl 1 1 15, rslO59849, rslO59855, rs5030386, rs5030339, rs5030387, rs5030388, rsl 799766, rs5030389, rs5490, rsl 1575070, rs5030340, rs5030390, rs5030391, rs3093035, rsl 1667983, rs5030341, rs5030342, rs5030343, rs5030344, rs5030347, rs5030348, rs5030349, rs5030350, rs5030351 , rs5491, rs5030352, rs5030353, rsl 0420063, rsl 1879117, rs5030354, rs5030355, rs281428, rs5030358, rs5030359, rs5030392, rs5030393, rs5030360, rs5030394, rs281429, rs5030361 , rs5030362, rs281430, rs281431 , rs5030395, rs5827095, rs281432, rs5030364, rs5030365, rs5030368, rs5030369, rs3073809, rs2358581, rs7258215, rs5030371 , rs5030372, rs281433, rs5030374, rs5030375, rs5030397, rs281434, rsl2462944, rs5030398, rs5030378, rsl2459133, rs5030399, rs5492, rsl800019, rsl 799969, rs5493, rs5030381, rs5494, rs3093033, rs5495, rsl 801714, rs2071441, rs5496, rs5497, rs5030382, rs5030400, rs2071440, rs5499, rs3093032, rslO57981, rs5500, rs5501, rs5030383, rs281436, rs923366, rs281437, rs3093030, rs5030384, rs5030385, rs3810159, rs281438, rs3093029, rs2735442, rs2569693, rs281439, rs281440, rs2569694, rsl 1575073, rs2569695, rs2075741, rsl 1575074, rs2569696, rs2735439, rs2569697, rs2075742, rs2569698, rsl 1669397, rs901886, rs885742, rs2569699, rsl 1549918, rs2569700, rs2228615, rs2569701, rs2569702, rs2735440, rs2569703, rslO418913, rslO56536, rs2569704, rsl 1673661 , rsl0402760, rs2569706, rs2569707, rs2436545, rs2436546, rs2916060, rs2916059, rs2916058, rs2569708, rs735747, rs885743, rs710845, rs2569709, rs2569710, rs256971 1 , rs2569712, rsl2610026, rs4804129, rsl2150978, rs439843, rs892188, rs2291473, rs281416, rs281417, rs882589, rslO48941, rs281418, rs430092, rs368835, rs2358583, rs378395, rs395782, rslO45384, rs281427, rs3745264, rs281426, rs281425, rs281424, rs281423, rs281422, rs281420, rs3745263, rs3745262, rs3745261 , rs3181049, rs281412, rs3181048, rs2230399, rs2278442, rs3181047, rs3181046, rs2304237, rs281413, rsl058154, rs3176769, rs2304238, rs2304239, rs2304240, rs3176768, rs3176767, rs3176766, rs281414, rs281415, position 45003 of SEQ ID NO: 1, and position 47504 of SEQ ID NO: 1 were tested for association with breast cancer or prostate cancer. The same polymorphic variant may be assigned two dbSNP numbers that correspond to the variant on both the forward and reverse nucleic acid strands (i.e., strands with opposite orientations). This is the case with rslO56538 and rsl 1549918, which correspond to the same polymorphic variant at position 44338 in SEQ ID NO:1. Polymorphic variants at the following positions in particular were associated with an increased risk of breast cancer: rs5030382, rs281439, rsl 1549918, and rs2228615. At these positions in SEQ ID NO: 1 , an adenine at position 37083, a guanine at position 41510, a cytosine at position 44338, and a guanine at position 44768, in particular were associated with risk of breast cancer.

Polymorphic variants at the following positions in particular were associated with an increased risk of prostate cancer: rsl l549918 and rs2228615. At these positions in SEQ ID NO: 1, a cytosine at position 44338, and a guanine at position 44768, in particular were associated with risk of prostate cancer.

Brief Description of the Figures

Figure 1 shows proximal SNPs in and around the ICAM region. The position of each SNP on the chromosome is presented on the x-axis. The y-axis gives the negative logarithm (base 10) of the p-value comparing the estimated allele in the case group to that of the control group.

Figure 2 shows genotype analysis of 15 SNPs in a 20-kb window around the ICAM region. A, P-values for each SNP in the breast cancer discovery sample. B, Meta-analysis P-values of the breast cancer replication samples (gray) and all three samples (black). C, P-values of the prostate cancer samples. D, Gene map and chromosomal positions as in Figure 1. SNP locations are indicated as tick marks. E, Estimates of linkage disequilibrium in the combined breast cancer sample. Gray-scale ranges from white (LD = 0) to black (LD = 1 ) with increments of 0.1.

Figure 3 shows ICAMl expression in normal and cancer cell lines and tissues. (A) Quantitation of ICAMl protein expression in human cell lines by flow cytometry. Values plotted were obtained from the background- subtracted mean fluorescence intensity (MFI) of at least two independent antibody staining experiments and normalized to the MFI of MDA-MB-231 cells. MDA-MB-231 and MDA-MB-435 data are from four independent experiments. Data shown represent means ± STD. (B) Quantitation of ICAMI mRNA expression in human tissues using QGE by MassARRAY. Expression was normalized to that of gamma 1 actin (ACTGl) as described in Materials and Methods. Data shown represent means of triplicate experiments ± STD.

Figure 4 shows suppression of ICAMl mRNA and protein levels by siRNA. (A) ICAMl mRNA expression in MCF-7 and MDA-MD-231 cells harvested two days post-siRNA-transfection. In Figure 4A, the top graph corresponds to MCF-7 cells, while the bottom graph corresponds to MDA-MD-231 cells. Expression was quantitated using QGE by MassARRAY. The homologous 1CAM5 mRNA was analyzed in parallel to determine target specificity of the siRNA. Values were normalized to ACTGl. NT, lipofectamine only. (B) ICAMl protein expression in MCF-7 and MDA-MD-231 cells harvested three days post-siRNA-transfection. Expression was quantitated by flow cytometry and expressed as background-subtracted mean fluorescence intensity. Figure 5 shows the effect of ICAMl siRNA on proliferation of MDA-MB-231 and MDA-MB-435 cells.

Cells were plated onto triplicate wells and proliferation assessed by metabolic activity based on WST-I absorbance. Values shown are normalized to values read on day 1 post-siRNA-transfection. Data from a^' representative siRNA transfection experiment out of at least three independent experiments with similar results are shown. Detailed Description

It has been discovered that polymorphic variations in the ICAM region described herein are associated with an increased risk of breast cancer or prostate cancer. In addition, it has been discovered that polymorphic variations in the ICAM region described herein are also associated with an increased risk of organ metastases, therefore the polymorphic variations also serve as valuable prognostic markers. More specifically, the susceptibility allele of the variant rs281439 in the 5' flanking region of ICAM5 correlated with a high rate of inter- organ metastases (/^>=0.003). A similar effect was observed for the non-synonymous SNP in the C-terminal immunoglobulin domain of ICAMl (K469E) where 7% of the individuals homozygous for the susceptibility (K) allele had organ metastases as opposed to 0% of the patients homozygous for the protective (E) allele. Further, it was demonstrated that siRNA duplexes specifically targeting endogenous human ICAMl substantially reduce invasion of human epithelial breast cancer cells in vitro. These results were further strengthened by complementary experiments employing ICAMl antibody. The effect on invasion was strong in MDA-MB-435 cells, which express a high level of ICAMl protein, demonstrating that dependence on ICAMl for invasion correlates with protein expression level. Altogether these findings indicate that ICAMl function is required for invasion of metastatic breast cancer cells.

All ICAM proteins are type I transmembrane glycoproteins, contain 2-9 immunoglobulin-like C2-type domains, and bind to the leukocyte adhesion LFA-I protein. The proteins are members of the intercellular adhesion molecule (ICAM) family. The gene ICAMl (intercellular adhesion molecule-1) is also known as human rhinovirus receptor, BB2, CD54. and cell surface glycoprotein P3.58. ICAMl has been mapped to chromosomal position 19pl3.3-pl 3.2. ICAMl (CD54) typically is expressed on endothelial cells and cells of the immune system. ICAMl binds to integrins of type CDl Ia / CD18, or CDl I b / CDl 8. ICAMl is also exploited by Rhinovirus as a receptor.

The gene ICAM4 (intercellular adhesion molecule 4) is also known as the Landsteiner-Wiener blood group or LW. ICAM4 has been mapped to 19pl3.2-cen. The protein encoded by this gene is a member of the intercellular adhesion molecule (ICAM) family. A glutamine to arginine polymorphism in this protein is responsible for the Landsteiner-Wiener blood group system (GLN=WB(A); ARG=WB(B). This gene consists of 3 exons and alternative splicing generates 2 transcript variants.

The gene ICAM5 (intercellular adhesion molecule 5) is also known as telencephalin. ICAM5 has been mapped to 19pl3.2. The protein encoded by the gene is expressed on the surface of telencephalic neurons and displays two types of adhesion activity, homophilic binding between neurons and heterophilic binding between neurons and leukocytes. It may be a critical component in neuron-microglial cell interactions in the course of normal development or as part of neurodegenerative diseases.

Breast Cancer and Sample Selection

Breast cancer is typically described as the uncontrolled growth of malignant breast tissue. Breast cancers arise most commonly in the lining of the milk ducts of the breast (ductal carcinoma), or in the lobules where breast milk is produced (lobular carcinoma). Other forms of breast cancer include Inflammatory Breast Cancer and Recurrent Breast Cancer. Inflammatory breast cancer is a rare, but very serious, aggressive type of breast cancer. The breast may look red and feel warm with ridges, welts, or hives on the breast; or the skin may look wrinkled. It is sometimes misdiagnosed as a simple infection. Recurrent disease means that the cancer has come back after it has been treated. It may come back in the breast, in the soft tissues of the chest (the chest wall), or in another part of the body.

As used herein, the term "breast cancer" refers to a condition characterized by anomalous rapid proliferation of abnormal cells in one or both breasts of a subject. The abnormal cells often are referred to as "neoplastic cells," which are transformed cells that can form a solid tumor. The term "tumor" refers to an abnormal mass or population of cells (i.e. two or more cells) that result from excessive or abnormal cell division, whether malignant or benign, and pre-cancerous and cancerous cells. Malignant tumors are distinguished from benign growths or tumors in that, in addition to uncontrolled cellular proliferation, they can invade surrounding tissues and can metastasize. In breast cancer, neoplastic cells may be identified in one or both breasts only and not in another tissue or organ, in one or both breasts and one or more adjacent tissues or organs (e.g. lymph node), or in a breast and one or more non-adjacent tissues or organs to which the breast cancer cells have metastasized.

The term "invasion" as used herein refers to the spread of cancerous cells to adjacent surrounding tissues. The term "metastasis" as used herein refers to a process in which cancer cells travel from one organ or tissue to another non-adjacent organ or tissue. Cancer cells in the breast(s) can spread to tissues and organs of a subject, and conversely, cancer cells from other organs or tissue can invade or metastasize to a breast. Cancerous cells from the breast(s) may invade or metastasize to any other organ or tissue of the body. Breast cancer cells often invade lymph node cells and/or metastasize to the liver, brain and/or bone and spread cancer in these tissues and organs. Breast cancers can spread to other organs and tissues and cause lung cancer, prostate cancer, colon cancer, ovarian cancer, cervical cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, bladder cancer, hepatoma, colorectal cancer, uterine cervical cancer, endometrial carcinoma, salivary gland carcinoma, kidney cancer, vulval cancer, thyroid cancer, hepatic carcinoma, skin cancer, breast cancer, ovarian cancer, neuroblastoma, myeloma, various types of head and neck cancer, acute lymphoblastic leukemia, acute myeloid leukemia, Ewing sarcoma and peripheral neuroepithelioma, and other carcinomas, lymphomas, blastomas, sarcomas, and leukemias. In an effort to detect breast cancer as early as possible, regular physical exams and screening mammograms often are prescribed and conducted. A diagnostic mammogram often is performed to evaluate a breast complaint or abnormality detected by physical exam or routine screening mammography. If an abnormality seen with diagnostic mammography is suspicious, additional breast imaging (with exams such as ultrasound) or a biopsy may be ordered. A biopsy followed by pathological (microscopic) analysis is a definitive way to determine whether a subject has breast cancer. Excised breast cancer samples often are subjected to the following analyses: diagnosis of the breast tumor and confirmation of its malignancy; maximum tumor thickness; assessment of completeness of excision of invasive and in situ components and microscopic measurements of the shortest extent of clearance; level of invasion; presence and extent of regression; presence and extent of ulceration; histological type and special variants; pre-existing lesion; mitotic rate; vascular invasion; neurotropism; cell type; tumor lymphocyte infiltration; and growth phase.

The stage of a breast cancer can be classified as a range of stages from Stage 0 to Stage IV based on its size and the extent to which it has spread. The following table summarizes the stages: TABLE 1

Stage 0 cancer is a contained cancer that has not spread beyond the breast ductal system. Fifteen to twenty percent of breast cancers detected by clinical examinations or testing are in Stage 0 (the earliest form of breast cancer). Two types of Stage 0 cancer are lobular carcinoma in situ (LCIS) and ductal carcinoma in situ (DCIS). LCIS indicates high risk for breast cancer. Many physicians do not classify LCIS as a malignancy and often encounter LCIS by chance on breast biopsy while investigating another area of concern. While the microscopic features of LCIS are abnormal and are similar to malignancy, LCIS does not behave as a cancer (and therefore is not treated as a cancer). LCIS is merely a marker for a significantly increased risk of cancer anywhere in the breast. However, bilateral simple mastectomy may be occasionally performed if LCIS patients have a strong family history of breast cancer. In DCIS the cancer cells are confined to milk ducts in the breast and have not spread into the fatty breast tissue or to any other part of the body (such as the lymph nodes). DCIS may be detected on mammogram as tiny specks of calcium (known as microcalcifications) 80% of the time. Less commonly DCIS can present itself as a mass with calcifications (15% of the time); and even less likely as a mass without calcifications (<5% of the time). Breast biopsy is used to confirm DCIS. Standard DCIS treatment is breast-conserving therapy (BCT): lumpectomy followed by radiation treatment or mastectomy. To date, DCIS patients have chosen equally among lumpectomy and mastectomy as their treatment option, though specific cases may sometimes favor lumpectomy over mastectomy or vice versa.

In Stage I, the primary (original) cancer is 2 cm or less in diameter and has not spread to the lymph nodes. In Stage IIA, the primary tumor is between 2 and 5 cm in diameter and has not spread to the lymph nodes. In Stage HB, the primary tumor is between 2 and 5 cm in diameter and has spread to the axillary (underarm) lymph nodes; or the primary tumor is over 5 cm and has not spread to the lymph nodes. In Stage HIA, the primary breast cancer of any kind that has spread to the axillary (underarm) lymph nodes and to axillary tissues. In Stage IHB, the primary breast cancer is any size, has attached itself to the chest wall, and has spread to the pectoral (chest) lymph nodes. In Stage IV, the primary cancer has spread out of the breast to other parts of the body (such as bone, lung, liver, brain). The treatment of Stage IV breast cancer focuses on extending survival time and relieving symptoms.

Based in part upon selection criteria set forth above, individuals having breast cancer can be selected for genetic studies. Also, individuals having no history of cancer or breast cancer often are selected for genetic studies. Other selection criteria can include: a tissue or fluid sample is derived from an individual characterized as Caucasian; the sample was derived from an individual of German paternal and maternal descent; the database included relevant phenotype information for the individual; case samples were derived from individuals diagnosed with breast cancer; control samples were derived from individuals free of cancer and no family history of breast cancer; and sufficient genomic DNA was extracted from each blood sample for all allelotyping and genotyping reactions performed during the study. Phenotype information included pre- or post-menopausal, familial predisposition, country or origin of mother and father, diagnosis with breast cancer (date of primary diagnosis, age of individual as of primary diagnosis, grade or stage of development, occurrence of metastases, e.g., lymph node metastases, organ metastases), condition of body tissue (skin tissue, breast tissue, ovary tissue, peritoneum tissue and myometrium), method of treatment (surgery, chemotherapy, hormone therapy, radiation therapy).

Provided herein is a set of blood samples and a set of corresponding nucleic acid samples isolated from the blood samples, where the blood samples are donated from individuals diagnosed with breast cancer. The sample set often includes blood samples or nucleic acid samples from 100 or more, 150 or more, or 200 or more individuals having breast cancer, and sometimes from 250 or more, 300 or more, 400 or more, or 500 or more individuals. The individuals can have parents from any place of origin, and in an embodiment, the set of samples are extracted from individuals of German paternal and German maternal ancestry. The samples in each set may be selected based upon five or more criteria and/or phenotypes set forth above.

Prostate Cancer and Sample Selection

Prostate cancer is the rapid proliferation of abnormal cells in the prostate gland. While normal prostate cells reproduce and develop into healthy prostate tissue, these abnormal cells proliferate rapidly and rarely form normal prostate tissue. Instead, the abnormal cells proliferate, form tumors, disrupt the prostate, and spread to surrounding tissues. As used herein, the term "prostate cancer" refers to a condition characterized by anomalous rapid proliferation of abnormal cells in the prostate gland of a subject. The abnormal cells often are referred to as "neoplastic cells," which are transformed cells that can form a solid tumor. The term "tumor" refers to an abnormal mass or population of cells (i.e. two or more cells) that result from excessive or abnormal cell division, whether malignant or benign, and pre-cancerous and cancerous cells. Malignant tumors are distinguished from benign growths or tumors in that, in addition to uncontrolled cellular proliferation, they can invade surrounding tissues and can metastasize. In prostate cancer, neoplastic cells may be identified in the prostate only and not in another tissue or organ, in the prostate and one or more adjacent tissues or organs (e.g. spine, lungs, liver or brain), or in a lung and one or more non-adjacent tissues or organs to which the lung cancer cells have metastasized. The term "invasion" as used herein refers to the spread of cancerous cells to adjacent surrounding tissues.

The term "metastasis" as used herein refers to a process in which cancer cells travel from one organ or tissue to another non-adjacent organ or tissue. Cancer cells in the prostate can spread to tissues and organs of a subject, and conversely, cancer cells from other organs or tissue can invade or metastasize to the prostate. Cancerous cells from the prostate may invade or metastasize to any other organ or tissue of the body. Prostate cancer cells often invade spine cells (e.g., vertebral column) and/or metastasize to the lungs, liver, and/or brain and spread cancer in these tissues and organs. Lung cancers can spread to other organs and tissues and cause breast cancer, lung cancer, colon cancer, ovarian cancer, cervical cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, bladder cancer, hepatoma, colorectal cancer, uterine cervical cancer, endometrial carcinoma, salivary gland carcinoma, kidney cancer, vulval cancer, thyroid cancer, hepatic carcinoma, skin cancer, melanoma, ovarian cancer, neuroblastoma, myeloma, various types of head and neck cancer, acute lymphoblastic leukemia, acute myeloid leukemia, Ewing sarcoma and peripheral neuroepithelioma, and other carcinomas, lymphomas, blastomas, sarcomas, and leukemias.

The prostate is about the size of a walnut and can be divided into two parts referred to as the right or left lobes. It lies just below the urinary bladder and surrounds the upper part of the urethra. The urethra is the tube that carries urine from the bladder and semen from the sex glands out through the penis. As one of a man's sex glands, the prostate is affected by male sex hormones (most notably testosterone). These hormones stimulate the activity of the prostate and the replacement of prostate cells as they wear out.

The prostate gland surrounds the neck of the bladder and urethra, and most prostate cancers initially occur in the peripheral zone of the prostate gland, away from the urethra. Tumors within this zone may not produce any symptoms and, as a result, most men with early-stage prostate cancer will not present clinical symptoms of the disease until significant progression has occurred. Tumor progression into the transition zone of the prostate may lead to urethral obstruction, thus producing the first symptoms of the disease. However, these clinical symptoms are indistinguishable from the common non-malignant condition of benign prostatic hyperplasia (BPH).

Early detection of the disease has proven to be critical for survival among prostate cancer patients. For example, there is 100% five-year survival rates for prostate cancer patients diagnosed at the local and regional stage; whereas, the five-year survival rate for prostate cancer patients in which the cancer has metastasized is only

34%. Early detection and diagnosis of prostate cancer currently relies on digital rectal examinations (DRE), prostate specific antigen (PSA) measurements, transrectal ultrasonography (TRUS), and transrectal needle biopsy

(TRNB). At present, serum PSA measurement in combination with DRE are the most common tools used to detect and diagnose prostate cancer. Both have major limitations which have fueled intensive research into finding better diagnostic markers for prostate cancer.

The most common method of staging prostate cancer is by using a system called the TNM system that stands for Tumor, Node, Metastases. Tables A, B and C below describe the characteristics of each stage and the available treatment options: TABLE A: size of the primary tumor

TABLE B: extent of lymph node involvement

TABLE C: presence or absence of metastases

In addition, the aggressiveness of prostate cancer may be measured using the Gleason scale (2-10), e.g., 2 = normal looking tumor, 10 = very abnormal looking tumor. Gleason grading system involves assigning numbers (called a Gleason grade) to cancerous prostate tissue, ranging from 1 through 5, based on how much the arrangement of the cancer cells mimics the way normal prostate cells form glands. Two grades are assigned to the most common patterns of cells that appear; these two grades (they can be the same or different) are then added together to determine the Gleason score (a number from 1 to 10).

A high Gleason score indicates that the prostate cancer is aggressive and likely to metastasize to surrounding organs or tissues. Prostate cancer most commonly spreads to the surrounding bones, including the pelvis, hips, pubic bone and spine. In 90% of prostate cancer metastasis, the cancer spreads to the spine, and often involves vertebral column. In 50% of prostate cancer metastasis, the cancer spreads to the either one or both of the lungs, while in 25% of prostate cancer metastasis, the cancer spreads to the liver. In rare cases, prostate cancer may spread to the brain, with a poor prognosis (average survival 7.6 months).

There is no available marker that can predict the emergence of the typically fatal metastatic stage of prostate cancer. Diagnosis of metastatic stage is presently achieved by open surgical or laparoscopic pelvic lymphadenectomy, whole body radionuclide scans, skeletal radiography, and/or bone lesion biopsy analysis. Clearly, identification of susceptibility genes and other less invasive diagnostic methods offer the promise of easing the difficulty those procedures place on a patient, as well as improving diagnostic accuracy and opening therapeutic options. A similar problem is the lack of effective prognostic markers for determining which cancers are indolent and which ones are or will be aggressive. PSA, for example, fails to discriminate accurately between indolent and aggressive cancers. Until there are prostate cancer markers capable of predicting susceptibility to development of the disease, reliably identifying early-stage disease, and predicting susceptibility to metastasis, the management of prostate cancer will continue to be extremely difficult.

Inclusion or exclusion of samples for a prostate cancer pool to be used in a genetic study may be based upon the following criteria: relevant phenotype information for the individual (e.g., case samples derived from individuals diagnosed with prostate cancer); or type of prostate cancer diagnosed. Control samples may be selected based on relevant phenotype information for the individual (e.g., derived from individuals free of any cancer); and no family history of cancer. Additional phenotype information collected for both cases and controls may include age of the individual, gender, date of primary diagnosis, age of individual as of primary diagnosis, age of individual when sample collected, or method of treatment, height, weight, disease status, such as heart disease, hypertension, vascular disease, CNS disease, gastrointestinal disease, urogenital disease, asthma, other cancers, diabetes, and also smoking status. In a preferred embodiment, the same phenotypic information may be collected for the parents of cases and controls, making additional phenotypic analysis possible.

Based in part upon selection criteria set forth above, individuals suffering from prostate cancer can be selected for genetic studies. Also, individuals having no history of cancer, particularly prostate cancer, often are selected for genetic studies as controls. Polymorphic Variants Associated with Breast Cancer and Prostate Cancer

A large-scale association study identified the ICAM gene region as a breast and prostate cancer susceptibility locus. As used herein, the term "polymorphic site" refers to a region in a nucleic acid at which two or more alternative nucleotide sequences are observed in a significant number of nucleic acid samples from a population of individuals. A polymorphic site may be a nucleotide sequence of two or more nucleotides, an inserted nucleotide or nucleotide sequence, a deleted nucleotide or nucleotide sequence, or a microsatellite, for example. A polymorphic site that is two or more nucleotides in length may be 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15 or more, 20 or more, 30 or more, 50 or more, 75 or more, 100 or more, 500 or more, or about 1000 nucleotides in length, where all or some of the nucleotide sequences differ within the region. A polymorphic site is often one nucleotide in length, which is referred to herein as a "single nucleotide polymorphism" or a "SNP."

Where there are two, three, or four alternative nucleotide sequences at a polymorphic site, each nucleotide sequence is referred to as a "polymorphic variant" or "nucleic acid variant." Where two polymorphic variants exist, for example, the polymorphic variant represented in a minority of samples from a population is sometimes referred to as a "minor allele" and the polymorphic variant that is more prevalently represented is sometimes referred to as a "major allele." Many organisms possess a copy of each chromosome (e.g., humans), and those individuals who possess two major alleles or two minor alleles are often referred to as being "homozygous" with respect to the polymorphism, and those individuals who possess one major allele and one minor allele are normally referred to as being "heterozygous" with respect to the polymorphism. Individuals who are homozygous with respect to one allele are sometimes predisposed to a different phenotype as compared to individuals who are heterozygous or homozygous with respect to another allele.

Furthermore, a genotype or polymorphic variant may be expressed in terms of a "haplotype," which as used herein refers to two or more polymorphic variants occurring within genomic DNA in a group of individuals within a population. For example, two SNPs may exist within a gene where each SNP position includes a cytosine variation and an adenine variation. Certain individuals in a population may carry one allele (heterozygous) or two alleles (homozygous) having the gene with a cytosine at each SNP position. As the two cytosines corresponding to each SNP in the gene travel together on one or both alleles in these individuals, the individuals can be characterized as having a cytosine/cytosine haplotype with respect to the two SNPs in the gene.

As used herein, the term "phenotype" refers to a trait which can be compared between individuals, such as presence or absence of a condition, a visually observable difference in appearance between individuals, metabolic variations, physiological variations, variations in the function of biological molecules, and the like. An example of a phenotype is occurrence of breast cancer.

Researchers sometimes report a polymorphic variant in a database without determining whether the variant is represented in a significant fraction of a population. Because a subset of these reported polymorphic variants are not represented in a statistically significant portion of the population, some of them are sequencing errors and/or not biologically relevant. Thus, it is often not known whether a reported polymorphic variant is statistically significant or biologically relevant until the presence of the variant is detected in a population of individuals and the frequency of the variant is determined. Methods for detecting a polymorphic variant in a population are described herein, specifically in Example 2. A polymorphic variant is statistically significant and often biologically relevant if it is represented in 5% or more of a population, sometimes 10% or more, 15% or more, or 20% or more of a population, and often 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, or 50% or more of a population. A polymorphic variant may be detected on either or both strands of a double-stranded nucleic acid. Also, a polymorphic variant may be located within an intron or exon of a gene or within a portion of a regulatory region such as a promoter, a 5' untranslated region (UTR), a 3 ' UTR, and in DNA (e.g., genomic DNA (gDNA) and complementary DNA (cDNA)), RNA (e.g., mRNA, tRNA, and rRNA), or a polypeptide. Polymorphic variations may or may not result in detectable differences in gene expression, polypeptide structure, or polypeptide function.

In the genetic analysis that associated the polymorphic variants set forth in Table 9 with breast cancer or prostate cancer, samples from individuals having breast cancer and individuals not having cancer were allelotyped and genotyped. The term "allelotype" as used herein refers to a process for determining the allele frequency for a polymorphic variant in pooled DNA samples from cases and controls. By pooling DNA from each group, an allele frequency for each SNP in each group is calculated. These allele frequencies are then compared to one another. Particular SNPs are considered as being associated with a particular disease when allele frequency differences calculated between case and control pools are statistically significant. The term "genotyped" as used herein refers to a process for determining a genotype of one or more individuals, where a "genotype" is a representation of one or more polymorphic variants in a population. Furthermore, a genotype or polymorphic variant may be expressed in terms of a "haplotype," which as used herein refers to two or more polymorphic variants occurring within genomic DNA in a group of individuals within a population. For example, two SNPs may exist within a gene where each SNP position includes a cytosine variation and an adenine variation. Certain individuals in a population may carry one allele (heterozygous) or two alleles (homozygous) having the gene with a cytosine at each SNP position. As the two cytosines corresponding to each SNP in the gene travel together on one or both alleles in these individuals, the individuals can be characterized as having a cytosine/cytosine haplotype with respect to the two SNPs in the gene.

It has been discovered that a polymorphic variation in the ICAM gene region is associated with the occurrence of breast cancer, and also associated with the occurrence of prostate cancer. Thus, featured herein are methods for identifying a risk of breast cancer or prostate cancer in a subject, which comprises detecting the presence or absence of one or more of the polymorphic variations described herein in a human nucleic acid sample.

Isolated Nucleic Acids

Featured herein are isolated nucleic acid variants depicted in SEQ ID Nos:l-4, and substantially identical nucleic acids thereof. A nucleic acid variant may be represented on one or both strands in a double-stranded nucleic acid or on one chromosomal complement (heterozygous) or both chromosomal complements (homozygous).

As used herein, the term "nucleic acid" includes DNA molecules (e.g., a complementary DNA (cDNA) and genomic DNA (gDNA)) and RNA molecules (e.g., mRNA, rRNA, siRNA and tRNA) and analogs of DNA or RNA, for example, by use of nucleotide analogs. The nucleic acid molecule can be single-stranded and it is often double-stranded. The term "isolated or purified nucleic acid" refers to nucleic acids that are separated from other nucleic acids present in the natural source of the nucleic acid. For example, with regard to genomic DNA, the term "isolated" includes nucleic acids which are separated from the chromosome with which the genomic DNA is naturally associated. An "isolated" nucleic acid is often free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5' and/or 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of 5' and/or 3' nucleotide sequences which flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived. Moreover, an "isolated" nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. As used herein, the term "gene" refers to a nucleotide sequence that encodes a polypeptide.

Also included herein are nucleic acid fragments. These fragments often are a nucleotide sequence identical to a nucleotide sequence in SEQ ID Nos:l-4, a nucleotide sequence substantially identical to a nucleotide sequence in SEQ ID Nos: l -4, or a nucleotide sequence that is complementary to the foregoing. The nucleic acid fragment may be identical, substantially identical or homologous to a nucleotide sequence in an exon or an intron in a nucleotide sequence of SEQ ID Nos: l-4. Further, the nucleic acid fragment may encode a full-length or mature polypeptide, or may encode a domain or part of a domain of a polypeptide. ICAM domains include, but are not limited to, transmembrane domains (520-591, 679-732, 952-1023, 1126-1179 base pairs of Figure 2), the protease domain (928-1461 base pairs of Figure 2), and the Zn-binding motif (HEXXH) present in the protease domain (1075-1089 base pairs of Figure 2). Sometimes, the fragment will comprises one or more of the polymorphic variations described herein as being associated with breast cancer. The nucleic acid fragment is often 50, 100, or 200 or fewer base pairs in length, and is sometimes about 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 2000, 3000, 4000, 5000, 10000, 15000, or 20000 base pairs in length. A nucleic acid fragment that is complementary to a nucleotide sequence identical or substantially identical to a nucleotide sequence in SEQ ID Nos:l-4 and hybridizes to such a nucleotide sequence under stringent conditions is often referred to as a "probe." Nucleic acid fragments often include one or more polymorphic sites, or sometimes have an end that is adjacent to a polymorphic site as described hereafter.

An example of a nucleic acid fragment is an oligonucleotide. As used herein, the term "oligonucleotide" refers to a nucleic acid comprising about 8 to about 50 covalently linked nucleotides, often comprising from about 8 to about 35 nucleotides, and more often from about 10 to about 25 nucleotides. The backbone and nucleotides within an oligonucleotide may be the same as those of naturally occurring nucleic acids, or analogs or derivatives of naturally occurring nucleic acids, provided that oligonucleotides having such analogs or derivatives retain the ability to hybridize specifically to a nucleic acid comprising a targeted polymorphism. Oligonucleotides described herein may be used as hybridization probes or as components of prognostic or diagnostic assays, for example, as described herein.

Oligonucleotides are typically synthesized using standard methods and equipment, such as the ABI™3900 High Throughput DNA Synthesizer and the EXPEDITE™ 8909 Nucleic Acid Synthesizer, both of which are available from Applied Biosystems (Foster City, CA). Analogs and derivatives are exemplified in U.S. Pat. Nos. 4,469,863; 5,536,821 ; 5,541 ,306; 5,637,683; 5,637,684; 5,700,922; 5,717,083; 5,719,262; 5,739,308; 5,773,601 ; 5,886,165; 5,929,226; 5,977,296; 6,140,482; WO 00/56746; WO 01/14398, and related publications. Methods for synthesizing oligonucleotides comprising such analogs or derivatives are disclosed, for example, in the patent publications cited above and in U.S. Pat. Nos. 5,614,622; 5,739,314; 5,955,599; 5,962,674; 6,1 17,992; in WO 00/75372; and in related publications.

Oligonucleotides may also be linked to a second moiety. The second moiety may be an additional nucleotide sequence such as a tail sequence (e.g., a polyadenosine tail), an adapter sequence (e.g., phage Ml 3 universal tail sequence), and others. Alternatively, the second moiety may be a non-nucleotide moiety such as a moiety which facilitates linkage to a solid support or a label to facilitate detection of the oligonucleotide. Such labels include, without limitation, a radioactive label, a fluorescent label, a chemiluminescent label, a paramagnetic label, and the like. The second moiety may be attached to any position of the oligonucleotide, provided the oligonucleotide can hybridize to the nucleic acid comprising the polymorphism.

Uses for Nucleic Acid Sequences

Nucleic acid coding sequences depicted in SEQ ID Nos:l-4 may be used for diagnostic purposes for detection and control of polypeptide expression. Also, included herein are oligonucleotide sequences such as antisense RNA, small-interfering RNA (siRNA) and DNA molecules and ribozymes that function to inhibit translation of a polypeptide. Antisense techniques and RNA interference techniques are known in the art and are described herein.

Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. The mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. For example, hammerhead motif ribozyme molecules may be engineered that specifically and efficiently catalyze endonucleolytic cleavage of RNA sequences corresponding to or complementary to the nucleotide sequences set forth in SEQ ID Nos:l-4. Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites which include the following sequences, GUA, GUU and GUC. Once identified, short RNA sequences of between fifteen (15) and twenty (20) ribonucleotides corresponding to the region of the target gene containing the cleavage site may be evaluated for predicted structural features such as secondary structure that may render the oligonucleotide sequence unsuitable. The suitability of candidate targets may also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using ribonuclease protection assays.

Antisense RNA and DNA molecules, siRNA and ribozymes may be prepared by any method known in the art for the synthesis of RNA molecules. These include techniques for chemically synthesizing oligodeoxyribonucleotides well known in the art such as solid phase phosphoramidite chemical synthesis. Alternatively, RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding the antisense RNA molecule. Such DNA sequences may be incorporated into a wide variety of vectors which incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Alternatively, antisense cDNA constructs that synthesize antisense RNA constitutively or inducibly, depending on the promoter used, can be introduced stably into cell lines.

DNA encoding a polypeptide also may have a number of uses for the diagnosis of diseases, including breast cancer and prostate cancer, resulting from aberrant expression of a target gene described herein. For example, the nucleic acid sequence may be used in hybridization assays of biopsies or autopsies to diagnose abnormalities of expression or function (e.g., Southern or Northern blot analysis, in situ hybridization assays).

In addition, the expression of a polypeptide during embryonic development may also be determined using nucleic acid encoding the polypeptide. As addressed infra, production of functionally impaired polypeptide is the cause of various disease states, breast cancer. In situ hybridizations using polypeptide as a probe may be employed to predict problems related to breast cancer. Further, as indicated infra, administration of human active polypeptide, recombinantly produced as described herein, may be used to treat disease states related to functionally impaired polypeptide. Alternatively, gene therapy approaches may be employed to remedy deficiencies of functional polypeptide or to replace or compete with dysfunctional polypeptide.

Expression Vectors, Host Cells, and Genetically Engineered Cells

Provided herein are nucleic acid vectors, often expression vectors, which contain a nucleotide sequence set forth in SEQ ID Nos:l-4 or a substantially identical sequence thereof. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked and can include a plasmid, cosmid, or viral vector. The vector can be capable of autonomous replication or it can integrate into a host DNA. Viral vectors may include replication defective retroviruses, adenoviruses and adeno-associated viruses for example. A vector can include a nucleotide sequence from SEQ ID Nos:l-4 in a form suitable for expression of an encoded ICAM polypeptide or ICAM nucleic acid in a host cell. A "ICAM polypeptide" is a polypeptide encoded by a nucleotide sequence from SEQ ID Nos:l-4 or a substantially identical nucleotide sequence thereof. The recombinant expression vector typically includes one or more regulatory sequences operatively linked to the nucleic acid sequence to be expressed. The term "regulatory sequence" includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence, as well as tissue-specific regulatory and/or inducible sequences. The design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of polypeptide desired, and the like. Expression vectors can be introduced into host cells to produce ICAM polypeptides, including fusion polypeptides. Recombinant expression vectors can be designed for expression of ICAM polypeptides in prokaryotic or eukaryotic cells. For example, ICAM polypeptides can be expressed in E. coli, insect cells (e.g., using baculovirus expression vectors), yeast cells, or mammalian cells. Suitable host cells are discussed further in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.

Expression of polypeptides in prokaryotes is most often carried out in E. coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion polypeptides. Fusion vectors add a number of amino acids to a polypeptide encoded therein, usually to the amino terminus of the recombinant polypeptide. Such fusion vectors typically serve three purposes: 1) to increase expression of recombinant polypeptide; 2) to increase the solubility of the recombinant polypeptide; and 3) to aid in the purification of the recombinant polypeptide by acting as a ligand in affinity purification. Often, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant polypeptide to enable separation of the recombinant polypeptide from the fusion moiety subsequent to purification of the fusion polypeptide. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith & Johnson, Gene 67: 31-40 (1988)), pMAL (New England Biolabs, Beverly, MA) and pRIT5 (Pharmacia, Piscataway, NJ) which fuse glutathione S-transferase (GST), maltose E binding polypeptide, or polypeptide A, respectively, to the target recombinant polypeptide.

Purified fusion polypeptides can be used in screening assays and to generate antibodies specific for ICAM polypeptides. In a therapeutic embodiment, fusion polypeptide expressed in a retroviral expression vector is used to infect bone marrow cells that are subsequently transplanted into irradiated recipients. The pathology of the subject recipient is then examined after sufficient time has passed (e.g., six (6) weeks).

Expressing the polypeptide in host bacteria with an impaired capacity to proteolytically cleave the recombinant polypeptide is often used to maximize recombinant polypeptide expression (Gottesman, S., Gene Expression Technology: Methods in Enzymology, Academic Press, San Diego, California 185: 1 19-128 (1990)). Another strategy is to alter the nucleotide sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E. coli (Wada et al, Nucleic Acids Res. 20: 2111-21 18 (1992)). Such alteration of nucleotide sequences can be carried out by standard DNA synthesis techniques. When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40. Recombinant mammalian expression vectors are often capable of directing expression of the nucleic acid in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Non-limiting examples of suitable tissue-specific promoters include an albumin promoter (liver-specific; Pinkert et al, Genes Dev. 1: 268-277 (1987)), lymphoid-specific promoters (Calame & Eaton, Adv. Immunol. 43: 235-275 (1988)), promoters of T cell receptors (Winoto & Baltimore, EMBO J. 8: 729- 733 (1989)) promoters of immunoglobulins (Banerji et al, Cell 33: 729-740 (1983); Queen & Baltimore, Cell 33: 741-748 (1983)), neuron-specific promoters (e.g., the neurofilament promoter; Byrne & Ruddle, Proc. Natl. Acad. Sci. USA 86: 5473-5477 (1989)), pancreas-specific promoters (Edlund et al, Science 230: 912-916 (1985)), and mammary gland-specific promoters (e.g., milk whey promoter; U.S. Patent No. 4,873,316 and European Application Publication No. 264,166). Developmentally-regulated promoters are sometimes utilized, for example, the murine hox promoters (Kessel & Gruss, Science 249: 374-379 (1990)) and the α-fetopolypeptide promoter (Campes & Tilghman, Genes Dev. 3: 537-546 (1989)).

A nucleic acid from SEQ ID Nos: l-4 may also be cloned into an expression vector in an antisense orientation. Regulatory sequences (e.g., viral promoters and/or enhancers) operatively linked to a nucleic acid of SEQ ID Nos:l-4 cloned in the antisense orientation can be chosen for directing constitutive, tissue specific or cell type specific expression of antisense RNA in a variety of cell types. Antisense expression vectors can be in the form of a recombinant plasmid, phagemid or attenuated virus. For a discussion of the regulation of gene expression using antisense genes see, e.g., Weintraub et al, Antisense RNA as a molecular tool for genetic analysis, Reviews - Trends in Genetics, Vol. 1(1) (1986).

Also provided herein are host cells that include a nucleotide sequence from SEQ ID Nos:l-4 within a recombinant expression vector or a fragment of a nucleotide sequence from SEQ ID Nos:l-4 which facilitate homologous recombination into a specific site of the host cell genome. The terms "host cell" and "recombinant host cell" are used interchangeably herein. Such terms refer not only to the particular subject cell but rather also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein. A host cell can be any prokaryotic or eukaryotic cell. For example, a ICAM polypeptide can be expressed in bacterial cells such as E. coli, insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells). Other suitable host cells are known to those skilled in the art. Vectors can be introduced into host cells via conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection" are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, transduction/infection, DEAE-dextran-mediated transfection, lipofection, or electroporation.

A host cell provided herein can be used to produce (i.e., express) a ICAM polypeptide. Accordingly, further provided are methods for producing a ICAM polypeptide using the host cells. In one embodiment, the method includes culturing host cells into which a recombinant expression vector encoding a ICAM polypeptide has been introduced in a suitable medium such that a ICAM polypeptide is produced. In another embodiment, the method further includes isolating a ICAM polypeptide from the medium or the host cell.

Also provided are cells or purified preparations of cells which include a transgene from SEQ ID Nos: l-4, or which otherwise misexpress ICAM polypeptide. Cell preparations can consist of human or non-human cells, e.g., rodent cells, e.g., mouse or rat cells, rabbit cells, or pig cells. In certain embodiments, the cell or cells include a transgene from SEQ ID Nos: l-4 (e.g., a heterologous form of a gene in SEQ ID Nos:l -4, such as a human gene expressed in non-human cells). The transgene can be misexpressed, e.g., overexpressed or underexpressed. In other embodiments, the cell or cells include a gene which misexpress an endogenous ICAM polypeptide (e.g., expression of a gene is disrupted, also known as a knockout). Such cells can serve as a model for studying disorders which are related to mutated or mis-expressed alleles or for use in drug screening. Also provided are human cells (e.g., a hematopoietic stem cells) transformed with a nucleic acid from SEQ ID Nos: l -4. Also provided are cells or a purified preparation thereof (e.g., human cells) in which an endogenous nucleic acid from SEQ ID Nos:l-4 is under the control of a regulatory sequence that does not normally control the expression of the endogenous gene corresponding to the sequence from SEQ ID Nos:l-4. The expression characteristics of an endogenous gene within a cell (e.g., a cell line or microorganism) can be modified by inserting a heterologous DNA regulatory element into the genome of the cell such that the inserted regulatory element is operably linked to the corresponding endogenous gene. For example, an endogenous corresponding gene (e.g., a gene which is "transcriptionally silent," not normally expressed, or expressed only at very low levels) may be activated by inserting a regulatory element which is capable of promoting the expression of a normally expressed gene product in that cell. Techniques such as targeted homologous recombinations, can be used to insert the heterologous DNA as described in, e.g., Chappel, US 5,272,071 ; WO 91/06667, published on May 16, 1991.

Transgenic Animals

Non-human transgenic animals that express a heterologous ICAM polypeptide (e.g., expressed from a nucleic acid from SEQ ID Nos:l-4 or substantially identical sequence thereof) can be generated. Such animals are useful for studying the function and/or activity of a ICAM polypeptide and for identifying and/or evaluating modulators of the activity of nucleic acids from SEQ ID Nos: l -4 and encoded polypeptides. As used herein, a "transgenic animal" is a non-human animal such as a mammal (e.g., a non-human primate such as chimpanzee, baboon, or macaque; an ungulate such as an equine, bovine, or caprine; or a rodent such as a rat, a mouse, or an Israeli sand rat), a bird (e.g., a chicken or a turkey), an amphibian (e.g., a frog, salamander, or newt), or an insect (e.g., Drosophila melanogaster), in which one or more of the cells of the animal includes a transgene. A transgene is exogenous DNA or a rearrangement (e.g., a deletion of endogenous chromosomal DNA) that is often integrated into or occurs in the genome of cells in a transgenic animal. A transgene can direct expression of an encoded gene product in one or more cell types or tissues of the transgenic animal, and other transgenes can reduce expression (e.g., a knockout). Thus, a transgenic animal can be one in which an endogenous nucleic acid homologous to a nucleic acid from SEQ ID Nos:l-4 has been altered by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell of the animal (e.g., an embryonic cell of the animal) prior to development of the animal.

Intronic sequences and polyadenylation signals can also be included in the transgene to increase expression efficiency of the transgene. One or more tissue-specific regulatory sequences can be operably linked to a nucleotide sequence of SEQ ID Nos:l-4 to direct expression of an encoded polypeptide to particular cells. A transgenic founder animal can be identified based upon the presence of a nucleotide sequence from SEQ ID Nos:l-4 in its genome and/or expression of encoded mRNA in tissues or cells of the animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying a nucleotide sequence from SEQ ID Nos:l-4 can further be bred to other transgenic animals carrying other transgenes. ICAM polypeptides can be expressed in transgenic animals or plants by introducing, for example, a nucleic acid from SEQ ID Nos:l-4 into the genome of an animal that encodes the ICAM polypeptide (SEQ ID Nos:5-7). In certain embodiments the nucleic acid is placed under the control of a tissue specific promoter, e.g., a milk or egg specific promoter, and recovered from the milk or eggs produced by the animal. Also included is a population of cells from a transgenic animal.

ICAM Polypeptides

Also featured herein are isolated ICAM polypeptides, which are encoded by a nucleotide sequence from SEQ ID Nos: l-4 or a substantially identical nucleotide sequence thereof, or alternatively are set forth in SEQ ID Nos:5- 7. Isolated ICAM polypeptides featured herein include both the full-length polypeptide and the mature polypeptide (i.e., the polypeptide minus the signal sequence or propeptide domain). An "isolated" or "purified" polypeptide or protein is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. In one embodiment, the language "substantially free" means preparation of a ICAM polypeptide having less than about 30%, 20%, 10% and more preferably 5% (by dry weight), of non-ICAM polypeptide (also referred to herein as a "contaminating protein"), or of chemical precursors or non-target chemicals. When the ICAM polypeptide or a biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, specifically, where culture medium represents less than about 20%, sometimes less than about 10%, and often less than about 5% of the volume of the polypeptide preparation. Isolated or purified ICAM polypeptide preparations are sometimes 0.01 milligrams or more or 0.1 milligrams or more, and often 1.0 milligrams or more and 10 milligrams or more in dry weight. Further included herein are ICAM polypeptide fragments. The polypeptide fragment may be a domain or part of a domain of a ICAM polypeptide. In addition, the polypeptide fragment may be a full-length ICAM polypeptide or a mature ICAM polypeptide (i.e., minus the signal peptide).

Substantially identical ICAM polypeptides may depart from the amino acid sequences of ICAM polypeptides in different manners. For example, conservative amino acid modifications may be introduced at one or more positions in the amino acid sequences of ICAM polypeptides. A "conservative amino acid substitution" is one in which the amino acid is replaced by another amino acid having a similar structure and/or chemical function. Families of amino acid residues having similar structures and functions are well known. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Also, essential and non-essential amino acids may be replaced. A "nonessential" amino acid is one that can be altered without abolishing or substantially altering the biological function of a ICAM polypeptide, whereas altering an "essential" amino acid abolishes or substantially alters the biological function of a ICAM polypeptide. Amino acids that are conserved among ICAM polypeptides are typically essential amino acids.

Also, ICAM polypeptides may exist as chimeric or fusion polypeptides. (See, for example, U.S. Patent No. 5,811,517). As used herein, a target "chimeric polypeptide" or target "fusion polypeptide" includes a ICAM polypeptide linked to a non-ICAM polypeptide. A "non-/G4M polypeptide" refers to a polypeptide having an amino acid sequence corresponding to a polypeptide which is not substantially identical to the ICAM polypeptide, which includes, for example, a polypeptide that is different from the ICAM polypeptide and derived from the same or a different organism. The ICAM polypeptide in the fusion polypeptide can correspond to an entire or nearly entire ICAM polypeptide or a fragment thereof. The non-ICAM polypeptide can be fused to the N-terminus or C-terminus of the ICAM polypeptide. Fusion polypeptides can include a moiety having high affinity for a ligand. For example, the fusion polypeptide can be a GST-target fusion polypeptide in which the target sequences are fused to the C-terminus of the GST sequences, or a polyhistidine-target fusion polypeptide in which the ICAM polypeptide is fused at the N- or C-terminus to a string of histidine residues. Such fusion polypeptides can facilitate purification of recombinant ICAM polypeptide. Expression vectors are commercially available that already encode a fusion moiety (e g., a GST polypeptide), and a nucleotide sequence from SEQ ID Nos:l-4, or a substantially identical nucleotide sequence thereof, can be cloned into an expression vector such that the fusion moiety is linked in-frame to the ICAM polypeptide. Further, the fusion polypeptide can be a ICAM polypeptide containing a heterologous signal sequence at its N-terminus. In certain host cells (e.g., mammalian host cells), expression, secretion, cellular internalization, and cellular localization of a ICAM polypeptide can be increased through use of a heterologous signal sequence. Fusion polypeptides can also include all or a part of a serum polypeptide (e.g., an IgG constant region or human serum albumin).

ICAM polypeptides can be incorporated into pharmaceutical compositions and administered to a subject in vivo. Administration of these ICAM polypeptides can be used to affect the bioavailability of a substrate of the ICAM polypeptide and may effectively increase ICAM polypeptide biological activity in a cell. Target fusion polypeptides may be useful therapeutically for the treatment of disorders caused by, for example, (i) aberrant modification or mutation of a gene encoding a ICAM polypeptide; (ii) mis-regulation of the gene encoding the ICAM polypeptide; and (iii) aberrant post-translational modification of a ICAM polypeptide. Also, ICAM polypeptides can be used as immunogens to produce anti-target antibodies in a subject, to purify ICAM polypeptide ligands or binding partners, and in screening assays to identify molecules which inhibit or enhance the interaction of a ICAM polypeptide with a substrate. Examples of ICAMl inhibitors are Celgene's Actimid and Millennium's Bortezomib. In addition, U.S. Patent No. 6,436,403 describes a conjugate between ICAMl and a virus for ICAMl delivery.

In addition, polypeptides can be chemically synthesized using techniques known in the art (See, e.g., Creighton, 1983 Proteins. New York, N.Y.: W. H. Freeman and Company; and Hunkapiller et al., (1984) Nature July 12 -18;310(5973): 105-1 1). For example, a relative short fragment can be synthesized by use of a peptide synthesizer. Furthermore, if desired, non-classical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the fragment sequence. Non-classical amino acids include, but are not limited to, to the D-isomers of the common amino acids, 2,4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, b-alanine, fluoroamino acids, designer amino acids such as b-methyl amino acids, Ca-methyl amino acids, Na-methyl amino acids, and amino acid analogs in general. Furthermore, the amino acid can be D (dextrorotary) or L (levorotary).

Polypeptides and polypeptide fragments sometimes are differentially modified during or after translation, e.g., by glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, and the like. Any of numerous chemical modifications may be carried out by known techniques, including but not limited, to specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH4; acetylation, formylation, oxidation, reduction; metabolic synthesis in the presence of tunicamycin; and the like. Additional post-translational modifications include, for example, N-linked or O-linked carbohydrate chains, processing of N- terminal or C-terminal ends), attachment of chemical moieties to the amino acid backbone, chemical modifications of N-linked or O-linked carbohydrate chains, and addition or deletion of an N-terminal methionine residue as a result of prokaryotic host cell expression. The polypeptide fragments may also be modified with a detectable label, such as an enzymatic, fluorescent, isotopic or affinity label to allow for detection and isolation of the polypeptide.

Also provided are chemically modified derivatives of polypeptides that can provide additional advantages such as increased solubility, stability and circulating time of the polypeptide, or decreased immunogenicity (see e.g., U.S. Pat. No: 4,179,337. The chemical moieties for derivitization may be selected from water soluble polymers such as polyethylene glycol, ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol and the like. The polypeptides may be modified at random positions within the molecule, or at predetermined positions within the molecule and may include one, two, three or more attached chemical moieties.

The polymer may be of any molecular weight, and may be branched or unbranched. For polyethylene glycol, the molecular weight often is between about 1 kDa and about 100 kDa (the term "about" indicating that in preparations of polyethylene glycol, some molecules will weigh more, some less, than the stated molecular weight) for ease in handling and manufacturing. Other sizes may be used, depending on the desired therapeutic profile (e.g., the duration of sustained release desired, the effects, if any on biological activity, the ease in handling, the degree or lack of antigenicity and other known effects of the polyethylene glycol to a therapeutic protein or analog). The polymers should be attached to the polypeptide with consideration of effects on functional or antigenic domains of the polypeptide. There are a number of attachment methods available to those skilled in the art (e.g., EP 0 401 384 (coupling PEG to G-CSF) and Malik et al. (1992) Exp Hematol. September;20(8):1028-35 (pegylation of GM-CSF using tresyl chloride)). For example, polyethylene glycol may be covalently bound through amino acid residues via a reactive group, such as a free amino or carboxyl group. Reactive groups are those to which an activated polyethylene glycol molecule may be bound. The amino acid residues having a free amino group may include lysine residues and the N-terminal amino acid residues; those having a free carboxyl group may include aspartic acid residues, glutamic acid residues and the C-terminal amino acid residue. Sulfhydryl groups may also be used as a reactive group for attaching the polyethylene glycol molecules. For therapeutic purposes, the attachment sometimes is at an amino group, such as attachment at the N-terminus or lysine group. Proteins can be chemically modified at the N-terminus. Using polyethylene glycol as an illustration of such a composition, one may select from a variety of polyethylene glycol molecules (by molecular weight, branching, and the like), the proportion of polyethylene glycol molecules to protein (polypeptide) molecules in the reaction mix, the type of pegylation reaction to be performed, and the method of obtaining the selected N-terminally pegylated protein. The method of obtaining the N-terminally pegylated preparation {i.e., separating this moiety from other monopegylated moieties if necessary) may be by purification of the N-terminally pegylated material from a population of pegylated protein molecules. Selective proteins chemically modified at the N-terminus may be accomplished by reductive alkylation, which exploits differential reactivity of different types of primary amino groups (lysine versus the N-terminal) available for derivatization in a particular protein. Under the appropriate reaction conditions, substantially selective derivatization of the protein at the N-terminus with a carbonyl group containing polymer is achieved.

Substantially Identical Nucleic Acids and Polypeptides

Nucleotide sequences and polypeptide sequences that are substantially identical to the nucleotide sequences in SEQ ID Nos:l -4 and the ICAM polypeptide sequences encoded by those nucleotide sequences, respectively, are included herein. The term "substantially identical" as used herein refers to two or more nucleic acids or polypeptides sharing one or more identical nucleotide sequences or polypeptide sequences, respectively. Included are nucleotide sequences or polypeptide sequences that are 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more (each often within a 1%, 2%, 3% or 4% variability) identical to the nucleotide sequences in SEQ ID Nos: 1 -4 or the encoded ICAM polypeptide amino acid sequences. One test for determining whether two nucleic acids are substantially identical is to determine the percent of identical nucleotide sequences or polypeptide sequences shared between the nucleic acids or polypeptides.

Calculations of sequence identity are often performed as follows. Sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). The length of a reference sequence aligned for comparison purposes is sometimes 30% or more, 40% or more, 50% or more, often 60% or more, and more often 70% or more, 80% or more, 90% or more, 95% or more, or 100% of the length of the reference sequence. The nucleotides or amino acids at corresponding nucleotide or polypeptide positions, respectively, are then compared among the two sequences. When a position in the first sequence is occupied by the same nucleotide or amino acid as the corresponding position in the second sequence, the nucleotides or amino acids are deemed to be identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, introduced for optimal alignment of the two sequences.

Comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. Percent identity between two amino acid or nucleotide sequences can be determined using the algorithm of Meyers & Miller, CABIOS 4: 11-17 (1989), which has been incorporated into the ALIGN program (version 2.0), using a PAM 120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. Also, percent identity between two amino acid sequences can be determined using the Needleman & Wunsch, J. MoI. Biol. 48: 444-453 (1970) algorithm which has been incorporated into the GAP program in the GCG software package (available at the http address www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1 , 2, 3, 4, 5, or 6. Percent identity between two nucleotide sequences can be determined using the GAP program in the GCG software package (available at http address www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1 , 2, 3, 4, 5, or 6. A set of parameters often used is a Blossum 62 scoring matrix with a gap open penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5. Another manner for determining if two nucleic acids are substantially identical is to assess whether a polynucleotide homologous to one nucleic acid will hybridize to the other nucleic acid under stringent conditions. As use herein, the term "stringent conditions" refers to conditions for hybridization and washing. Stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. , 6.3.1-6.3.6 (1989). Aqueous and non-aqueous methods are described in that reference and either can be used. An example of stringent hybridization conditions is hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 50⁰C. Another example of stringent hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 55°C. A further example of stringent hybridization conditions is hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 60°C. Often, stringent hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45⁰C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 65°C. More often, stringency conditions are 0.5M sodium phosphate, 7% SDS at 65°C, followed by one or more washes at 0.2X SSC, 1% SDS at 65°C.

An example of a substantially identical nucleotide sequence to a nucleotide sequence in SEQ ID Nos:l-4 is one that has a different nucleotide sequence but still encodes the same polypeptide sequence encoded by the nucleotide sequence in SEQ ID Nos: l-4. Another example is a nucleotide sequence that encodes a polypeptide having a polypeptide sequence that is more than 70% or more identical to, sometimes more than 75% or more, 80% or more, or 85% or more identical to, and often more than 90% or more and 95% or more identical to a polypeptide sequence encoded by a nucleotide sequence in SEQ ID Nos: l-4. Nucleotide sequences from SEQ ID Nos: l-4 and amino acid sequences of encoded polypeptides can be used as "query sequences" to perform a search against public databases to identify other family members or related sequences, for example. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul et a!.. J. MoI. Biol. 215: 403-10 (1990). BLAST nucleotide searches can be performed with the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to nucleotide sequences from SEQ ID Nos: l-4. BLAST polypeptide searches can be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to polypeptides encoded by the nucleotide sequences of SEQ ID Nos: l-4. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al, Nucleic Acids Res. 25(17): 3389-3402 (1997). When utilizing BLAST and Gapped BLAST programs, default parameters of the respective programs {e.g., XBLAST and NBLAST) can be used {see the http address www.ncbi.nlm.nih.gov).

A nucleic acid that is substantially identical to a nucleotide sequence in SEQ ID Nos: l-4 may include polymorphic sites at positions equivalent to those described herein when the sequences are aligned. For example, using the alignment procedures described herein, SNPs in a sequence substantially identical to a sequence in SEQ ID Nos: l-4 can be identified at nucleotide positions that match {i.e., align) with nucleotides at SNP positions in each nucleotide sequence in SEQ ID Nos:l-4. Also, where a polymorphic variation results in an insertion or deletion, insertion or deletion of a nucleotide sequence from a reference sequence can change the relative positions of other polymorphic sites in the nucleotide sequence.

Substantially identical nucleotide and polypeptide sequences include those that are naturally occurring, such as allelic variants (same locus), splice variants, homologs (different locus), and orthologs (different organism) or can be non-naturally occurring. Non-naturally occurring variants can be generated by mutagenesis techniques, including those applied to polynucleotides, cells, or organisms. The variants can contain nucleotide substitutions, deletions, inversions and insertions. Variation can occur in either or both the coding and non-coding regions. The variations can produce both conservative and non-conservative amino acid substitutions (as compared in the encoded product). Orthologs, homologs, allelic variants, and splice variants can be identified using methods known in the art. These variants normally comprise a nucleotide sequence encoding a polypeptide that is 50%, about 55% or more, often about 70-75% or more, more often about 80-85% or more, and typically about 90-95% or more identical to the amino acid sequences of ICAM polypeptides or a fragment thereof. Such nucleic acid molecules can readily be identified as being able to hybridize under stringent conditions to a nucleotide sequence in SEQ ID Nos:l-4 or a fragment of this sequence. Nucleic acid molecules corresponding to orthologs, homologs, and allelic variants of a nucleotide sequence in SEQ ID Nos:l-4 can further be identified by mapping the sequence to the same chromosome or locus as the nucleotide sequence in SEQ ID Nos:l-4.

Also, substantially identical nucleotide sequences may include codons that are altered with respect to the naturally occurring sequence for enhancing expression of a ICAM polypeptide in a particular expression system. For example, the nucleic acid can be one in which one or more codons are altered, and often 10% or more or 20% or more of the codons are altered for optimized expression in bacteria {e.g., E. coli.), yeast (e.g., S. cervesiae), human {e.g., 293 cells), insect, or rodent {e.g., hamster) cells.

Methods for Identifying Subjects at Risk of Breast Cancer or Prostate Cancer in a Subject Methods for determining whether a subject is at risk of breast cancer or prostate cancer are provided herein. These methods include detecting the presence or absence of one or more polymorphic variations associated with breast cancer or prostate cancer in an ICAM nucleotide sequence, or substantially identical sequence thereof, in a sample from a subject, where the presence of such a polymorphic variation is indicative of the subject being at risk of breast cancer or prostate cancer. These genetic tests are useful for prognosing and/or diagnosing breast cancer or prostate cancer and often are useful for determining whether an individual is at an increased, intermediate or decreased risk of developing or having breast cancer or prostate cancer. Thus, featured herein is a method for identifying a subject at risk of breast cancer or prostate cancer, which comprises detecting in a nucleic acid sample from the subject the presence or absence of a polymorphic variation associated with breast cancer or prostate cancer at a polymorphic site in an ICAM nucleotide sequence. The nucleotide sequence often is selected from the group consisting of: (a) a nucleotide sequence set forth in SEQ ID Nos:l-4; (b) a nucleotide sequence which encodes a polypeptide consisting of an amino acid sequence encoded by a nucleotide sequence set forth in SEQ ID Nos: l -4; (c) a nucleotide sequence which encodes a polypeptide that is 90% or more identical to an amino acid sequence encoded by a nucleotide sequence set forth in SEQ ID Nos: l-4 or a nucleotide sequence about 90% or more identical to the nucleotide sequence set forth in SEQ ID Nos: l-4; and (d) a fragment of a nucleotide sequence of (a), (b), or (c), where the fragment comprises a polymorphic site; whereby the presence of the polymorphic variation is indicative of the subject being at risk of breast cancer or prostate cancer. A polymorphic variation assayed in the genetic test often is located in an intron, sometimes in a region surrounding the ICAM open reading frame (e.g., within 50 kilobases (kb), 40 kb, 30 kb, 20 kb, 15, kb, 10 kb, 5 kb, 4 kb, 3 kb, 2 kb, or 1 kb of the open reading frame initiation site or termination site), and sometimes in an exon. Sometimes the polymorphic variation is not located in an exon. In an embodiment, a polymorphic variant at NCBI SNP ID rsl 1549918 or rs2228615 is detected for determining a risk of breast cancer or prostate cancer. In some embodiments, one or more polymorphic variants at the following positions are detected for determining risk of breast cancer, prostate cancer, or an aggressive or metastatic form of the foregoing: rslO56538, rs2228615, rs5030382. In related embodiments, a polymorphic variant at a corresponding position in the encoded protein is detected, such as at amino acid position 301 and/or 348 in ICAM5 and/or amino acid position 469 in ICAMl . Specific examples of alleles associated with breast cancer and prostate cancer are described in greater detail hereafter.

A risk of developing aggressive forms of breast cancer likely to metastasize or invade surrounding tissues (e.g., Stage IIIA, IHB, and IV breast cancers), and subjects at risk of developing aggressive forms of breast cancer also may be identified by the methods described herein. Similarly, a risk of developing aggressive forms of prostate cancer likely to metastasize or invade surrounding tissues (e.g., stage T2, T3, T4, Nl or Ml as defined in Tables A-C), and subjects at risk of developing aggressive forms of prostate cancer also may be identified by the methods described herein. These methods include collecting phenotype information from subjects having breast cancer or prostate cancer, which includes the stage of progression of the breast cancer or prostate cancer, and performing a secondary phenotype analysis to detect the presence or absence of one or more polymorphic variations associated with a particular stage form of breast cancer or prostate cancer. Thus, detecting the presence or absence of one or more polymorphic variations in an ICAM nucleotide sequence associated with a late stage form of breast cancer or prostate cancer often is diagnostic of an aggressive form of the cancer.

Results from prognostic tests may be combined with other test results to diagnose breast cancer or prostate cancer. For example, prognostic results may be gathered, a patient sample may be ordered based on a determined predisposition to breast cancer or prostate cancer, the patient sample is analyzed, and the results of the analysis may be utilized to diagnose breast cancer or prostate cancer. Also breast cancer or prostate cancer diagnostic methods can be developed from studies used to generate prognostic/diagnostic methods in which populations are stratified into subpopulations having different progressions of breast cancer or prostate cancer. In another embodiment, prognostic results may be gathered; a patient's risk factors for developing breast cancer or prostate cancer analyzed (e.g., age, race, family history, age of first menstrual cycle, age at birth of first child); and a patient sample may be ordered based on a determined predisposition to breast cancer or prostate cancer. In an alternative embodiment, the results from predisposition analyses described herein may be combined with other test results indicative of breast cancer or prostate cancer, which were previously, concurrently, or subsequently gathered with respect to the predisposition testing. In these embodiments, the combination of the prognostic test results with other test results can be probative of breast cancer or prostate cancer, and the combination can be utilized as a breast cancer or prostate cancer diagnostic. The results of any test indicative of breast cancer or prostate cancer known in the art may be combined with the methods described herein. Examples of such tests are mammography (e.g., a more frequent and/or earlier mammography regimen may be prescribed); breast biopsy and optionally a biopsy from another tissue; breast ultrasound and optionally an ultrasound analysis of another tissue; breast magnetic resonance imaging (MRI) and optionally an MRI analysis of another tissue; electrical impedance (T-scan) analysis of breast and optionally of another tissue; ductal lavage; nuclear medicine analysis (e.g., scintimammography); BRCAl and/or BRCA2 sequence analysis results; and thermal imaging of the breast and optionally of another tissue. Testing may be performed on tissue other than breast to diagnose the occurrence of metastasis (e.g., testing of the lymph node).

Risk of breast cancer or prostate cancer sometimes is expressed as a probability, such as an odds ratio, percentage, or risk factor. The risk is based upon the presence or absence of one or more polymorphic variants described herein, and also may be based in part upon phenotypic traits of the individual being tested. Methods for calculating risk based upon patient data are well known (see, e.g., Agresti, Categorical Data Analysis, 2nd Ed. 2002. Wiley). Allelotyping and genotyping analyses may be carried out in populations other than those exemplified herein to enhance the predictive power of the prognostic method. These further analyses are executed in view of the exemplified procedures described herein, and may be based upon the same polymorphic variations or additional polymorphic variations. Risk determinations for breast cancer or prostate cancer are useful in a variety of applications. In one embodiment, breast cancer or prostate cancer risk determinations are used by clinicians to direct appropriate detection, preventative and treatment procedures to subjects who most require these. In another embodiment, breast cancer or prostate cancer risk determinations are used by health insurers for preparing actuarial tables and for calculating insurance premiums.

The nucleic acid sample typically is isolated from a biological sample obtained from a subject. For example, nucleic acid can be isolated from blood, saliva, sputum, urine, cell scrapings, and biopsy tissue. The nucleic acid sample can be isolated from a biological sample using standard techniques, such as the technique described in Example 2. As used herein, the term "subject" refers primarily to humans but also refers to other mammals such as dogs, cats, and ungulates (e.g., cattle, sheep, and swine). Subjects also include avians (e.g., chickens and turkeys), reptiles, and fish (e.g., salmon), as embodiments described herein can be adapted to nucleic acid samples isolated from any of these organisms. The nucleic acid sample may be isolated from the subject and then directly utilized in a method for determining the presence of a polymorphic variant, or alternatively, the sample may be isolated and then stored (e.g., frozen) for a period of time before being subjected to analysis. The presence or absence of a polymorphic variant is determined using one or both chromosomal complements represented in the nucleic acid sample. Determining the presence or absence of a polymorphic variant in both chromosomal complements represented in a nucleic acid sample from a subject having a copy of each chromosome is useful for determining the zygosity of an individual for the polymorphic variant (i.e., whether the individual is homozygous or heterozygous for the polymorphic variant). Any oligonucleotide-based diagnostic may be utilized to determine whether a sample includes the presence or absence of a polymorphic variant in a sample. For example, primer extension methods, ligase sequence determination methods (e.g., U.S. Pat. Nos. 5,679,524 and 5,952,174, and WO 01/27326), mismatch sequence determination methods (e.g., U.S. Pat. Nos. 5,851,770; 5,958,692; 6,1 10,684; and 6,183,958), microarray sequence determination methods, restriction fragment length polymorphism (RFLP), single strand conformation polymorphism detection (SSCP) (e.g., U.S. Pat. Nos. 5,891,625 and 6,013,499), PCR-based assays (e.g., TAQMAN® PCR System (Applied Biosystems)), and nucleotide sequencing methods may be used.

Oligonucleotide extension methods typically involve providing a pair of oligonucleotide primers in a polymerase chain reaction (PCR) or in other nucleic acid amplification methods for the purpose of amplifying a region from the nucleic acid sample that comprises the polymorphic variation. One oligonucleotide primer is complementary to a region 3' of the polymorphism and the other is complementary to a region 5' of the polymorphism. A PCR primer pair may be used in methods disclosed in U.S. Pat. Nos. 4,683,195; 4,683,202, 4,965,188; 5,656,493; 5,998,143; 6,140,054; WO 01/27327; and WO 01/27329 for example. PCR primer pairs may also be used in any commercially available machines that perform PCR, such as any of the GENEAMP® Systems available from Applied Biosystems. Also, those of ordinary skill in the art will be able to design oligonucleotide primers based upon a nucleotide sequence set forth in SEQ ID Nos:l, 2, 3 or 4 without undue experimentation using knowledge readily available in the art.

Also provided is an extension oligonucleotide that hybridizes to the amplified fragment adjacent to the polymorphic variation. As used herein, the term "adjacent" refers to the 3' end of the extension oligonucleotide being often 1 nucleotide from the 5' end of the polymorphic site, and sometimes 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from the 5' end of the polymorphic site, in the nucleic acid when the extension oligonucleotide is hybridized to the nucleic acid. The extension oligonucleotide then is extended by one or more nucleotides, and the number and/or type of nucleotides that are added to the extension oligonucleotide determine whether the polymorphic variant is present. Oligonucleotide extension methods are disclosed, for example, in U.S. Pat. Nos. 4,656,127; 4,851,331 ; 5,679,524; 5,834,189; 5,876,934; 5,908,755; 5,912,118; 5,976,802; 5,981 ,186; 6,004,744; 6,013,431 ; 6,017,702; 6,046,005; 6,087,095; 6,210,891 ; and WO 01/20039. Oligonucleotide extension methods using mass spectrometry are described, for example, in U.S. Pat. Nos. 5,547,835; 5,605,798; 5,691,141 ;

5,849,542; 5,869,242; 5,928,906; 6,043,031 ; and 6,194,144, and a method often utilized is described herein in Example 2. Multiple extension oligonucleotides may be utilized in one reaction, which is referred to herein as "multiplexing."

A microarray can be utilized for determining whether a polymorphic variant is present or absent in a nucleic acid sample. A microarray may include any oligonucleotides described herein, and methods for making and using oligonucleotide microarrays suitable for diagnostic use are disclosed in U.S. Pat. Nos. 5,492,806; 5,525,464; 5,589,330; 5,695,940; 5,849,483; 6,018,041 ; 6,045,996; 6,136,541 ; 6,142,681 ; 6,156,501 ; 6,197,506; 6,223,127; 6,225,625; 6,229,911 ; 6,239,273; WO 00/52625; WO 01/25485; and WO 01/29259. The microarray typically comprises a solid support and the oligonucleotides may be linked to this solid support by covalent bonds or by non-covalent interactions. The oligonucleotides may also be linked to the solid support directly or by a spacer molecule. A microarray may comprise one or more oligonucleotides complementary to a polymorphic site set forth in Figure 1 or below.

A kit also may be utilized for determining whether a polymorphic variant is present or absent in a nucleic acid sample. A kit often comprises one or more pairs of oligonucleotide primers useful for amplifying a fragment of an ICAM nucleotide sequence or a substantially identical sequence thereof, where the fragment includes a polymorphic site. The kit sometimes comprises a polymerizing agent, for example, a thermostable nucleic acid polymerase such as one disclosed in U.S. Pat. Nos. 4,889,818 or 6,077,664. Also, the kit often comprises an elongation oligonucleotide that hybridizes to an ICAM nucleotide sequence in a nucleic acid sample adjacent to the polymorphic site. Where the kit includes an elongation oligonucleotide, it also often comprises chain elongating nucleotides, such as dATP, dTTP, dGTP, dCTP, and dITP, including analogs of dATP, dTTP, dGTP, dCTP and dITP, provided that such analogs are substrates for a thermostable nucleic acid polymerase and can be incorporated into a nucleic acid chain elongated from the extension oligonucleotide. Along with chain elongating nucleotides would be one or more chain terminating nucleotides such as ddATP, ddTTP, ddGTP, ddCTP, and the like. In an embodiment, the kit comprises one or more oligonucleotide primer pairs, a polymerizing agent, chain elongating nucleotides, at least one elongation oligonucleotide, and one or more chain terminating nucleotides. Kits optionally include buffers, vials, microtiter plates, and instructions for use.

An individual identified as being at risk of breast cancer or prostate cancer may be heterozygous or homozygous with respect to the allele associated with a higher risk of breast cancer or prostate cancer. A subject homozygous for an allele associated with an increased risk of breast cancer or prostate cancer is at a comparatively high risk of breast cancer or prostate cancer, a subject heterozygous for an allele associated with an increased risk of breast cancer or prostate cancer is at a comparatively intermediate risk of breast cancer or prostate cancer, and a subject homozygous for an allele associated with a decreased risk of breast cancer or prostate cancer is at a comparatively low risk of breast cancer or prostate cancer. A genotype may be assessed for a complementary strand, such that the complementary nucleotide at a particular position is detected.

Also featured are methods for determining risk of breast cancer or prostate cancer and/or identifying a subject at risk of breast cancer or prostate cancer by contacting a polypeptide or protein encoded by an ICAM nucleotide sequence from a subject with an antibody that specifically binds to an epitope associated with increased risk of breast cancer or prostate cancer in the polypeptide.

Applications of Prognostic and Diagnostic Results to Pharmacogenomic Methods

Pharmacogenomics is a discipline that involves tailoring a treatment for a subject according to the subject's genotype. For example, based upon the outcome of a prognostic test described herein, a clinician or physician may target pertinent information and preventative or therapeutic treatments to a subject who would be benefited by the information or treatment and avoid directing such information and treatments to a subject who would not be benefited (e.g., the treatment has no therapeutic effect and/or the subject experiences adverse side effects). As therapeutic approaches for breast cancer or prostate cancer continue to evolve and improve, the goal of treatments for cancer related disorders is to intervene even before clinical signs (e.g., identification of a detectable tumor) first manifest. Thus, genetic markers associated with susceptibility to breast cancer or prostate cancer prove useful for early diagnosis, prevention and treatment of breast cancer or prostate cancer.

The following is an example of a pharmacogenomic embodiment. A particular treatment regimen can exert a differential effect depending upon the subject's genotype. Where a candidate therapeutic exhibits a significant interaction with a major allele and a comparatively weak interaction with a minor allele (e.g., an order of magnitude or greater difference in the interaction), such a therapeutic typically would not be administered to a subject genotyped as being homozygous for the minor allele, and sometimes not administered to a subject genotyped as being heterozygous for the minor allele. In another example, where a candidate therapeutic is not significantly toxic when administered to subjects who are homozygous for a major allele but is comparatively toxic when administered to subjects heterozygous or homozygous for a minor allele, the candidate therapeutic is not typically administered to subjects who are genotyped as being heterozygous or homozygous with respect to the minor allele.

The methods described herein are applicable to pharmacogenomic methods for detecting, preventing, alleviating and/or treating breast cancer or prostate cancer. For example, a nucleic acid sample from an individual may be subjected to a genetic test described herein. Where one or more polymorphic variations associated with increased risk of breast cancer or prostate cancer are identified in a subject, information for detecting, preventing or treating breast cancer or prostate cancer and/or one or more breast cancer or prostate cancer detection, prevention and/or treatment regimens then may be directed to and/or prescribed to that subject.

In certain embodiments, a detection, preventative and/or treatment regimen is specifically prescribed and/or administered to individuals who will most benefit from it based upon their risk of developing breast cancer or prostate cancer assessed by the methods described herein. Thus, provided are methods for identifying a subject at risk of breast cancer or prostate cancer and then prescribing a detection, therapeutic or preventative regimen to individuals identified as being at risk of breast cancer or prostate cancer. Thus, certain embodiments are directed to methods for treating breast cancer or prostate cancer in a subject, reducing risk of breast cancer or prostate cancer in a subject, or early detection of breast cancer or prostate cancer in a subject, which comprise: detecting the presence or absence of a polymorphic variant associated with breast cancer or prostate cancer in a nucleotide sequence set forth in SEQ ID Nos: l, 2, 3 or 4 in a nucleic acid sample from a subject, where the nucleotide sequence comprises a polynucleotide sequence selected from the group consisting of: (a) a nucleotide sequence set forth in SEQ ID Nos:l, 2, 3 or 4; (b) a nucleotide sequence which encodes a polypeptide having an amino acid sequence encoded by a nucleotide sequence in SEQ ID Nos:l, 2, 3 or 4; (c) a nucleotide sequence which encodes a polypeptide that is 90% or more identical to an amino acid sequence encoded by a nucleotide sequence in SEQ ID Nos:l, 2, 3 or 4 or a nucleotide sequence about 90% or more identical to the nucleotide sequence set forth in SEQ ID Nos:l, 2, 3 or 4; and (d) a fragment of a nucleotide sequence of (a), (b), or (c), sometimes comprising a polymorphic site associated with breast cancer or prostate cancer; and prescribing or administering a breast cancer or prostate cancer treatment regimen, preventative regimen and/or detection regimen to a subject from whom the sample originated where the presence of one or more polymorphic variations associated with breast cancer are detected in the nucleotide sequence. In these methods, genetic results may be utilized in combination with other test results to diagnose breast cancer as described above. Other test results include but are not limited to mammography results, imaging results, biopsy results and results from BRCAl or BRAC2 test results, as described above.

Detection regimens for breast cancer include one or more mammography procedures, a regular mammography regimen (e.g., once a year, or once every six, four, three or two months); an early mammography regimen (e.g., mammography tests are performed beginning at age 25, 30, or 35); one or more biopsy procedures (e.g., a regular biopsy regimen beginning at age 40); breast biopsy and biopsy from other tissue; breast ultrasound and optionally ultrasound analysis of another tissue; breast magnetic resonance imaging (MRI) and optionally MRI analysis of another tissue; electrical impedance (T-scan) analysis of breast and optionally another tissue; ductal lavage; nuclear medicine analysis (e.g., scintimammography); BRCAl and/or BRCA2 sequence analysis results; and/or thermal imaging of the breast and optionally another tissue.

Treatments sometimes are preventative (e.g., is prescribed or administered to reduce the probability that a breast cancer associated condition arises or progresses), sometimes are therapeutic, and sometimes delay, alleviate or halt the progression of breast cancer. Any known preventative or therapeutic treatment for alleviating or preventing the occurrence of breast cancer is prescribed and/or administered. For example, certain preventative treatments often are prescribed to subjects having a predisposition to breast cancer and where the subject is not diagnosed with breast cancer or is diagnosed as having symptoms indicative of early stage breast cancer (e.g., stage I). For subjects not diagnosed as having breast cancer, any preventative treatments known in the art can be prescribed and administered, which include selective hormone receptor modulators (e.g., selective estrogen receptor modulators (SERMs) such as tamoxifen, reloxifene, and toremifene); compositions that prevent production of hormones (e.g., aramotase inhibitors that prevent the production of estrogen in the adrenal gland, such as exemestane, letrozole, anastrozol, groserelin, and megestrol); other hormonal treatments (e.g., goserelin acetate and fulvestrant); biologic response modifiers such as antibodies (e.g., trastuzumab (herceptin/HER2)); surgery (e.g., lumpectomy and mastectomy); drugs that delay or halt metastasis (e.g., pamidronate disodium); and alternative/complementary medicine (e.g., acupuncture, acupressure, moxibustion, qi gong, reiki, ayurveda, vitamins, minerals, and herbs (e.g., astragalus root, burdock root, garlic, green tea, and licorice root)).

The use of breast cancer treatments are well known in the art, and include surgery, chemotherapy and/or radiation therapy. Any of the treatments may be used in combination to treat or prevent breast cancer (e.g., surgery followed by radiation therapy or chemotherapy). Examples of chemotherapy combinations used to treat breast cancer include: cyclophosphamide (Cytoxan), methotrexate (Amethopterin, Mexate, Folex), and fluorouracil (Fluorouracil, 5-Fu, Adrucil), which is referred to as CMF; cyclophosphamide, doxorubicin (Adriamycin), and fluorouracil, which is referred to as CAF; and doxorubicin (Adriamycin) and cyclophosphamide, which is referred to as AC. For prostate cancer, examples of prophylactic regimens include reducing environmental risks known to cause cancer. In addition the following may be recommended or administered: Exercise (walking); Soy Protein; Flaxseeds (Phytoestrogens); Lycopones (tomatoes); Selenium; Green tea; Vitamin D Supplementation; Calcium Supplementation; Vitamin E Supplementation; Garlic; PC-SPES; Grape seed extract; and Zinc.

In another embodiment, additional testing for clinical signs of prostate cancer may be ordered when one or more polymorphic variations associated with increased risk of prostate cancer are identified in a subject. The two best known additional tests for clinical signs of prostate cancer are a digital rectal examination (DRE) and test to measure prostate-specific antigen (PSA) in the blood. A DRE is a quick and safe screening technique in which a doctor inserts a gloved, lubricated finger into the rectum to feel the size and shape of the prostate (See picture below). The prostate should feel soft, smooth, and even. The doctor examines for lumps or hard, irregular areas of the prostate that may indicate the presence of prostate cancer. The entire prostate cannot be felt during a DRE, but most of it can be examined, including the area where most prostate cancers are found. PSA is a substance produced by both normal and cancerous prostate cells. When prostate cancer grows or when other prostate diseases are present, the amount of PSA in the blood often increases.

Preventative regimens for prostate cancer include, but are not limited to, methods of reducing or removing environmental and behavioral risks known to cause prostate cancer, including changing diet and increasing exercise.

Pharmacogenomic methods described herein can help professionals recognize the early onset of prostate cancer and subsequently allow for therapeutic intervention at the first clinical signs of prostate cancer. As there are currently no cures for prostate cancer, the objective of treatment is to reduce the severity of the symptoms, if possible to the point of remission. Surgical prostatectomy, radiation therapy, hormone ablation therapy, and chemotherapy continue to be the main treatment modalities. Surgery is often effective for early stage or non- aggressive prostate cancer. Radiation can be used for early stage prostate cancer, and in advanced prostate cancer. Hormonal therapy may be used to remove androgens (LHRH agonists, anti-androgens); however, all prostate cancer becomes resistant to hormonal therapy eventually. Chemotherapy may be effective in advanced cases non-responsive to hormonal therapy. Chemotherapy agents used to treat prostate cancer include mitoxantrane plus corticosteroids; and estramustane plus taxanes. Any of the treatments may be used in combination with one another to treat or prevent prostate cancer.

As breast cancer or prostate cancer preventative and treatment information can be specifically targeted to subjects in need thereof (e.g., those at risk of developing breast cancer or prostate cancer or those that have early signs of breast cancer or prostate cancer), provided herein is a method for preventing or reducing the risk of developing breast cancer or prostate cancer in a subject, which comprises: (a) detecting the presence or absence of a polymorphic variation associated with breast cancer or prostate cancer at a polymorphic site in a nucleotide sequence in a nucleic acid sample from a subject; (b) identifying a subject at risk of breast cancer or prostate cancer, whereby the presence of the polymorphic variation is indicative of a risk of breast cancer or prostate cancer in the subject; and (c) if such a risk is identified, providing the subject with information about methods or products to prevent or reduce breast cancer or prostate cancer or to delay the onset of breast cancer or prostate cancer. Also provided is a method of targeting information or advertising to a subpopulation of a human population based on the subpopulation being genetically predisposed to a disease or condition, which comprises: (a) detecting the presence or absence of a polymorphic variation associated with breast cancer or prostate cancer at a polymorphic site in a nucleotide sequence in a nucleic acid sample from a subject; (b) identifying the subpopulation of subjects in which the polymorphic variation is associated with breast cancer or prostate cancer; and (c) providing information only to the subpopulation of subjects about a particular product which may be obtained and consumed or applied by the subject to help prevent or delay onset of the disease or condition.

Pharmacogenomics methods also may be used to analyze and predict a response to a breast cancer or prostate cancer treatment or a drug. For example, if pharmacogenomics analysis indicates a likelihood that an individual will respond positively to a breast cancer or prostate cancer treatment with a particular drug, the drug may be administered to the individual. Conversely, if the analysis indicates that an individual is likely to respond negatively to treatment with a particular drug, an alternative course of treatment may be prescribed. A negative response may be defined as either the absence of an efficacious response or the presence of toxic side effects. The response to a therapeutic treatment can be predicted in a background study in which subjects in any of the following populations are genotyped: a population that responds favorably to a treatment regimen, a population that does not respond significantly to a treatment regimen, and a population that responds adversely to a treatment regiment (e.g., exhibits one or more side effects). These populations are provided as examples and other populations and subpopulations may be analyzed. Based upon the results of these analyses, a subject is genotyped to predict whether he or she will respond favorably to a treatment regimen, not respond significantly to a treatment regimen, or respond adversely to a treatment regimen.

The methods described herein also are applicable to clinical drug trials. One or more polymorphic variants indicative of response to an agent for treating breast cancer or prostate cancer or to side effects to an agent for treating breast cancer or prostate cancer may be identified using the methods described herein. Thereafter, potential participants in clinical trials of such an agent may be screened to identify those individuals most likely to respond favorably to the drug and exclude those likely to experience side effects. In that way, the effectiveness of drug treatment may be measured in individuals who respond positively to the drug, without lowering the measurement as a result of the inclusion of individuals who are unlikely to respond positively in the study and without risking undesirable safety problems. In certain embodiments, the agent for treating breast cancer or prostate cancer described herein targets ICAM or a target in the ICAM pathway (e.g., Rho GTPase).

Thus, another embodiment is a method of selecting an individual for inclusion in a clinical trial of a treatment or drug comprising the steps of: (a) obtaining a nucleic acid sample from an individual; (b) determining the identity of a polymorphic variation which is associated with a positive response to the treatment or the drug, or at least one polymorphic variation which is associated with a negative response to the treatment or the drug in the nucleic acid sample, and (c) including the individual in the clinical trial if the nucleic acid sample contains said polymorphic variation associated with a positive response to the treatment or the drug or if the nucleic acid sample lacks said polymorphic variation associated with a negative response to the treatment or the drug. In addition, the methods for selecting an individual for inclusion in a clinical trial of a treatment or drug encompass methods with any further limitation described in this disclosure, or those following, specified alone or in any combination. The polymorphic variation may be in a sequence selected individually or in any combination from the group consisting of (i) a polynucleotide sequence set forth in SEQ ID Nos:l, 2, 3 or 4; (ii) a polynucleotide sequence that is 90% or more identical to a nucleotide sequence set forth in SEQ ID Nos:l , 2, 3 or 4; (iii) a polynucleotide sequence that encodes a polypeptide having an amino acid sequence identical to or 90% or more identical to an amino acid sequence encoded by a nucleotide sequence set forth in SEQ ID Nos:l, 2, 3 or 4; and (iv) a fragment of a polynucleotide sequence of (i), (ii), or (iii) comprising the polymorphic site. The including step (c) optionally comprises administering the drug or the treatment to the individual if the nucleic acid sample contains the polymorphic variation associated with a positive response to the treatment or the drug and the nucleic acid sample lacks said biallelic marker associated with a negative response to the treatment or the drug.

Also provided herein is a method of partnering between a diagnostic/prognostic testing provider and a provider of a consumable product, which comprises: (a) the diagnostic/prognostic testing provider detects the presence or absence of a polymorphic variation associated with breast cancer or prostate cancer at a polymorphic site in a nucleotide sequence in a nucleic acid sample from a subject; (b) the diagnostic/prognostic testing provider identifies the subpopulation of subjects in which the polymorphic variation is associated with breast cancer or prostate cancer; (c) the diagnostic/prognostic testing provider forwards information to the subpopulation of subjects about a particular product which may be obtained and consumed or applied by the subject to help prevent or delay onset of the disease or condition; and (d) the provider of a consumable product forwards to the diagnostic test provider a fee every time the diagnostic/prognostic test provider forwards information to the subject as set forth in step (c) above.

Compositions Comprising Breast cancer or prostate cancer-Directed Molecules Featured herein is a composition comprising a breast cancer or prostate cancer cell and one or more molecules specifically directed and targeted to a nucleic acid comprising an ICAM nucleotide sequence or a ICAM polypeptide. Such directed molecules include, but are not limited to, a compound that binds to a ICAM nucleic acid or a ICAM polypeptide; a RNAi or siRNA molecule having a strand complementary to an ICAM nucleotide sequence; an antisense nucleic acid complementary to an RNA encoded by a ICAMONA sequence (see, for example, PCT Publication No. WO9961462-A1); a ribozyme that hybridizes to an ICAM nucleotide sequence; a nucleic acid aptamer that specifically binds a ICAM polypeptide; and an antibody that specifically binds to a ICAM polypeptide or binds to a ICAM nucleic acid. In specific embodiments, the breast cancer or prostate cancer directed molecule interacts with a ICAM nucleic acid or polypeptide variant associated with breast cancer or prostate cancer. In other embodiments, the breast cancer or prostate cancer directed molecule interacts with a polypeptide involved in the ICAM signal pathway, or a nucleic acid encoding such a polypeptide.

Compositions sometimes include an adjuvant known to stimulate an immune response, and in certain embodiments, an adjuvant that stimulates a T-cell lymphocyte response. Adjuvants are known, including but not limited to an aluminum adjuvant (e.g., aluminum hydroxide); a cytokine adjuvant or adjuvant that stimulates a cytokine response (e.g., interleukin (IL)-12 and/or γ-interferon cytokines); a Freund-type mineral oil adjuvant emulsion (e.g., Freund's complete or incomplete adjuvant); a synthetic lipoid compound; a copolymer adjuvant (e.g., TitreMax); a saponin; Quil A; a liposome; an oil-in-water emulsion (e.g., an emulsion stabilized by Tween 80 and pluronic polyoxyethlene/polyoxypropylene block copolymer (Syntex Adjuvant Formulation); TitreMax; detoxified endotoxin (MPL) and mycobacterial cell wall components (TDW, CWS) in 2% squalene (Ribi Adjuvant System)); a muramyl dipeptide; an immune-stimulating complex (ISCOM, e.g., an Ag-modified saponin/cholesterol micelle that forms stable cage-like structure); an aqueous phase adjuvant that does not have a depot effect (e.g., Gerbu adjuvant); a carbohydrate polymer (e.g., AdjuPrime); L-tyrosine; a manide-oleate compound (e.g., Montanide); an ethylene-vinyl acetate copolymer (e.g., Elvax 40Wl , 2); or lipid A, for example. Such compositions are useful for generating an immune response against a breast cancer or prostate cancer directed molecule (e.g., an HLA-binding subsequence within a polypeptide encoded by a nucleotide sequence in SEQ ID NO: 1). In such methods, a peptide having an amino acid subsequence of a polypeptide encoded by a nucleotide sequence in SEQ ID Nos: l, 2, 3 or 4 is delivered to a subject, where the subsequence binds to an HLA molecule and induces a CTL lymphocyte response. The peptide sometimes is delivered to the subject as an isolated peptide or as a minigene in a plasmid that encodes the peptide. Methods for identifying HLA-binding subsequences in such polypeptides are known (see e.g., publication WO02/20616 and PCT application US98/01373 for methods of identifying such sequences).

The breast cancer or prostate cancer cell may be in a group of breast cancer or prostate cancer cells and/or other types of cells cultured in vitro or in a tissue having breast cancer or prostate cancer cells (e.g., a melanocyte lesion) maintained in vitro or present in an animal in vivo (e.g., a rat, mouse, ape or human). In certain embodiments, a composition comprises a component from a breast cancer or prostate cancer cell or from a subject having a breast cancer or prostate cancer cell instead of the breast cancer or prostate cancer cell or in addition to the breast cancer or prostate cancer cell, where the component sometimes is a nucleic acid molecule (e.g., genomic DNA), a protein mixture or isolated protein, for example. The aforementioned compositions have utility in diagnostic, prognostic and pharmacogenomic methods described previously and in breast cancer or prostate cancer therapeutics described hereafter. Certain breast cancer or prostate cancer molecules are described in greater detail below.

Compounds

Compounds can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; peptoid libraries (libraries of molecules having the functionalities of peptides, but with a novel, non-peptide backbone which are resistant to enzymatic degradation but which nevertheless remain bioactive (see, e.g., Zuckermann et al., J. Med. Chem.37: 2678-85 (1994)); spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; "one-bead one-compound" library methods; and synthetic library methods using affinity chromatography selection. Biological library and peptoid library approaches are typically limited to peptide libraries, while the other approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam, Anticancer Drug Des. 12: 145, (1997)). Examples of methods for synthesizing molecular libraries are described, for example, in DeWitt et al., Proc. Natl. Acad. Sci. U.S.A. 90: 6909 (1993); Erb et al, Proc. Natl. Acad. Sci. USA 91 : 1 1422 (1994); Zuckermann et al., J. Med. Chem. 37: 2678 (1994); Cho et al., Science 261 : 1303 (1993); Carrell et al., Angew. Chem. Int. Ed. Engl. 33: 2059 (1994); Carell et al, Angew. Chem. Int. Ed. Engl. 33: 2061 (1994); and in Gallop et al, J. Med. Chem. 37: 1233 (1994).

Libraries of compounds may be presented in solution (e.g., Houghten, Biotechniques 13: 412-421 (1992)), or on beads (Lam, Nature 354: 82-84 (1991)), chips (Fodor, Nature 364: 555-556 (1993)), bacteria or spores (Ladner, United States Patent No. 5,223,409), plasmids (Cull et al, Proc. Natl. Acad. Sci. USA 89: 1865-1869

(1992)) or on phage (Scott and Smith, Science 249: 386-390 (1990); Devlin, Science 249: 404-406 (1990); Cwirla et al, Proc. Natl. Acad. Sci. 87: 6378-6382 (1990); Felici, J. MoI. Biol. 222: 301-310 (1991); Ladner supra.).

A compound sometimes alters expression and sometimes alters activity of a ICAM polypeptide and may be a small molecule. Small molecules include, but are not limited to, peptides, peptidomimetics (e.g., peptoids), amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, nucleotides, nucleotide analogs, organic or inorganic compounds (i.e., including heteroorganic and organometallic compounds) having a molecular weight less than about 10,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 5,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 1 ,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 500 grams per mole, and salts, esters, and other pharmaceutically acceptable forms of such compounds.

Antisense Nucleic Acid Molecules. Ribozvmes. RNAi. siRNA and Modified Nucleic Acid Molecules An "antisense" nucleic acid refers to a nucleotide sequence complementary to a "sense" nucleic acid encoding a polypeptide, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence. The antisense nucleic acid can be complementary to an entire coding strand in SEQ ID Nos: l, 2, 3 or 4, or to a portion thereof or a substantially identical sequence thereof. In another embodiment, the antisense nucleic acid molecule is antisense to a "noncoding region" of the coding strand of a nucleotide sequence in SEQ ID NO: 1 (e.g., 5' and 3' untranslated regions). An antisense nucleic acid can be designed such that it is complementary to the entire coding region of an mRNA encoded by a nucleotide sequence in SEQ ID NO: 1 (e.g., SEQ ID Nos: 2-4), and often the antisense nucleic acid is an oligonucleotide antisense to only a portion of a coding or noncoding region of the mRNA. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of the mRNA, e.g., between the -10 and +10 regions of the target gene nucleotide sequence of interest. An antisense oligonucleotide can be, for example, about 7, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or more nucleotides in length. The antisense nucleic acids, which include the ribozymes described hereafter, can be designed to target a nucleotide sequence in SEQ ID Nos: l , 2, 3 or 4, often a variant associated with breast cancer or prostate cancer, or a substantially identical sequence thereof. Among the variants, minor alleles and major alleles can be targeted, and those associated with a higher risk of breast cancer or prostate cancer are often designed, tested, and administered to subjects. An antisense nucleic acid can be constructed using chemical synthesis and enzymatic ligation reactions using standard procedures. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used. Antisense nucleic acid also can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).

When utilized as therapeutics, antisense nucleic acids typically are administered to a subject (e.g., by direct injection at a tissue site) or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a polypeptide and thereby inhibit expression of the polypeptide, for example, by inhibiting transcription and/or translation. Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then are administered systemically. For systemic administration, antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, for example, by linking antisense nucleic acid molecules to peptides or antibodies which bind to cell surface receptors or antigens. Antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. Sufficient intracellular concentrations of antisense molecules are achieved by incorporating a strong promoter, such as a pol II or pol III promoter, in the vector construct.

Antisense nucleic acid molecules sometimes are alpha-anomeric nucleic acid molecules. An alpha-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual beta-units, the strands run parallel to each other (Gaultier et al., Nucleic Acids. Res. 15: 6625-6641 (1987)). Antisense nucleic acid molecules can also comprise a 2'-o-methylribonucleotide (Inoue et al., Nucleic Acids Res. 15: 6131-6148 (1987)) or a chimeric RNA-DNA analogue (Inoue et al., FEBS Lett. 215: 327-330 (1987)). Antisense nucleic acids sometimes are composed of DNA or PNA or any other nucleic acid derivatives described previously.

In another embodiment, an antisense nucleic acid is a ribozyme. A ribozyme having specificity for an ICAM nucleotide sequence can include one or more sequences complementary to such a nucleotide sequence, and a sequence having a known catalytic region responsible for mRNA cleavage (see e.g., U.S. Pat. No. 5,093,246 or Haselhoff and Gerlach, Nature 334: 585-591 (1988)). For example, a derivative of a Tetrahymena L-19 IVS RNA is sometimes utilized in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in a mRNA (see e.g., Cech et al. U.S. Patent No. 4,987,071 ; and Cech et al. U.S. Patent No. 5,1 16,742). Also, target mRNA sequences can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules (see e.g., Bartel & Szostak, Science 261 : 141 1-1418 (1993)). Breast cancer or prostate cancer directed molecules include in certain embodiments nucleic acids that can form triple helix structures with an ICAM nucleotide sequence or a substantially identical sequence thereof, especially one that includes a regulatory region that controls expression of a polypeptide. Gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of an ICAM nucleotide sequence or a substantially identical sequence (e.g., promoter and/or enhancers) to form triple helical structures that prevent transcription of a gene in target cells (see e.g., Helene, Anticancer Drug Des. 6(6): 569-84 (1991); Helene et al., Ann. N.Y. Acad. Sci. 660: 27-36 (1992); and Maher, Bioassays 14(12): 807-15 (1992). Potential sequences that can be targeted for triple helix formation can be increased by creating a so-called "switchback" nucleic acid molecule. Switchback molecules are synthesized in an alternating 5'-3', 3 '-5' manner, such that they base pair with first one strand of a duplex and then the other, eliminating the necessity for a sizeable stretch of either purines or pyrimidines to be present on one strand of a duplex.

Breast cancer or prostate cancer directed molecules include RNAi and siRNA nucleic acids. Gene expression may be inhibited by the introduction of double-stranded RNA (dsRNA), which induces potent and specific gene silencing, a phenomenon called RNA interference or RNAi. See, e.g., Fire et al., US Patent Number 6,506,559; Tuschl et al. PCT International Publication No. WO 01/75164; Kay et al. PCT International Publication No. WO 03/010180A1 ; or Bosher JM, Labouesse, Nat Cell Biol 2000 Feb;2(2):E31-6. This process has been improved by decreasing the size of the double-stranded RNA to 20-24 base pairs (to create small- interfering RNAs or siRNAs) that "switched off genes in mammalian cells without initiating an acute phase response, i.e., a host defense mechanism that often results in cell death (see, e.g., Caplen et al. Proc Natl Acad Sci U S A. 2001 Aug 14;98(17):9742-7 and Elbashir et al. Methods 2002 Feb;26(2): 199-213). There is increasing evidence of post-transcriptional gene silencing by RNA interference (RNAi) for inhibiting targeted expression in mammalian cells at the mRNA level, in human cells. There is additional evidence of effective methods for inhibiting the proliferation and migration of tumor cells in human patients, and for inhibiting metastatic cancer development (see, e.g., U.S. Patent Application No. US2001000993183; Caplen et al. Proc Natl Acad Sci U S A; and Abderrahmani et al. MoI Cell Biol 2001 Nov21(21):7256-67).

An "siRNA" or "RNAi" refers to a nucleic acid that forms a double stranded RNA and has the ability to reduce or inhibit expression of a gene or target gene when the siRNA is delivered to or expressed in the same cell as the gene or target gene. "siRNA" refers to short double-stranded RNA formed by the complementary strands. Complementary portions of the siRNA that hybridize to form the double stranded molecule often have substantial or complete identity to the target molecule sequence. In one embodiment, an siRNA refers to a nucleic acid that has substantial or complete identity to a target gene and forms a double stranded siRNA.

When designing the siRNA molecules, the targeted region often is selected from a given DNA sequence beginning 50 to 100 nucleotides downstream of the start codon. See, e.g., Elbashir et al,. Methods 26:199-213 (2002). Initially, 5' or 3' UTRs and regions nearby the start codon were avoided assuming that UTR-binding proteins and/or translation initiation complexes may interfere with binding of the siRNP or RISC endonuclease complex. Sometimes regions of the target 23 nucleotides in length conforming to the sequence motif AA(Nl 9)TT (N, an nucleotide), and regions with approximately 30% to 70% G/C-content (often about 50% G/C- content) often are selected. If no suitable sequences are found, the search often is extended using the motif

NA(N21). The sequence of the sense siRNA sometimes corresponds to (N 19) TT or N21 (position 3 to 23 of the 23-nt motif), respectively. In the latter case, the 3' end of the sense siRNA often is converted to TT. The rationale for this sequence conversion is to generate a symmetric duplex with respect to the sequence composition of the sense and antisense 3' overhangs. The antisense siRNA is synthesized as the complement to position 1 to 21 of the 23-nt motif. Because position 1 of the 23-nt motif is not recognized sequence-specifically by the antisense siRNA, the 3'-most nucleotide residue of the antisense siRNA can be chosen deliberately. However, the penultimate nucleotide of the antisense siRNA (complementary to position 2 of the 23-nt motif) often is complementary to the targeted sequence. For simplifying chemical synthesis, TT often is utilized. siRNAs corresponding to the target motif NAR(N 17) YNN, where R is purine (A,G) and Y is pyrimidine (C,U), often are selected. Respective 21 nucleotide sense and antisense siRNAs often begin with a purine nucleotide and can also be expressed from pol III expression vectors without a change in targeting site. Expression of RNAs from pol III promoters often is efficient when the first transcribed nucleotide is a purine.

The sequence of the siRNA can correspond to the full length target gene, or a subsequence thereof. Often, the siRNA is about 15 to about 50 nucleotides in length (e.g., each complementary sequence of the double stranded siRNA is 15-50 nucleotides in length, and the double stranded siRNA is about 15-50 base pairs in length, sometimes about 20-30 nucleotides in length or about 20-25 nucleotides in length, e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. The siRNA sometimes is about 21 nucleotides in length. Methods of using siRNA are well known in the art, and specific siRNA molecules may be purchased from a number of companies including Dharmacon Research, Inc. Antisense, ribozyme, RNAi and siRNA nucleic acids can be altered to form modified nucleic acid molecules.

The nucleic acids can be altered at base moieties, sugar moieties or phosphate backbone moieties to improve stability, hybridization, or solubility of the molecule. For example, the deoxyribose phosphate backbone of nucleic acid molecules can be modified to generate peptide nucleic acids (see Hyrup et al., Bioorganic & Medicinal Chemistry 4 (1): 5-23 (1996)). As used herein, the terms "peptide nucleic acid" or "PNA" refers to a nucleic acid mimic such as a DNA mimic, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained. The neutral backbone of a PNA can allow for specific hybridization to DNA and RNA under conditions of low ionic strength. Synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described, for example, in Hyrup et al., (1996) supra and Perry-O'Keefe et al., Proc. Natl. Acad. Sci. 93: 14670-675 (1996). PNA nucleic acids can be used in prognostic, diagnostic, and therapeutic applications. For example, PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, for example, inducing transcription or translation arrest or inhibiting replication. PNA nucleic acid molecules can also be used in the analysis of single base pair mutations in a gene, (e.g., by PNA-directed PCR clamping); as "artificial restriction enzymes" when used in combination with other enzymes, (e.g., Sl nucleases (Hyrup (1996) supra)); or as probes or primers for DNA sequencing or hybridization (Hyrup et al., (1996) supra; Perry-O'Keefe supra).

In other embodiments, oligonucleotides may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across cell membranes (see e.g., Letsinger et al., Proc. Natl. Acad. Sci. USA 86: 6553-6556 (1989); Lemaitre et al., Proc. Natl. Acad. Sci. USA 84: 648-652 (1987); PCT Publication No. W088/09810) or the blood-brain barrier (see, e.g., PCT Publication No. W089/10134). In addition, oligonucleotides can be modified with hybridization-triggered cleavage agents (See, e.g., Krol et al., Bio-Techniques 6: 958-976 (1988)) or intercalating agents. (See, e.g., Zon, Pharm. Res. 5: 539- 549 (1988) ). To this end, the oligonucleotide may be conjugated to another molecule, (e.g., a peptide, hybridization triggered cross-linking agent, transport agent, or hybridization-triggered cleavage agent).

Also included herein are molecular beacon oligonucleotide primer and probe molecules having one or more regions complementary to a nucleotide sequence of SEQ ID Nos: l, 2, 3 or 4 or a substantially identical sequence thereof, two complementary regions one having a fluorophore and one a quencher such that the molecular beacon is useful for quantifying the presence of the nucleic acid in a sample. Molecular beacon nucleic acids are described, for example, in Lizardi et al., U.S. Patent No. 5,854,033; Nazarenko et al., U.S. Patent No. 5,866,336, and Livak et al., U.S. Patent 5,876,930. Antibodies

The term "antibody" as used herein refers to an immunoglobulin molecule or immunologically active portion thereof, i.e., an antigen-binding portion. Examples of immunologically active portions of immunoglobulin molecules include F(ab) and F(ab')2 fragments which can be generated by treating the antibody with an enzyme such as pepsin. An antibody sometimes is a polyclonal, monoclonal, recombinant (e.g., a chimeric or humanized), fully human, non-human (e.g., murine), or a single chain antibody. An antibody may have effector function and can fix complement, and is sometimes coupled to a toxin or imaging agent. For example, antibodies that specifically bind to ICAMl are disclosed in U.S. Patent Nos. 5,475,091 and 5,773,293. ICAMl antibodies are available from R&D Systems, including the ICAMl antibody described in Example 8. MorphoSys also has a series of fully human antibodies binding human ICAM-I in the Fab format (MORlOl) and the IgG4 format (MORI 02). An antibody may have effector function and can fix complement, and is sometimes coupled to a toxin or imaging agent.

A full-length polypeptide or antigenic peptide fragment encoded by an ICAM nucleotide sequence can be used as an immunogen or can be used to identify antibodies made with other immunogens, e.g., cells, membrane preparations, and the like. An antigenic peptide often includes at least 8 amino acid residues of the amino acid sequences encoded by a nucleotide sequence of SEQ ID NO: 1 , 2 or 3, or substantially identical sequence thereof, and encompasses an epitope. Antigenic peptides sometimes include 10 or more amino acids, 15 or more amino acids, 20 or more amino acids, or 30 or more amino acids. Hydrophilic and hydrophobic fragments of polypeptides sometimes are used as immunogens. Epitopes encompassed by the antigenic peptide are regions located on the surface of the polypeptide (e.g., hydrophilic regions) as well as regions with high antigenicity. For example, an Emini surface probability analysis of the human polypeptide sequence can be used to indicate the regions that have a particularly high probability of being localized to the surface of the polypeptide and are thus likely to constitute surface residues useful for targeting antibody production. The antibody may bind an epitope on any domain or region on polypeptides described herein.

Also, chimeric, humanized, and completely human antibodies are useful for applications which include repeated administration to subjects. Chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, can be made using standard recombinant DNA techniques. Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in Robinson et al International Application No. PCT/US86/02269; Akira, et al European

Patent Application 184,187; Taniguchi, M., European Patent Application 171 ,496; Morrison et al European Patent Application 173,494; Neuberger et al PCT International Publication No. WO 86/01533; Cabilly et al U.S. Patent No. 4,816,567; Cabilly et al European Patent Application 125,023; Better et al., Science 240: 1041-1043 (1988); Liu et al., Proc. Natl. Acad. Sci. USA 84: 3439-3443 (1987); Liu et al., J. Immunol. 139: 3521-3526 (1987); Sun et al., Proc. Natl. Acad. Sci. USA 84: 214-218 (1987); Nishimura et al., Cane. Res. 47: 999-1005 (1987); Wood et al., Nature 314: 446-449 (1985); and Shaw et al., J. Natl. Cancer Inst. 80: 1553-1559 (1988); Morrison, S. L., Science 229: 1202-1207 (1985); Oi et al., BioTechniques 4: 214 (1986); Winter U.S. Patent 5,225,539; Jones et al., Nature 321 : 552-525 (1986); Verhoeyan et al., Science 239: 1534; and Beidler et al., J. Immunol. 141 : 4053- 4060 (1988). Completely human antibodies are particularly desirable for therapeutic treatment of human patients. Such antibodies can be produced using transgenic mice that are incapable of expressing endogenous immunoglobulin heavy and light chains genes, but which can express human heavy and light chain genes. See, for example, Lonberg and Huszar, Int. Rev. Immunol. 13: 65-93 (1995); and U.S. Patent Nos. 5,625,126; 5,633,425; 5,569,825; 5,661,016; and 5,545,806. In addition, companies such as Abgenix, Inc. (Fremont, CA) and Medarex, Inc. (Princeton, NJ), can be engaged to provide human antibodies directed against a selected antigen using technology similar to that described above. Completely human antibodies that recognize a selected epitope also can be generated using a technique referred to as "guided selection." In this approach a selected non-human monoclonal antibody (e.g., a murine antibody) is used to guide the selection of a completely human antibody recognizing the same epitope. This technology is described for example by Jespers et al., Bio/Technology 12: 899-903 (1994). An antibody can be a single chain antibody. A single chain antibody (scFV) can be engineered (see, e.g., Colcher et al., Ann. N Y Acad. Sci. 880: 263-80 (1999); and Reiter, Clin. Cancer Res. 2: 245-52 (1996)). Single chain antibodies can be dimerized or multimerized to generate multivalent antibodies having specificities for different epitopes of the same target polypeptide.

Antibodies also may be selected or modified so that they exhibit reduced or no ability to bind an Fc receptor. For example, an antibody may be an isotype or subtype, fragment or other mutant, which does not support binding to an Fc receptor (e.g., it has a mutagenized or deleted Fc receptor binding region).

Also, an antibody (or fragment thereof) may be conjugated to a therapeutic moiety such as a cytotoxin, a therapeutic agent or a radioactive metal ion. A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1 dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thiotepa chlorambucil, melphalan, carmustine (BCNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis- dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine and vinblastine).

Antibody conjugates can be used for modifying a given biological response. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a polypeptide such as tumor necrosis factor, γ-interferon, α-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator; or, biological response modifiers such as, for example, lymphokines, interleukin-1 ("IL-I"), interleukin-2 ("IL- 2"), interleukin-6 ("IL-6"), granulocyte macrophage colony stimulating factor ("GM-CSF"), granulocyte colony stimulating factor ("G-CSF"), or other growth factors. Also, an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Patent No. 4,676,980, for example.

An antibody (e.g., monoclonal antibody) can be used to isolate target polypeptides by standard techniques, such as affinity chromatography or immunoprecipitation. Moreover, an antibody can be used to detect a target polypeptide (e.g., in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression of the polypeptide. Antibodies can be used diagnostically to monitor polypeptide levels in tissue as part of a clinical testing procedure, e.g., to determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance (i.e., antibody labeling). Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include '²⁵I, ¹³¹1, ³⁵S or ³H. Also, an antibody can be utilized as a test molecule for determining whether it can treat breast cancer or prostate cancer, and as a therapeutic for administration to a subject for treating breast cancer or prostate cancer.

An antibody can be made by immunizing with a purified antigen, or a fragment thereof, e.g., a fragment described herein, a membrane associated antigen, tissues, e.g., crude tissue preparations, whole cells, preferably living cells, lysed cells, or cell fractions.

Included herein are antibodies which bind only a native polypeptide, only denatured or otherwise non-native polypeptide, or which bind both, as well as those having linear or conformational epitopes. Conformational epitopes sometimes can be identified by selecting antibodies that bind to native but not denatured polypeptide. Also featured are antibodies that specifically bind to a polypeptide variant associated with breast cancer or prostate cancer.

Screening Assays

Featured herein are methods for identifying a candidate therapeutic for treating breast cancer or prostate cancer. The methods comprise contacting a test molecule with a target molecule in a system. A "target molecule" as used herein refers to a nucleic acid of SEQ ID Nos:l , 2, 3 or 4, a substantially identical nucleic acid thereof, or a fragment thereof, and an encoded polypeptide of the foregoing. The method also comprises determining the presence or absence of an interaction between the test molecule and the target molecule, where the presence of an interaction between the test molecule and the nucleic acid or polypeptide identifies the test molecule as a candidate breast cancer or prostate cancer therapeutic. The interaction between the test molecule and the target molecule may be quantified.

Test molecules and candidate therapeutics include, but are not limited to, compounds, antisense nucleic acids, siRNA molecules, ribozymes, polypeptides or proteins encoded by a ICAM nucleic acids, or a substantially identical sequence or fragment thereof, and immunotherapeutics (e.g., antibodies and HLA-presented polypeptide fragments). A test molecule or candidate therapeutic may act as a modulator of target molecule concentration or target molecule function in a system. A "modulator" may agonize (i.e., up-regulates) or antagonize (i.e., down- regulates) a target molecule concentration partially or completely in a system by affecting such cellular functions as DNA replication and/or DNA processing (e.g., DNA methylation or DNA repair), RNA transcription and/or RNA processing (e.g., removal of intronic sequences and/or translocation of spliced mRNA from the nucleus), polypeptide production (e.g., translation of the polypeptide from mRNA), and/or polypeptide post-translational modification (e.g., glycosylation, phosphorylation, and proteolysis of pro-polypeptides). A modulator may also agonize or antagonize a biological function of a target molecule partially or completely, where the function may include adopting a certain structural conformation, interacting with one or more binding partners, ligand binding, catalysis (e.g., phosphorylation, dephosphorylation, hydrolysis, methylation, and isomerization), and an effect upon a cellular event (e.g., effecting progression of breast cancer or prostate cancer).

As used herein, the term "system" refers to a cell free in vitro environment and a cell-based environment such as a collection of cells, a tissue, an organ, or an organism. A system is "contacted" with a test molecule in a variety of manners, including adding molecules in solution and allowing them to interact with one another by diffusion, cell injection, and any administration routes in an animal. As used herein, the term "interaction" refers to an effect of a test molecule on test molecule, where the effect sometimes is binding between the test molecule and the target molecule, and sometimes is an observable change in cells, tissue, or organism.

There are many standard methods for detecting the presence or absence of interaction between a test molecule and a target molecule. For example, titrametric, acidimetric, radiometric, NMR, monolayer, polarographic, spectrophotometric, fluorescent, and ESR assays probative of a target molecule interaction may be utilized.

Test molecule/target molecule interactions can be detected and/or quantified using assays known in the art. For example, an interaction can be determined by labeling the test molecule and/or the target molecule, where the label is covalently or non-covalently attached to the test molecule or target molecule. The label is sometimes a radioactive molecule such as '²⁵1, ¹³¹1, ³⁵S or ³H, which can be detected by direct counting of radioemission or by scintillation counting. Also, enzymatic labels such as horseradish peroxidase, alkaline phosphatase, or luciferase may be utilized where the enzymatic label can be detected by determining conversion of an appropriate substrate to product. In addition, presence or absence of an interaction can be determined without labeling. For example, a microphysiometer (e.g., Cytosensor) is an analytical instrument that measures the rate at which a cell acidifies its environment using a light-addressable potentiometric sensor (LAPS). Changes in this acidification rate can be used as an indication of an interaction between a test molecule and target molecule (McConnell, H. M. et al, Science 257: 1906-1912 (1992)).

In cell-based systems, cells typically include a nucleic acid from SEQ ID Nos: 1 -4, an encoded polypeptide, or substantially identical nucleic acid or polypeptide thereof, and are often of mammalian origin, although the cell can be of any origin. Whole cells, cell homogenates, and cell fractions {e.g., cell membrane fractions) can be subjected to analysis. Where interactions between a test molecule with a ICAM polypeptide are monitored, soluble and/or membrane bound forms of the polypeptide may be utilized. Where membrane-bound forms of the polypeptide are used, it may be desirable to utilize a solubilizing agent. Examples of such solubilizing agents include non-ionic detergents such as n-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N- methylglucamide, decanoyl-N-methylglucamide, Triton® X-100, Triton® X-114, Thesit®, Isotridecypoly(ethylene glycol ether)_n, 3-[(3-cholamidopropyl)dimethylamminio]-l -propane sulfonate (CHAPS),

3-[(3-cholamidopropyl)dimethylamminio]-2-hydroxy-l-propane sulfonate (CHAPSO), or N-dodecyl-N,N- dimethyl-3-ammonio-l -propane sulfonate. An interaction between a test molecule and target molecule also can be detected by monitoring fluorescence energy transfer (FET) {see, e.g., Lakowicz et al, U.S. Patent No. 5,631,169; Stavrianopoulos et al. U.S. Patent No. 4,868,103). A fluorophore label on a first, "donor" molecule is selected such that its emitted fluorescent energy will be absorbed by a fluorescent label on a second, "acceptor" molecule, which in turn is able to fluoresce due to the absorbed energy. Alternately, the "donor" polypeptide molecule may simply utilize the natural fluorescent energy of tryptophan residues. Labels are chosen that emit different wavelengths of light, such that the "acceptor" molecule label may be differentiated from that of the "donor". Since the efficiency of energy transfer between the labels is related to the distance separating the molecules, the spatial relationship between the molecules can be assessed. In a situation in which binding occurs between the molecules, the fluorescent emission of the "acceptor" molecule label in the assay should be maximal. An FET binding event can be conveniently measured through standard fluorometric detection means well known in the art (e.g., using a fluorimeter).

In another embodiment, determining the presence or absence of an interaction between a test molecule and a target molecule can be effected by monitoring surface plasmon resonance (see, e.g., Sjolander & Urbaniczk, Anal. Chem. 63: 2338-2345 (1991) and Szabo et al, Curr. Opin. Struct. Biol. 5: 699-705 (1995)). "Surface plasmon resonance" or "biomolecular interaction analysis (BIA)" can be utilized to detect biospecific interactions in real time, without labeling any of the interactants (e.g., BIAcore). Changes in the mass at the binding surface

(indicative of a binding event) result in alterations of the refractive index of light near the surface (the optical phenomenon of surface plasmon resonance (SPR)), resulting in a detectable signal which can be used as an indication of real-time reactions between biological molecules.

In another embodiment, the target molecule or test molecules are anchored to a solid phase, facilitating the detection of target molecule/test molecule complexes and separation of the complexes from free, uncomplexed molecules. The target molecule or test molecule is immobilized to the solid support. In an embodiment, the target molecule is anchored to a solid surface, and the test molecule, which is not anchored, can be labeled, either directly or indirectly, with detectable labels discussed herein.

It may be desirable to immobilize a target molecule, an anti-target molecule antibody, and/or test molecules to facilitate separation of target molecule/test molecule complexes from uncomplexed forms, as well as to accommodate automation of the assay. The attachment between a test molecule and/or target molecule and the solid support may be covalent or non-covalent (see, e.g., U.S. Patent No. 6,022,688 for non-covalent attachments). The solid support may be one or more surfaces of the system, such as one or more surfaces in each well of a microtiter plate, a surface of a silicon wafer, a surface of a bead (see, e.g., Lam, Nature 354: 82-84 (1991)) that is optionally linked to another solid support, or a channel in a microfluidic device, for example. Types of solid supports, linker molecules for covalent and non-covalent attachments to solid supports, and methods for immobilizing nucleic acids and other molecules to solid supports are well known (see, e.g., U.S. Patent Nos. 6,261,776; 5,900,481 ; 6,133,436; and 6,022,688; and WIPO publication WO 01/18234).

In an embodiment, target molecule may be immobilized to surfaces via biotin and streptavidin. For example, biotinylated ICAM polypeptide can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, IL), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical). In another embodiment, a ICAM polypeptide can be prepared as a fusion polypeptide. For example, glutathione-S-transferase//C4M polypeptide fusion can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione derivitized microtiter plates, which are then combined with a test molecule under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components, or the matrix is immobilized in the case of beads, and complex formation is determined directly or indirectly as described above. Alternatively, the complexes can be dissociated from the matrix, and the level of target molecule binding or activity is determined using standard techniques. In an embodiment, the non-immobilized component is added to the coated surface containing the anchored component. After the reaction is complete, unreacted components are removed (e.g., by washing) under conditions such that a significant percentage of complexes formed will remain immobilized to the solid surface. The detection of complexes anchored on the solid surface can be accomplished in a number of manners. Where the previously non-immobilized component is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the previously non-immobilized component is not pre-labeled, an indirect label can be used to detect complexes anchored on the surface, e.g., by adding a labeled antibody specific for the immobilized component, where the antibody, in turn, can be directly labeled or indirectly labeled with, e.g., a labeled anti-Ig antibody.

In another embodiment, an assay is performed utilizing antibodies that specifically bind target molecule or test molecule but do not interfere with binding of the target molecule to the test molecule. Such antibodies can be derivitized to a solid support, and unbound target molecule may be immobilized by antibody conjugation.

Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with the target molecule, as well as enzyme- linked assays which rely on detecting an enzymatic activity associated with the target molecule.

Cell free assays also can be conducted in a liquid phase. In such an assay, reaction products are separated from unreacted components, by any of a number of standard techniques, including but not limited to: differential centrifugation (see, e.g., Rivas, G., and Minton, Trends Biochem Sci Aug;18(8): 284-7 (1993)); chromatography (gel filtration chromatography, ion-exchange chromatography); electrophoresis (see, e.g., Ausubel et al., eds. Current Protocols in Molecular Biology , J. Wiley: New York (1999)); and immunoprecipitation (see, e.g., Ausubel et al., eds., supra). Media and chromatographic techniques are known to one skilled in the art (see, e.g., Heegaard, JMo/. Recognit. Winter; 11(1-6): 141-8 (1998); Hage & Tweed, J. Chromatogr. B Biomed. Sci. Appl Oct 10; 699 (1-2): 499-525 (1997)). Further, fluorescence energy transfer may also be conveniently utilized, as described herein, to detect binding without further purification of the complex from solution.

In another embodiment, modulators of target molecule expression are identified. For example, a cell or cell free mixture is contacted with a candidate compound and the expression of target mRNA or ICAM polypeptide is evaluated relative to the level of expression of target mRNA or ICAM polypeptide in the absence of the candidate compound. When expression of target mRNA or ICAM polypeptide is greater in the presence of the candidate compound than in its absence, the candidate compound is identified as an agonist of target mRNA or ICAM polypeptide expression. Alternatively, when expression of target mRNA or ICAM polypeptide is less (e.g., less with statistical significance) in the presence of the candidate compound than in its absence, the candidate compound is identified as an antagonist or inhibitor of target mRNA or ICAM polypeptide expression. The level of target mRNA or ICAM polypeptide expression can be determined by methods described herein.

In another embodiment, binding partners that interact with a target molecule are detected. The target molecules can interact with one or more cellular or extracellular macromolecules, such as polypeptides in vivo, and these interacting molecules are referred to herein as "binding partners." Binding partners can agonize or antagonize target molecule biological activity. Also, test molecules that agonize or antagonize interactions between target molecules and binding partners can be useful as therapeutic molecules as they can up-regulate or down-regulated target molecule activity in vivo and thereby treat breast cancer or prostate cancer.

Binding partners of target molecules can be identified by methods known in the art. For example, binding partners may be identified by lysing cells and analyzing cell lysates by electrophoretic techniques. Alternatively, a two-hybrid assay or three-hybrid assay can be utilized (see, e.g., U.S. Patent No. 5,283,317; Zervos et al, Cell 72:223-232 (1993); Madura et al., J. Biol. Chem. 268: 12046-12054 (1993); Bartel et al., Biotechniques 14: 920- 924 (1993); Iwabuchi et ai, Oncogene 8: 1693-1696 (1993); and Brent WO94/10300). A two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. The assay often utilizes two different DNA constructs. In one construct, a nucleic acid from SEQ ID Nos:l-4 (sometimes referred to as the "bait") is fused to a gene encoding the DNA binding domain of a known transcription factor (e.g., GAL-4). In another construct, a DNA sequence from a library of DNA sequences that encodes a potential binding partner (sometimes referred to as the "prey") is fused to a gene that encodes an activation domain of the known transcription factor. Sometimes, a nucleic acid from SEQ ID Nos:l-4 can be fused to the activation domain. If the "bait" and the "prey" molecules interact in vivo, the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ) which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to identify the potential binding partner.

In an embodiment for identifying test molecules that antagonize or agonize complex formation between target molecules and binding partners, a reaction mixture containing the target molecule and the binding partner is prepared, under conditions and for a time sufficient to allow complex formation. The reaction mixture often is provided in the presence or absence of the test molecule. The test molecule can be included initially in the reaction mixture, or can be added at a time subsequent to the addition of the target molecule and its binding partner. Control reaction mixtures are incubated without the test molecule or with a placebo. Formation of any complexes between the target molecule and the binding partner then is detected. Decreased formation of a complex in the reaction mixture containing test molecule as compared to in a control reaction mixture indicates that the molecule antagonizes target molecule/binding partner complex formation. Alternatively, increased formation of a complex in the reaction mixture containing test molecule as compared to in a control reaction mixture indicates that the molecule agonizes target molecule/binding partner complex formation. In another embodiment, complex formation of target molecule/binding partner can be compared to complex formation of mutant target molecule/binding partner (e.g., amino acid modifications in a ICAM polypeptide). Such a comparison can be important in those cases where it is desirable to identify test molecules that modulate interactions of mutant but not non-mutated target gene products.

The assays can be conducted in a heterogeneous or homogeneous format. In heterogeneous assays, target molecule and/or the binding partner are immobilized to a solid phase, and complexes are detected on the solid phase at the end of the reaction. In homogeneous assays, the entire reaction is carried out in a liquid phase. In either approach, the order of addition of reactants can be varied to obtain different information about the molecules being tested. For example, test compounds that agonize target molecule/binding partner interactions can be identified by conducting the reaction in the presence of the test molecule in a competition format. Alternatively, test molecules that agonize preformed complexes, e.g., molecules with higher binding constants that displace one of the components from the complex, can be tested by adding the test compound to the reaction mixture after complexes have been formed.

In a heterogeneous assay embodiment, the target molecule or the binding partner is anchored onto a solid surface (e.g., a microtiter plate), while the non-anchored species is labeled, either directly or indirectly. The anchored molecule can be immobilized by non-covalent or covalent attachments. Alternatively, an immobilized antibody specific for the molecule to be anchored can be used to anchor the molecule to the solid surface. The partner of the immobilized species is exposed to the coated surface with or without the test molecule. After the reaction is complete, unreacted components are removed (e.g., by washing) such that a significant portion of any complexes formed will remain immobilized on the solid surface. Where the non-immobilized species is pre- labeled, the detection of label immobilized on the surface is indicative of complex. Where the non-immobilized species is not pre-labeled, an indirect label can be used to detect complexes anchored to the surface; e.g., by using a labeled antibody specific for the initially non-immobilized species. Depending upon the order of addition of reaction components, test compounds that inhibit complex formation or that disrupt preformed complexes can be detected.

In another embodiment, the reaction can be conducted in a liquid phase in the presence or absence of test molecule, where the reaction products are separated from unreacted components, and the complexes are detected (e.g., using an immobilized antibody specific for one of the binding components to anchor any complexes formed in solution, and a labeled antibody specific for the other partner to detect anchored complexes). Again, depending upon the order of addition of reactants to the liquid phase, test compounds that inhibit complex or that disrupt preformed complexes can be identified.

In an alternate embodiment, a homogeneous assay can be utilized. For example, a preformed complex of the target gene product and the interactive cellular or extracellular binding partner product is prepared. One or both of the target molecule or binding partner is labeled, and the signal generated by the label(s) is quenched upon complex formation (, e.g., U.S. Patent No. 4,109,496 that utilizes this approach for immunoassays). Addition of a test molecule that competes with and displaces one of the species from the preformed complex will result in the generation of a signal above background. In this way, test substances that disrupt target molecule/binding partner complexes can be identified.

Identification of Candidate Therapeutics

Candidate therapeutics for treating breast cancer or prostate cancer are identified from a group of test molecules that interact with a target molecule. Test molecules are normally ranked according to the degree with which they modulate (e.g., agonize or antagonize) a function associated with the target molecule (e.g., DNA replication and/or processing, RNA transcription and/or processing, polypeptide production and/or processing, and/or biological function/activity), and then top ranking modulators are selected. Also, pharmacogenomic information described herein can determine the rank of a modulator. The top 10% of ranked test molecules often are selected for further testing as candidate therapeutics, and sometimes the top 15%, 20%, or 25% of ranked test molecules are selected for further testing as candidate therapeutics. Candidate therapeutics typically are formulated for administration to a subject.

Therapeutic Formulations

Formulations and pharmaceutical compositions typically include in combination with a pharmaceutically acceptable carrier one or more target molecule modulators. The modulator often is a test molecule identified as having an interaction with a target molecule by a screening method described above. The modulator may be a compound, an antisense nucleic acid, a ribozyme, an antibody, or a binding partner. Also, formulations may comprise a ICA M polypeptide or fragment thereof in combination with a pharmaceutically acceptable carrier.

As used herein, the term "pharmaceutically acceptable carrier" includes solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the compositions. Pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

A pharmaceutical composition typically is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral {e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Oral compositions generally include an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules, e.g., gelatin capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the methods of preparation often utilized are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer. Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art. Molecules can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery. In one embodiment, active molecules are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. Materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,81 1.

It is advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD₅₀ (the dose lethal to 50% of the population) and the ED₅₀ (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀. Molecules which exhibit high therapeutic indices often are utilized. While molecules that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such molecules lies preferably within a range of circulating concentrations that include the ED₅₀ with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any molecules used in methods described herein, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC₅₀ (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography. As defined herein, a therapeutically effective amount of protein or polypeptide (i.e., an effective dosage) ranges from about 0.001 to 30 mg/kg body weight, sometimes about 0.01 to 25 mg/kg body weight, often about 0.1 to 20 mg/kg body weight, and more often about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight. The protein or polypeptide can be administered one time per week for between about 1 to 10 weeks, sometimes between 2 to 8 weeks, often between about 3 to 7 weeks, and more often for about 4, 5, or 6 weeks. The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a protein, polypeptide, or antibody can include a single treatment or, preferably, can include a series of treatments.

For antibodies, a dosage of 0.1 mg/kg of body weight (generally 10 mg/kg to 20 mg/kg) is often utilized. If the antibody is to act in the brain, a dosage of 50 mg/kg to 100 mg/kg is often appropriate. Generally, partially human antibodies and fully human antibodies have a longer half-life within the human body than other antibodies. Accordingly, lower dosages and less frequent administration is often possible. Modifications such as lipidation can be used to stabilize antibodies and to enhance uptake and tissue penetration (e.g., into the brain). A method for lipidation of antibodies is described by Cruikshank et ai, J. Acquired Immune Deficiency Syndromes and Human Retrovirology 14: 193 (1997).

Antibody conjugates can be used for modifying a given biological response, the drug moiety is not to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a polypeptide such as tumor necrosis factor, alpha- interferon, beta-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator; or, biological response modifiers such as, for example, lymphokines, interleukin-1 ("IL-I "), interleukin-2 ("IL-2"), interleukin-6 ("IL-6"), granulocyte macrophage colony stimulating factor ("GM-CSF"), granulocyte colony stimulating factor ("G-CSF"), or other growth factors. Alternatively, an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Patent No. 4,676,980.

For compounds, exemplary doses include milligram or microgram amounts of the compound per kilogram of subject or sample weight, for example, about 1 microgram per kilogram to about 500 milligrams per kilogram, about 100 micrograms per kilogram to about 5 milligrams per kilogram, or about 1 microgram per kilogram to about 50 micrograms per kilogram. It is understood that appropriate doses of a small molecule depend upon the potency of the small molecule with respect to the expression or activity to be modulated. When one or more of these small molecules is to be administered to an animal (e.g., a human) in order to modulate expression or activity of a polypeptide or nucleic acid described herein, a physician, veterinarian, or researcher may, for example, prescribe a relatively low dose at first, subsequently increasing the dose until an appropriate response is obtained. In addition, it is understood that the specific dose level for any particular animal subject will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, any drug combination, and the degree of expression or activity to be modulated.

With regard to nucleic acid formulations, gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see, e.g., U.S. Patent 5,328,470) or by stereotactic injection (see e.g., Chen et al, (1994) Proc. Natl. Acad. ScL USA 97:3054-3057). Pharmaceutical preparations of gene therapy vectors can include a gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells (e.g., retroviral vectors) the pharmaceutical preparation can include one or more cells which produce the gene delivery system. Examples of gene delivery vectors are described herein.

Therapeutic Methods

A therapeutic formulation described above can be administered to a subject in need of a therapeutic for treating breast cancer or prostate cancer. Therapeutic formulations can be administered by any of the paths described herein. With regard to both prophylactic and therapeutic methods of treatment, such treatments may be specifically tailored or modified, based on knowledge obtained from pharmacogenomic analyses described herein. As used herein, the term "treatment" is defined as the application or administration of a therapeutic formulation to a subject, or application or administration of a therapeutic agent to an isolated tissue or cell line from a subject with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect breast cancer or prostate cancer, symptoms of breast cancer or prostate cancer or a predisposition towards breast cancer or prostate cancer. A therapeutic formulation includes, but is not limited to, small molecules, peptides, antibodies, ribozymes and antisense oligonucleotides. Administration of a therapeutic formulation can occur prior to the manifestation of symptoms characteristic of breast cancer or prostate cancer, such that breast cancer or prostate cancer is prevented or delayed in its progression. The appropriate therapeutic composition can be determined based on screening assays described herein.

As discussed, successful treatment of breast cancer or prostate cancer can be brought about by techniques that serve to agonize target molecule expression or function, or alternatively, antagonize target molecule expression or function. These techniques include administration of modulators that include, but are not limited to, small organic or inorganic molecules; antibodies (including, for example, polyclonal, monoclonal, humanized, anti- idiotypic, chimeric or single chain antibodies, and FAb, F(ab')₂ and FAb expression library fragments, scFV molecules, and epitope-binding fragments thereof); and peptides, phosphopeptides, or polypeptides. Further, antisense and ribozyme molecules that inhibit expression of the target gene can also be used to reduce the level of target gene expression, thus effectively reducing the level of target gene activity. Still further, triple helix molecules can be utilized in reducing the level of target gene activity. Antisense, ribozyme and triple helix molecules are discussed above. It is possible that the use of antisense, ribozyme, and/or triple helix molecules to reduce or inhibit mutant gene expression can also reduce or inhibit the transcription (triple helix) and/or translation (antisense, ribozyme) of mRNA produced by normal target gene alleles, such that the concentration of normal target gene product present can be lower than is necessary for a normal phenotype. In such cases, nucleic acid molecules that encode and express target gene polypeptides exhibiting normal target gene activity can be introduced into cells via gene therapy method. Alternatively, in instances in that the target gene encodes an extracellular polypeptide, it can be preferable to co-administer normal target gene polypeptide into the cell or tissue in order to maintain the requisite level of cellular or tissue target gene activity.

Another method by which nucleic acid molecules may be utilized in treating or preventing breast cancer or prostate cancer is use of aptamer molecules specific for target molecules. Aptamers are nucleic acid molecules having a tertiary structure which permits them to specifically bind to ligands (see, e.g., Osborne, et al., Curr. Opin. Chem. Biol. l(l): 5-9 (1997); and Patel, D. J., Curr. Opin. Chem. Biol. Jun;l(l): 32-46 (1997)). Yet another method of utilizing nucleic acid molecules for breast cancer or prostate cancer treatment is gene therapy, which can also be referred to as allele therapy. Provided herein is a gene therapy method for treating breast cancer or prostate cancer in a subject, which comprises contacting one or more cells in the subject or from the subject with a nucleic acid having a first nucleotide sequence. Genomic DNA in the subject comprises a second nucleotide sequence having one or more polymorphic variations associated with breast cancer or prostate cancer (e.g., the second nucleic acid is selected from SEQ ID Nos:l-4). The first and second nucleotide sequences typically are substantially identical to one another, and the first nucleotide sequence comprises fewer polymorphic variations associated with breast cancer or prostate cancer than the second nucleotide sequence. The first nucleotide sequence may comprise a gene sequence that encodes a full-length polypeptide or a fragment thereof. The subject is often a human. Allele therapy methods often are utilized in conjunction with a method of first determining whether a subject has genomic DNA that includes polymorphic variants associated with breast cancer or prostate cancer.

In another allele therapy embodiment, provided herein is a method which comprises contacting one or more cells in the subject or from the subject with a polypeptide encoded by a nucleic acid having a first nucleotide sequence. Genomic DNA in the subject comprises a second nucleotide sequence having one or more polymorphic variations associated with breast cancer or prostate cancer (e.g., the second nucleic acid is selected from SEQ ID Nos:l-4). The first and second nucleotide sequences typically are substantially identical to one another, and the first nucleotide sequence comprises fewer polymorphic variations associated with breast cancer or prostate cancer than the second nucleotide sequence. The first nucleotide sequence may comprise a gene sequence that encodes a full-length polypeptide or a fragment thereof. The subject is often a human.

For antibody-based therapies, antibodies can be generated that are both specific for target molecules and that reduce target molecule activity. Such antibodies may be administered in instances where antagonizing a target molecule function is appropriate for the treatment of breast cancer or prostate cancer.

In circumstances where stimulating antibody production in an animal or a human subject by injection with a target molecule is harmful to the subject, it is possible to generate an immune response against the target molecule by use of anti-idiotypic antibodies {see, e.g., Herlyn, Ann. Med;31(l): 66-78 (1999); and Bhattacharya-Chatterjee & Foon, Cancer Treat. Res.; 94: 51 -68 (1998)). Introducing an anti-idiotypic antibody to a mammal or human subject often stimulates production of anti-anti-idiotypic antibodies, which typically are specific to the target molecule. Vaccines directed to breast cancer or prostate cancer also may be generated in this fashion. In instances where the target molecule is intracellular and whole antibodies are used, internalizing antibodies often are utilized. Lipofectin or liposomes can be used to deliver the antibody or a fragment of the Fab region that binds to the target antigen into cells. Where fragments of the antibody are used, the smallest inhibitory fragment that binds to the target antigen often is utilized. For example, peptides having an amino acid sequence corresponding to the Fv region of the antibody can be used. Alternatively, single chain neutralizing antibodies that bind to intracellular target antigens can also be administered. Such single chain antibodies can be administered, for example, by expressing nucleotide sequences encoding single-chain antibodies within the target cell population (see, e.g., Marasco et ai. Proc. Natl. Acad. ScL USA 90: 7889-7893 (1993)).

Modulators can be administered to a patient at therapeutically effective doses to treat breast cancer or prostate cancer. A therapeutically effective dose refers to an amount of the modulator sufficient to result in amelioration of symptoms of breast cancer or prostate cancer. Toxicity and therapeutic efficacy of modulators can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD₅₀ (the dose lethal to 50% of the population) and the ED₅₀ (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LO₅₀ZEO₅₀. Modulators that exhibit large therapeutic indices often are utilized. While modulators that exhibit toxic side effects can be used, care should be taken to design a delivery system that targets such molecules to the site of affected tissue in order to minimize potential damage to uninfected cells, thereby reducing side effects.

Data obtained from cell culture assays and animal studies can be used in formulating a range of dosages for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED₅₀ with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in a method described herein, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC₅₀ (i.e., the concentration of the test compound that achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography.

Another example of effective dose determination for an individual is the ability to directly assay levels of "free" and "bound" compound in the serum of the test subject. Such assays may utilize antibody mimics and/or "biosensors" that have been created through molecular imprinting techniques. Molecules that modulate target molecule activity are used as a template, or "imprinting molecule", to spatially organize polymerizable monomers prior to their polymerization with catalytic reagents. The subsequent removal of the imprinted molecule leaves a polymer matrix which contains a repeated "negative image" of the compound and is able to selectively rebind the molecule under biological assay conditions. A detailed review of this technique can be seen in Ansell et al., Current Opinion in Biotechnology 7: 89-94 (1996) and in Shea, Trends in Polymer Science 2: 166-173 (1994). Such "imprinted" affinity matrixes are amenable to ligand-binding assays, whereby the immobilized monoclonal antibody component is replaced by an appropriately imprinted matrix. An example of the use of such matrixes in this way can be seen in Vlatakis, et al., Nature 361: 645-647 (1993). Through the use of isotope-labeling, the "free" concentration of compound which modulates target molecule expression or activity readily can be monitored and used in calculations OfIC₅₀. Such "imprinted" affinity matrixes can also be designed to include fluorescent groups whose photon-emitting properties measurably change upon local and selective binding of target compound. These changes readily can be assayed in real time using appropriate fiberoptic devices, in turn allowing the dose in a test subject to be quickly optimized based on its individual IC₅₀. An example of such a "biosensor" is discussed in Kriz et al., Analytical Chemistry 67: 2142-2144 (1995). The examples set forth below illustrate but not limit the invention.

Examples In the following studies a group of subjects were selected according to specific parameters pertaining to breast cancer or prostate cancer. Nucleic acid samples obtained from individuals in the study group were subjected to genetic analyses that identified associations between breast cancer or prostate cancer and certain polymorphic variants in human genomic DNA. Test molecules identified as being interactors with ICAM polypeptides can be screened further as breast cancer or prostate cancer therapeutics. Example 1 Samples and Pooling Strategies

Sample Selection

Blood samples were collected from individuals diagnosed with breast cancer, which were referred to case samples. Also, blood samples were collected from individuals not diagnosed with breast cancer or any form of cancer or a history of breast cancer or prostate cancer; these samples served as gender and age-matched controls. All of the samples were of German/German descent. A database was created that listed all phenotypic trait information gathered from individuals for each case and control sample. Genomic DNA was extracted from each of the blood samples for genetic analyses.

DNA Extraction from Blood Samples

Six to ten milliliters of whole blood was transferred to a 50 ml tube containing 27 ml of red cell lysis solution (RCL). The tube was inverted until the contents were mixed. Each tube was incubated for 10 minutes at room temperature and inverted once during the incubation. The tubes were then centrifuged for 20 minutes at 3000 x g and the supernatant was carefully poured off. 100-200 μl of residual liquid was left in the tube and was pipetted repeatedly to resuspend the pellet in the residual supernatant. White cell lysis solution (WCL) was added to the tube and pipetted repeatedly until completely mixed. While no incubation was normally required, the solution was incubated at 37°C or room temperature if cell clumps were visible after mixing until the solution was homogeneous. 2 ml of protein precipitation was added to the cell lysate. The mixtures were vortexed vigorously at high speed for 20 sec to mix the protein precipitation solution uniformly with the cell lysate, and then centrifuged for 10 minutes at 3000 x g. The supernatant containing the DNA was then poured into a clean 15 ml tube, which contained 7 ml of 100% isopropanol. The samples were mixed by inverting the tubes gently until white threads of DNA were visible. Samples were centrifuged for 3 minutes at 2000 x g and the DNA was visible as a small white pellet. The supernatant was decanted and 5 ml of 70% ethanol was added to each tube. Each tube was inverted several times to wash the DNA pellet, and then centrifuged for 1 minute at 2000 x g. The ethanol was decanted and each tube was drained on clean absorbent paper. The DNA was dried in the tube by inversion for 10 minutes, and then 1000 μl of I X TE was added. The size of each sample was estimated, and less TE buffer was added during the following DNA hydration step if the sample was smaller. The DNA was allowed to rehydrate overnight at room temperature, and DNA samples were stored at 2-8°C.

DNA was quantified by placing samples on a hematology mixer for at least 1 hour. DNA was serially diluted (typically 1 :80, 1 :160, 1 :320, and 1 :640 dilutions) so that it would be within the measurable range of standards. 125 μl of diluted DNA was transferred to a clear U-bottom microtiter plate, and 125 μl of IX TE buffer was transferred into each well using a multichannel pipette. The DNA and IX TE were mixed by repeated pipetting at least 15 times, and then the plates were sealed. 50 μl of diluted DNA was added to wells A5-H12 of a black flat bottom microtiter plate. Standards were inverted six times to mix them, and then 50 μl of IX TE buffer was pipetted into well Al , 1000 ng/ml of standard was pipetted into well A2, 500 ng/ml of standard was pipetted into well A3, and 250 ng/ml of standard was pipetted into well A4. PicoGreen (Molecular Probes, Eugene, Oregon) was thawed and freshly diluted 1 :200 according to the number of plates that were being measured. PicoGreen was vortexed and then 50μl was pipetted into all wells of the black plate with the diluted DNA. DNA and PicoGreen were mixed by pipetting repeatedly at least 10 times with the multichannel pipette. The plate was placed into a Fluoroskan Ascent Machine (microplate fluorometer produced by Labsystems) and the samples were allowed to incubate for 3 minutes before the machine was run using filter pairs 485 nm excitation and 538 πm emission wavelengths. Samples having measured DNA concentrations of greater than 450 ng/μl were re-measured for conformation. Samples having measured DNA concentrations of 20 ng/μl or less were re-measured for confirmation.

Pooling Strategies

Samples were placed into one of two groups based on disease status. The two groups were female case samples and female control samples. A select set of samples from each group were utilized to generate pools, and one pool was created for each group. Each individual sample in a pool was represented by an equal amount of genomic DNA. For example, where 25 ng of genomic DNA was utilized in each PCR reaction and there were 200 individuals in each pool, each individual would provide 125 pg of genomic DNA. Inclusion or exclusion of samples for a pool was based upon the following criteria: the sample was derived from an individual characterized as Caucasian; the sample was derived from an individual of German paternal and maternal descent; the database included relevant phenotype information for the individual; case samples were derived from individuals diagnosed with breast cancer; control samples were derived from individuals free of cancer and no family history of breast cancer; and sufficient genomic DNA was extracted from each blood sample for all allelotyping and genotyping reactions performed during the study. Phenotype information included pre- or postmenopausal, familial predisposition, country or origin of mother and father, diagnosis with breast cancer (date of primary diagnosis, age of individual as of primary diagnosis, grade or stage of development, occurrence of metastases, e.g., lymph node metastases, organ metastases), condition of body tissue (skin tissue, breast tissue, ovary tissue, peritoneum tissue and myometrium), method of treatment (surgery, chemotherapy, hormone therapy, radiation therapy). Samples that met these criteria were added to appropriate pools based on gender and disease status. The discovery sample comprised 254 breast cancer patients attending the Frauenklinik Innenstadt, Munich,

Germany. Lymph node status was positive at time of assessment in 94 cases (37%), and 18 cases (7%) had known distant metastases. Twenty- four cases (1 1%) reported a positive family history of breast cancer. The median age at diagnosis was 56 yr (range = 23-87 yr). 268 controls with a median age of 57 yr (range = 17-88 yr) were recruited from patients with benign disease attending the clinic during the same period. Controls with a family history of breast or ovarian cancer were excluded. Both parents of each study participant were reported to be of German descent. The selection process yielded the pools set forth in Table 3, which were used in the studies that follow:

TABLE 3

Example 2 Association of Polymorphic Variants with Breast cancer

A whole-genome screen was performed to identify particular SNPs associated with occurrence of breast cancer. As described in Example 1, two sets of samples were utilized, which included samples from female individuals having breast cancer (breast cancer cases) and samples from female individuals not having cancer (female controls). The initial screen of each pool was performed in an allelotyping study, in which certain samples in each group were pooled. By pooling DNA from each group, an allele frequency for each SNP in each group was calculated. These allele frequencies were then compared to one another. Particular SNPs were considered as being associated with breast cancer when allele frequency differences calculated between case and control pools were statistically significant. SNP disease association results obtained from the allelotyping study were then validated by genotyping each associated SNP across all samples from each pool. The results of the genotyping were then analyzed, allele frequencies for each group were calculated from the individual genotyping results, and a p value was calculated to determine whether the case and control groups had statistically significant differences in allele frequencies for a particular SNP. When the genotyping results agreed with the original allelotyping results, the SNP disease association was considered validated at the genetic level.

SNP Panel Used for Genetic Analyses

A case-control study design using a whole genome association strategy involving approximately 28,000 single nucleotide polymorphisms (SNPs) was employed. Approximately 25,000 SNPs were evenly spaced in gene-based regions of the human genome with a median inter-marker distance of about 40,000 base pairs.

Additionally, approximately 3,000 SNPs causing amino acid substitutions in genes described in the literature as candidates for various diseases were used. The case-control study samples were of female German origin (German paternal and maternal descent) 548 individuals were equally distributed in two groups (female controls and female cases). The whole genome association approach was first conducted on 2 DNA pools representing the 2 groups. Significant markers were confirmed by individual genotyping.

TABLE 4

Allelotvping and Genotyping Results

The genetic studies summarized above and described in more detail below identified allelic variants associated with breast cancer. The allelic variants identified from the SNP panel described in Table 4 are summarized below in Table 5.

TABLE 5

Table 5 includes information pertaining to the incident polymorphic variant associated with breast cancer identified herein. Public information pertaining to the polymorphism and the genomic sequence that includes the polymorphism are indicated. The genomic sequences identified in Table 3 may be accessed at the http address www.ncbi.nih.gov/entrez/query.fcgi, for example, by using the publicly available SNP reference number (e.g., rsl541998). The chromosome position refers to the position of the SNP within NCBI's Genome Build 34, which may be accessed at the following http address: www.ncbi.nlm.nih.gov/mapview/rnap_search.cgi?chr=hum_chr.inf&query=. The "Contig Position" provided in Table 5 corresponds to a nucleotide position set forth in the contig sequence, and designates the polymorphic site corresponding to the SNP reference number. The sequence containing the polymorphisms also may be referenced by the "Sequence Identification" set forth in Table 5. The "Sequence Identification" corresponds to cDNA sequence that encodes associated target polypeptides (e.g., ICAMl) of the invention. The position of the SNP within the cDNA sequence is provided in the "Sequence Position" column of Table 5. Also, the allelic variation at the polymorphic site and the allelic variant identified as associated with breast cancer is specified in Table 5. All nucleotide sequences referenced and accessed by the parameters set forth in Table 5 are incorporated herein by reference. The positions for these SNPs are indicated in the tables below and in SEQ ID NO:1.

Assay for Verifying. Allelotvping. and Genotvping SNPs

A MassARRAY™ system (Sequenom, Inc.) was utilized to perform SNP genotyping in a high-throughput fashion. This genotyping platform was complemented by a homogeneous, single-tube assay method (hME™ or homogeneous MassEXTEND™ (Sequenom, Inc.)) in which two genotyping primers anneal to and amplify a genomic target surrounding a polymorphic site of interest. A third primer (the MassEXTEND™ primer), which is complementary to the amplified target up to but not including the polymorphism, was then enzymatically extended one or a few bases through the polymorphic site and then terminated. For each polymorphism, SpectroDESIGNER™ software (Sequenom, Inc.) was used to generate a set of PCR primers and a MassEXTEND™ primer was used to genotype the polymorphism. Table 6 shows PCR primers and Table 7 shows extension primers used for analyzing polymorphisms. The initial PCR amplification reaction was performed in a 5 μl total volume containing I X PCR buffer with 1.5 mM MgCl₂ (Qiagen), 200 μM each of d ATP, dGTP, dCTP, dTTP (Gibco-BRL), 2.5 ng of genomic DNA, 0.1 units of HotStar DNA polymerase (Qiagen), and 200 nM each of forward and reverse PCR primers specific for the polymorphic region of interest. Table 6: PCR Primers

Samples were incubated at 95°C for 15 minutes, followed by 45 cycles of 95°C for 20 seconds, 56°C for 30 seconds, and 72°C for 1 minute, finishing with a 3 minute final extension at 72⁰C. Following amplification, shrimp alkaline phosphatase (SAP) (0.3 units in a 2 μl volume) (Amersham Pharmacia) was added to each reaction (total reaction volume was 7 μl) to remove any residual dNTPs that were not consumed in the PCR step. Samples were incubated for 20 minutes at 37⁰C, followed by 5 minutes at 85°C to denature the SAP.

Once the SAP reaction was complete, a primer extension reaction was initiated by adding a polymorphism- specific MassEXTEND™ primer cocktail to each sample. Each MassEXTEND™ cocktail included a specific combination of dideoxynucleotides (ddNTPs) and deoxynucleotides (dNTPs) used to distinguish polymorphic alleles from one another. In Table 7, ddNTPs are shown and the fourth nucleotide not shown is the dNTP.

Table 7: Extend Primers

The MassEXTEND™ reaction was performed in a total volume of 9 μl, with the addition of 1 X ThermoSequenase buffer, 0.576 units of ThermoSequenase (Amersham Pharmacia), 600 nM MassEXTEND™ primer, 2 mM of ddATP and/or ddCTP and/or ddGTP and/or ddTTP, and 2 niM of d ATP or dCTP or dGTP or dTTP. The deoxy nucleotide (dNTP) used in the assay normally was complementary to the nucleotide at the polymorphic site in the amplicon. Samples were incubated at 94⁰C for 2 minutes, followed by 55 cycles of 5 seconds at 94°C, 5 seconds at 52°C, and 5 seconds at 72⁰C. Following incubation, samples were desalted by adding 16 μl of water (total reaction volume was 25 μl), 3 mg of SpectroCLEAN™ sample cleaning beads (Sequenom, Inc.) and allowed to incubate for 3 minutes with rotation. Samples were then robotically dispensed using a piezoelectric dispensing device (SpectroJET™ (Sequenom, Inc.)) onto either 96-spot or 384-spot silicon chips containing a matrix that crystallized each sample (SpectroCHIP^® (Sequenom, Inc.))- Subsequently, MALDI-TOF mass spectrometry (Biflex and Autoflex MALDI-TOF mass spectrometers (Bruker Daltonics) can be used) and SpectroTYPER RT™ software (Sequenom, Inc.) were used to analyze and interpret the SNP genotype for each sample.

Genetic Analysis

Variations identified in the target genes are provided in SEQ ID NO:1. Minor allelic frequencies for these polymorphisms was verified as being 10% or greater by determining the allelic frequencies using the extension assay described above in a group of samples isolated from 92 individuals originating from the state of Utah in the United States, Venezuela and France (Coriell cell repositories).

Genotyping results are shown for female pools in Table 8. In the subsequent tables, "AF" refers to allelic frequency; and "F case" and "F control" refer to female case and female control groups, respectively.

TABLE 8

The single marker alleles set forth in Table 5 were considered validated, since the genotyping data for the females, males or both pools were significantly associated with breast cancer, and because the genotyping results agreed with the original allelotyping results. Particularly significant associations with breast cancer are indicated by a calculated p-value of less than 0.05 for genotype results, which are set forth in bold text.

Odds ratio results are shown in Table 8. An odds ratio is an unbiased estimate of relative risk which can be obtained from most case-control studies. Relative risk (RR) is an estimate of the likelihood of disease in the exposed group (susceptibility allele or genotype carriers) compared to the unexposed group (not carriers). It can be calculated by the following equation:

RR = /A//a

/A is the incidence of disease in the A carriers and /a is the incidence of disease in the non-carriers.

RR > 1 indicates the A allele increases disease susceptibility.

RR < 1 indicates the a allele increases disease susceptibility. For example, RR = 1.5 indicates that carriers of the A allele have 1.5 times the risk of disease than non- carriers, i.e., 50% more likely to get the disease.

Case-control studies do not allow the direct estimation of /A and /a, therefore relative risk cannot be directly estimated. However, the odds ratio (OR) can be calculated using the following equation:

OR = (nDAnda)/(nd AnDa) = /»DA(l - pdA)//>dA(l - pDA), or OR = ((case 0 / (1- case f)) / ((control 0 / (l-control T)), where f = susceptibility allele frequency.

An odds ratio can be interpreted in the same way a relative risk is interpreted and can be directly estimated using the data from case-control studies, i.e., case and control allele frequencies. The higher the odds ratio value, the larger the effect that particular allele has on the development of breast cancer. Possessing an allele associated with a relatively high odds ratio translates to having a higher risk of developing or having breast cancer. Example 3 Samples and Pooling Strategies for the Replication Samples

The SNPs of Table 5 were genotyped again in two collections of replication samples (the German Replication sample and the Australian Replication Sample) to further validate its association with breast cancer. Like the original study population described in Examples 1 and 2, the replication samples consisted of females diagnosed with breast cancer (cases) and females without cancer (controls). The case and control samples were selected and genotyped as described below.

Pooling Strategies

Samples were placed into one of two groups based on disease status. The two groups were female case groups and female control groups. A select set of samples from each group were utilized to generate pools, and one pool was created for each group. Each individual sample in a pool was represented by an equal amount of genomic DNA. For example, where 25 ng of genomic DNA was utilized in each PCR reaction and there were 190 individuals in each pool (i.e., 190 cases and 190 controls), each individual would provide 125 pg of genomic DNA. Inclusion or exclusion of samples for a pool was based upon the following criteria: the sample was derived from a female individual characterized as Caucasian from Australia; case samples were derived from individuals diagnosed with breast cancer; control samples were derived from individuals free of cancer and no family history of breast cancer; and sufficient genomic DNA was extracted from each blood sample for all allelotyping and genotyping reactions performed during the study. Samples in the pools also were age-matched. Samples that met these criteria were added to appropriate pools based on gender and disease status. The German replication sample consisted of 188 cases and 150 controls recruited at the Department of

Obstetrics and Gynecology, Technical University of Munich. The majority of breast cancer cases were recruited at pre-operative visits, and female controls were recruited from healthy individuals or patients with non-malignant diagnoses. Median age of diagnosis for cases was 59 yr (range = 22-87 yr) and median age of controls was 50 yr (range = 19-91 yr). All but two participants reported both parents were of German descent. The two exceptions each reported one parent of non-German, Eastern European origin. No information was available regarding cancer family history.

The Australian replication sample comprised 180 breast cancer cases recruited by the Pathology Department of Gold Coast Hospital or by the Genomics Research Center, Southport. Median age of diagnosis was 50 yr (range = 24-74 yr). Controls consisted of 180 healthy volunteers recruited through the Genomics Research Center. Only controls with no family history of cancer or pre-cancerous conditions were included. Controls were individually age matched to cases (±5 yr). Median age for controls was 60 yr (range = 28-94 yr).

The replication genotyping results are shown in Table 9. The odds ratio was calculated as described in Example 2.

TABLE 9

P- P-

Sample N^a MAF" OR^C value" Genotype Frequencies⁸ value' rs 1056538 ICAM5 Exon 5 (V3011) A AA AG GG

German Controls 266 0.44 1.54 0.001 0.21 0.47 0.32 0.006

(Discovery) Cases 231 0.34 0.13 0.42 0.45

The marker SNP rsl 1549918 {alias, rs 1056538), a non-synonymous variation (V301I) in exon 5 of intercellular adhesion molecule 5 (ICAM5), had a/?-value of P = 0.001 (OR = 1.5) in the discovery sample and of P = 0.03 (OR = 1.4) and P = 0.07 (OR = 1.3) in the German and Australian replication samples, respectively (Table 1). The analysis of all three samples resulted in a combined significance of P = 0.0001 (OR = 1.4) and a significance of P = 0.01 (OR = 1.3) within the replication samples only.

The absence of a statistically significant association in the replication cohort should not be interpreted as minimizing the value of the original finding. There are many reasons why a biologically derived association identified in a sample from one population would not replicate in a sample from another population. The most important reason is differences in population history. Due to bottlenecks and founder effects, there may be common disease predisposing alleles present in one population that are relatively rare in another, leading to a lack of association in the candidate region. Also, because common diseases such as breast cancer are the result of susceptibilities in many genes and many environmental risk factors, differences in population-specific genetic and environmental backgrounds could mask the effects of a biologically relevant allele. For these and other reasons, statistically strong results in the original, discovery sample that did not replicate in the replication sample may be further evaluated in additional replication cohorts and experimental systems.

Example 4 ICAM Region Proximal SNPs

It has been discovered that a polymorphic variation (rsl 1549918) in a region that encodes ICAMl, ICAM2 and ICAM5 is associated with the occurrence of breast cancer (see Examples 1 and 2). Subsequently, SNPs proximal to the incident SNP (rsl 1549918) were identified and allelotyped in breast cancer sample sets and control sample sets as described in Examples 1 and 2. Approximately 195 allelic variants located within the ICAM region were identified and allelotyped. The polymorphic variants are set forth in Table 10. The chromosome positions provided in column four of Table 10 are based on Genome "Build 34" of NCBI's GenBank.

TABLE 10

Assay for Verifying and Allelotyping SNPs

The methods used to verify and allelotype the proximal SNPs of Table 10 are the same methods described in Examples 1 and 2 herein. The PCR primers and extend primers used in these assays are provided in Table 1 1 and Table 12, respectively. TABLE 11

TABLE 12

W

Genetic Analysis of Allelotvping Results

Allelotyping results are shown for cases and controls in Table 13. The allele frequency for the A2 allele is noted in the fifth and sixth columns for breast cancer pools and control pools, respectively, where "AF" is allele frequency. SNPs with blank allele frequencies were untyped.

TABLE 13

Figure 1 shows association fine mapping of breast cancer susceptibility region on chromosome 19pl3.2. Sixty public domain SNPs in a 100-kb window around the incident SNP (bold "+" in center) were compared between pools of cases and controls. The x-axis corresponds to their chromosomal position and the y-axis to the test P-values (shown on the -logio scale). The continuous dark line presents the results of a goodness-of-fit test for an excess of significance (compared to 0.05) in a 10 kb sliding window assessed at 1 kb increments. The continuous light gray line is the result of a nonlinear smoothing function showing a weighted average of the P- values across the region. The darkness of each point corresponds to the minor allele frequency of each SNP in the control sample (see legend below graph). The LocusLink gene annotations for NCBI genome build 34 are included.

Additional Genotvping

In addition to the ICAM region incident SNP, fourteen other SNPs were genotyped in the discovery cohort. The discovery cohort is described in Example 1. The SNP rs2228615 is located in the ICAM5 encoding portion of the sequence, and is associated with breast cancer with a p-value of 0.00236, and encoded non-synonymous amino acids (see Table 15).

The methods used to verify and genotype the two proximal SNPs of Table 15 are the same methods described in Examples 1 and 2 herein. The PCR primers and extend primers used in these assays are provided in Table 14 and Table 15, respectively.

TABLE 14

TABLE 15

Table 16, below, shows the case and control allele frequencies along with the p-values for the SNPs genotyped. The disease associated allele of column 4 is in bold and the disease associated amino acid of column 5 is also in bold. The chromosome positions provided correspond to NCBI's Build 34.

The SNPs most strongly associated in the discovery (Fig. 2a) and combined samples (Fig. 2b) were two non- synonymous SNPs in ICAM5, the original marker rsl 1549918 (V301I) and rs2228615 (A348T), which were in near complete linkage disequilibrium (Fig. 2e). A non-synonymous SNP in ICAMI, rs5030382 (K469E), was significantly associated in the discovery sample but not in the replication samples. Analyses of haplotypes consisting of subsets of the 15 genotyped SNPs did not reveal any haplotype with stronger association than individual SNPs (data not shown).

Secondary Phenotvpe Analysis The discovery collection of German breast cancer patients included information related disease onset and severity. The genotyped SNPs were tested for association with these variables. The SNP rslO56538 (and rs2228615) was mostly strongly associated with a positive family history of breast cancer (P = 0.0065, Fisher's exact test). 15% of those homozygous for the susceptible allele (C) had a positive family history, compared to 9% of the heterozygotes and none of those homozygous for the protective allele. There was no association between rslO56538 and age of diagnosis. Also noteworthy, the SNP most strongly associated with breast cancer status in the replication samples, rs281439 located 542 bp 5' of ICAM5 (Table 17), was also associated with indicators of cancer severity (Table 18). Patients carrying the allele associated with breast cancer susceptibility (G) had a significantly shorter time span between diagnosis and recruitment, suggesting shorter survival time, and higher rates of metastases to other organs. These results suggest that one or more variants in this region are risk factors for breast and prostate cancer and may influence disease progression and prognosis. TABLE 17

P- P-

Sample N^a MAF* OR^C value" Genotype Frequencies⁶ value^f rs 1056538 /C/W5 Exon 5 (V301 l) A AA AG GG

German Controls 266 0.44 1.54 0.001 0.21 0.47 0.32 0.006

(Discovery) Cases 231 0.34 0.13 0.42 0.45

German Controls 143 0.43 1.35 0.07 0.18 0.50 0.32 0.15

(Replication) Cases 181 0.36 0.14 0.43 0.43

Australian Controls 175 0.41 1.27 0.03 0.19 0.43 0.38 0.21

(Replication) Cases 175 0.35 0.17 0.36 0.47

Replication 1.30 0.01 Total 1.39 0.0001 rs5030382 ICAM1 Exon 6 (K469E) G GG GA AA

German Controls 265 0.46 1.39 0.01 0.22 0.49 0.29 0.04

(Discovery) Cases 242 0.38 0.16 0.45 0.39

German Controls 142 0.48 1.27 0.07 0.25 0.47 0.28 0.34

(Replication) Cases 178 0.42 0.19 0.48 0.34

Australian Controls 170 0.41 1.14 0.20 0.19 0.44 0.36 0.73

(Replication) Cases 167 0.38 0.17 0.43 0.40

Replication 1.20 0.05 Total 1.28 0.004 rs281439 ICAM5542 bp Upstream G GG GC CC

German Controls 257 0.23 1.09 0.57 0.04 0.37 0.59 0.74

(Discovery) Cases 242 0.24 0.06 0.37 0.57

German Controls 143 0.19 1.52 0.02 0.06 0.26 0.69 0.03

(Replication) Cases 179 0.26 0.06 0.39 0.55

Australian Controls 174 0.21 1.33 0.05 0.05 0.33 0.63 0.25

(Replication) Cases 176 0.26 0.09 0.35 0.56

Replication 1.41 0.004 Total 1.27 0.02 ^a Number of subjects with genotypes.

" Minor relative allele frequency.

⁰ Odds ratio with reference to allele that increases frequency in cases. ^d P-value for test comparing allele frequencies between cases and controls. ^e Relative genotype frequencies.

' P-value for tests comparing genotype frequencies between cases and controls.

TABLE 18: Association of rs281439 with clinical indicators of breast cancer severity

* Quantitative variables are expressed as 1^st quartile / median / 3^rd quartile. Categorical variables are expressed as column percentage (count). Example 5 Prostate Cancer Samples and Pooling Strategies

Since there are several common features between breast and prostate cancer, this locus was also tested for association with prostate cancer susceptibility in an independent collection of German cases and controls.

Prostate Cancer Samples

The prostate cancer sample consisted of 368 German patients with a median age of diagnosis of 65 yr (range = 43-90 yr) recruited at the Urology Clinic Munich-Planegg, and 368 controls without symptomatic prostate disease with a median age of 68 yr (range = 25-92 yr) collected at the University Hospital Tuebingen. All subjects involved reported both parents to be of German ancestry. All subjects involved in our studies signed a written informed consent and the institutional ethics committees of participating institutions approved the experimental protocols. For discovery samples, DNA from 5 ml blood of each subject was extracted using a desalting method and quantitated fluorimetrically (Fluoroskan Ascent CF, Labsystems) using Pico green. DNA pools were generated by combining equimolar amounts of each sample as described elsewhere. See, Buetow, K. H., Edmonson, M., MacDonald, R., Clifford, R., Yip, P., Kelley, J., Little, D. P., Strausberg, R., Koester, H., Cantor, C. R., and Braun, A. High-throughput development and characterization of a genomewide collection of gene- based single nucleotide polymorphism markers by chip-based matrix-assisted laser desorption/ionization time-of- flight mass spectrometry. Proc Natl Acad Sci U S A, 98: 581-584, 2001. Bansal, A., van den Boom, D., Kammerer, S., Honisch, C, Adam, G., Cantor, C. R., Kleyn, P., and Braun, A. Association testing by DNA pooling: an effective initial screen. Proc Natl Acad Sci U S A, 99: 16871-16874, 2002.

Genotyping Results

Thirteen of the 15 SNPs genotyped in the breast cancer sample were genotyped in the prostate cancer sample. The resulting association pattern was similar to that observed for breast cancer. See Tables 19 and 20 below:

TABLE 19 l.bl-icaiiisnps SNKI D.Oiϊg SJXR ID liS/O Chromosome Ijiic.ii ii Ciciicpce ΛΛ.changp

I I0.Ϊ9&I0 X 1U.-JDS.IU I O59N49 19 102 J 1654 UΎIΪ NA

2 X3003035.2 3093035 1 9 10243973 II II ion ic NA

.ϊ 25MH2 X28 M32.2 2S l 432 1 9 1025165 A liil roiiic NA

'I SNPCID0.-ii|CI7n SNPQ00.VIQ7O.2 17999«« 19 10255792 Nniisyii R2"l IG

S ICAM IJSOI ^"Ul ICΛM I . IS0I 7M IS0 I7 I4 19 1025620» Nniisyn Pi-ι2l.

(J PCII-Orøl PCII .Ofiil l 5O303K2 19 I Q2S66&3 N oi is yi i K'lCtlK

7 ClHllSl lp I S.", 3093030 19 1025*403 linorgeiiic NA

S tκ8277Sfi rcS277SC.3 281439 19 10261 1 IO Intragenic NA

9 207:1741 X2D757-I I .2 2075741 19 I 0262O9X liil roiiic NA

10 FCII-OKM PCILOD-U 1O5653X 19 1026393 A Nniiεyn V30 I I

I l ICΛM!\_222S6I5 ICAM5.222SOIS 222X615 19 I 026436S Nniisyn THiISA

12 2f.GD702 X2.ϊfi9702 2569702 19 10264947 liil roii ic NA

1.4 cponαnno ι.10 Cl' GOOOOCI M0.2 2569703 19 102 «5227 l iil roi iic NA

U S92 IS5 xsnaiss X92 ISX 19 10270793 l iil C'igoiiit NA

I. Ti 2SUI I7 X28 WI 7 2S I4I7 19 I 02S0I3O Syu NA TABLE 20 t bl-apvals-K icchle N CtISC(N = 390) Cυnti'ϋl(N = 300) δ OR P-value Siftiiif

X 1059849 : G 666 58% (21S) 52% (150) 5.6 1 .250 0.15 10

X3093035.2 : A 66S 6% (22) 5% (14) 0.9 1 .200 0.5990

X28 L432.2 : G 65S 43% ( ICj) 50% (1421 -6.4 0.774 0. 1040

SNP00054070.2 : A 670 12% (44) 10% (29) 1 .6 I . I SO 0.5160

ICAMl. J 801714 : T 66S 3% (L2) 4% (U) 0.6 0.832 0.6640

FCH .0G91 : G 656 43% (J58) 48%) (13Gl -4.8 O.S22 0.21 50 cainsnplS5.2 : T 66S 42% ( loo) 47%, (1361 -4.6 0.83 I 0.2390 reS27786.3 : C 658 74% (277) 81 % (232) -6.7 0.67'J ιUι43 1 *

X2075741.2 : C4 646 57%, (210) 53% (146) 4.5 1 .20⁽1 H.25O0

FCH .0994 : T 662 30%. (134) 43% (122) -7.0 0.744 0.0662 -

ICAM5.2228G 15 : G 656 65%, (245) 57% (158) 8.0 1.400 0.0380 *

X25G9702 : C 592 35% (no) 41 % (10?) -5.7 0.785 0.1560

CPGOOOOO 140.2 : C 652 46% ( 170) 41 % (lie) 4.x 1 .220 0.2200

X892 I 8S : C 664 64% (240) 57% (io4) 6.9 1 330 11.07 16 -

X28 L417 : C 668 67% (253) GG% (192) 0.7 I .030 H.M40

The non-synonymous SNPs rslO56538 and rs2228615 in ICAM5 were most strongly associated. These two SNPs are in close linkage disequilibrium, and accordingly have nearly identical odds ratios and p-values (OR = 1.4, P = 0.002). See Figure 2C.

Discussion

The identification of a susceptibility region influencing both breast and prostate cancer is particularly interesting since they have many common features, such as hormone-sensitivity, parallel incidence rates in various countries, and common genetic alterations. The role of intercellular adhesion molecules in cancer has been described. ICAMl is constitutively expressed on cells involved in immune response and induced on other cells, including endothelial and epithelial cells. It has a well-known role in inflammation related processes and immune surveillance, and derangements of its expression have been implicated in the development of a variety of inflammatory diseases as well as in tumor progression of several cancers, including breast cancer. ICAM l is also involved in transmembrane signal transduction upon binding to beta 2 integrin ligands and multimer formation, activating the mitogen-activated protein kinase pathway and eventually transcription factors like AP-I that regulate cell proliferation events. The inhibition of AP-I has been shown to inhibit breast cancer cell growth. Relatively little is known about the roles of ICAM4 and ICAM5 in cell signaling events and tumor surveillance but their involvement in similar pathways is likely. ICAM4 has been reported to be exclusively expressed in erythrocytes and has a suggested role in cell interaction events, including hemostasis and thrombosis. ICAM5 is mainly expressed in specific areas of the brain and has been implicated in dendritic outgrowth and rapid cell spreading of microglia. Although the reported expression patterns and described functions of ICAM4 and ICAM5 are not indicative of a role in breast and prostate cancer susceptibility, their roles in cell adhesion and cell signaling together with their low level expression in cancer-relevant tissues leave the possibility that their dysregulation or dysfunction may increase cancer risk.

These findings are in agreement with previous reports on the involvement of ICAMl in tumor progression and the K.469E variant is a potential candidate to influence this process. However, the data also suggests an influence of other variants in this region on breast and prostate cancer susceptibility. While the genetic evidence favors ICAM5, higher relative expression levels of ICAMl make it a more favorable candidate for predisposition and potentially tumor progression. However, current data cannot exclude any of the three ICAM genes as biologically responsible for the observed association.

The route by which these genetic associations were arrived at and the potential for spurious association must certainly be considered. Recent published work has brought much needed light to the need for proper validation to verify genetic findings for complex traits. In the current study, the initial association found between the ICAM5 marker and breast cancer status was one result from over 25,000 hypothesis tests. A conservative Bonferroni adjustment to yield an experiment-wide type I error rate of 0.05 would demand a test-wise p-value on the order of 10^'6. Given the modest sample size, only common variations with relatively large effects (OR > 2) would reach such significance levels. Instead, the role of type II error rates and apply a more liberal set of criteria in the initial phases of the study was considered and true genetic effects were identified by independent replication. The analysis of 52 selected markers in the German and Australian replication samples resulted in multiple associations of continuing interest, with the ICAM5 providing the most consistent and statistically significant association (P = 0.01, OR = 1.3). This would not be considered significant on an experiment-wide level after Bonferroni adjustment, which would require a test-wise p-value on the order of 0.001. Indeed, if the true size of the genetic effect is 1.4, then the total replication sample size of 368 cases and 330 controls has less than 50% power to reject the false null hypothesis at this level. However, the independent identification of the same variation with remarkably similar effects observed in a larger prostate cancer study gives us added reason to believe that one or more variations in these ICAM genes are influencing breast and prostate cancer susceptibility in populations of European origin.

Example 6

In Vitro Production of ICAM Polypeptides cDNA is cloned into a pIVEX 2.3-MCS vector (Roche Biochem) using a directional cloning method. A cDNA insert is prepared using PCR with forward and reverse primers having 5' restriction site tags (in frame) and 5-6 additional nucleotides in addition to 3' gene-specific portions, the latter of which is typically about twenty to about twenty-five base pairs in length. A Sal I restriction site is introduced by the forward primer and a Sma I restriction site is introduced by the reverse primer. The ends of PCR products are cut with the corresponding restriction enzymes (i.e., Sal I and Sma I) and the products are gel-purified. The pIVEX 2.3-MCS vector is linearized using the same restriction enzymes, and the fragment with the correct sized fragment is isolated by gel- purification. Purified PCR product is ligated into the linearized pIVEX 2.3-MCS vector and E. coli cells transformed for plasmid amplification. The newly constructed expression vector is verified by restriction mapping and used for protein production.

E. coli lysate is reconstituted with 0.25 ml of Reconstitution Buffer, the Reaction Mix is reconstituted with 0.8 ml of Reconstitution Buffer; the Feeding Mix is reconstituted with 10.5 ml of Reconstitution Buffer; and the Energy Mix is reconstituted with 0.6 ml of Reconstitution Buffer. 0.5 ml of the Energy Mix was added to the Feeding Mix to obtain the Feeding Solution. 0.75 ml of Reaction Mix, 50 μl of Energy Mix, and 10 μg of the template DNA is added to the E. coli lysate.

Using the reaction device (Roche Biochem), 1 ml of the Reaction Solution is loaded into the reaction compartment. The reaction device is turned upside-down and 10 ml of the Feeding Solution is loaded into the feeding compartment. All lids are closed and the reaction device is loaded into the RTS500 instrument. The instrument is run at 30⁰C for 24 hours with a stir bar speed of 150 rpm. The pIVEX 2.3 MCS vector includes a nucleotide sequence that encodes six consecutive histidine amino acids on the C-terminal end of the ICAM polypeptide for the purpose of protein purification. ICAM polypeptide is purified by contacting the contents of reaction device with resin modified with Ni²⁺ ions. ICAM polypeptide is eluted from the resin with a solution containing free Ni²⁺ ions.

Example 7

Cellular Production of ICAM Polypeptides

Nucleic acids are cloned into DNA plasmids having phage recombination cites and ICAM polypeptides are expressed therefrom in a variety of host cells. Alpha phage genomic DNA contains short sequences known as attP sites, and E. coli genomic DNA contains unique, short sequences known as attB sites. These regions share homology, allowing for integration of phage DNA into E. coli via directional, site-specific recombination using the phage protein Int and the E. coli protein IHF. Integration produces two new att sites, L and R, which flank the inserted prophage DNA. Phage excision from E. coli genomic DNA can also be accomplished using these two proteins with the addition of a second phage protein, Xis. DNA vectors have been produced where the integration/excision process is modified to allow for the directional integration or excision of a target DNA fragment into a backbone vector in a rapid in vitro reaction (Gateway™ Technology (Invitrogen, Inc.)).

A first step is to transfer the nucleic acid insert into a shuttle vector that contains attL sites surrounding the negative selection gene, ccdB (e.g. pENTER vector, Invitrogen, Inc.). This transfer process is accomplished by digesting the nucleic acid from a DNA vector used for sequencing, and to ligate it into the multicloning site of the shuttle vector, which will place it between the two attL sites while removing the negative selection gene ccdB. A second method is to amplify the nucleic acid by the polymerase chain reaction (PCR) with primers containing attB sites. The amplified fragment then is integrated into the shuttle vector using Int and IHF. A third method is to utilize a topoisomerase-mediated process, in which the nucleic acid is amplified via PCR using gene-specific primers with the 5' upstream primer containing an additional CACC sequence (e.g., TOPO^® expression kit (Invitrogen, Inc.)). In conjunction with Topoisomerase I, the PCR amplified fragment can be cloned into the shuttle vector via the attL sites in the correct orientation.

Once the nucleic acid is transferred into the shuttle vector, it can be cloned into an expression vector having attR sites. Several vectors containing attR sites for expression of ICAM polypeptide as a native polypeptide, N- fusion polypeptide, and C-fusion polypeptides are commercially available (e.g., pDEST (Invitrogen, Inc.)), and any vector can be converted into an expression vector for receiving a nucleic acid from the shuttle vector by introducing an insert having an attR site flanked by an antibiotic resistant gene for selection using the standard methods described above. Transfer of the nucleic acid from the shuttle vector is accomplished by directional recombination using Int, IHF, and Xis (LR clonase). Then the desired sequence can be transferred to an expression vector by carrying out a one hour incubation at room temperature with Int, IHF, and Xis, a ten minute incubation at 37°C with proteinase K, transforming bacteria and allowing expression for one hour, and then plating on selective media. Generally, 90% cloning efficiency is achieved by this method. Examples of expression vectors are pDEST 14 bacterial expression vector with att7 promoter, pDEST 15 bacterial expression vector with a T7 promoter and a N-terminal GST tag, pDEST 17 bacterial vector with a T7 promoter and a N- terminal polyhistidine affinity tag, and pDEST 12.2 mammalian expression vector with a CMV promoter and neo resistance gene. These expression vectors or others like them are transformed or transfected into cells for expression of the ICAM polypeptide or polypeptide variants. These expression vectors are often transfected, for example, into murine-transformed a adipocyte cell line 3T3-L1, (ATCC), human embryonic kidney cell line 293, and rat cardiomyocyte cell line H9C2.

Example 8

Biological Evidence of ICAM's Role in the Invasion of Human Breast Cancer Cells

A region was identified on chromosome 19pl3.2 spanning the genes encoding the intercellular adhesion molecules ICAMl, ICAM4, and ICAM5 as a breast cancer susceptibility locus. Genetic variants in this region are associated with indicators of disease severity, including higher rates of metastases to other organs (See Examples 1-4). Based on this association, the role of ICAMl in proliferation and invasion of human breast cancer cells was determined. It was observed that ICAMl down-regulation at the mRNA and protein level led to a strong suppression of human breast cell invasion through a matrigel matrix. Under the same conditions, no significant effect on cell proliferation in vitro was seen. Incubation of cells with an antibody against ICAMl blocked invasion of the highly metastatic MDA-MB-435 cell line in a dose-dependent manner without affecting cell migration. It was also demonstrated that the level of ICAMl protein expression on the cell surface positively correlated with metastatic potential of five human breast cancer cell lines and that ICAMl mRNA levels were elevated in breast tumor compared to adjacent normal tissue. These results showed variations in the ICAM region are associated with occurrence of metastases and establish a causal role of ICAMl in invasion of metastatic human breast carcinoma cell lines.

Materials and Methods

Cell culture and siRNA-mediated gene silencing

MDA-MB-231 and MDA-MB-435 breast cancer cell lines were obtained from the Developmental Therapeutics Program, NCI/NIH (Frederick, MD) and were cultured in DMEM and RPMI media, respectively, supplemented with 10% fetal calf serum (FCS, Omega Scientific, Tarzana, CA). NCI-H460, A375, and PC3 cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and were cultured according to ATCC recommendations. Normal human dermal fibroblasts (NHDF) were obtained from BioWhittaker (Walkersville, MD) and cultured according to the manufacturer's instructions. Small inhibitory RNA duplexes were designed according to the guidelines of Elbashir et al. (Elbashir, S.M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K. and Tuschl, T. (2001) Duplexes of 21 -nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature, 41 1 , 494-8) and were synthesized by Dharmacon Research Inc. (Lafayette, CO). Oligonucleotide siRNA sequences for human ICAMl were designed as follows: siICAMl -1 , 5'- AAACAACCGGAAGGUGUAUGA; siICAMl-2, 5'-AAGCCAACCAAUGUGCUAUUC; siICAMl -3, 5'- AAGAUCACCAUGGAGCCAAUU; siICAMl -4, 5'-AACUGUCACUCGAGAUCUUGA. A control siRNA siGL2 (5'-AACGUACGCGGAAUACUUCGA), which is non-homologous to any human sequence, was obtained from Dharmacon. Additional control siRNA were designed to target human ICAM5 and RAD21 mRNA (siICAM5, 5'-UAAAUGCCACCGAGAACGA; siRAD21, 5'-AAGAGUUGGAUAGCAAGACAA). Cells were plated in 6-well culture dishes to achieve 70-85% confluency on the following day. A mixture of siRNA (35-150 nM) and Lipofectamine 2000 (Invitrogen, Carlsbad, CA) at a ratio of 1/3 (μg/μl) in a total volume of 1.25 ml OptiMEM I (Invitrogen) was incubated for 30 min at room temperature to form liposomes. This mixture was then added to cells that were pre-washed with OptiMEM I medium. After incubating for 5 hr at 37°C, 1.25 ml of standard medium containing 20% FCS was added. The final concentration of siRNA during the overnight incubation was 18 to 75 nM. The cells were incubated for additional 16 hr at 37°C, washed with complete medium, and replated either on 96-well plates for determination of proliferation or on chambered glass slides for an apoptosis assay. For preparation of mRNA or determination of invasion potential, cells were replated on 6- well plates to achieve 70-90% confluency 2-3 days post-transfection.

cDNA preparation and quantitative gene expression (QGE) by MassARRA Y

Human normal breast, lung, heart, and skin tissue total RNA samples were purchased from Ambion (Austin, TX) and used for cDNA synthesis. To assess cellular expression of mRNA, 5O x IO⁶ cells were collected and mRNA isolated using Dynalbead mRNA Direct (Dynal, Oslo, Norway) according to manufacturer's protocols. Frozen human clinical breast tumor samples were purchased from ProteoGenex (Los Angeles, CA). Total RNA from tissues was extracted using TRIzol (Invitrogen, Carlsbad, CA). For siRNA experiments, cells were harvested on day 2 post-transfection with siRNA, and total RNA extracted using TRIzol. cDNA was prepared using random hexamers or oligo-dT primers and Superscript II reverse transcriptase (Invitrogen) and these preparations were pooled. Levels of transcripts were assessed using competitive RT-PCR and mass spectrometry (QGE by MassARRA Y™ assay, Sequenom, San Diego, CA). The competitive PCR step of QGE by MassARRAY includes a synthetic competitor oligonucleotide that differs at one base position from the cDNA target. This competitor is used to calibrate the assay and quantitate the genes of interest at an absolute level by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS). The principle of QGE by MassARRAY™ assay was described elsewhere (Ding, C. and Cantor, CR. (2003) A high-throughput gene expression analysis technique using competitive PCR and matrix-assisted laser desorption ionization time- of-flight MS. Proc Natl Acad Sci USA, 100, 3059-64). Levels of gene-specific mRNA were normalized against levels of gamma 1 actin (ACTGJ) mRNA by dividing the observed transcript concentration of each target gene (ICAMl or ICAMS) by the observed transcript concentration of ACTGl for each respective sample. Normalized data were expressed as ratios. All QGE oligonucleotides used in this study are shown in Table 21. QGE analysis included triplicate experiments with quadruplicate spotting of reaction products onto SpectroCHIPs (Sequenom).

Protein expression analysis by flow cytometry

Cells were removed from tissue culture plates using Cellstripper, a non-enzymatic cell dissociation reagent containing a mixture of chelators (Mediatech, Herndon, VA). Next, cells were brought to a concentration of 10⁵ cells per 100 μl of PBS containing 1% FCS, followed by incubation with monoclonal anti-human ICAMl antibody (2 mg/ml, clone BBIG-Il (1 1C81) IgGl , R&D Systems, MN) or isotype control mouse IgGl antibody (2 mg/ml, Jackson ImmunoResearch Labs, West Grove, PA) for 30 min at 4⁰C. Cells were washed twice in PBS/1% FCS and then incubated with a secondary goat anti-mouse antibody conjugated with phycoerythrin (Jackson ImmunoResearch). Recombinant human ICAMl (rhICAMI, BD Biosciences, San Jose, CA) was used to determine the specificity of the anti-ICAMl staining in each of the cell lines assayed. Flow cytometry was performed using a Guava personal flow cytometer (Hayward, CA). For cells treated with siRNA, ICAMl staining was performed 2 to 3 days post-transfection. Cell proliferation and apoptosis assays

Cell proliferation was measured using the WST-I assay kit (Roche Diagnostics, Indianapolis, IN) at designated time points, and relative proliferation calculated by normalizing to day 1 values. Experiments were performed at least three times. Apoptosis was measured on day 2 using the Vybrant apoptosis assay kit 3 (Molecular Probes, Eugene, OR) as directed by the manufacturer.

Boyden chamber invasion and migration assays

Invasion assays were performed using porous (8 μm) filters coated with growth-factor reduced matrigel (BD Biosciences, San Diego, CA) to occlude the pores. The lower chamber contained 750 μl of conditioned medium from a 24-hr confluent culture of the corresponding cells. 30,000 to 50,000 MDA-MB-231 or MDA-MB-435 cells per well, respectively, were added to the upper chamber in 500 μl of serum-free medium containing 0.1% bovine serum albumin (Sigma-Aldrich, St. Louis, MO). After 16-18 hr incubation, cells were briefly washed with PBS, fixed in 2% glutaraldehyde for 10 min, and stained in 0.2% crystal violet. Chambers were photographed before and after the upper chambers were scraped to remove cells that did not invade. The photographed cells were counted to quantify percent invasion. "No treatment" siRNA control samples were mock transfected in the absence of siRNA. The total cell numbers for both chambers were the same in control (no treatment, siGL2, or IgGl ) and experimental (silCAM or anti-ICAMl) wells. The migration assays were performed in a similar fashion except that the filters were not coated with matrigel and therefore the pores were opened for cell passage. For ICAMl antibody experiments, migration and invasion assays were performed after pretreatment of cells with 2 or 10 μg/ml antibody for 30 min at 4°C.

Table 21. Oligonucleotides used for QGE by MassARRAY expression analysis (5' to 3')

VO

Results

ICAMl expression is elevated in several cancer cell lines and tissues

ICAMl mRNA expression was first characterized in a panel of human cancer cell lines using semi- quantitative RT-PCR (data not shown). The cell surface expression of ICAMl protein was quantitated in five breast cancer cell lines using an antibody against the extracellular N-terminal region of this molecule (Fig. 3A, first five cell lines, left to right). The ICAMl protein expression levels positively correlated with the reported metastatic potential of each of the cell lines. The most metastatic MDA-MB-435 cells showed the highest level of expression. The expression levels in a normal skin fibroblast line and in the metastatic lines NCI-H460, A375, and PC3 derived from lung, melanoma, and prostate, respectively, are shown for comparison (Fig. 3A). To confirm whether expression of ICAMl correlates with certain pathological phenotypes in clinical breast tumor samples, quantitative gene expression (QGE) by MassARRAY was performed on patient-matched normal and tumor adjacent tissue pairs. Analysis of these human tissues showed a trend towards elevated ICAMl mRNA levels in the tumor versus the adjacent normal tissue. The stage 3B infiltrating ductal carcinoma tissue had the greatest mRNA expression difference of 15-fold relative to the normal adjacent breast tissue (Fig. 3B). These data demonstrate the involvement of ICAMl in breast tumorigenesis. Low levels of ICAMl mRNA expression also were observed in most normal tissues except lung (Fig. 3B). It should be noted that possible monocytic infiltration of clinical samples could complicate proper analysis of ICAMl expression in frozen tissue specimens. The high expression in the lung could be explained by the fact that ICAMl is the major receptor for rhinoviruses in lung epithelial cells and its expression in this tissue is very sensitive to the infection state of the individual from which the sample was derived.

Suppression of ICAMl in breast cancer cells by RNAi

To investigate whether ICAMl could serve as a therapeutic target in breast cancer, synthetic siRNA duplexes were used to deplete ICAMl expression in three human breast cancer cell lines: the non-metastatic cell line MCF- 7, the metastatic cell line MDA-MB-231, and the highly metastatic cell line MDA-MB-435. Four siRNA sequences were designed targeting the coding region of human ICAMl mRNA. These were transfected into MCF-7 and MDA-MB-231 cells. In some experiments, siRNA against human ICAM5 (siICAM5) and a non- human siRNA (siGL2) were included as controls. Two days after transfection, cell lysates were harvested and cDNA prepared and analyzed for ICAMl gene expression by QGE. Fig. 4A shows results of these experiments using individual siRNA duplexes. ICAMl gene expression was significantly suppressed by siICAMl -1. ICAMl mRNA levels were reduced by about 75% in MCF-7 cells and more than 90% in MDA-MB-231 cells as compared to the cells treated with siICAM5 or siGL2, respectively. The inhibitory effect of siICAMl -1 was likely to be specific because it did not cause a non-specific down-regulation of the human homologous ICAM5 gene (Fig 4A). Expression of human RAD21 and the housekeeping genes ACTGl and HMBS was also not affected (data not shown). SiICAMl-2 down-regulated ICAMl in both cell lines but up-regulated ICAM5 in

MDA-MB-231. SiICAM 1-3 inhibited ICAMl expression in MCF-7 slightly but had no effect in MDA-MB-231 cells where, interestingly, 1CAM5 expression was down-regulated. SiICAM 1-4 strongly inhibited ICAMl in MDA-MB-231 cells but had no effect in MCF-7 cells. At the same time, siICAMl-4 significantly up-regulated ICAM5 in MCF-7 cells while inhibiting this gene in MDA-MB-231 cells. It is likely that siICAMl-2, silCAMl- 3, and siICAMl-4, in addition to targeting ICAMl, also caused off-target effects. Therefore, the inhibitory effect of siICAMl-2, -3, and -4 on ICAMl gene expression in MDA-MB-231 as well as MCF-7 cells was non-specific, whereas the effect of siICAMl-1 in both cell lines was specific. In MDA-MB-435 cells, siICAMl -1 inhibited ICAMl gene expression by 75±12%. Similar results were also obtained in MDA-MB-435 cells for siICAM l -3 and siICAMl -4 (data not shown). Based on these data, in all subsequent experiments siICAMl-1 was selected for the specific down-regulation of ICAMl expression.

Next, an analysis of cell surface ICAMl antibody staining was performed in transfected MCF-7 and MDA- MB-231 cells. The mean fluorescence intensity (MFI) values of anti-ICAMl antibody staining from a representative experiment were measured. The specificity of the test system was established using untreated cells with and without pre-incubation of antibodies with human recombinant ICAMl (hrlCAMl). Pre-incubation with hrICAMl almost completely abolished immuno-fluorescence in experiments with both cell lines. It should be mentioned that rhICAM l reduced the fluorescence to the same level as that of the isotype control IgGl plus secondary antibody (data not shown). Therefore, the system is appropriately specific. Significant down- regulation of ICAMl protein expression in both cell lines after siICAMl-1 treatment was demonstrated. This effect was more pronounced in the metastatic MDA-MB-231 cell line. MFI values from three independent experiments showed an average suppression of ICAMl surface protein of 60 ± 12% in MDA-MB-231 cells and 42 ± 9% in MCF-7 cells.

Cell proliferation is not affected by the specific ICAMl-I siRNA It was examined whether a suppression of ICAMl affects proliferation of breast cancer cell lines in vitro.

MDA-MB-231 and MDA-MB-435 cells transfected with siICAMl-1 siRNA grew at a rate similar to cells transfected with control siGL2 (Fig. 5). Therefore, selective inhibition of ICAMl did not affect proliferation of these cells growing under regular tissue culture conditions. Similar results were obtained for MCF-7 breast cells also indicating that ICAMl is not required for the proliferation of these cells in culture (data not shown). Consistent with the absence of an effect of siICAMl-1 on cell proliferation, staining of cells for the early apoptotic marker, Annexin V, in conjunction with the DNA dye propidium iodide, showed negligible induction of apoptosis (data not shown). In contrast, siRAD21 used as a positive control showed a strong inhibition of cell proliferation (Fig. 5) and induction of apoptosis (data not shown).

Inhibition of ICAMl reduces human breast cancer cell invasion in vitro

To determine whether the loss of ICAMl expression can affect the ability of cells to invade in vitro, the cell line MDA-MB-435 was used, which was derived from a breast cancer with high metastatic potential and has high expression levels of ICAM 1. The ability of these cells to invade the matrigel after transfection with specific siICAMl-1, non-specific siICAMl -3 and siICAMl-4, and control siGL2 was compared. Only treatment with siICAMl-1 effectively and significantly inhibited invasion. Non-specific ICAMl siRNA species (siICAMl -3 and siICAMl -4) and control siGL2 did not influence invasion as compared to mock-transfected cells. Quantitative estimates of invasion inhibition by siICAMl-1 were derived from analysis of microphotographs based on three independent experiments. SiICAMl -1 alone inhibited invasion by 85 ± 10% relative to the siGL2 control (0% inhibition). Given the fact that the suppression of ICAMl protein expression was below 60% in the majority of cells and that MDA-MB-435 cells express very high levels of ICAMl, detecting strong inhibition of invasion was significant. These results indicate that for a particular cell type, a threshold level of ICAMl protein is required for invasion to occur.

Further proof of ICAMl playing a role in invasion was obtained from the antibody blocking experiments. Incubation with 2 μg/ml anti-ICAMl antibody led to 10% inhibition of invasion (data not shown). Increasing antibody concentration to 10 μg/ml caused almost complete inhibition of invasion similar to the effect of siICAMl-1 siRNA transfection. Control IgG, had no effect on invasion MDA-MB-435 cells, thus indicating the specificity of the result. The following quantitative data were calculated from two independent experiments with MDA-MB-435 cells: at 2 μg/ml, anti-ICAMl antibody inhibited invasion by 15 ± 6%, whereas at 10 μg/ml, invasion was inhibited by 90 ± 4%, relative to the IgG] control. The finding that targeting ICAMl with siRNA or antibody did not affect cell migration was significant (data not shown). Thus, these data indicate that ICAMl plays a causal role in breast cancer cell invasion but does not appear to be involved in cell migration in vitro.

Representative Nucleic Acid and Amino Acid Sequences Provided hereafter are ICAM genomic sequences (SEQ ID NO: 1 , 2, 3, 4, 5, 6 and 7, respectively).

Polymorphic variants are designated in IUPAC format. The following nucleotide representations are used throughout the specification and figures: "A" or "a" is adenosine, adenine, or adenylic acid; "C" or "c" is cytidine, cytosine, or cytidylic acid; "G" or "g" is guanosine, guanine, or guanylic acid; "T" or "t" is thymidine, thymine, or thymidylic acid; and "I" or "i" is inosine, hypoxanthine, or inosinic acid. SNPs are designated by the following convention: "R" represents A or G, "M" represents A or C; "W" represents A or T; "Y" represents C or T; "S" represents C or G; "K" represents G or T; "V" represents A, C or G; "H" represents A, C, or T; "D" represents A, G, or T; "B" represents C, G, or T; and "N" represents A, G, C, or T.

Following is an ICAM genomic nucleotide sequence that encodes ICAMl, ICAM4 and ICAMS (SEQ ID NO: 1). >19 : 10219601-10312200

1 aggaggctga ggtgggagaa tggcgtgaac ctgggaggcg gagcttgcag tgagccaaga

61 tcacgccact gcactccagc ctgggcgaca gagcgagact ccgtctcaaa aaaaaaaaaa

121 aaaaaaaaaa agccaggcga ggtgactcac acccataatc gcagcacttt gggaggccaa

181 ggcgggcaga tcgcttgagc ccagaagttt gagatctgcc tgggcaacaY ggcgagaccc 241 tgtctctatg aaacaagaaa aaagaaagaa acgaaatact gttccatcat ttgccacata

301 aaatctaaac tctctggtgc agaacagact tgcaaatctg cctgtaaaat catacaccgg

361 attttggagt agacacacag gaggctgatt gtagtggtgg cttctgggag aagaactggg

421 gggtaggggg cacagataag agtagacaga attttcactc tagtaatctt tgtaattttt

481 gaaattctct ctaccaggtg tatgaatgac caatgaaaat atacaatagg ccaggcgcag 541 tggctcacgc ctataatccc agcactttgg gaggctgagg cgggtggatc acatgaggtc

601 aggagtccga ggccagcctg accaacatgg tgaaaccttg tctctacgaa aaatacaaac

661 attagtcagg tagggagcat cacttgagaa caggagttca agaccagcct ggacaacaaa

721 gcaagacctc tgcctctaca aaaaaaaaaa aaaaaaaaaa aaattgctgg gcatggtgga

781 acatgcctgt ggcctcagct acttaggtgg ctgcagcaga ggatcatctg aggccaggaa 841 ttcaagacta cagtgaacta taatcgttac actgcactac agactggtaa cagggtgaga

901 tcctgtctca aaaacaacag caacaaaata aacgattaaa agacctttta aagttgcaaa

961 atacatagag tcttaaacat gcatccacag ttatttattt atttatttag aaacggagtt

1021 tcactcttgt tgcccaggct ggagtgcaat ggtgcggtat cagctcactg caacctccgc

1081 ctcccaagtt aaaggaattc tgcctcagcc ttccaagtag ctggaattac aggcatgcgc 1141 caccatgctc ggctaatttt gtatttttag taaagatggg gttttggcat attggccagg

1201 atggtttcga acccctgacc tcaggtgatc cacccgcctc ggactcccaa agtgctagga

1261 ttataggcat gagccaccac gcccggccgc attcagttat tcttgcaggg ataaaataca

1321 caatgaaata cactatactt tttgttttct ttctttcttt tttttttttt tttttgagac

1381 agagtctcac tcgtcgcatg ggctggagtg cggtgcggga tctcggctca ctgcaacctc 1441 tgcctcctgg gttgaagcga ttctcctgcg tcagcctccc gagtagctgg gattacaggt

1501 gcccgccact acgcccagct aatttttttg tatttttagt agagataagg tttcaccatg

1561 ttggccaggc tggtctcgaa ctcctgactc gtgattcgcc caccttggcc tcccaaagtg 1621 ctgggattac aggcgtgagc caccgcaccc ggccacattt tttttttttt ttttttgaga 1681 caggatctta gtctgtcacc caggctggag tacagtggca tgaacatggc tcactgcagc 1741 ctcaatctct tgggttcagg tgatccttct gccttagcct ccccattagc tgggaccaca 1801 ggcatatacc atcacaccta gctaattttt aaatttttgg tagaggccag gcgcggtggc 1861 tcacgcctgt aatcccagca ctttgggagg ccgagtcggg tggatcacct gaggtcggga 1921 gttcgagacc agcctggcca aaatggtgaa accctgtctc tactaaaaat aaaaaataaa 1981 aataaaaatt agctgggcgt agtggcgggc gcctgtaatc ccagctactc gggaggctga 2041 ggcaggagaa tcgcttgaac ccaggaggca gaggttgcag tgagccaaga tcgcgctact 2101 gcactccagc ctgggcaaca gagtgagatt ctgtctcaaa aaaaaaataa taataatgct 2161 atttattgac tttacacctt gtaccaggca tgggaagctt tgcctccatt acgtcactga 2221 atctcataac ctccttttcc agcagaggaa aatgaggttg gttcacagac cacgttgtca 2281 gctgtgctct gtccagaacg cactggcctc caagtagaca gccctggact ggtagggaag 2341 ccggctatga tccggtggcg ccccctggag gtctatcggg aacatggtaa agaacctaaa 2401 aatgggtggg ccacagtagc tcatgcctgt aatcccagca ctttggggga ccaatgcggg 2461 agaattgctt gagcccagga gttcaagacc agcctgggca acattgggag acccaccccc 2521 cgccatctct acaaaaaaaa aattaggccg ggcgcggtgg ctcaggcctg taatcccagc 2581 actttgggag gccgaggcgg gtggatcacc tgaggtcagg agttcaagac cagcctggcc 2641 aacgtggtgc cacactgtct ctaataaaaa cacgaaaatt agggccgggc tcggtggctc 2701 acgcctgtaa tcccagcact ttgggaagcc gaggcgggcg gatcacgagg tcaggagatt 2761 gagaccatcc tggctaacac ggtgaaaccc cctctctact aaaaatacaa aaagttagcc 2821 aggtgtggtg gcgggcgcct gtagtcccag ctactcggta agctgaggca gggaatcgct 2881 tgaacccggg aggccgagat gtgcgatctc acaccactgc actccggcct gggcgacaga 2941 gcgatactcc atctcaaaat acaaatacaa aataaaaaaa tacaatacaa aaatgagccg 3001 ggcttgcgta cctgtagacc agctactcag gagacagagg caggagaatc ccttgagctc 3061 tggaggtcga ggctgcagta agccatgatc ttgcccattg cactccagcc tgggcgacag 3121 aggaagacct tgtctctaaa aaccaaaaca aagaaactaa aaataagcat tcggatttgt 3181 tagggggcga caagggaggc actccaggat ctgtggactc cccactttgt tctgtccttg 3241 gagagccctg gaaggtctga gaggggacgg gacctggttt aagggggtag gggagaggac 3301 cctggtctag ggggtaaggg acacaaatac ctaatctgag gggtttcggg gggacatggc 3361 cctgccctgg ggaatctaaa ctgggaggca tggtctggca ggcccaatcc tgaaggcttt 3421 ctgagggcaa taaggccctg cctttaaaca aatgaaaaaa cgccaggcac ggtggctcac 3481 gcctgtaatc ccagcacttt ggaaggctga ggcgggagga tcacgaggtc aggagatcga 3541 gaccatcctg gctaacgcgg tgatacccgt ctctactaaa aatacaaaaa attagccagg 3601 cgtggtggcg ggcgcctgca gtcccagcta ctcgggaggc tgaggcagga gaatggcgtg 3661 aacccaggag gcggagcttg cagtgagccg agatcgcgcc actgcactcc agcctgggca 3721 acagagcaag actccgtctc aaaaaaaaca aaaacaaaaa caaaaacaaa aacaaaaaaa 3781 caaataaaaa aacaaaacca cataactgga taaagaaaaa accatctccg acagccttgg 3841 aggcgggatg aaggcgtggc ccggtgggcg tgaccaacag caaaagttta agcgttattg 3901 gctgtattcc ttagttgctc actccagaac tgcccactta tgggcggggg tcactccttc 3961 aggcttaaca gtcattggct gaattgggcc agagaggtct cattggctga attcctgcac 4021 cggctcgtcg gaggcgggac ccaaagtagg ctaggcctac ggaagctggg tcttcttgct 4081 gtgaggtcgc gttccccagt gttacggagg gtccttgagg caggagtgaa aattgggtct 4141 gggggttagt cctggggtgg aggtctgggc acgccgggtc ggaccccctc catcttcggt 4201 tttgcacacc ccgctttcca gcgcggagtc gcggcgggta gggcggcgtc gcgtgcgtga 4261 cgtcatccag cggcgcctcg cRaggctcca gtggccttga cctcccgcgg cgtgggaggc 4321 tgcgcggcga tgctgcagtt cgtccgggcc ggggcgcggg cctggcttcg gcctaccggc 4381 agccaggtga ggccaggggc tggaggcgtg gtcgaaggat gaaatttggg ggtgtccagg 4441 ggtcgtctct cactttcgcc caacccttgc agggcctgag ttccctggcg gaagaggcag 4501 cgcgtgcgac cgagaacccg gagcaggtgg cgagcgaggg taaggcaacc ggggtggctc 4561 caggaggggc ggcgacagag aggtctgacc cttgacccta acctctgacc cccgcaatcg 4621 ctccaggtct cccggagccc gtgctgcgca aagtcgagct cccggtaccc actcatcgac 4681 gcccagtgca ggcctgggtc gagtccttgc ggggcttcga gcaggagcgc gtgggcctgg 4741 ccgacctgca ccccgatgtt ttcgccaccg cgcccaggtg agcgagggct gtaatggtga 4801 actgagtggc agagggatga agagcgggat ttcaggagtc acgatgactt tgggcttgta 4861 cccttgggaa agtgctgtat ttctacagcc tccgtttctc cacctgtcaa aggggaatga 4921 tgacagtttc ccctgctgta gcgctgtgtg agattgaagc ctgagaggtg acatcatcta 4981 agggttaggg agacagaatt ctggagcccg actgattagg ttcaaatcct gccttcccct 5041 cttgtccctc agtgtcccta ttttgtcagc ggtcgggagg ttgctgtgat gaataaatga 5101 cttaattctg gcacataata agttctatag aaatgttgat aatctttgtt aactggtttt 5161 tgcaaataag agcactaaaa agactaaacc attcctcggt gcctggaaga ggctgtttgc 5221 attttagtta ccctgctgtt cataacatct ctaagaaaat gtaggggcca ccctgggcgc 5281 agtggctcac gcctgtaatc ccagcacttt gggaggccaa ggcgggcgga tcacgaggtc 5341 aggagatcga gaccatcctg gctaacatgg tgaaaccccg tctctactaa aaatacaaaa 5401 aaaaattagc cgggcgtggt ggcgggcgtc tgtagtccca ggtactctgg aggctgaggc 5461 aagagaatgg cgtgaacccg ggaggcggag cttgcagtga gccgagatcg cgccactgca 5521 ctccatcctg ggcaacactc tgtctcaaaa aaaaaaaaaa aaaagaaaga aaagaaaatg 5581 taggggccag ttactgtggc tcacatctgt aatcccagca cattgggagg ccgaggtggg 5641 cggatcactt gaaaccagga gttgcagacc agcctggcca acatgatgaa accccgtctc 5701 taccaaaaat acaaaaatta gccggacgtc ttagtgcaag cctgtagtcc cagctactca 5761 ggaggctgag gcatgagaat cgcttgcacc tggggagatg gaggttgcag tgagccgtga 5821 ttatactact gcactccagc ctgggcgaca gagacagact ccatctcaaa aaaaaaaaca 5881 ggtgaaattg atttcaataa tgtatttaac ctgtatttaa aactatgtcg aaatcacatt 5941 gtagcatggg gcactggcca tgtttcagat gttgagtatg tgactgtatg gaatggtata 6001 gaactagaga ggaaaaccag tccctgaaga aggtggcaat aagtgaagtg taatagcagg 6061 aaaaaagtaa tggtaggaaa aacaaggaag aaggggtggc tttttttttc tgagatgaag 6121 tttcgctctt gtcgcccagg ctggagtgca atggcatgct ctcagctcac ttcaacctcc 6181 gcctcctggg ttctactaat tctcatgcct cagcctcccg agtagccaga atgacagaca 6241 tgtaccaccg tgcccagcta atctttgtat attttggaga gacatcactt tgccatgttg 6301 cccaggctgg tcttactcct tgcctcaagt gatccacctg ccttggcctc ccaaagtgcc 6361 aggattccag gcatgagcca ctgcacctgg tcagggtggc tctttcttta gaaggtacct 6421 ttcagcagtt attggagctg ctacctgtag gctgagaaag aaccatccag gagaagagtg 6481 tttcaggcag agggaacagc aagtgccaag gccctgaggc agaatttcaa gatggggtca 6541 gtgaggggca gaggcaaatc gcccagggcc ctggaggcag aagggagaat cctgggtttt 6601 cctatagtgg ggtgggagcg tttgaagcag agttgggctt ctcatgtgtc cttcctcccc 6661 gcaggctgga catactgcac caggttgcta tgtggcagaa gaacttcaag agaattgtga 6721 gtgcctaaat ggagcaaggt ggtgggaagg agcttcctgg ggaggttggg gataggaccc 6781 agaggaagcc catcgctggg ttttctctgg actgctcggc tggggcctca tctgtctcct 6841 gaactattca ccgatgggtc catttttggt tctctttttt ttgtttgttt ttgagatgga 6901 gatggagtct cactctgtca cccaggctgg agtgcagtgg cgcaatcttg gttcactgca 6961 acctccgctt cctgggttca agcgattctc ctgcctctgc ctcctgagta gctgggatta 7021 caggtgtgca ccaccatgca ggctaatttt tgtattttta gtagagatgg agtttcacca 7081 tgttggtcag gctggtctca aactcctgac ctcaaggaac ctacctgcct tggcctccca 7141 aagtgctggg attacaggcg tgagccaccg tgcccagctc attttcagtt gttttctgat 7201 cactcactga tgtgggcatt gtggtgggtg agaggatata gcagggacca cgaggacaaa 7261 acaggcaagg tccggctgtg tgcaagtggc tcaggcctgt aatcccagca ctggaagctg 7321 aggtgggtgg gttgcttggg gccaggaaag accagcctgg gaaacagcaa gacccagtct 7381 ctaccccttc ccccacaacc ccaagaaaaa gatgggcaaa gtccctattc taatgaaggt 7441 cacagtgtca tgggaggaga agtgaaggtg agtcagatgg tcatacgtaa tgtgtaatta 7501 tgagcatgtc ccagagaatg ggacgttcaa ccttgggtct actgtagaag ctaaccagat 7561 caaggaggca gtggggctag tgtggagata acaggaacag ggtgtgcaaa ggccctgtgg 7621 cagggaccac aaggaagcca gtgttgccac agaggccagt gggagcagtg gggaggagtc 7681 tgggttttgt gccaagaaca ttgaagaatc cattaccagt cagggtctta tccatccacc 7741 ccagaggtct ggccagtcct ccccctgctc acaccttccc tggctcccca tcaccctagg 7801 aataaagtca tcagtctgct attctgagtt cttcctgatt tccttgccct gctcctcttt 7861 cctgattctc cctgtctgtg tcatttccca tggccctccc tccttgtggc tacaaccaca 7921 ttaaaattgt tcaaaaaata ggccggcacg gtggctcaca cctgtaatct cagcactttg 7981 ggaggccaag gcaggtggat cacttgaggc caggagttcc aaaccagcct agtcaacgtg 8041 gcaaaacccc atctctactg aaaatacaaa aaattagctg gggccaggtg cggtggctca 8101 cacctgtaat cccagtactt tgggaggccg aggcggatgg atcacgaggt cgggagatcg 8161 agaccagcct gaccaatatg gtgaaacccc atctctacta aaaatacaaa tattagttgg 8221 gtgtggcagc gggcgcctgt agtcccagct actctggagg ctgagacagg agaattgctt 8281 gaacctggga ggcggaggtt gcagtgagcc gagatcgcgc cactgcactc cagcgtgggc 8341 gacagagcga gactccatct caaaaaaaaa aattagccgg gcatggtgac aggcacccgt 8401 aatctcagct actagggagg ctgaggcagg agaatcgctt gaatctagga atcagaggtt 8461 gcagtgagcc aaggtcacac cacttgcact ccacagcctg ggtgacagag tgagactgcc 8521 tcaaaaaaaa aaatcaatag ttcagaaaat accgagcacc ccctgcgtgc ccggtggcag 8581 ggcccctgcc tttgtgaggc tggcatgggg caggagctgt gacttcttta ccctccccct 8641 cgctccccaa gtactctgtg cttggccaga gagagcccgt catcatggtg cctcctgtct 8701 gacttccccg tggcaggact gatctgcccg gctccctgac acctgcctcg tggacctgac 8761 ccccctcctc tttgtgcctc cagagctatg ccaagaccaa gacgagagcc gaggtgcggg 8821 gcggtggccg gaagccttgg ccgcagaaag gcactgggcg ggcccggcat ggcagcatcc 8881 gctctccgct ctggcgagga ggtaacagga cagggtggag ggggcgggga ggggtggggg 8941 ggccagggaa gggcctgggt gtttactcac acacagctgc gcacatctgg catggtatta 9001 tgtcagccct gttccctcca cctcatggaa ccacctgggc tggtgacatc ggaactgagg 9061 cccttggacc tcactaccca tataagggga cagagatctg ggagccatcc actcctccct 9121 ctcgcatgcc ctgtctcttc agcctgtggc cacagcccct cttgacccca cctcctgtca 9181 ccctctcctg acccacactt cccacagcac ccagagagct cttctaaatc gtggaacctg 9241 aactccggac ctcggccttt gtgtggaacc tgaactcctg accttgtcat tgtgggccct 9301 ggtggctgca cacctttccc cctcatcccc tcctttccct ctctgaccag gcagagatga 9361 ctctctcttg ttttttcggt tgtgtttgtt tttaactttt tattttcttg aatgctaaca 9421 agatgactca gggtggtgcc agccacccca tcacctgttt acctggccag ctctcctcac 9481 ctttcagggt tctgggccac acctcctcca ggaagccccc cttgatctcc tttccttcca 9541 catcccccgg agctaccctg atttcttcta cagctgagcc tcttttctgc cctgccggaa 9601 tgtgaatggc atgagggcag ggaccatgtc tgttgtcttc tctgctgcat ttccaaggcc 9661 caggcgaggg cagacaccaa cacatggtgc ttgcaggggt ctccctgact gttgttgctc 9721 ccagatcata ctgcttgctc caccggagca tgtgcctgat gccttcctct cccgcttgac 9781 ctgaaagttc gaacctcctg ataacttcag cattaacagc gtgcttgagt taagttcaca 9841 ctctagccac tctatggaat ccacaccata actcatggtg tcctatgggg caggaactgt 9901 ccgatctcaa ggtggtttgt tttttttatt gtttgctttt gagacaagat ctcgctctgt

9961 tgcccaggct gtagtgcagt ggtgcaatca tagctcactg cagccttgag ctcctgggct

10021 caagtgatcc tccatcctca gcctcccaaa atgctggtgt gagccacctt cgccagccct

10081 gtctcagagt gtgacagctg ggaaaactga agcccagtga agcaaagtca ctggtcccat

10141 gttgtccctg atccagcctc ccctgaggcc ccaccctctc tgccttgttc ctggcagcgc

10201 cccctctctc gtcaccccat gccagccact gcagcagatt gctgacctcc aggctctgca

10261 gtggccccta cctgctcaca tgcctctgtc cccgcaggag gtgttgccca tggcccccgg

10321 ggccccacaa gttactacta catgctgccc atgaaggtgc gggcgctggg tctcaaagtg

10381 gcactgaccg tcaagctggc ccaggtacag ccatgggggg gcccagacag ctgctagagg

10441 tggggctgct ctggacccag ggttcaaacc atcctttcct tccaccagga cgacctgcac

10501 atcatggact ccctagagct gcccaccgga gacccacagt acctgacaga gctggcgcac

10561 taccgccgct ggggggactc cgtactcctc gtggacttgt gagggcacag ggcagagcag

10621 gggcaggggg ccctgagctc cgtactctga gggttcaacc cccactccct ggcctctctt

10681 acagaacaca cgaggagatg ccacagagca tcgtggaggc cacctctagg cttaagacct

10741 tcaacttgat cccggctgtt ggtgagcaaa gagcccaggc ccctagagtg cgcatgtgca

10801 ggctccgctg ttagaatcac agcggttcaa atccggcatc tggtcgctga gtggcctcag

10861 gcagtgacca cgctcccgga cccaaccttc agcttgccca aagcaataat ctttcctaaa

10921 gaagtgcttg gctggggatg gtggctcacg cttgtaatcc cagtactttg ggaggccaag

10981 gcagtctggg caatatagtg aggctcccat ctgtactaaa aataaaaaag ttaggcgtgg

11041 cgatgtgcac ctgtagtccc agctactcgg gggctgagcc aggcggatag cttgagctca

11101 gggggccaag gctgcagtga gtcatgatcg caccactgca cccaaacctg gggagagagc

11161 tagactcttg tctcaaaaaa aaaaaaaaaa aaaaaaaaaa agctccaaag tcacctctgt

11221 caaagccaca gtctgttcct tccacagcca caagatggcg acatgagcct aagtcaagtc

11281 ctgtccctca acccacgccc ctcgctggct tccccacctc tctcaggatg aaagcccaag

11341 tcatcagggt ggcacgtcag gccctgcacg atgtgccccg tcacctccct gccctcccgt

11401 catttgccct gggtctcccc caccagaatg ggagccagga gtcccagcca ggcacaggac

11461 cgagcctggc tctcggtcag caggtgatga gctgggagca gctccttggc cagaggctta

11521 caggcaacaa gcggccaggc agggtctggc cccgggctgc tgggctcaca aagtcacact

11581 agaccacagt gacgatctct gtaagcacaa agggactccg atgtgggtgg ggtgaggaga

11641 gaggcagccc cggcctgacc ggccccccgc cccgccccca ccccgccccc aggcctaaat

11701 gtgcacagca tgctcaagca ccagacgctg gtcctgacgc tgcccaccgt cgccttcctg

11761 gaggacaagc tgctctggca ggactcacgt tacagacccc tctacccctt cagcctgccc

11821 tacagcgact tcccccgacc cctMccccac gctacccagg gcccagcggc caccccgtac

11881 cactgttgaW gtgaagcacc tcttYtgagc caggccgagc ccctggccga cttgggagcc

11941 tYaggcccac gcccaccctt cgaggaaggt gtcacctgga ccccttcatt ccacggagga

12001 agctgaggcc acagggagcg gccatcgcca ttgggaaggg gcgactccac ggaRagccca

12061 gacgggcttY tgcatccatt ccctcttttt gtttttaaaa taaattgtat ttttgaatca

12121 aggaggataa agataacttc tcagtgtcat ttttgataat tgcattgaga acgatgagct

12181 ccttcccagg ttctgggcac tgttggggat ccgccgtctc aagaggctca cggtctggtt

12241 taggggaacc ccagggctgt ttgtggaaat tacaagaatt cacttacccg gtgagtcaga

12301 ctccaggagc tcacaggtgc gtgggcgctg gcacttccta ggagctgact cctgccacat

12361 ccctctcctg agcactgctg ccagccattc tcacccctga gagggtttgc agtcctgtct

12421 gcgacagtac ttcatgcagc ctgagagtgt gtgcttgatt gtacaagtag atgttgtgta

12481 aacctgagtt ttcagagtga cttcctgtaa gcacagtcag aaaagtaaat gtctctctgt

12541 aagtgagcag ataagcacat gcttaaaaac accagctggg cgtggtgact cgcacctgta

12601 accctagcac tttgggaggt caaggtggaa ggatcgtttg agctcagagc ttccagacca

12661 gcctgggcaa catggtgaaa ccccatctct gcaaaaaata taaaaattaa gtgggcgtgt

12721 tggcgtccac ctgtggtccc agctacttgg gaggctgagg tgggaggatc acctgagccc

12781 aggaagcaga ggttgcagtg agctgagact gagccaccac acaccagcct gggtgacaga

12841 gtgagacact gtcttaaaac cccaatatcc caatttttga gtttgctgag gttgccagcc

12901 aagttaattt ttcttaacag ccataacaga ctataatata gacaatgtga tcgatttatt

12961 taaagtcaac cagctgggcg cggtggctca cgcctataat cccagaattt taggaggctg

13021 agccaggtgg atcaccggaa gtcaggagtt cgagaccagc ctggccaagc cgggagtggt

13081 ggcgggcacc tgtaatccca gctacttagg aggctgaggt gggagaatcg cttgaaccca

13141 ggaggcagag gttgcagtga gccaagatag cgccactgcg ctccagcctg ggtgacagag

13201 caagattcca tctataaata aataaataaa taaagtcaat caggactgcc ttctgcctgt

13261 gtgggcctgg ccaggctaag cagccacaca accctcccta cctgctgggc cacccctggc

13321 tgaaaacctc tctggaatgc ctctcttggg attgccttca gagattgcaa cacattctcc

13381 aaacaccctc agtggacata gactttgatc cttgaagcag cagatttgac taagtaaaaa

13441 agaaaaagct attaaaacca gttggctggg cgcggtggct cacgcctgta atcccagcac

13501 tttgggaggc cgaggcaggc ggatcacgag gtcaggagat cgagactatc ctggctaaca

13561 cagtgaaacc ccatctctac caaaaataca aaaaattagc cgggcgtggt ggtgggcgcc

13621 tgtagtccca gctactcagg agactgaggc aggagaatgg tgtgaacccg ggaggtggag

13681 cttgcagtga gccgagatcg cgccactgca ctccactcca gcctggcgac agagctagac

13741 tgtctcaaaa aaaaaaacag aggcgtgcca aagctcagca gaaaatgccg cctcatcact

13801 cttcctcttg ccagtcttgt gggggcacca aggcctagag taacacccag ctgttggcct

13861 gacagtgcct ggcccagcct ggagagttgc agccagaagt ataaatgtgg ttcagtctgc

13921 gtcatacctg tcaggaaaca tggtgagacc aactgctgcc cagacggatt ttcaaaagaa

13981 atgggtcaga acgtattccc ccacactgga atccctcagc cagattgagg ataaaaacag 14041 gcatcagaaa aaaatgatac aggcaggcct ggtggctcat gcctgtaatc ccagcacttt 14101 gggaggctga ggcgggagga tcgcctgagc acaggagttt gagaccagcc tgggcaacac 14161 agtgagaacc tatctctact aaaaatagaa caattagcca ggcacggtgg tgtggctgtg 14221 gtcccagcta ctctggaggc tgaggtggga ggatcacttg agccctaggg gtagaggctg 14281 cagtgagcgg agatcacccc actgcaatcc agcctgggca acagatcaag accctgtctc 14341 aaaaaaaaaa aaaagaaaag aaaacaaaag aaaaatatga tacactgact agaatgtgct 14401 ttaaaataat tggcatagtt gggtatggtg gcatgcacct gcagtctcac ctacttggaa 14461 agctgtggcc aggagtttga gaccagcctg ggcaacacag caagacctca tctctataaa 14521 aaataggcgg ggagcagtgg ctcacgcctg taatcccagc actttgggag gccaaggcag 14581 gcggatcact agaggacagg agttcaagac cagcctggcc aacatggtga aaccccatct 14641 ctactaaaaa tacaaaaatt ggcaggatgt ggtggcgggt gcctataatc ccagctactt 14701 gggaggctga ggcaggagaa tcgcttgaac ctgggaggca gaggttgcag tgagccgagg 14761 tcatgacact gccctcaaac ctgggtgaca gagcaagact cggtctcgaa aaaaaaaata 14821 atgaatgaat gaattaattc attaattaaa tagggggggt atatgagttt gttagggctg 14881 ccgtaggagt gccacaaact gcaggggttg ggggggttag tcaacagaaa tttattctgt 14941 cctgtttctg gaggctggaa gtccaaggtg aagaacaggg ttagctcctt ctaagggaaa 15001 atctgttcca gatccctctc ctagcgtctg gtggtttgct ttgctgacta tctttgacat 15061 tccttggctt gtagccacac tgcttcagtc tccaccttca tctttacatg atgttgtccc 15121 tgtgggtatg tgtctgtttc tgtgtctaaa tttctccttt ttattttttt cttttttctc 15181 tatctttttt tttagacgga gtcgcgctct gttgcccagg ctggagtgca gtggctcgat 15241 ctcggctcac tgcaacttct gcctcctggg ttcaagcgat tctcctgcct cagcctccca 15301 agtagctggg attacaggcg cccaccacca tgcctggcta atttttgtgt ttttagtaga 15361 gaaggggttt cgccatattg gccaggctgg taacctcagg tgatccaccc gctttggcct 15421 cccaaagtac tgggattaca agcgtgagcc accgaacctg gcctattttt tcatttattt 15481 ttagacagag ttttgctctt cttgtccagg ctagagtaca atggcgcaat ctcggctaat 15541 ctcaacctcc gcctcccggg ttcaggcgat tctcctgcct cagcctccca agtagctggg 15601 attacaggca tacgccacaa tgcctggcta attctgtttt gttagcagag atggggtttc 15661 accatgttgg tcaggctggt ctcgaactcc cgacctcagg tgatccaccc acctcagcct 15721 cccaaagtgc tgagattaca gacacgagcc actgagcctg gccctctcct ttttattttt 15781 aaaatatatt ttgtggacgg cagcagtggc tcacacctgt aatcccagca ctttgggagg 15841 ccgaggcggg tggatcacaa ggtcaggaac tcgagaccag cctggccaat atggtgaagc 15901 cccatctcta ctaaaaatat aaaaattagc caggcatggc cgggcgcggt ggctcacgcc 15961 tgtaatccca gcactttggg aggccgaggc gggcggatca cgaggtcagg agatcgagac 16021 catcctggtt aatacggtga aaccccgtct ctactaaaaa tacaaaaaaa aaattagctg 16081 ggcgaggtgg caggtgcctg tagtcccagc tactcgggag gctgaggcat gagaatggcg 16141 tgaacccggg aggcggagct tgcagtgagc tgagatcgcg ccactgcact ccagcctggg 16201 tgacagagtg agactccatc tcaaaaaaaa aaaaaaaaaa tttagccagg cgtggtgtca 16261 gaagcctgta gtcgcaacta cttgggaggc tgaggcagga gaatcacttg aacccaggag 16321 gtggaggttg cagtgagccg agaccacatc acggcacatc taaaaaaaaa aaaagttttc 16381 tgtttttgtt ttttgcaaaa ctactgaaat aaatacagtg agatatttat ttataaatga 16441 gaacgaatta ataatgagcc gtaggctggg tgtggtggct catgcctata atcccagcat 16501 tttggggggc caaggcaggt ggaacacttg aggtcaagag ttcgagacca ggctgaccaa 16561 tatagtggaa ccccatctct actaaaaata caaaagaatt agccgggcat ggtgccgggc 16621 gcctgtaatc tcagctatgg gaggctgagg taggagaatc gcttaaaccc tggaggcgga 16681 ggttgcagtg aggcgatatc acgccactgc actccagcct gggggacaga gcgagactcc 16741 atctctaaat aaataaataa ggagctgtat ttcaaaattt ggagaaggtg acactgagag 16801 tactgaatac acagtttttt gtttttttgg ttttttttga gacagagtct cgctctgtca 16861 cccaggctgg agtgcagtgg tgcaacctcg gctcactgca atctctgcct cccggttcaa 16921 gcaaatctcc tgcctcagcc tcccgcgtag ctgggattac aggcacgcat ccccatgcct 16981 ggctaatttt tgtattttta gtagagacgg gatttcacca tattggtcag gctgatctca 17041 aactcctgac ctcgtgatct gcctgcctcg gcctcccaaa gtgctgggat tacaggcgtg 17101 agccaccgag cctggcccca aaaaatattt atcaaaacta tgttaatgct ggccgggtgc 17161 ggtggctcat gcctgtaatc ccagcacttt gggaggccga ggcaggtgga acacgaggtc 17221 aggagattga gaccatcctg gctaacacgg tgaaaccccg tctctactaa aaaatacaaa 17281 aaattagccg ggcgcggtgg tgggtgcctg tagtcccagc tactcaggag gctgaagcag 17341 gagaatggca ggaaccgggg aggcagaggt tgtagtgagc tgagatcgcg ccattgcact 17401 ccagcctggg cgacagagcg agaatccgtc tcgaaaaaaa aaaaaaaata cacacacaca 17461 cacaaaaact gtgttaatgc ttaactacac aaaaatgata atcagataaa tatgcattta 17521 tttagagaac tgcatgttgg tcagtccagt ccctgcagag ggaattccca gcatgacctc 17581 attcacttgt gaagacagag caatccttgt gttttatttt tttaagatgg atctcactct 17641 gttgcccaga ctggagtgca gtggcatgat ctcagccctc tgccacctcc accttccggg 17701 ttcaagagat tctcatgcct cagcctcctg agtagctgag attacaggct tgtgcctcca 17761 tgcccagcta atttttttat ttttactaga gatgaggttt caccagtttg ggcaggccgg 17821 tctcaaactc ctgacctcaa gtgatccacc cacctcggcc tcccgaagtg ctgggatgac 17881 aggtgcctgg tcagcaactg ttgtttagac atacacattt tatctgctcg tccagcatgg 17941 tcagccctcc actttttaaa ttttatttta tttatttttt tgagacagag tctcactctg 18001 ttgtccaggt tggagtccag tggcgtgatt tcggctcact gcaacctcta cttcccaggt 18061 tcgagcaatt ctcctgcctc agcttcccga gtagctggga ttacaggccc gcgtccccac 18121 acctagctaa tttttgtatt tttagtagag acagggtttc accatgttgg ccaggctggt 18181 ctcgaactcc tgacctcagg tgattctcct gccttggcct cccaaagtgc tgagattaca 18241 ggtgtgagcc actgcacacg gccttaaatt ttatttatta tttatttatt tatttattta 18301 gagacttagt ctcactctgt tgcccaggct ggagtgcagt ggcatggtct cggctcactg 18361 cactccacct cctgggttca cgccattctc ctgcctcagc ctcccgagta gctgggacta 18421 caggcgccca ccaccactcc cggctaattt ttgtattttt agtagagatg gggtttcact 18481 gtgttagcca ggatagtctc gatctcctga cctcgtgatc cgcctgcctc ggcctcccaa 18541 agtgctggga ttacttattt tgttttttgt agagacaggt tctcactgtg ttgcccaggc 18601 tggtcttgaa ctcctgatct caagtgatct tcccacctca gtctctcaaa gggctgggat 18661 tacaggggtg agccactgca ccccaccttc cctctacttt ttgacggttt ccttctgcta 18721 tgaatgtgca tgtccagttg tctgcttctt agaactgata tttaccttcc tcatccatca 18781 gccattggag gaggactggg accgctcaga ttattgatct gacccattct ttcggcaggg 18841 tttcctggtg gctgtcttcc atcaccaaaa ctggaatcag aagagtttcc atagcccttt 18901 ttttttcccc acatctttgc tgaagcagag ttttgaaaaa caaaaccaca aactaagcta 18961 ttccccagaa gaaatctgta atcaaagata agctctgccg ggcacagtgg ctcacgcctt 19021 ttggaggcca aggcgggcgg atcacctgag gtcaggagtt ctagacctgc caggccaaca 19081 tggtaaaacc tcatctctac taaaaataca aaaattagct agatgtggtg gtgggtacct 19141 gtagtctcag ctacctggga ggctgaggca agagaatcgc ttgaacctgg gaagtagagg 19201 ttgcagtgag ccgagattgc accactgcac tccagcctgg gcgacggagt gagacgacct 19261 cacaaaaatt tacataaata aaatgaaaag taaaataaaa atacaaaagt tggccgggtg 19321 cgtttgctca cgcctgtaat cccagcactt tgggagggtg aggcaggcag ataatgaggt 19381 aagaagatcg agaccatcct ggctaacacg gtgaaaccct gtctctacta aaaatacaaa 19441 aaattagctg tgcgtggtga cacgcacctg tagtcccagc tatttgggag gctgaggcag 19501 gagaatcact tgaacctggg aggtggaggt tgcagtgagc cgagatcgca ccactgcact 19561 ccagcctggg ccacagagtg agactccatc ttgaaaaaaa aaaaaaatac aaaagttagc 19621 caggggtgtt ggtgggtgcc tgtaatccca gctatttggg aggctaaggc agaagaattt 19681 cttgaaccta ggaaacggag gttgcagtga gccgagatca cacctctgta ctccagcctg 19741 gacaacagag cgagactttg tctcaaaaaa aaaaaaaaaa aaaaaactaa ataggccggg 19801 agcagtggct catgcctata atcccagccc tttgggaggc caaggcaggt ggatcacttg 19861 aggtcaggag tttgagacca ggctggccaa catggtgtaa ccccgtctct actaaaaaca 19921 caaaaattag ccgggtctgg tggcgtatgt ctgtaatccc agctactcgg gaggctgagg 19981 caggagaatc acttaaacct gggaggcagg ggttgcagtg agctgagatc gtgccactgc 20041 actctagcca gggtgacaga gtgaaactct gtctcaaaaa attaaaaaag aaattcagca 20101 agtaatgagt taaggaattc gaatattaag gcgagtgaca aggaacgccc aggatgtggc 20161 ccaggatgga gtagggggga cactcattta ggagaaagct caggccacaa gacaggagga 20221 gccagccttg ttggggttga agggaagagc attccaggct gagggaactg caaggcgttt 20281 gcatgggaca ctatgggatg gcttctgccc ttggtgggca gcctctggtc tgaggccatt 20341 ctttggcctg cctgactgtc tggcaaccgg gaggaagccc tgcccttcct ggagacagaa 20401 acaaaggtct aggaaatatc tgcttccctt ttccttgaaa aacgcttaag ggaacggagg 20461 actgggaggt gccgtctctc tctgccagcc tgccccctac catagccatc ccactcccat 20521 ctcagaaagt gacccgccat cctccaaaag gctcggaccc tgatcaagga gtcatccccc 20581 ttgtcccagc acctccagtt ggcccagcct ccaaaacgga tgtcaaattc agccctttct 20641 ccaaggacac tgcccagtcc aggccccact atcattcatc tggactagaa cagtcacctc 20701 ctctcccatc tcctggctgc agctcttgaa gcctcaactg ggcccctgtg aacacttgag 20761 ttagggcaag gtccttcctc tgctcagaac cctctatacc tcccacctcg ctgggcataa 20821 aagccaaagt cctggccagg cacggtggct cacatctgtt atcccagcac tttgggaggc 20881 caaggggggc ggatcactag aggtcaggag ttagagacca acatggtgaa accccatctc 20941 tactaaaaat acaaaaatta gctaggcgtg gtgacgcacc cctgtagtac cagctactcg 21001 gtaggctgag gtgggagaat cgcttgaacc tgggaggcag agtttgcagt gagccgagat 21061 cacaccactg tgctccagcc tgggtgacag aacgagactg gggttcagaa acaaacaaac 21121 aaaacaacaa agtcctcctc aggtgacagg aacttgcacc tatctgccct gtcatctccc 21181 tgcccgctcc tctcctcgaa tctctccttt gctaagcctg ctccagccac actgttctcc 21241 tggctgttcc tttttttttt tttttttttt tttttttgag tctcactctc acccaggctg 21301 gagtgcagtg cctctatctt ggctcactgc aacctccgcc tgccgggttc aagagattct 21361 cctgcatcag cctcccaagt aggtggaatt acaggtgtgc accaccacac ccggctaatt 21421 tttgtatttt gcatagagat gggggtctcc ctatgttgcc caggctggtc ttgaactcct 21481 gggctcaagt gatcctScca tctcggcctc ccaaaatgct gggattacag gtgggaRccg 21541 cgcccaggtg gatttttgtc tgactctgtt cattcctgtg tccccagtac ctggaaggac 21601 gccaagcaca cagtaggcgc ttaaaaaaca ttgagccaca tgttgagaaa agaacggcac 21661 cattgtggct gcaagtggga cKtgggccgc gcgggggagc tcgcgcacct cgggccgggg 21721 caagagctca gtggaacccg cccgaggaag aacccgtggc gcaggatttt cccaggcctt 21781 ctgaggacca ggggcgtccc ccgtcccacc ctgtgacttt gctcaggccg ttccggggcg 21841 ggaattcaga actcctcagc cccccaagaa aaaaatatcc ccgtggaaat tccttgggaa 21901 tgaccgaggc gggggaaata tgcgtctctg gatggccagt gactcgcagc ccccttcccc 21961 gataggaagg gcctgcgcgt ccggggaccc ttcgcttccc cttctgctgc gcgacctccc 22021 tggcccctcg gagatctcca tggcgacgcc gcgcgcgccc cacaacagga aaRccttagg 22081 cggcgcggct tggtgctcgg agacttaaga gtacccagcc tcgacgtggt ggatgtcgag 22141 tcttggggtc acacgcacag gcggtggcca agcaaacacc cgctcatatt tagtgcatga 22201 gcctgggttc gagttgccgg agcctcgcgc gtagggcagg ggttcgagcg ccccttctcc 22261 ctgcctcgcc tctgcgcctg ggggctgctg cctcagtttc ccagcgacag gcagggattt 22321 cgagcgtccc cctcccctcc ctcgtcaaga tccaagctag ctgcctcagt ttccccgcgg 22381 agcctgggac gcNNgcggag gggctcggcg cgtagggatc acgcagcttc cttccttttt 22441 ctgggagctg taaagacgcc tccgcggcca aggccgaaag gggaagcgag gaggccgccg 22501 gggtgagtgc cctcgggtgt agagagagga cgccgatttc cccggacgtg gtgagaccgc 22561 gcttcgtcac tcccacggtt agcggtcgcc gggaggtgcc tggctctgct ctggccgctt 22621 ctcgagaaat gcccgtgtca gctaggtgtg gacgtgacct agggggaggg gcatccctca 22681 gtggagggag cccggggagg attcctgggc ccccacccag gcagggggct catccactcg 22741 attaaagagg cctgcgtaag ctggagaggg aggacttgag ttcggacccc ctcgcagcct 22801 ggagtctcag tttaccgctt tgtgaaatgg acacaataac agtctccact ctccggggaa 22861 gttggcagta tttaaaagta cttaataaac cgcttagcgc ggtgtagacc gtgattcaag 22921 cttagcctgg ccgggaaacg ggaggcgtgg aggccgggag cagcccccgg ggtcatcgcc 22981 ctgccaccgc cgcccgattg ctttagcttg gaaattccgg agctgaagcg gccagcgagg 23041 gaggatgacc ctctcggccc gggcaccctg tcagtccgga aataactgca gcatttgttc 23101 cggaggggaa ggcgcgaggt ttccgggaaa gcagcaccgc cccttggccc ccaggtggct 23161 agcgctataa aggatcacgc gccccagtcg acgctgagct cctctgctac tcagagttgc 23221 aacctcMgcc tcgctatggc tcccagcagc ccccggcccg cgctgcccgc actcctggtc 23281 ctgctcgggg ctctgttccc aggtgagtcg gggtggggat tgccgtcggg ccagttctcc 23341 gaagccccgg gaggaccggc tcccgggtca ggtcatgcat gcttaggtag ctgtttatgg 23401 gaaggagggg ctagagacag cgattgaaag gcaacagcca gtaggttcga atccagaccc 23461 tgcatacctc cacgtgtggc cttgggctat agattgcagc tttaaaaaag ggtagggggt 23521 tggagatgga ggggaggggc gggcctcgtt ttgttgccca ggccggtctt gaactccggg 23581 ggtctagcct tacctcctgc ctcagcctcc cgaStagctg ggatgagagg tgtgaaccac 23641 cgccttgctt ggctagattg cgtctcttac agtttctcag Ytgtaaaacg ggaaacgtta 23701 tagcggccac ctggcagggt atcttggccc agcgcagcac ctggccccag gactcgatca 23761 tgatggtttg ggaacttggc tctgtgccaa cccaacaagg cttaagggac ccccaccccc 23821 ctcaagatgt atattctgtt cctcatcctc tctgcccctg gggaagtcca gggctgcttc 23881 tacttggggg aattccagag ctgacttatc cgtggcccaa agctgagaag tgggacRccc 23941 cagcacaccc tcccccagct ccagcccagc tagggaagag ggaaggggtc agagggtctt 24001 tcatggtggt gtaagtttgg ggaaccagga gggtggRaga ttgacagctt ggttaacagc 24061 tcaacaaagc ctgagatcca ggccagcacg gtagttcatg ccagtaatcc caacacttta 24121 ggagccccag gcgggcgaat cacttaaggt caggagtttg agaccagcct ggccaacatg 24181 gcaacatccc gtctctacta aaaatacaaa aattagctgg catggtggtg ggcgcctgtg 24241 atcccagctg ctcgggaggc tgagggagga aaatccctta agcccacgag gctgaggttg 24301 cagtgaacca agattgtgcc actgcactcc agcctgggag acatagcgag attctgtctc 24361 aaaaaacaaa gcRttctgat ccggactcag acccagatcg cactgctttc tagctgagta 24421 accatttctc tctatgaaat gggaatggtc ccagaatctc ccttggagaa tgtatggagc 24481 cagtgtcctc acacccccat ccaagataga acaaatctga gacaggaatc tttgagtgag 24541 gcagtgctgg gctcagacat tttttcccac cttcggaggc agcagaatYt gagggacctg 24601 atccaaataa gccccttctt tctttctttt cttttctttt tttttttttt ttttttttga 24661 gacggagtct cactNNgtcK cccaggctgg agtgcagtgg cgtgatctcg gctcactgca 24721 acctctgcct cccaggttca aacgattctc ctgYctcagc ctccctgagt agctgggact 24781 acaggcatgt gccatcacac ccggctaatc actgtgttag ccaggatggt ctcgatttcc 24841 tgacctcatg atctgcccac cctgcctccc aaagtgctgg gattacatgc gtgagccaca 24901 gtgcccaccc cgtaagcccc ttctttctta cctgcaaggt agccagttgc tacccatcct 24961 gtgctgagtt acttgtatta gcaagggatg gggtggctat actcacccac cttacagatg 25021 gggaaattga ggcccaaaga gggggaaact acRtgtctca gggagtgagg agccagtctg 25081 attcctggag ggctgactgt ctccacctga cttcttagga gggaggaggg caccaacttc 25141 acattaaaat ctggttggac acagtggctc acacctgtaa tcctggcatt ttgggaggct 25201 taggcgggag gatcacttga ggccaggagt ttgagaccag ccttagcaac atagtgagac 25261 cccatctcta caaaaatgtt tttcagggcc aggcgcggtg gctcacacct ataatcccag 25321 cactttggga ggctgaggcg ggcggattac ctgaggtcag gagtttgaga ccagcctgac 25381 caacatggag aaaccccgtc tctactaaaa gtacaaaatt acccgggcgt ggtggcgcat 25441 gcctgtaatc ccagctactc gggaggctga ggcaggagaa tcgcttgaac ctgggaggcg 25501 gaggttgcgg tgaactgaga tcgtgccatt gcactccagc ctgggcaaca agagctaaac 25561 tccgtctcaa aaaaaaaaaa atgtttttca aatattagcc gggtatggtg gtgtcctgta 25621 gtcccagcta cttgggaggc tgagatggga ggatcacttg agcccaggag ttcaaggtta 25681 cagtgagcta tgattgtgcc actgtattcc agcctgggta acagagggag acccgtttaa 25741 aaaaaaaaaa gtgatggcta aagtccttcc atggctccct attgccctca gtataaagaa 25801 cacatgtggc tgggcgtggt ggttcacgcc tgtaatccca gcactttggg aggctgaggc 25861 gggcggatca cttgaggcca ggagtttgag agcaggctgg ccgacgtggc gaaaccccgt 25921 ctctattaaa aatacaaaaa ttagctgggc gtggtggtgc ttgcctgtaa tcccagatac 25981 tctggaggct gaggcaggag aatMacttga acccgggagg caRaggttgc agtgagctga 26041 gattgYgcca ctgcactcca gtctgagtga caaagcgaga ctccatctca aaaaaaaaaW 26101 aaataaaaga acacatcttt agcatggcct tcagtgctca cgggatcttc ctgaattaat 26161 ctccccctct tcatccttgc tcactcagct ccagccaccc tgccccggga catctgtact 26221 tgcctggaac ttatttccct tttctccgga cagccagccc tttctcgtca tttagatctc 26281 tgctgaaaca ttaccctgtc accaaagcac tgtctattct atcaccctgt tttgtttttg 26341 tcaaagctca tattaacatc agttattaat tatcttgttt gctcataatt tttttttttt 26401 tttttttgga gacagagtct cgttctgttg ctcaggctgg agtgcagtgg cacaatcttg 26461 gctcactgta acctccacct cccaggttca agtgattctt gtgcctcagc ctcccaaata 26521 gctaggacta caggcacgtc ccaccatgcc cagctaattt ttgtattttt agtggagacg 26581 gggctttgtc atgttggcca ggctgatctc aaattcctga cctcaagtga tctgcccgcc 26641 ttggcctccc aaagtgctgg gattacaggc gtgagccacc acacccggcc tgctcatgaa 26701 ttttctcttt aacttccaca tcgaaggcaa agtattgtct tgttaaggct gtgcctccag 26761 cacccagcac aggctgggcg cacattccct tgatgaacct gatttgtaat gcctgtYgcc 26821 tcttccctcg tttcttctag gacctggcaa tgcccagaca tctgtgtccc cctcaaaagt 26881 catcctgccc cggggaggct ccgtgctggt gacatgcagc acctcctgtg accagcccaW 26941 gttgttgggc atagagaccc cgttgcctaa aaaggagttg ctcctgcctg ggaacaaccg 27001 gaaggtgtat gaactgagca atgtgcaaga agatagccaa ccaatgtgct attcaaactg 27061 ccctgatggg cagtcaacag ctaaaacctt cctcaccgtg tactgtgagt aactgagccc 27121 ggagggctgg actaggcaga ccSggtggga gagacgtgca gRggcacctg cagaggcctg 27181 ggggaatctt tgccacttgc tcgtagggtc aaggaggggc tccttgcagg gcaggtgggg 27241 acatccttgg aaagtccctt tgtgaatttc tttgggtaca attaaagtat ttacaggctg 27301 ggtgcggtgg ctcatgcctg taatcccagc actttgagag gctgaggctg gcggatcacc 27361 tgagatcagg agtttaagtt tcgccaacat ggcgaaaccc tgtctctgct gaaaatacaa 27421 aaatcagccg ggcatggtgt caagcgcctg taatcccagc tacttggaag gctgaggcag 27481 gagaacgctt gaacctgaga ggcagagatt gcagtgagcc gcgatcgtgc cagtgcactc 27541 cagtctggat aacagagcaa gattccatct caagaaaaaa aaaatgccat ctctctatgc 27601 ctcactcttt gaacatatga cacggtcctg cttcagacac tttaataaaa gatgcaaatt 27661 aagccaagtg tggtggcttg tacctataat cccaactact ccagaggctg aggcagaagg 27721 atggtttgag cccaggagtt tgagaccagc ctgggcaaca gagtgagacc ctgtttcttt 27781 cttttttttt tttttttttt tgagacggag tctcactctg tcgcccaggc tggagtgcag 27841 tggtgtgatc tcggctcact gcaagctccg cctcccgggt tcacgccatt ctcctgcctc 27901 agcctcccga gtagctggga ctacaggcgc ctgccaccat gcccggctca tttttttgta 27961 tttttagtag agacggggtt tcactgtgtt agccaggatg gtctcaatct cctgacctag 28021 tggtccgccc gcctcggcct cccaaagtgc tgggattaca ggtgtaagcc actgtgccca 28081 tccaagaccc tgtttctacc ggaaaaaaaa agtaaataat ttagctgggc atcgtggtgt 28141 gcacctgtaa tcccagctgc tcctgaggct gtgatgggag gattgcttta acccaggggt 28201 tcgaatccta ggagttcgaa tccatcctag gcaacatagc aaaaccccat ttttatttaa 28261 aaaaaaaaaa aaagatatga gttaaaacta gccctgggat ggcatttttc acatattggt 28321 aacaaacRaa agaattgatg gccgggcgca gtggctcacg cctgtaatcc cagcactttg 28381 ggaggccgag gcgggcggat cacaaggtca ggagatcgag accatcctgg ctaacatggt 28441 gaaaccccgt ctctactaaa actacaaaaa attagccggg catggtggca ggcgcctgtg 28501 gtcccagcta ctcaggaggc tgaggcagga gaatggcatg aacccgggag gcagagcttg 28561 cagtgagcca agatcgtgcc actgcactcc agcctgggcg acagagcaag actccatctc 28621 aaaaaaaaaa aaaaaaaaaa gaattgataa cagctgtgct gccaaggcta ttggaacgta 28681 ggaggtccta ggacagtgct gttgggagca taaataagcc caaccctgtg gcgggaaatt 28741 gggcatcagt tctcaaaatg tcatgggctg ggcacggtgg ctcacgcctg taatcccaSc 28801 actttgggag gctgagggag gcggatcact tgaggtcagg agttcgagac cagcctgacc 28861 accatggaga aaccccgtct ctattaaaaa tacaaaaaaa actagccagg catagtggca 28921 catacctgta atcccagcta ctcgggaggc tgaggcagga gaatctcttg aaactgggag 28981 gcagaggttg cggtgagctg agattgcgcc actgcactcc agcttgggca acaagagcaa 29041 aactccatct caaaaaaaaa aaaaaaagaa ataaaagaag gtatgttgaa tatgagtggt 29101 atgccaccct cacattaggg aagggcagtt tcggggaggc tgtatttatg tataaaatag 29161 ccctaMaagg aagtgggaga aatgacaata ttagctggct atgagaagag aggctgggag 29221 gctgtgggag agggcttggg tgtggagaat tctttttgtt ttttcctttt tttgagacag 29281 agtttcactc ttgttgccca ggctggagtg caatggcacg atctcagctc Mccgcaacct 29341 tcacctcctg agtttaagtg attctccggc ctcagcctcc cgactagctg ggattacagg 29401 catgggccac tacgcctggc aaattttgta tttttagtag agacagggtt tctccatgtt 29461 ggtcaggctg gtcttgaact ctgacctcag gtgatccgcc cgcctcggcc tcccaaagtg 29521 ctgggattac aacgtgagcc actttgcctg gctgagaatt ctttttttgt tgttgtcttt 29581 ttgagatgga gttttgctgt gtccccagcc tggagtgcaa tggtgtaatc tcagctcact 29641 gcaacctctc cctcccgggt tcaagcaatt ctcctgcctc agcctcccaa gtagctggaa 29701 ttacaggcgc ccagcaccac gccYggctaa tttttgtatt tttagtagag acgggatttc 29761 atcatgttgg ccRggctggt cttgatctcc tgaccttgtg atctgcccgc ctcggcctcc 29821 caaagtgctg ggattacagg cgtgagccac tgcccccagc cRagaatttc tctttgYgtc 29881 cttcctactt tggggacttc gaatggtggg aaagagttat caaggccaaa ataaggaatt 29941 caaatgaaaa caaaacaaaa tcaaagaaga aaaaacagaa gagcactggg caggctaggc 30001 acgtggctca tgcctataat cccagtgatt tagaaggccg aagtagaagg atcgcttgga 30061 ggccaggagt tggacaccag cctgggcaac atagcaagac cccatatcta caaaaaataa 30121 aaaacctaac caagcgtgct ggcatactag tagtcccagc tactcaggag gctgaggtgg 30181 gaggatcacc tgaKcctggg aggtccaggc tgtagtgagc cgtgatgaca ccactgcact 30241 ccagcctggg tgacagaKaa agaccctgtc tctaaaaaat aaaaactggc caagtagctt 30301 tgggattagc cttgggttcc agtcccagca aggcctttaa tagcttggga catgacttct 30361 gcatttactt tgcaatcagg tgagacctcc tctgatgggg aaaatgacac ggtgagtgac 30421 aaaggatgtt ctcctatcat tgtgtcaggg caaggaagcc tctgggtaaa tgatcaaatg 30481 atcagctttg cttctgattt ggagggtggg tgagcagatg ctgaccttcc caggtgaggg 30541 aagtccccRa acattcccag cagcttctgg aaaccccagg gaaacctctt tgaaggtctt 30601 ttctgcatct ctgcctgata ggtctttttt tttttttttt tttttttYtt ttttttgaca 30661 cacagtcttg ctctgtcgcc caggctggat cactgcaacc tccatctccc gggttcaagc 30721 agttctcctg cctcatcctc tccagtagct gggattacag gcacctggca ccacgcctgg 30781 ctaatttttg tatttttagt agagacaggg tttcgccaag ttggccaggc tggtctcgaa 30841 ctctagacct ctggtgatcc acccgcctca gcctcccgaa gtgctgggat tacaggcttg 30901 agccaccaYg cgcggctctg cctgatagct gagagcatag aactccaggt ttgagacctg 30961 gMtctgccac atttctccct ctatgactgt gggtgcccca ctttgcctta gtttttacct 31021 ctgtgaaatg gagcagatgg ctggcacagg tagcaaagga gtaaaagtta tgtgggaggg 31081 tggtacctga gagagactct agcttggtct tgccccaccc ctggtgtaaa cataaagaag 31141 cctccctgga tggctcaatc ttctccaaaa aggttagagg tgtaattcct agaggaggcg 31201 accactagct gggctttgaa ggatgtgtag gagttcataa ggacaggcat tctgggcagg 31261 aggaacagcc tgggcaaaag ttgggagcag ggagaaatct tgatggaggc aggaggagga 31321 ggaggtaggt tggtgtRggc caggYgcagt ggctcacacc tgtaatccca gtgctttggg 31381 aggccaaagc aagaggattg tttgagccca ggagttcgag accagcctgg gcaacatagc 31441 gagaccctgt ctctaagaaa aaataaaaaa attagggtac agtggcatat gcttgtattc 31501 tcaactactc tggaggctca agtgggagga tcaYttgaac ccaggaattt ttttgttttt 31561 gtttttgttt ttttgagatg gagtctcact ctgtggccca ggctggagtg cagtggtgcc 31621 atcttggctc actgcagcct cctccacctc ctgggttcaa ccgattctgc tgcctgagcc 31681 tcccgagttg ctgggattaa ggtgcccacc atcatgccca gctaattttt ttgtattttt 31741 agtagagata gggtttacca tgttagccag gctggtcttg aacgcctgac cgcaagagat 31801 cctcctgcct cagtttccca aagtgctggg attataggtg tgagccactg agcctggtca 31861 agcccaggaa tttgaggtta cagtcagcta tgattgcacc actgcattcc agcccaggtg 31921 acagagagag acactgcccc tNaaaaaaaa aaaaaaattg attgatggga ggaagggtga 31981 ggttggcaga gccttgaatg ccaggtggag gagctgggac tttccttctt ggggtgatag 32041 ggagtcatgg agggtttStg agcaggccag ggattagata gctgaaggct ggatttactg 32101 gaagccaaWg agcagttggc tatggtcctt gtccacgcgg cccatgttgt gggcagtgac 32161 cgtattcaag aagggaagga cagacaagta tttgaatact tcagtgacca ggatttggta 32221 aaggactgca ggtcagggtc aagaagaggt gagagcagga cagacttcct ccccgctgca 32281 ccaggcagct gagctgggtt tcctctaggg gctgaggttt gagggtacct caagttctgc 32341 aagagtctat aggaggtggt aagagagaag agctggaggt cagagttttc ttgactatat 32401 Rtatatatat ttttttgttt ttgtttttaa cagcttaaca gctttctgtt ttatttttag 32461 agacagggtc tcagggtctc actttgtcac ccaggctgga gtgcagtggt acaatcgtag 32521 ctgactgcag cctcaaactc ccaggctcaa gaaatcctcc tcccaccctt agcctcctga 32581 gtagcaggga ctacaggtgt gagccagcag gaagcccagc tggttttttt tttttctttg 32641 gtgttttttg tttgtttgtt tgagaccgga gtttcgctct tattgcccag gctgaagtgc 32701 aatggcagga tcttagctca ccacaacctc cgacccccag gatcaagcta ttctcttgcc 32761 tcagccacct gagtagctgg gattacaggc atgcgacacc acacaaggct aattttgtat 32821 ttttagtaaa gacagggttt ctccRtgttg gtcaggctgg tctYgaactc ccaacctcag 32881 atgatccacc tgcctcggcc tcccaaagtg ctgagattac aggcatgagc caccgtgccc 32941 ggcctttttt tttttttttt ttttNNtgag acagagtctc actctgtcgc ccaggcagga 33001 gtgcagtggt Kcgatctggg ctcactgcaa gctccgcctc ccgggttcac tccatcctcc 33061 tgccttagcc tcctgagtag ctgggactac aggcgcccac caccacgcct ggctaatttt 33121 ttgtattttt agtagagacg gggtttcacc gtgttagcca ggatggtctY gatctcctga 33181 cctcgtgatc cgcccgcctt ggcctcccaa agtgctggga ttacaggcgt gagccaccat 33241 gtctggcctg gccaggctgg tcttgaactc ctgacttccg gtgatccatc tgttctggcc 33301 tcccaaagtg ctgggattac aggcataagc caccacgcca tgccgaagcc cagcttgttt 33361 ttaatttttt tttttttttt tggagaaatg aggtcttgca atgttgccca agctagcctt 33421 gaactcctgg cctcaaatga ccccgccttg gcatcccaaa gtactgggat tacagatgtg 33481 agccaccatg ccccagcctt gctttcttga gatacRattt agaataccat aagattcatc 33541 ccttttaagc acataattca atgacttctg tacaaacaac catgactaca atctaatttt 33601 aaaatatYtc aatcactcta aaMaagaaac ctcctgctta tgtacagcga ctctgtctac 33661 ctcttaagtg aMttctccta cctttaatag ccctatttta cagttcagga aactgaggtt 33721 cagNgagaca aagtcactta cccacagcaa agaagcaagg ctgggtatca aatgcaggac 33781 ccccccKgtc ctgatgcttt tttttttttt ttttttttcc tctgagagag actctcactc 33841 tgtcactcag tctagagtac agtggcgcga tctcagttca ctgcaatctc tgcctcctgg 33901 gctgaagtaa tcctttcctc acaagtaaac ctcagcctct caagtagctg ggactacagg 33961 cacacaacac cacgcctggc taatttttgt atttttaggt agagacgggg tttcactatR 34021 ttggccaggc tggtcttSaa ctcctgacct caggtgatcc gcctgcctcg gccccccaaa 34081 gtgttgggat tacaggcgtg agccaccaca cgcagccttt ttgttattag actctgtcat 34141 tactgacttt tttttttttt taatagaaac agggtctttc tttcccaggS taaagtacag 34201 tggcatgatc acagttcact atagccttaa actcctgggc tcaagtgatc ctcctgcctc 34261 agcctcccaa gtagcaggga ctacaggtgt gcaccaccac acccagttaa ccattcattc 34321 attcattcat tcatttattt tgagatggag tctcgctctg tcacctaggc tggagtgcag 34381 tggcacgatc tcagctcact gcaacctcca cctcccaggt tcaagagatt ctcctgcctc 34441 agcctcccga gtagctgaga ctacaggcgt gcaccaccat gccagactaa tttttgtatt 34501 tttaatagag acggggtttc actctgttgg tcaggcttat ctcgaactcc tgacttcgtg 34561 gatccaccct ccttggcctc ccaaagtgct gggattaaag gcgtgagcca ccgcgccctg 34621 ccaacctttt tttaattttt cttagagatg ggggtctccc tatgttgccc aggcttgtct 34681 tgaactcctg gcctcaagtg accctSttgc cttggcctca caaagtgcta ggattacagc 34741 ctgagccatc acacctggcc aacaggtttt tttttttKtt ttgttttgtt ttttaaagaa 34801 tgtctaggcc aggctcattt actttcacct gtaatcccag cactttggga ggccggggtg 34861 ggcagatcac ttgaggtcag gaattcgaga ccagcctggg caacatgctg aaaccccgtt 34921 tctactaaaa atacaaaaat tagctgggtg tggtgacacg tgcctgtaat cccagctact 34981 caggaggttg aggcaggaga attgcttgaa cccaggaggc agaggttgca gtgagccaag 35041 atcatgccat caccctccag cctgggcgac agaaggagac tcagtctaaa aacttaatta 35101 attaattaat taaaaataaa aatacaaaaa ttaacctggt gtggtggtgt gtgcctgtaa 35161 tctaagctac tcaggaggct gaggcaggag aatcccctga atcccagagg cagaggttgc 35221 agtgagccaa gatcgagcca ctgtttgccc agtctagtgc actgggctgc tgaatttatt 35281 tgaccagaca cctagcaata gactttgaag ttcttttcca cttttcactc taagatgctg 35341 ctgtcatgaa taaggaatat tttgatcccc ttcacaaaca ctcggggccc tcttaccagt 35401 tttcactgaa gatcttgaca ttcctatctg cttaggtgtc tgggcgtgtt tgggggagat 35461 actgaagagg tagggctccc aggcaggtgc agttcgtctg ttaggcaggc agcaaggtcc 35521 acttcaccag acacccccac ctctgttttc ctRcagggac tccagaacgg gtggaactgg 35581 cacccctccc ctcttggcag ccagtgggca agaaccttac cctacgctgc caggtggagg 35641 gtggggcacc ccgggccaac ctcaccgtgg tgctgctccg tggggagaaS gagctgaaac 35701 gggagccagc tgtgggggag cccgctgagg tcacgaccac ggtgctggtg aggagagatc 35761 accatggagc caatttctcg tgccgcactg aactggacct gcggccccaa gggctggagc 35821 tgtttgagaa cacctcggcc ccctaccagc tccagacctt tggtgaggat tgaagaagcc 35881 agcagggaga aggtgggggt ggggtatcct gcaatgcggt gcctgtggcc acaggatctt 35941 ttgagatggg tgtggccccg gctaaggggt gcatgtgttc taggcgtatg tgacctaggc 36001 tgctgagtgg ccctggaaga ggatctcgca ggagggggaa tgaaatgccc cagagaaggg 36061 cttcgggacg tccatccctg tctgctcaca cctttcttct ctccctagtc ctgccagcga 36121 ctcccccaca acttgtcagc ccccgggtcc tagaggtgga cacgcagggg acMgtggtct 36181 gttccctgga cRgKctgttc ccagtctcgg aggcccaggt ccacctggca ctgggggacc 36241 agaggttgaa ccccacagtc acctatggca acgactcctt ctcggccaag gcctcagtca 36301 gtgtgacYgc agaggaYgag ggcacccagc ggctgacgtg tgcagtaata ctggggaacc 36361 agagccagga gacactgcag acagtgacca tctacagtaa gaaggggcag gggcggagtg 36421 gggcttcttg Rgggtgtgac ctgaacccgg ggcggggctc actgtgtgcc tattccaggc 36481 tttccggcgc ccaacRtgat tctgacgaag ccagaggtct cagaagggac cgaggtgaca 36541 gtgaagtgtg aggcccaccc tagagccaag gtgacgctga atggggttcc agcccagcca 36601 ctgggccYga gggcccagct cctgctgaag gccaccccag aggacaacgg gcgcagcttc 36661 tcctgctctg caaccctgga ggtggccggc cagcttatac acaagaaYca gacccgggag 36721 cttcgtgtcc tgtgtgagtg gggctgctgg tcaatggccc ctatccccca aggcccaatc 36781 tccctgaagg tcccataagg tcttgcctcc aagtcctgcc cccacccacc tccatgtcat 36841 ctcatcRtgt ttttccagat ggcccccRac tggacgagag ggattgtccg ggaaactgga 36901 cgtggccaga aaattcccag cagactccaa tgtgccaggc ttgggggaac ccattgcccg 36961 agctcaagtg tctaaaggat ggcactttcc cactgcccat cggggaatca gtgactgtca 37021 ctcgagatct tgagggcacc tacctctgtc gggccaggag cactcaaggg gaggtcaccc 37081 gcRaggtgac cgtgaatgtg ctctgtgagt gagccggcgg gcagagctgg gtgggggcag 37141 gggccatgga cctaatgcaa tcctcaccgc ctgttgtatc ctccccacag cccccYggta 37201 tgagattgtc atcatcactg tggtagcagc cgcagtcata atgggcactg caggcctcag 37261 cacgtacctc tataaccgcc agcggaagat caagaaatac agactacaac aggcccaaaa 37321 agggaccccc atgaaaccga acacacaagc cacgcctccc tgaacctatc ccRggacagg 37381 gcctcttcct cggccttccc atattggtgg cagtggtgcc acactgaaca gagtggaaga 37441 catatgccat gcagctacac ctacYggccc tgggacgccg gaggacaggg cattgtcctc 37501 agtcagatac aacagcattt ggggccatgg tacctgcaca cctaaaacac taggccacgc 37561 atctgatctg tagtcacatg actaagccaa gaggaaggag caagactcaa gacatgattg 37621 atggatgtta aagtctagcc tgatgagagg ggaagtggtg ggggagacat agccccacca 37681 tgaggacata caactgggaa atactgaaac ttgctgccta ttgggtatgc tgaggYccca 37741 cagacttaca gaagaagtgg ccctccatag acatgtgtag catcaaaaca caaaggccca 37801 cacttcctga cggatgccag cttgggcact gctgtctact gaccccaacc cttgatgata 37861 tgtatttatt catttgttat tttaccagct atttattgag tgtcttttat gtaggctaaa 37921 tgaacatagg tctctggcct cacggagctc ccagtcctRa tcacattcaa ggtcaccagg 37981 tacagttgta caggttgtac actgcaggag agtgcctggc aaaaagatca Ratggggctg 38041 ggacttctca ttggccaacc tgcctttccc cagaaggagt gatttttcta Ycggcacaaa 38101 agcactatat ggactggtaa tggttacagg ttcagagatt acccagtgag gccttattcc 38161 tcccttcccc ccaaaactga cacctttgtt agccacctcc ccacccacat acatttctgc 38221 cagtgttcac aatgacactc agYggtcatg tctggacatg agtgcccagg gaatatgccc 38281 aagctatgcc ttgtcctctt gtcctgtttg catttcactg ggagcttgca ctatgcagct 38341 ccagtttcct gcagtgatca gggtcctgca agcagtgggg aagggggcca aggtattgga 38401 ggactccctc ccagctttgg aagcctcatc cgcgtgtgtg tgtgtgtgta tgtgtagaca 38461 agctctcgct ctgtcaccca ggctggagtg cagtggtgca atcatggttc actgcagtct 38521 tgaccttttg Rgctcaagtg atcctcccac ctcagcctcc tgagtagctg ggaccatagg 38581 ctcacaacac cacacctggc aaatttgatt tttttttttt ttYcagagac ggggtctYgc 38641 aacattgccc agacttcctt tgtgttagtt aataaagctt tctcaactgc ctcagccttg 38701 tgtgagttga ggggaggtgt cacatccagc tggagtcctt tctaagcagc cacagcctga 38761 tcctcccact tcctccccca agaaaacatt gtgggttgat ggYcataccc tgaggttctg 38821 gtccaaatcg gactttctat gaccttctgg gtctctagtg aaaactaaag actcctctcc 38881 agaaaaaaac atttggtttc taatgaggcc tggaatctta ttcttgacct ggggagcgga 38941 atcccttttt gcagtactcc cgggccctct gttggggcct ccccttcctc tccagggtgg 39001 agtcgaggag gcggggctgc gggcctcctt atctctagag ccggccctgg ctctctggcg 39061 cggggcccct tagtccgggc tttttgccat ggggtctctg ttccctctgt cgctgctgtt 39121 ttttttggcg gccgcctacc cgggagttgg gagcgcgctg ggacgccgga ctaagcgggc 39181 gcaaagcccc aagggtagcc ctctcgcgcc ctccgggacc tcagtgccct tctgggtgcg 39241 catgagcccg gagttcgtgg ctgtgcagcc ggggaagtca gtgcagctca attgcagcaa 39301 cagctgtccc cagccgcaga attccagcct ccgcaccccg ctgcggcaag gcaagacgct 39361 cagagggccg ggttgggtgt cttaccagct gctcgacgtg agggcctgga gctccctcgc 39421 gcactgcctc gtgacctgcg caggaaaaac acgctgggcc acctccagga tcaccgccta 39481 cagtgaggRa caggggctcg gtcccggctg gggtgagggg agggggctgg aagaggtggg 39541 ggaagggtag ttgacagtcg ctctataggg agcgcccgcg gacctcactc agaggctccc 39601 ccttgcctta gaaccgcccc acagcgtgat tttggagcct ccggtcttaa agggcaggaa 39661 atacactttg cgctgccacg tgacgcaggt gttcccggtg ggctacttgg tggtgaccct 39721 gaggcatgga agccgggtca tctattccga aagcctggag cgcttcaccg gcctggatct 39781 ggccaacgtg accttgacct acgagtttgc tgctggaccc cgcgacttct ggcagcccgt 39841 gatctgccac gcgcgcctca atctcgacgg cctggtggtc cgcaacagct cggcacccat 39901 tacactgatg ctcggtgagg cacccctgta accctgggga ctaggaggaa gggggcagag 39961 agagttatga ccccgagagg gcgcacagac caagcgtgag ctccacgSgg gtcgacagac 40021 ctccctgtgt tccgttccta attctcgcct tctgctccca gcttggagcc ccgcgcccac 40081 agctttggcc tccggttcca tcgctgccct tgtagggatc ctcctcactg tgggcgctgc 40141 gtacctatgc aagtgcctag ctatgaagtc ccaggcgtaa agggggatgt tctatgccgg 40201 ctgagcgaga aaaagaggaa tatgaaacaa tctggggaaa tggccataca tggtggctga 40261 cgcctgtaat cccagcactt tgggaggccg aggcaggaga atcgcttgag cccaggagtt 40321 cgagaccagc ctggacaaca tagtgagacc ccgtctatgc aaaaaataca caaattagcc 40381 tggtgtggtg gcccgcacct gtggtcccag ctacccggga ggctgagttg ggaggatcct 40441 ttgagccctg aaagtcgagg ttgcagtgag ccttgatcgt gccactgcac tccagcctgg 40501 gggacagagc acgaccctgt ctccaaaaat aaaataaaaa taaaaataaa tattggcggg 40561 ggaaccctct ggaatcaata aaggcttcct taaccagcct ctgtcctgtg acctaagggt 40621 ccgcattact gcccttcttc ggaggaactg gtttgttttt gttgttgttg ttgtttttgc 40681 gatcactttc tccaagttcc ttgtctccct Ragggcacct gaggtttcct cactcagggc 40741 ccacctgggg tcccgaagcc ccagactctg tgtaKcccca gcgggtgtca cagaaacctc 40801 tccttctgct ggccttatcg agtgggatca gcgcgggccg gggagagcca cgggcagggg 40861 cggggtgggg ttcatggtat ggctttcctg attggcgccg ccgccaccac gcggcagctc 40921 tgattggatg ttaagtttcc tatcccagcc Scaccttcag accctgtgct ttcctggagg 40981 ccaaacaact gtggagcgag aactcatctc caaaataact taccacgctg gagtgagacc 41041 acgaatggtg gggaggggag ggtcccacgg acatattgag ggacgtggat acgcagaaga 41101 ggtatccatg tggtggcagc cgggaagggg tgatcagatg gtccacaggg aatatcacaa 41161 actcgaattc tgacgatgtt ctggtagtca cccagccaga tgagcgcatg gagttggSgg 41221 tggggggtgt caaagcttgg ggcccggaag cggagtcaaa agcatcaccc tcggtccctt 41281 gttctcgcgt ggatgtcagg gccYccaccc accgagcaga aggcggactc aggggcgctc 41341 cagggtggct cgagctcaca cacgctgagt agacacgtgc ccgctgcacc ctgggtaaat 41401 acagacccgg agccgagcgg attctaattt agacgcccgc gaacgctgcg cgcacgcaca 41461 cgtgtcctcg gctcgctggc actttcgtcc cgccccctcc gtcgcgtgcS ggagctgacc 41521 cggaggggtg cttagaggta tggctccgcg gggtcaaaag gagaaggatc agtgagagag 41581 gcatccccac accctcccct agaactgtcc tttccccatc cagtgcctcc caaatctctc 41641 ttagtcccca aatgtatccc cgccctaagg ggcgctggtg ggaggagcta aatgtggggg 41701 cggRgctcgg agtccagctt attatcatgg catctcagcc agggctgggg taggggtttg 41761 ggaagggcaa cccagcatcc cccgatccca gagtcgcggc cggggatgac gcgagagagc 41821 gtggtcgccc ccagaaggcc ctgggccatc atgccggcct ccacgtagac cccaggggtc 41881 gctcactcct gccagctcgc cttcaccaag gccaggagct tagcgcacgc tcgcctcccg 41941 cccccccgcc gcctctgccg ccgccccctc cttggaaacc aagttaccaa cgttaaacca 42001 atccccaagc gcaactctgY ctcccccaca ccccacccgc cgcgccgcgc ggagccgtcc 42061 tctagcccag ctcctcggct cgcgctctcc tcgcctcctg tgctttcccc gccgcggcga 42121 tgccagggcc ttcgccaggg ctgcgccggg cgctactcgg cctctgggct gctctgggcc 42181 tggggctctt cggcctctca ggtaagagcY ccgctctggt tcggggtgga cagggcgggg 42241 gcggagtccc tggacctgag aaacggcctc ctgtccctcc cagctctgcc ctcgcctcgc 42301 tSccacgcct ctgcccccac ctcgagcctg agtcttctcc ccttccccct cctccccaac 42361 acacacccga gccccagctt cctgactcct cgatagcccc tacccgcttc gagatcctag 42421 gtgttcttcc gcacccaacc cttcgccctg gagacccagt gtctctcctg tccgctcccc 42481 gggtacctcc ttacgctStg ctgtgcacca tggtccacgR actggcatct tccccactcg 42541 cggtccgcga agactccatc tctccaacta ccctgactca gaggcgctgt tcccgctcca 42601 cccagaRccc tggcaaccgg gtccctcagc tgttcccgcg gttccttcca tgagcccagc 42661 cttgcgtccc ggcKccgtgt tcttcaccgg gtgtagggtc cttcctgatc cttgacccag 42721 cctcgtctct cctttgccct tgccgcaaac gcactctcct cgtatcccgg cattctgcca 42781 ggaccctgag aactggttca tcccccatcc cccatcccgg gtccccttct ctcagccttg 42841 ctgtgttcat ccaagaaccc acctttcctc tcctctacgc cctcccccca tgctttcccg 42901 ccgctccatc ggcgctttgg agaccatgSc tctctgctac cacgtcccag agacaccctc 42961 gaggtttaga ctctgggagt gcgcctttaa accggaggcc tgggcaSgac gcgggacgcc 43021 tgggttcttt ccctggctgS agcctctcct cctcctcccc gccctctgag aacccttgac 43081 tcgacatagg ggcgctaaga tgtcagggag ttggctcccc aggctcagcc cgcgtttccc 43141 tgggcagcgg tctcgcagga gcccttctgg gcggacctgc agcctcgcgt ggcgttcgtg 43201 gagcgcgggg gctcgctgtg gctgaattgc agcaccaact gccctYggcc ggagcgcggt 43261 ggcctggaga cctcgctgcg ccgaaacggg acccagaggg gtttgcgttg gttggcgcgg 43321 cagctggtgg acattcgcga gccggagact cagcccgtct gcttcttccg ctgcgcgcgg 43381 cgcacactac aggcgcgtgg gctcattcgc actttccgtg agttctgggt ggccacgcgc 43441 gtactccact actctccctc cctcccaggc cccgccccct gggtcccagg gtcctcccct 43501 tcaggcccca ccttctgttc caagtcccgg Ygttcaaaga gctgcggact cttccccctt 43561 gcagagcgac cagatcgcgt agagctgatg ccgctgcctc cctggcagcc ggtgggcgag 43621 aacttcaccc tgagctgtag ggtccccggc gccgggcccc gtgcgagcct cacgctgacc 43681 ctgctgcggg gcgcccagga gctgatccgc cgcagcttcg ccggtgaacc accccgagcg 43741 cggggcgcgg tgctcacagc cacggtactg gctcggaggg aggaccatgg agccaatttc 43801 tcgtgtcgcg ccgagctgga cctgcggccg cacggactgg gactgtttga aaacagctcg 43861 gcccccagag agctccgaac cttctgtgag tgggtgtggg gaggagatgg ggacccagtg 43921 gggtcggtcg gtgtttagga ggtttagagg tagatacatc tgaatgctga ccccgacttc 43981 aaccctcgcc ggctgagctg tttccccctc cgtgccttga ggRKgataat acgataagat 44041 agtgcatgtc aagtgcttag gacatattga gcgctcggag ttagttcaaa cttggttctt 44101 cgacccctag ccctgtctcc ggatgccccg cgcctcgctg ctccccggct cttggaagtt 44161 ggctcggaaa ggcccgtgag ctgcactctg gacggactgt ttccagcctc agaggccagg 44221 gtctacctcg cactggggga ccagaatctg agtcctgatg tcaccctcga aggggacgca 44281 ttcgtggcca ctgccacagc cacagctagc gcagagcagg agggtgccag gcagctgRtc 44341 tgcaacgtca ccctgggggg cgaaaaccgg gagacccggg agaacgtgac catctacagt 44401 aaggaaggag gcggggtctc cgcggctccg aggtgggacc agaggaatgc gaaggcgggg 44461 cgaagagtgg gcgggacctc agtaccggaa caggcgtgKc ccgaggggcg gggcaggtgg 44521 gggcggagac gtaatcgctg gggaggagga gcctgtacag cctgagaggc ggggcgccgt 44581 accctagttc gttctcagca ccccgaggat tcgggcagat aaggggcggg ccttgaccgg 44641 agggaggggt atggtcagta tactacgacc aaatgctccg cccccaggct tcccggcacc 44701 actcctgacc ctgagcgaac ccagcgtctc cgaggggcag atggtgacag taacctgcgc 44761 agctgggRcc caagctctgg tcacactgga gggagttcca gccgcggtcc cggggcagcc 44821 cgcccagctt cagctaaatg ccaccgagaa cgacgacaga cgcagcttct tctgcgacgc 44881 caccctcgat gtggacgggg agaccctgat caagaacagg agcgcagagc ttcgtgtcct 44941 atgtgagttg gtgataaccc ctcgcccccc accttctggt gacttccaag gacccgcctg 45001 ctYcctcacc gtgtcgtgga ggcggagcca tttcttacgt ctaagcctct gtaaccccac 45061 gccctgcccg cagacgctcc ccggctagac gattcggact gccccaggag ttggacgtgg 45121 cccgagggcc cagagcagac gctgcgctgc gaggcccgcg ggaacccaga accctcagtg 45181 cactgtgcgc gctccgacgg cggggccgtg ctggctctgg gcctgctggg tccagtcact 45241 cgggcgctct caggcactta ccgctgcaag gcggccaatg atcaaggcga ggcggtcaag 45301 gacgtaacgc taacggtgga gtgtgagtgg gggtgcgcag RgtgcaYttc tatctggttc 45361 aaggtctgga gggtggccag cctccaggga agagtaggag tagggtatga ggtgtccctt 45421 tgggtgaggt tttgggaaag ggaagaggct ggttagtggg gttggagaaa gatcttggag 45481 gatggaaggg accgggtggg cgtgccccta gcctagggcg tggtatttgg gcggagtcgt 45541 ggaaaggcgg gcagtccaga gtgtttaagt ttttagRcga aaaaggcgcc actggtggct 45601 caggaagctc ccagacagag tgcatgSctc gactagcgtg acacctcctt ggatcggcgt 45661 ccaagggtta tgcagggaca acacttcgtg gaagccttgc cgcgccaagg agggtctagg 45721 gacgtcagat ttgcccccaa accccaaagc caacaataca ctccctcctc cagacgcacc 45781 agcgctggac agcRtgggct gcccagaacg cattacttgg ctggagggaa cagaagcctc 45841 gctgagctgt gtggcgcacg gggtaccgcc gcctgatgtg atctgcgtgc gctctggaga 45901 actcggggcc gtcatcgagg ggctgttgcg tgtggcccgg gagcatgcgg gcacttaccg 45961 ctgcgaagcc accaaccctc ggggctctgc ggccaaaaat gtggccgtca cggtggaatg 46021 tgagtagggg caccgcggag ttaggcagga tctgtgggac aaccccggct ggacttcctg 46081 gcccccgtgt gagcccctgc aatcctgttt cccagatggc cccaggtttg aggagccgag 46141 ctgccccagc aattggacat 999tggaagg atctgggcgc ctgttttcct gtgaggtcga 46201 tgggaagcca cagccaagcg tgaagtgcgt gggctccggg ggcRccactg agggggtgct 46261 gctgccgctg gcacccccag accctagtcc cagagctccc agaatcccta gagtcctggc 46321 acccggtatc tacgtctgca acgccaccaa ccgccacggc tccgtggcca aaacagtcgt 46381 cgtgagcgcg gagtgtgagc gaggcccagg cgggtaggga gcaggggtgc cccacggtcc 46441 aggcactccc tgacatcccc catggctgYt ttgcagcgcc accggagatg gatgaatcta 46501 cctgcccaag tcaccagacg tggctggaag gggctgaggc ttccgcgctg gcctgcgccg 46561 cccggggtcg cccttcccca ggagtgcgct gctctcggga aggcatccca tggcctgagc 46621 agcagcgcgt gtcccgagag gacgcgggca cttaccactg tgtggccacc aatgcgcatg 46681 gcacggactc ccggaccgtc actgtgggcg tggaatgtga gtgggggcag caccggatgg 46741 aggggacacg gtcctcggaa gaatgactcg cagcggtggg agcattcaag ggcacctctc 46801 ccaatcccat tctcggggac agggaattcc agcctaaacc agggggtaat gaaaattcta 46861 gccaggcgca gtggctcagg tctgtaatcc caacactttg gaaggttgag gcggatgaat 46921 cacttgaggc caggagttcg agaccagccc ggccaacatg gcgaaaaccc gtctctacaa 46981 aaattagccg gtcgtggtSg tgggcgcctg tggtcccagc tacttgggag gctgaggcag 47041 gagaatcgct tgagcctggg aggcagaggt tgcagggagc cgagatcccg ccactgcact 47101 ccagcctggg caacagagtg agactctttc ttagaaaaac agaaaaagaa aattataggg 47161 aataggagca cggccctcct caaatcctgg attagaacac tgacctgggc ttcacctcct 47221 tccattcggt gaagaagggc gaggaatttt agccgcaaca gcagYctgat tgtcggggaa 47281 ggaggctctg ataggaggca ggatcctctt ctgcccatca gaggcgcggt ggtctcccat 47341 cgatcgttgt gggccggagc agggcatttg atcagtggct gggccggcgc taagccccac 47401 ttcaccttct gtgcccttca gaccggccag tggtggccga acttgctgcc tcgccccctg 47461 gaggcgtgcg cccaggagga aacttcacgt tgacctgccg cgcRgaggcc tggcctccag 47521 cccagatcag ctggcgcgcg cccccgRggg ccctcaacat cggcctgtcg agcaacaaca 47581 gcacactgag cgtggcaggc gccatgggaa gccacggcgg cgagtacgag tgcgcaScca 47641 ccaacgcgca cgggcgccac gcgcggcgca tcacggtgcg cgtggccggt MMgtggcagc 47701 tggggagagg cggggcgagg tatctgagag ggggcgtgac ctgggtcttg gggcggccgg 47761 ccccgcctgc ctccctctcg gtcccggKag actagRcggW agtgggacag aRtagaagtc 47821 aaaggtgcct tagcgggtgg ggctgttgat cgcactttga ggggtgggag atggaggtgg 47881 cagggggact gaattcaagg ggagggaccc tccggggctg tcactgctgc aggaaaggac 47941 tttaaaactt gggctggatt ctgcgtggYg gaaacttgac ccaattcaca atccactgtg 48001 gggaagattg gggagggacc aaagtcattg gatgWtggag ggagagaggg gtcaagatcg 48061 ccttctctca cttcctgaag ctcctggggc tgggttctct ctggggtcgg gggaacctga 48121 cctggtcctg ccggtcctca ttttttgggg gatggggaga gttgacttgt cgccaggggg 48181 ctaagtgtgg tcacgggttt atatccccat gggctgaagt gcgatataaa tcgggactcg 48241 ggtcgccctg gcactgggag tggccttatt gcatgggttg tctcccttct cccaaggcca 48301 gacagtgagc tccccggggc atgaggactg acttcgtgcc tgtgggagtg cggggggcgg 48361 tgggagaagc cccccggagc ttggccaccc tccgcgaggg tctccacccc gcaggtccgt 48421 ggctatgggt cgccgtgggc ggcgcggcgg ggggcgcggc gctgctggcc gcgggggccg 48481 gcctggcctt ctacgtgcag tccaccgcct gcaagaaggg cgagtacaac gtgcaggagg 48541 ccgagagctc aggcgaggcc gtgtgtctSa acggagcggg cggcggcgct ggcggggcgg 48601 caggcgcgga gggcggaccc gaggcggcgg ggggcgcggc cgagtcgccg gcggagggcg 48661 aggtcttcgc catacagctg acatcggcgt gagcccgctc ccctctcccc gcgggccggg 48721 ggacgccccc cagactcaca cgggggctta tttattgctt tatttattta cttattcatt 48781 tatttatgta ttcaactcca agggcgtcac ccccattttc taSccatccc ctcaataRag 48841 tttttataaa ggaactccct gtctccgctt ctgtttctgc aaggtggaac aagSccaggg 48901 ttccagtcgt gacctcccga gttattcatt caaWaagcga tttttgagag ggcctggggt 48961 ggtggcttag gcctgtaatc ccaggacttg tgggaggccc aggtgggagg atcccttgca 49021 cccaagagtt tcagaccagc ctgggcaaca tagggagacc ctacaaaaaa tatttttcaa 49081 aaattagcca aggccggtcg cggtggctca tgcgtgtaat cccaggactt tggaaggccg 49141 aggcggccag atcacgaggt cgaggtcatg agatcgagac catcctggcc aatatggtga 49201 aaccctgtct ctactaaaaa tacaaaaatt agctgggcat ggtggtgcRc gcctgtagtc 49261 ccagctattc gggaggctga ggcaggagaa tcgctttaac cccggaggca gaggttgcag 49321 tgagccgaga tcgtgccact gaactccagc ctggcgacag agcgagattc cgtcttaaaa 49381 aaaaaaccag gcgtggtggc tcacgcctgt aatcccagca ctttgggagg ccgaggcggg 49441 cggatcacga ggtcgggaga tcgagaccat cctcgctaac acggtgaaac cccgtctcta 49501 ctaaaaatMc aaaaaaaaaa aaaaaaaaaa aattagccga gcgtggtggc gggcgcctgt 49561 agtcccagct actcaggagg ctgaggcagg agaatggcat gaacccgggg agcagagctt 49621 gcagtgagcc gagatcgcgc cactgcactc caccctggca gacagcaaga ctccgtctca 49681 aaaaaaaaaa aaaaaattag ccagggccga gcacagtRct ctcgcttgaa tcccagaact 49741 ttgggaaact aaggcggggg gggggggtca cttgaggtca ggagttcgag accaccctgg 49801 ccaacgtggt gaaacacctt ctctaccaaa aatacaaaaa aattaaggcc gggtgaagtg 49861 gctcacgtct gtaatcccag cactttgcga ggctgaggcg ggcggatcat ggggttagaa 49921 gatcaagact gtcctggcta acatggtgaa accccgtcac taMtaaaaac acaaaaatta 49981 gccgggcgtg gtggcacatg cctgtagtcc cagctactca agaggctgag gcaggagaat 50041 cgctttaacc ccggaggcag aggttgcagt gagccaagat cgcaccactg cactccagcc 50101 tgggcaatag agcgagactc tgtctcaaaa aaaaaccaaa aacaaaaaca aaaaaataca 50161 aaaatttagc tgggctgtag tggcacacgc cctgtaatcc cagctactcg tgaggctgag 50221 gcaggagaat tgctagaacc cgggaggtag agatcttgcc attgcactcc agcctgggca 50281 acagaactag actctgtctc aaaaaaaaaa aaaaaaaaaa aaaagtgttt tttgaggggg 50341 ctgggcatgg tggctcaagc ctgtaatccc aggacttttg ggaggcccag gtgggaggat 50401 cccttgtgcc cacgagtttc agaccagcct gggcaacata gcgagaccct acaaaaaaac 50461 atgtttcaaa aaaaaatttt tttttttgag atggagtctc gctctgtcgc ccaggctgga 50521 cagcagtggc actatctctg ctcactgcaa gctccgtctc ccgggttcat gccattctcc 50581 tgcctcagcc tcccaagtag ctgggattac aggcgcctgc caccacaccc agctaatttt 50641 tttttgtatt tttagtagag atgggatttc accgtgttcg ccaggatggt ctctattcct 50701 gacctcgaga tccacccgcc tcagcctccc aaagtgctgg gattacaggc gtgagccacc 50761 gcgcccggcc tcaaaaaaaa aatttttttt ttgagatgga gtcttgctct gtcacccagg 50821 ctggagtgct gtggtgtgat cttggctcac tgcaacctct gcctcctggg ttcaagcaat 50881 tcccctgcct tggcttccca aatagctggg actataggca tgcaccacca cacccagcta 50941 atttttgtat ttttagtaga gacagggttt caccatgttg gccaggctgg tctcgaactc 51001 ctgacctaag gtgatccacc tgcctcggtc tcccaaagtg ctgggattac agctgtaagc 51061 caccatgcct ggtgtatttt tcaaaaatta gccagacatg gtggtgcatg cctgtagtcc 51121 cagctacttg ggaggctgag gcaggaggat cctttgagcc taggaggtca aagccacttc 51181 cagctatgat atYgtcattg tgctccagcc caggtgacag aaccagaccc tgtctctaaa 51241 aacaaacaac gaaaaaatcc aaaaactttt tttgaacacc tactatgcat caggcacaat 51301 ctaagctgtc tttttctttt tctttttttt ttttttttga gacggagtct ctctgtcgcc

51361 caggctggag tgcagtggtg taatctcagc tcactgcaag ctctgcctcc cacgttcaag

51421 cgattttcct gcctcagcct cctgagtagc tgggattaca ggcgcgcgcc accactacgc

51481 ccggctaatt tttgtatttt tagtagagac agggtttcac catgttggtc aggctagtct 51541 ggaactactg acctcgtgat tcacctgcct cggtctccca acgtgctgga attacaagcc

51601 tgagccactg tgcctggccc ggctggctgt cttttctcag tgtgacctag agctcgtcct

51661 ctctcagtgc ctgttttctg ctctcttcac acccttgggc aggggtgggg gcctcactcc

51721 ttcctcctac ttatctcttc cacccagcac tgccctctga tccgggtcac tcttggaggt

51781 gggggctagg tgcttccccc aggcctggtt accggcagag ctgaggccgc cttgccaggg 51841 tgggtggagg ccctctccgt tgtgcccctg ccctgagact tcggtgagac cttcctgggc

51901 aggcactggc tgatgctatg ggtgtcagcc cttagcatgt cctccctgtt aaagggggca

51961 cccctgcccc gccctcaatg cctcctttgt cctgtgtcca cccacagtgc ttcagtttac

52021 ccccaccctg cctttccact cagcccatca tgattcagaa cagacacaca cagggtatcc

52081 gctgcaaccc ccactacacg gcaccttccc ccctcatccc caacagcata aggcacagca 52141 gctgggcctc ctccccacag cctcctcctc tgcccctcct ccctcaaaag caggaacacg

52201 gagccctaga gaaggaagcc agtcccatgc aaacatgtaa tgagcaaaga tgagacgggg

52261 aaatggcaca agagggcagg ccagtgctcg gtgctggagc caggggccag gtataacacc

52321 caaaaaggca ccagccaggt gcagtccctg ccactaacag cacctcacct tttctgcctg

52381 tgccctccct acttaatgat ctctctccgc tcctactgac ctcaaggtct ctttcagagc 52441 cttcagatga gatatttatt ttagtaggtt ggtgcaaaag taattgctgt ttttgccatt

52501 gaaagtaatg gcaggcaggg catggtggct cacgcctgta atcccagcac tttgggaggc

52561 cgaggtgggc agatcacgag gtcaggagtt caagaccagc ctggctaaca tggtgaaacc

52621 ctgtctctac taacaataca aaaaaaatta gccggacatg gtggcacacc cctgtagtct

52681 cagctacttg ggaggctgag gcaggagaac tgcttgaacc ctgaagcgga ggctgcagtg 52741 agctgaaatc acgccactgc actccagcct gggccacaga gtgagactgt ctcagaggaa

52801 aaaaaaaaaa aaaaagtaat ggcaaaaact gcaattactt tttctttttc tttctgtttt

52861 tttttttgtt tgtttgtttg tttgtttgtt tgtttgagac ggagtctcac tgtgtggccc

52921 aggctggagt gcagtggcgc aatcttggct tactgcagcc tccgcctccc aggttcaagt

52981 gattctctcc tgcctcagcc tcccaatagg attacaggca cccaccacta tgcctggcta 53041 atttttgtat ctttagcaga gatggggttt caccatgttt gccaggctgg tctcgaactc

53101 ctgatccacc cacctcagct tcccaaagtg ctgggattac aggcaggcat gagccatggt

53161 gcccgacatt tttttttttt tttctttttt tttttgagac agagtctcac tttgtcaccc

53221 aggctggagt gcaaaggcaa gatctcactg cagcctctgc ctcctgggtt aaagcgattc

53281 tcccacctta gccccctgag taggtgagat tacaggcaca tgccaccaca cctgcctaat 53341 ttttgtattt ttaatagaga gggggtttca ccattttgtc cacgctggtc tcgaactcct

53401 gacctcaagg gattcgccca cctcagcctc ccaaagtgct gggattacag gcatgagcca

53461 ccacgcccga cctccttaac tcttttattt ccttgcacac tttttctctg tagcccagct

53521 cacaacttaa catcctctta gacatacacc tgaggttttt tttttttttt tttttgagag

53581 ggagtctctc tctgtcaccc aggatggaat gcagtggtgt gctctcagct cactgcaacc 53641 ttgcaaccac tgcctcccag gttcaagcaa ttctcctgcc tcagcctccc gagtagctgg

53701 gactacagac acatgccgcc acacctggct gattttttgt tttttagtag agacagggtt

53761 tcaccgtgtt gcccaggctg gtctcaaact cctgagttca ggcagtccgc ctgccttagc

53821 cttccaaaat gctgggatta caggcatgag gcaccgcgtc ctgcctctga gttttttttt

53881 tttttttttt tttactgtct cctagaacat cagccccagt agagcaggga tctttgttct 53941 gttcaccact gagggtcctc attgggtcct agcacacaca gttgctcaat aaatgttgaa

54001 taagtgggta aagacagcca cgagcttgca gatatgtgtt caaggtgtgt ccttgcagag

54061 agcttctcta tacttggcac tggagaggcc tgtcgtgggc aaggacatag atgtggcgac

54121 ctcagacttg agaactcctg gggcagtagg ggagatggat gtggataatg taaccataag

54181 ccttcctttc tattttagac tgagtggtca aggaaggctt ccagacaatg gaaattctga 54241 taggttctaa gagggagacc agcaaggagc tgaaccggag gctgacacag ggtgagagtg

54301 ggaatgtcta ttttattttt tttttggaga cagggtctca ctctgttgct caggcagcag

54361 tgcagtggca cgatcgtggc tcactgcagc ttcaacttcc cagactcaag tgatcctccc

54421 acctcagcct cccaagcagc tgggaatata ggtgcatgcc accacatccg gctaattttt

54481 gtatttttgg tagaaacggg gtttcaccat gttgcccagg ctggtcttga actcctgggt 54541 tcaagtgatc ttgctgcctt gggctcccaa agtgcttgga ttacaggtgt gagcctgtga

54601 gccacatctg gcctatttta ttttttaatt agttttttgt ttttgttttt gttttgagat

54661 ggagtctctg tctccaaggc tggagtgcag tggcacgatc tcggctcact gtaacctcca

54721 cctcccaagt agctgggatt acaggcacat gccaccacgc ctggctaatt tttgtatctt

54781 tagtagagac agggtttccc catgttggcc aggctggtct cgaactcctg acctccggtg 54841 atccacctgc ctcagtttcc caaagtgctg ggattatagg aatgagccac tctgcctggc

54901 tgggcatgtg tcttgttttt ttcattctgt agataacaag caattgtcct ccctcagcct

54961 cccaagtagc tggtactaca ggcgcctgcc actacgcccg gctatttttt gtagttttag

55021 cagaggtggg gtttcaccat gttaaccagt ctctaactcc tgacctcagg tgatcctcct

55081 gccttggcct cccaaagtgc tggaattaca ggtgtgagcc accaagccca gcctcaaact 55141 cctgagttta agtgatcctc acacctcagc ctccctgagt gctgggatta caggtgtgag

55201 ccaccacacc cggcctgagt cgggggaggt gtctatttta gtctgagtgg tcaaggaagg

55261 cttctctgaa gagctgatgt tggggccagg cctcctcaag cgattagaag gtgtcagcca

55321 tggggagatg tggagggagg gtgtttcagg ccaaggaagg gtaagatcaa ataattcagg

55381 gatctgaggg cagaggaatc tggactccag ttctcccatt cagggctgcc caggagagac 55441 aaagaggatc tcataagggt gtgtggcttt gtggctcact cacgcccaca gcaaggacca 55501 tgcccaccct actttttttt ttttccccaa gtctccctct attacccagg ctggagtaca 55561 gtggtacgat cacggttcac tgcagtcttg acctccaggg ctcaagtgat cctcccacct 55621 cagtgtcctg agtagctggc actacaggca tgcaccacca cccctggctc attttttgat 55681 tttttggtag agacggggtc tcactgtgtt acccaggctg gtctcaaact cctgggctca 55741 agcgatcctc ccacctctgc cttctgaagt gttgggatta caggtgtgag ccaccatgcc 55801 cagcccgcca ccagtcttga ctctccatcc tccctccctc cctgtgagtg ctggtggcct 55861 gtgtgtcccg cctgctgaaa gatgcctctg acttggggct tttgacttca ggcccgggag 55921 acacattttg gctggattag ggagatctta aaagggggta ggggacacag caggggctaa 55981 ggagagtgac acactgcagg caggggttgg gtgacagatg aaggatccac tggtatatgc 56041 tgccagtgtg tgccatctac ttgtgtgttt tacgtgtgtc tggtgcatgt gtcctgtgtg 56101 ggctgtatct gtgtgtgact cagctacgtg tcagcatatc cctgtcgacg tgtgtctctg 56161 ctgatgtgtc tgtcagcacg cacggctgtc ttgcatgtgt cttttttgag atagagtctc 56221 gctctgtcac ccaggctgga gtgcagtggc acgatcttgg ctcattgcaa cctcaacctc 56281 ccaggttcaa gcgattctcc tgcctgagcc tcccaagtag ctcggattac aggcacatgc 56341 taccacgcct ggctaatttt tgcatattta gtagagacgg ggtttcatca tgttggccag 56401 gctggtctcg atctcctgac ctcaggtgat ccacctgcat cagcctccca aagtgctggg 56461 attacagaaa tgagccactg tgcctggcta gacatgtgtc tttttttttc ccttctgtat 56521 ataacagggt ctcactatgt tgcccaggct ggtctcaaac tcgtggcctc aagcaatcct 56581 cctgcctcag cctcccaaag tgttgggatt acaggcgtga gccactgcac ccggcttgtg 56641 tgtgtctttc tgtatctgtt gtgtgtgtct gtgggtagtg gctgtttccc tgcatgggac 56701 attgtggtaa atgtggctaa ttccctgtgt gggggctgct gcctgtggat aagactgtgt 56761 ctctgtgcat gcgcacgtgt gtgcacgccc ctcacaaccc caacgagaaa acacctgtcc 56821 accttcctgg gctggcacag gggcacagga ggcgggatcc caaatcacag gctttttctc 56881 tcggcatatc tctgtacttt attgtccctg ctgtaacaat gctcatcctt cctgagagcg 56941 tctcctgagg gggcctcggc caaggctgac tggagaaggg gctggtgacc cctagcaggc 57001 tctgccacct gaggcctggg tcttcccccg ggatccttga atctggagat ggcagagagg 57061 aggcaggccg gctctttccc caatcctcct aggagagctg cttctgccca ttctcccact 57121 ggtgaaacgg aggcagaagc agcagctcag cagggtgaag ctgggtttaa ccttcctgaa 57181 Yggggtccag ggacactcca catctgccac atagcctctt gggctggaca tttttcctgg 57241 gcaccagcca gcagctggag cagcgagtgc cgtatttctt gtacctaggg ttgggggaca 57301 agaaactgcc atttaggatg cagtgggggc ccggaaaccg ccacaaggaa accacttttc 57361 ccaagaggta ggtgttttgc tttttgcttt ccccacaggc catcctggtt acacgtggac 57421 tgatttgggg acccccgccc caactccctc ctccattcta aggacctgat cccacaggcg 57481 ttgcagagag gggtcccatc ttcagcgtct ctccagagcg gggtcctctg ggtccgacag 57541 gaagcacagc gccggggctc tgaagggtca gaggtcaaag ggcaggggtc agaggccaag 57601 catgtgagct cgggatgcct gtgcggagtc gggcattttg tgggggtctc agtaaaggcc 57661 ccacctagct ctggatcagc cctgggagtt gccagctcta agccacagca aagccaggaa 57721 gggagacaga tggcagcatt ccgcagagga ggaagctggg ggtgggggtg actcagccca 57781 agtggagggg ggtgctgcga ctcctccctg agggctctaa atggggaagc aagatggaga 57841 aggggggggc agggagaaag gcagggaaga caggaaattg gcccccaaaa tatttatagc 57901 tcttgggttt tcaggactca cccagggcct cgctgcctgc tgagtgggcc tcggtgcctc 57961 ctgggtgggc tgcagggccc ccaacagcat ctgcagggga ttcctgagaa cggctactgc 58021 agggcaggct gtggggcaga caaggtatta gcactggggg ggatctgtag cttgtcccca 58081 acagccctcc gaaatgaaga tttagtatca aggtatcagc catcccccga ggaggcaact 58141 aatatctgaa tggcctggct ttgcctctca ttagtaatat tattattatt attattatta 58201 tttttgcaga tggagtctcg ctctgttgcc caggctggag tgtagtggtg tcatctcagc 58261 tcactacaac ctccacctcc cgggttcaag caattcttga gcctcagcct cccaagcagc 58321 tgggactaca ggcgcgcgac accacgccca gctaattttt gtatttttag tacagacggg 58381 gtttcaccat gttggccagg ctggtctcga actcctgacc tcaagtgatc cacccacctc 58441 ggcctcccaa agtgcaggga ttacaggcat gagctaccgt gcccgaccta attattatta 58501 ttattatttg aaaaatagta agcacaggga cagcctgcca ggttccaatc ccagttctcc 58561 atgtccttgc tgtgtgagcc tggacacatt atctcctact ctgtgcctca gtttcctcat 58621 ctgtaaaatg ggcttcccaa tacaacagtt tttttttctt tgaaaatttg atttattgat 58681 ttttaaaaag agatgggagt ctgggcaata tagggagacc ccgtctctac aaacaaaaaa 58741 aatagccagg agtggtggta caggcctgca ctcccagcta gttgggaggc tgaggaggga 58801 ggatggcttg ggcctgggag gtccaggctg cagtgagcca tgattgcgcc actgcactcc 58861 accctgggtg acagagcaag accctgtctc aaaaacaaac aaacaaacaa aagcaaaaaa 58921 aagcagccag gcatggtggc ggctcactcc tgtaatccca acactttggg agggcaaggc 58981 agatggatca cctgaggtca agagatcgag accatcccgg ccaacatggc aaaaccccat 59041 ctctactaaa aatacaaaaa ttagccaggc gtggtagcag gcacctgtaa tcccagctac 59101 tccagaggct gaggcaggag aatcgtttga atccgggagg cagaggttgc agtgagccga 59161 gattgtgtca ttgcactcca gcctgaacga cagagtgcaa ctccatctca aaaaaaaaaa 59221 aaaatattgt tttggcggca tgattaaaaa agtcataatt ataattttta taataacaat 59281 aaataggtaa aaattagagc tgggggtcat actatgttgc ccaggctggt cttgaactcc 59341 tgggctcatg caatcctccc accttggcct cccaaagtgc tgggattaca ggtgtgagcc 59401 accaggccca gcttcccaaa gctttttttt tttttttttt aaggggatgg agtcttgctc 59461 tgtcacccag gctggagtgc agtggtgcca tctctgctca gtgcaacctc cacctcctgg 59521 gttcaagcga ttctcctgcc tcagcctccc aagtagctgg gattacaggt gcccattacc 59581 attcccagct agtttttttt ttttttttga gactgagtcc cactctgtgg ctcaggctgg 59641 agtgaggtgg cgctatctct gcttactgaa gacttcacct cctgagttca agcgattctc 59701 ctgcctcagc ctcctgagca gctgggatta caggcaccca ctacaacgcc cagctaattt 59761 tttgtatttt tagtagacat ggggtttcac tatgttggcc aggctgatct caaactcctg 59821 acttcaggtg atctgctcgc cttggcctcc caaagtgatg ggattatagg cgtgagccac 59881 tgtgcccggc ctccccaggc ttttgaatga gcccatacac ataaaatact gcctgttaac 59941 ctcttattgt tgtcactacc aacaaatagt agctgttagc attattggga aaaatcaccg 60001 cagccgcagc tgcagcctaa gcaaggccag gcctgattcc taaagagacc aggtctcaca 60061 ttgggagtgg ggagccactg agtcaggagc ggcaggactg tatctggcac cagctcaagt 60121 tgtcaaccag agactgggcc acgtgtaacg tagagcacag gtgagggtcc acaccgacgc 60181 cagRgagcac ccctacctcc cacactggtg tttgcgttgt aactgggaga aagggcggcc 60241 tggccccagc cggcccctac ctgtacgtgg ggatgatctg caggctggag tccggcttta 60301 tctgaaactt cagagtcacc ccctcgaacc cagggtccac tctctcggtg ccccggcagg 60361 ggttcagctg tttccggggc cttctctggg gtggggccga gggagtcccc gggggctgca 60421 gacggcggcc cttttggctg atcctggtct gtgtgtcctt ggaatccctg tccagcagca 60481 ggcggccctg agtccccaga gccatcgggt cccagcaagg ccccaggacY ggggtgtcct 60541 gggcagggga ctgacccagc ctctccactg tctcttggag gaagcacagg gcggtgaccg 60601 actcctggca tgcaggccag agggacctgg ggagaagggc agtggggtca cgttttcttg 60661 ggtgactcct gggtttagcc ggccaggggg ttccaacttg ggccggctct ttgtgtaccc 60721 ttggacaggc tacacacccc acctgaggtt cagtcatttc taactctggg tggaaggttt 60781 aagagttgca gtaattgcaa ctcacctcgc ccactgtggg cctcccaggc acgcccagcc 60841 ctccacatca atcaacccct tacggcaccc gtagatcacc caggcaggga ccgccctagg 60901 tgaccgaggc aggatatcaa gcctctggct gcaggatcga gaaaaaggtt actttttttc 60961 tttgtgtttc ttttagagac cgagcctcac tctgtacccc cgcctggagt gcaggggcgt 61021 gatcaggact cagtgcagcc tagaactcct gggctcgacc gatccttccg cctcagcctc 61081 ccgagtagct gggacttaca ggcacgcgcc accacgccgc tggcttgcca taatatgatt 61141 acgattttta tcattttcat tattattgag ttgccctcca taacccctgg atgatccctg 61201 gggagtgcct acatcagccc cagagacatg gaattgcatg gcctctgatc cctagatgac 61261 cccaagtgtc tctgggcacc agaacctctt tgtaagtttc agtaactatt ataaaattgc 61321 cacttctcga taaccccagc aaagagaggt catRacccct tgatcactga gccaggtggt 61381 gaagtgctag cggggtccgg ggaatggggt ttggccttac gcacctctca ctgattctgc 61441 agcatcctgg ggtcctcggc ttctcctggg agggggatcc tcccagccgc tccaggttca 61501 gacagggcgg cgccagaagc tttgctggca ttgaggagtc cgggattggc tcggcctcca 61561 ttgcgcccca gcccagcccc ctccctaatt tcacccggcc ctgtcccgct gggcccaccc 61621 gatgaaaccc acctgggagg ggcgggacct gtgtggtggg agcgctcttg gtagaggatg 61681 cgatgagccc cagctgagag cgaccagcct cggcggcacc tttcctcttt ctggcttttt 61741 tcaggtcatc cgcgcacccc tgcgtggcaa gggcggcctg gaccccacca aatccctccg 61801 cagctgccgg ggcggggcct tccacgctag ccctccccct cggaaatggg ggctcccagg 61861 cctctgcctt ggatccccag cggcatctta gatcccagag ggaaaagaga accgggggcc 61921 cagcacagca gatgcaggtg tggtttgttt ttgtttttgt tttttttttt tttgagacag 61981 ggcctcactc tgtcacccag gctggagtgg aatggcctga tcaccgctcc actgcagcct 62041 aaatcccctg ggctcaagtg atcctcccac ctcagtctcc cgagtagctg ggactacagg 62101 cacttaccac cactccacgc taatttttac tgtttttata gagacgaggt ctcgccatgt 62161 tgcccaggat ggtcttaagc tcctggcctc aagtgatcct cccatcttgg cttcccaaag 62221 tgctaggatt acaggtgtga gccacctgta gaccccagat ctgtccccca aggacactca 62281 acatgctggg gcctgtgtta tcgatttatt gcagctccaa tatgagtcca ctcctactca 62341 aaccctgatt ctcaggggcc tggggaaggc caccccacta ggggccctgt ccccaccaga 62401 gcctggacat gggactctct cccaagcagg ggtgttgtcc ttcacagggg cttccacgtc 62461 tctcccgggc ctgtccgcac tctgggcagg gctggcacct ggctattccc tcaatctggg 62521 ccctggggcc atggcaatgt ggaatggtcc aggtgttcat gtcagtgggg cttggggaca 62581 tggccatcca cgtagaagtt cctggtgatc ttgggMaggg tgaattccgc tccttccagc 62641 tccggtgtca gcacaatctg gcagcccagc cgcgagttct cctggaggag gggggccatg 62701 tctagcatgt cgtcttccct agggtggtga cacgggcagt gttagccgag ggcaagctag 62761 ctgggtgggt ggggggccag aggtSgggga gggaggaagc tgactgagcg cagggggtag 62821 tggtggggga ggcagctccc attccaggaa ctggaagtgg ggaggatggt gggggctgct 62881 gaatgctcca gggtgaagct gccagctaga cagggctgac ccactcacct ctcctcggga 62941 ggaggcagga gatccaggtg gtcttcactc acatacacat ggcaggtgga gcaggccagg 63001 gaggcttcac aggcccctag gggtgggaag tgaagggggc gcaggtgagt ggcagagtca 63061 ggaaggggct gctatgcgaa tggcgtagag gagtagacct ctcttccacc acgtgcctca 63121 cgtctccctc ctgggcctgc agctccactg tgtgtaccag gcactgtgtc atgtcctcaa 63181 tgtcctcagc tagctgttcc ccagtgtgta gggtttacaa acggacatct gggaggcccc 63241 atttcccagg gactccctag caggctgaga tgcagaacca ttaaggagca gggacttggc 63301 tgccaggtag ctgagagggg aaaggccagg caggctcccc gcagtctgtg agcccttgct 63361 gtgaccctat cactgtgctg ggctaatgtg tccccacctc tccactgctc ttggaggatg 63421 gaccggctga ctcagcagtc agtgtggtgg gggaaggaat caggcattag gattttgttt 63481 tttttttttt tgagatggag tcttgttctg ttgcccaggc tggagtgcag tggcacgatc 63541 tcggctcact gcaagctctg cctcccgggt tcacgccatt ctcctgcctc agcctcccaa 63601 gtagctggga ctacaggtgc ctgccaccac gcctggctaa tttttttata tttttttagt 63661 agagctgggg tttcaccatg ttagccagga tggtcttgat ctcctgacct catgatctgc 63721 ccgcctcagc ttcccaaagt gctgggatta caggcgtaag ccactgcacc cggccaggat 63781 tttttttttt tttttttgag acagggtctc actctgttgc ccagcctgga gtgccatggc 63841 tcagtcatag cttactgcct caacctcccg gctcaagcga tcctcccacc ccagcctccc 63901 gagtagctgg gactacatgt gcgcaccacc acacctggct aatttttttt tttttttttt 63961 tttgaggtgg agtttcgctc tctttgccca ggctggagtg caatggtatg atctcggctc 64021 aatgcaacct ctgcctccca ggttcaagtg attctcctgc ctcagcctcc caagtagctg 64081 ggattacagg tgcccaccat cacgcccagc taattttgta tttttagtag agatggggtt 64141 ttgccatgct ggccaggctg gtcttgaact cctgacctca ggtgatccac ctatctcggc 64201 ctccctaagt gctgggatta caggtgtgag ccaccgcacc tggccatatt tttttttttt 64261 tttttttttt ttgtagagac gaggttcacc atgtggccca ggctggtctc aaactcctaa 64321 gctcaagcaa tctgcctgcc ttggcttccc aaaatgctgg gactacaggc atatgccact 64381 gcaccagacc aggaattagg gttgaaagag acagaacctg aagttttctg ctgaccacct 64441 gcccttggtc acacccccac atcctctgat cacagagacc ggtgcttggg gatcatgtcc 64501 ctcctggcct aaaactttcc aaagggtttt cacactcaga ataaaatcca aactccttca 64561 cttagtcttg cagttccccc atccccctca ttcattcctt tgttttcctt tgtaattttt 64621 ttcttagaga tggagtcccg ctctgttgcc caggctggac tgcagtggcg tgatctcggc 64681 tcactacaag ctccgcttcc cggattcacg ccattctcct gcctccgcct cccagagcag 64741 ctgggactac aggcgcctgc caccatgcct ggctaatttt gtttttgtat ttttagtaga 64801 gacagggttt caccatgtta gccaggatgg tcttgatctc ctgacctcgt gatccgctcg 64861 cctcagcctc ccaaagtgct gggattacag gcgtgagcca ccacaccttg ccaggccact 64921 tacctctact agcccggcac agtggctcat gtctgtaatc ccagcacttt gggaggccaa 64981 ggcaggcaga tcaactgtgg tcaggagttt gagaccaccc tggccaacat ggcgaaaccc 65041 catctctatt aaaaatacaa aaacttagcc gggcatggtg gcagttgcct gtaatcccag 65101 ctactcggga ggctaaggca ggagaatcac ttgaaccctg gaggtagagg ttgcagtaag 65161 ccaagatcat gccattgcag tccagcctgg gtgatagagc gagactctgt ctcagataaa 65221 taaatgaaca aataaatcta tctcacattc caatccctgt cctagactat tggctgctgg 65281 attccagaac agaactttag gtaggccttt cataacccct cccaacatat cttccccttt 65341 aatgataaaa tcaggatccc tgatatattc atcccaaaag caaaccctat atattttctt 65401 ccagacaacc cttcccaagt tggtcataat tatgtattta cctgtgggat tattgtccct 65461 attagaccga aatggatgtg agatgacaga ggccttattt gttttgtata ttgctgcctg 65521 acgcgcagta ggtattcata gaaaggcagc acagattagt ggctacaatg gactggtgag 65581 tctagtattg tcactcactt gcccttagac caggtctact taacctgggg tttagttgcc 65641 tcatctataa tagggaataa cagtctctac tactcaagtt gtcatgaaga ttcagtcagt 65701 gaatatttat taacatggtt aaagacatat atatatatat atatatattt tttttttttt 65761 tttttttttt tttttttttt gagacagtct cactctgtcg cccaggctgg agtgcagtgg 65821 cgtgatcttg gctcactgca agctccacct cccgggttca tgccattctc ctgcctcagc 65881 ctcccgagta gctgggacta caggcgccca ccaccacgcc cacctaattt tttgtatttt 65941 tattagagac ggggtttcac cattttagcc aggatggtct ctatctcctg acctcgtgat 66001 ccgcccgcct cggcctccca aagtgctggg attacaggcg tgagccaccg cgcctggcct 66061 tttttttttg tttgtttgag acagagtccc gctctgtcat gcagcctgga gtgcagtggt 66121 gccatcttgg ctcactgcaa cctctgcctc ccaagttcaa gtgattcttg tgcctcagcc 66181 tcccaagtag ctgggattat atgcatgtgc caccacgccc agctaatttt tgtgtttctt 66241 gtagagacag ggtttcgcca tgttggccag actagtctca aactcctgtc ctcaagtgag 66301 tcgcctgcct cggcctccca aagtgttggg attacaggca tgagccactg Ygcctggaca 66361 agaaatttct attattattt ttctctcaac aaattatcaa acatctgtta catcttgaca 66421 ggcaacagat aagcatgaat aaggacatgg gtcaccaatc tcactggggg acagatgaca 66481 aagtaggaaa caaaacagcc tggcattgtg gcttgtgcct ataatcccag ctacttggga 66541 ggctgaggtg ggaggatcac ttgaacccag gagttcaagg ctgcagtgag gtatgattgc 66601 atcatggcac tccagcattg gcgacacagc aaggctccgt ctcttgaaaa aaaaaaagtg 66661 agaagtgcta tatatcatat cacactgata agacagagta actagggcat gggtacttca 66721 gctgggctga ggggaatggg attgttaggg acatgacatt tacactgagg tctgaaggat 66781 gagaaagagc cagaatgttt gctgggaaag cacttcagac agagggaaca gcaaatgtaa 66841 aggccccgaa gcaggaatga gcttggttgt ttgaggaacc acaagaaagc cagtgtgggc 66901 tgggcgcggt ggctcatgcc tgtaatccca gcattttggg aggctgaggc gggtggatca 66961 cctttcagga gtttgagacc agcctggcca atgtggtgaa accccgtctc tactaaaaat 67021 acaaaaatta gctgggcttg gtggtgggcg cctgtaatcc cagctagtct ggaggctggg 67081 gcaggagaaa cgcttgaacc cgggaggtgg aggttgcagt gagctgaggt gcagtaagcc 67141 attgcactcc agcctgggca acaagagtga aactccatct caaaacaaca acaacaaaaa 67201 gaaagccaat gtggctagaa tacagggagg aagggggaag agaacacaaa atcctgggag 67261 ccaggagtcc tagtgatctc taataaatgc ttaacaaata agagcttggc aacagatgag 67321 gccatggtga ggggtttgga ttttagtcca tatttggtgg gaaaaagacg tgaagagaca 67381 cacctRattt gtggKttttt tttttttttt ttttggacaa atgtataagg acctccttaa 67441 ttccctctgc ccggttccta ccttccaggt ccaccccgtg gcgctgggcc aggtgaagaa 67501 cattgtcccc gactctgcca ctcactggga tccgctggcc tgagcggtct acgaacacca 67561 cgttcaccct ggggacagat ggaagtgcga atgccgaact ggatgatggg ctcagggccc 67621 gggtagaatc tagggttagg aagtaggaat tgaggcttga cgcacaggta ctgaataggg 67681 caaagcagaa gggggacctc gagatgaagc tacagggcaa gatggggacc agaggaattc 67741 cgtgggatga ggaatggggg acttggcccc ctgcactcac acgtccccgg gccgctccgg 67801 gccgcccgcg tcctcctctc cMgccgggcg cgagcctgga aaacacggtt cggtgagcgg 67861 ctgcgccgag ccccgccccg ccggcatctg acggattaac gctgcccgcc ccgggcgccc 67921 caggtacctg tcgcttgaaa ctttctggtt gtccccagcg ccaccccctc ccccgacccg 67981 gaagtgcccc caggtctgtt ccaccaggtg cccctggcag cctgcagYag aaccctggca 68041 ctcacgcctc cccgggccat ggaggcggcc atgacatgca tcacgtgact caccgactga 68101 gcatgcgccg cgccagggag gcgagggaaa gcccataatc aatggaacat aagcgcagat 68161 gtttgcttag ggcctgcggt gaccaggtgg gaagagcagg gcggaatcca tctcaggtaa 68221 acccgcgggg ccatcccaga cctcccactc aagcccactg tttgtcccaa gaaccagcca 68281 actctgattt ctttaaaacc atttacttac aaactttaat tcagcaaagg tccgtgtggg 68341 gagactgggg tggggtcggg ggaatagtcc ccttggagtg gatgtggacc cccagagtca 68401 agggagggaa gctggtggcc cagttggctg ggggcaaggc ccagggtcac ctcaggtcga 68461 caggtcctgc tggtgggcgg gcccagagtt tatcttcatg gagtgctggt ttctggcact 68521 gggctggaag gaggccagct ccagggatct ggccgggggt gggcaggcag aattcaagaa 68581 ttcatcttca acaagcgagt gacagcagag gctccgggag atgggcacaa tgtccgactc 68641 ccacaKacag acagcagggg actggcagag aaagcccatc tctgcacgga ggcccgggta 68701 ggagggggtg gtggggccgg ttcgccaaga tgaaggcttt ccccttctac tgtccccagg 68761 gtggagatcc tgggtagggt ggcccaatcc ctaggccaga gctgtttggt ccatagtcaa 68821 gctcccagag cttggcatct gtggctctgg ccagcagggc ctggggccca gcttttaagg 68881 catcagaagg ggagggggct gcggcaggga ccccgggccc cacggctggg gtataggcca 68941 gatgggcagg caggggcagg agacagccat gcctgcagca aatgtgggaa aaacagctgt 69001 ttcaaaccgc aaggtgtgga atggtggccc ggacaggccg ggccttgaag gaatcagagc 69061 tgggggctgt ccggggtggt ttgaaaaata aaacttagaa aaggaaacag aagtcagttg 69121 tcaaagttaa aaaaaaagga gacagtctct gtatcttcac gggaggtcag ggaaacctcc 69181 aaggcactcg aaaggccaaa attacaggag caatgaggca ggagggctcc ggagagacgg 69241 gcacagcgga ggaggagatg gggggaggga gggagagcag gccggggccc ctctctctta 69301 aggctgcagg gtttcagcgt ggggagcaag ccagagacat aatgaggcct ccagactccc 69361 ccacacccct tgggctcctg gggctccggc tgattggtca gtaaagtctt tcagagattt 69421 ttctattacc gaaagagaga aaatggttta aaaaaaacac aaaacaaaac atcagaaaac 69481 ccaaaagcga tttggtgcag gcccttgagt tatctctggt gccagccact tagaaaatcc 69541 tcttccgctt caggtaggag tccgcgtagt ggccgccgag gccctgggag tgctggccca 69601 catagctgcc ttctgggctg ggctcgggcg agggcagcag gtgggcaaag ctgcgtttct 69661 ggccgcccag tggggtctgg agacagaggg cagggcgggg cgggtcaggg gccgctgggg 69721 ggccggggct tcccagccct gcatgtcccc accccctgcc cgtaccttca gcaggtggct 69781 gtgaccatta ggcccggggc tgaggcccag gagcccttct ccggacccca gcggggaaga 69841 gccgattgcc tgggagaaat ggagaggtgg aacaggtcac tccctggggg ccagaggaag 69901 cacccacccc gcccccctgc cactgccccc cagccctgag ggaaagcgaa cagtttcagg 69961 ttcaaatctt gcttctgcta ctttctagct ataagaccta gggccaattg ctccatttta 70021 tttgagcctc ggttttctca tctgtcagat ggggccagta gcacccacag cctgggctgt 70081 agagagagga agtgacagaa agcccggaat gctggcagtg ctaggtgctt gatgcagagt 70141 ggatggagga caataggctg ttgctactcc accctgagca agaccaaccc ctgccaccac 70201 cccagctttg gcctgggcac cgagggttac agagccccgg tggcaggcac ccccaaatcc 70261 tagctgtccc agggggcctg tgcttggcca gagaggttcc tcaccttact gaggtggctc 70321 tgcttgaggc cagcctgcag gccgctggtg aagtaggaag tgggcgagcc tgaatagaag 70381 tgagacaggg ggcccccgcc actcccaccg ctgctccgtt cgccgaagcc actgggcggg 70441 ggggacatct gtagaagaag ggccggtgga gtgaagcaga caattgtagt acccagccct 70501 ttgtatacaa tcttccctcY gcctcccagg ttcaagcgat tctcctgcct cagcctcccg 70561 agtagctggg attacaggca tgcgccacca tgcccagcta attttttgta tttttagtag 70621 agacggggtt tctccatgtt ggtcagactg gtctcacact cacgacctca ggtgatccgc 70681 ccacctcggc ctcccaaagt gctgggatta caggcgtgag ccaccatgcc cggcagttct 70741 gggctttgca aagactagct ttggctaata ggactgagga gagctggcac gtgggtaccc 70801 gcctcctctt cctcctccag gaacccaggg atcatcaccg cacgaatggg cacacagaaa 70861 ctgttccagc ctgtggacgc caaatatgtg aggccacata gctcactgag acccaggtga 70921 gtgccgggga ccgcccactg ccccatagag tggcaagaag taataMaatg cctggcatgg 70981 tatggtggct catgcctgtg atcctagcac tttgggaggc caaagtggga ggattgcttg 71041 agcccaggag tttgaggcca cagtgagcta tcatggagcc actgcactcc agcctgggca 71101 acacagtgag atcctgtctt aaaaaaaccc acaaagaaca cacaaaatac aaggtctgct 71161 gttccaggct actaagtcat ggcacagcct gtcatgtagt aaaagctgat tgctacacat 71221 gccataccag gtggctctta ccttgtgtcg gctgggtccg tgaggcccaa gggctggttc 71281 ccgtgggtgg gagaagagcc gccgaggtcc catgtcctga atgaaagcgg gagaagcaca 71341 ttgggttgga gtctgtccca gcctcacccc tggctgaact ccatggcaga gctgtcatca 71401 gggaagacag tccccaccac tctgggacca ggaagaggca aatttggggc tggatgcagc 71461 ttggcaggga tggctgctgt gaaggggggt gccccccatg ggcctaagtg tttggagttt 71521 cccaggcaag ctgggaattc gagctgtgtg gactcttgca gttttcctgt gttggcgtta 71581 gttcctgttg ttatttgttt gaaacacaga gggggccaaa catcacccag gtgacctgtg 71641 ggcggccagt ttgcaatttc tttctttttt ttttttaaac ggagttttgc tcttgttgcc 71701 caggctggag tgtaatggca caatcttggc tcaccacaac ctccgcctcc caggttcaag 71761 tgattctcct gcctcagcct ccctagtaac tgggattaca ggcatgtcta ccacacctgg 71821 ctaattttgt atttttagta taaaacataa attatatttt atattatata tgaaatatat 71881 attttattat atattttata atatataatt tctccatgtt ggtcaggctg gtcttgaact 71941 cttgacctca ggtgatctgc ccgcctcggc ctcccaaagt gctgggatta taggcatgag 72001 ccaccgtgcc tggcccagtt tgcaatttct gacctgccaa ggaggttggc atcatagaag 72061 gaccaaatct ccaggggcac agacaggaat caaaggtggg agagagggat agaaagggca 72121 agccctgtct ctggggagag acagtgtggc ctaagtcctc ttgggagaga aagacagacg 72181 caggatgaca gacagactga gacccaagat cctccagggc tcgagaatgg acagacaggc 72241 aggtggaccg gaagcagggg gtgatgtgca gacagacggg aaactttaag ggcatgaggc 72301 tgggcgcggt ggctcttgcc tgtaatccca gtactttggg aggctgaggc gggtggctca 72361 cttgaggtca ggagtttgag accagcctgg acaacacaat gagactccgt ctctaccaca 72421 aatttaaaaa ttagctgggc atggaggtgc Rtgcctgtgg tcccagctcc tcaggagact 72481 gaggtgtgag gatcgcttga gcccaagagg ttgaggctgc agtgagctat gatcgcacca 72541 ctgcactcca gccagggcag cagagggaga ccctgtctct aaaaaaaaaa aaaaaaaaaa 72601 gcagagacca tatctggcac gttccctgcc gcctccctag cacatggcct ggcatagtca 72661 atcctagaga aatatcagtt tggagaatga tttggcctta ggggacagag agctgcagcc 72721 aggcactcac cgaggggtag tcgaagctgt agctgtcaga cagtcctggt tcggggggca 72781 ggcgggcgct gctgaggggg ctgagcaggc gggacttgag ctggaaggct ttgctgctgc 72841 tgctgccacc tccaggggct ggggggttag ttggggggcc ctcagctggg cggcggcttc 72901 tcccggcgcc tccccagccc cgggcctcct tacccgccag gttgctggcc gggagcaggc 72961 tgtgtaggtt caggtaggga ttcaggggaa tccggtagtc cttggggggc tcccccagca 73021 gtgagacctg tggcgggaag gcaggattcg agagggcccc cgggagaggg aaccctgcgg 73081 aacaggaggc tggtgtggtg ccgcgcgcag ggcagggcct taccccaggg ggcgtcggca 73141 gaggcccact gtctttctgg aggcctctga ggccagagcc tcggagcccc acaggggcKg 73201 ggggaggagt gagctgggcc gctgggggac ccaagcccag ggcctcccgg tcacccccag 73261 cagggccaag cacggatggc agcaggggtg gtggcttccc tcgccggggc ggcagctccg 73321 ggggcaggcc ccctcctagg gagggaagga ggatttcagg ggcccaggct cacaggaccc 73381 ctctccccag ggccctccag ctcccacctg gaccagggcc caggggtcaa gagacaaatc 73441 catgctggcc actgagcaga gctcatccct gggccctgag tggagggcac ccaactgatg 73501 gacaagggcg ctgggaccca gaggttgggt cactcacccg aggccacgta gcagggaagg 73561 aaggggaagt ggctggggga caccgtgtgg gcagggtctg cgttacctgc aggcagctcc 73621 ccgaggaggg ggttggctgg ctgggcccca ggctggaggg cgcccagggg tgagtctccc 73681 aggatgccgg gcttctaggg acagagcaca gacagttgca gatgggggca gggtggccca 73741 gggaagcaga caggtgtggc ccagagtctt ggcagacaca ccccaactcc agggcacggc 73801 cgatcctggg cagacaggca cagggttccc cagtgcccgc cacactccaa ctctatcact 73861 gtcccatcag ccatggtgac cgagggtctc ctgagctcca ggccccagtt cccCggccaa 73921 tgattgcagc acagtatcaa ctgcagccac atggaatggg acagccacac agtgtttcag 73981 ccacttttag tcacagaagg ccagacccac aggctcacat attctgtcag ttaccaccag 74041 acagacacac ctgacttaga atccagggaa ccacaccaga caggacggag catgggtgta 74101 tgtctatgtg tgtgtgtgtc tgtgtgtgtg tgtgcgtgta tatctgtgtc tttgtttctg 74161 tgtgcatgag tctgtcttgt gtctatatgt gtgtctgtgt ctatgtgtgt gtctttgtct 74221 tatgtctttg tgtgtctatg tctctgtgtg tatatttgca tctttgtatg tgtgagtcta 74281 tatgtatacc tgtgtttcta ggtgtgcgtg tgtgtgtgYg cacacactga ggggatagac 74341 ctggagtttt ttcccaggca tgtctataga gcttccgagc agagtctatt cacaaactgg 74401 tgcccagcag cggcgctccc agcccacacc cagtttcagg ctgtaaatgg gcgtgtccag 74461 gtcccctgac cccaccccag gcaccaagtc caggggcgct ctcagaatgg agggaaagat 74521 ggcgaaccat catgagcaat tgccagaggg ggatggggat gtgggtggga aaggcagggg 74581 tttgggggag tcgccctcac cctggttctg cgcccccagg gcacagccag aggtcccccc 74641 accccaccga tcgcaccagc tgtgttgaaa agagcctgta ggggctgtag agcagtgggt 74701 gagggggatg cgtgtgttac cttctggccc tgggtctgca gggcgagctg caacagcgcc 74761 gtggacaggg ctggcccatt gagcagcggc atggctgggg gggcacccag gaggcctgga 74821 gggagacata ggaggatgtg tgggggtccc tgtgtcctcc ctgccccacc tcacgaaaca 74881 cagccctgaa cccattgatg gggcaggaaa ctgacactct ggggccgggg ggtgggcagt 74941 ggacttgccc aaaggaaggg cctagaaggg gcagaggcta ctccagtgaa ggtcagcccc 75001 ctcttgtcag caccccacct cactgcaggc tgctgtgtgg ccctggtccc ggcccccagt 75061 tgtggacagc agcagccccc gctatgactc catactcccc ctcggcccta ctcctggggc 75121 tcctgtcact gcagccaaga tgagcagttt cgggaagtgc cgggggcagc gctgcagccc 75181 ctgttctgca cagtggggcc cctgtccagc cccaccttac cctgcttccc ccccgcactg 75241 ccatggagca gggggttgag cagcagctgg agggacgcgg atgggcccag gttgttgagc 75301 agctgcagga tgttgggctc ggggaggagt cccttccccc gattgagggc ctgcaggaag 75361 gaacagaagc agctcagagt gccgctgggg ccctgacatc cccatggggc tcccatcccc 75421 accccgccac ctctgcccgc acaggctcac cgtggcctgg gcagcgatga gagcggccag 75481 catactgcgg ccggggggcc caggggcgca gaaggagact cgcaggtggc tgccccccag 75541 ggacaggccg tccgcctgct gctgtgcctc ctccgccatc tcagccgtct catactccag 75601 caccgcgaag cccttcagct gcccatcctg gccgcacgcc agctgccgga ggaaggcagg 75661 cagtcatgag catctgggca gcagtgactg caccaccccg ggaagtgaca accgtaacca 75721 gactcagctg ccccataagg gtgaactcca cgccaggcct ccactgccct gtgcctcatt 75781 cctgcatcag ctcatctgat tttcccaatg accctgggag gctgcatcat acccatttct 75841 cagagaaggc aactgaggca cggagaggtc agcgcaccca aggccacaca gctggaggga 75901 gggcggagct gggagccacc tcaccaacaa agccaaggcc acgggcggat gcggtggctc 75961 acacctgtaa tcccagcact ttgggaggcc gaggcgggca gataacctga ggtcgggagt 76021 tcgagaccag cctgaccaac atggagaaac cccgtctcta ctaaaaatac aaaattagcc 76081 aggcgtggtg gcacatgcct gtattcccag ctacttggga ggctgaggca gaggcaagag 76141 agtcacttga acctgtgagg cagaggttgc ggtgggccga gatcgcgcca ctgcactcca 76201 gcctgggcaa caagagcaaa actccatctc aaaaaaaaaa aaaaaaaaga gataatcagc 76261 aacgggtggg gaaggaaggg ctctcgttcc agacagtggc caagccaggt agcagtgcta 76321 agggaagtgg ttgggctggg caccggcact tgctgaatgc ccgggcagca aaggtgcagg 76381 tagcagccct tcacctctgc ccacgccctg tgccaaggtc tgggcacaga tctcccataa 76441 ccaaacctgt cggcttttgt tttgttttgt tttttttgag acagagtctt gctctgtcac 76501 cccggagtgc agtgatgtga acttagctta ccgtaacctc cacctcctgg gttcaagtga 76561 ttctcctgcc tcagcctcct ggtagctggg attacaggtg tgcaccacca cgcccagcta 76621 atttttgtat ttttagtaga aatgaggttt caccatgtcg gcaaggctgg tcttgaactc 76681 ctgacctcaa gcgatccgcc tgccttggcc tcccaaagtg ctggattaca ggcgtgagcc 76741 actgtgcccg gccataaaca cacccatctt agggatgaac agagagagac tcagaggggt 76801 cacagagtga tcaaggccac acaattccta agtcgaggga ctgatgtgtg aacctcaagc 76861 tgcttcactg tcaagcccac cctccattca gcYgggtcac ctaacattaa aggtgggctc 76921 ctcggaggac atcacttcaa cgcctgctac tcatcagatg ctcaacaaat gtttggtgag 76981 accaggccca gtggctcgaa tctataatcc cagctctttg ggaggcaaag gcaggaggat 77041 cgtttgagca caggagtttc agaccagcct gggcaacata gggagacccc atcttcacat 77101 aatttttttt ttttttgagg gagtctcgct ctgttgccca ggctggagtg cggtggcatg 77161 atcttggctc actgcaacct ccacctccct ggttcaatca attctcctgc ctcagcttcc 77221 cgagtagctg ggattaaagg cacctaccac cacgcctggc taattttttt tctattttta 77281 atagagacag ggtttttgtt ttttgttttg ttttttttga gacggagtct cgctctgcca 77341 cccagactgg aatgcagtag tgggatcttg gctcaccaca acctctgtct cccaggttca 77401 ggtgattctt ctgcctcagc ctcccaaata gctgggacca caggcacatg ccaccacacc 77461 cagctaattt ttttttttgt atttttaatg gagatagggt ttcaccatgt tagccaggat 77521 ggcctcgatc tccagacctc gtgatgcacc cacctcggcc tcccaaagtg ctgggattac 77581 aggcgtgagc caccgtgcca tgagacaggg ttttaccatg ttggccaggc tggtttcgaa 77641 ctcctgacct caagtgatct gcccgccgca gcctcccaaa gtgctgggat tacaggcatg 77701 agccactgtg cccggcctct acataaaatt ttaaaaatca gctgggcatg gtgaYgcatt 77761 cctgtagttc cccagctaca tgggaggcca aggtaggagg atttcttgaa cccaggaggt 77821 cgaggccgga gtgtactcct ccagcctggg tgattgagaa cctgtctcaa aaaaaaaaaa 77881 aattaagttt agtgaatgtt cgagtggagg aatgaatggg cagctgttca caacaacctg 77941 atcatgaagc actgccagcc ccattttgag atgtggaaac aggctaggag agtacctgta 78001 gctggtcatg accctgtgac ctttgatctc cacataagaa ggtaaggcca ggccagcgca 78061 gtggctcaca cctgtaatct cagcacttgg ggaggccgag gcaggtgtat cacctgaagt 78121 caggagttca agaccagcct ggccaacacg gcgaaacccc atctctatta aaaacacaaa 78181 aattagctgg gtgtggtgtc aggcgctgtc atcccagcta ctcgggaggc tgaagctgga 78241 gaatcacttg aacccaggag gcggaggatg cagtgagcca agatcgcgcc cctgcactcc 78301 agactgggtg acagagcaag actctgtctt aaaaaaatat aaaataaaat aaaagaaagg 78361 tgaggccatt gtgctaactc atcttctgtc ctgagcccgc acaggcggcc tggtacatag 78421 caggcctggt acgtagcagg tgtccaagag aaagcagcag aatggatgaa tgataagtaa 78481 acagagaggg ggttcccgtg gctcatggat actcagtttt tgtttttttt gtttctttga 78541 gacggagtct cgctctgtcg cccaggctgg agtgcagcgg cacgatctcc actaaccgca 78601 accgcagtct tctgggttca agcaattctc ctgtctcagc ctcctgagta gctgagatta 78661 caggcgcgtg gcgccatgcc tggctacctt tttttttttg ttgtattttt agtagagacg 78721 ggatttctcc atgtttgtca ggctggtctc aaactcctga cctcaggtga tccgtccgtc 78781 tcggcctcca aagagctggc attacaagcg tgagccactg cgcctggcct gttttttttt 78841 tttttttttt ttctgttttt gagatgcagt gttgctttgt cacccaggct ggagtacagt 78901 ggcgtgatct cagctcactg caacctccac ctcccaggta caagcgattc tcctgcctca 78961 gcctcctgag tagctgggat tacaggcctg caccaccaca cctggctaat tttttttgta 79021 tttttagtag agacagggtt tcaccatgtt ggccaggctg gtcttgaact cttgacctca 79081 agtgatccac ctgcctcggc ctctcaaagt gctgggatta catgcatgag ccacctcacc 79141 cagcctgttt cttgttttaa atatagagat ggtgtcccac tatcttgccc aggctagtct 79201 tgaactcctg gtctcgagtg atcctcctgc ctctgcctcc caacagacac ctagtttaaa 79261 tggggcacca ataccccact tacacgagag ggacccactc aagcccacag agctgggggt 79321 ccgagcccag gactgtctct ctgtttttga gatRagggtc tagctctgtt gctcaggctg 79381 tagtgtcatg gtgctgtggt gcgatcatgg ctcactgcag cctcggactc ccagaatcag 79441 gcaatcctcc cacctcagcc tcctgagtag ctgggactac aggcacacca tcatacctat 79501 ctaatttttg tatagagagg atttcaccat attgcccagg ctggtctcga actcctgggc 79561 tcgagggatc cgcccgcctc agcctcccaa agtgttaggg ttacaggtgt gagtcactgc 79621 acctggccag gcctacccct tttaagcctc tgcactggcc tccgctcact ggcccaagct 79681 ctggagctag gctgcggtgt tctgatccca gctctgccag accctggctg tttaactcta 79741 ggcaagttac ttgacccctc tgtgccttgg tatcctcacc tgaaaatggg agtgatacct 79801 gcctctcagg gttgctacag gatgaagcaa ctgaaggtta cgtactcaga gtccacagcg 79861 agtgctgggg gaatgtcccc tttgggatct ctggggatca agaccttctg ggggctctta 79921 gagagtgagt ctggcctgtt ctcgccccac tgcacctgtt acagggcgag gcacacacac 79981 agaagtgcct ggcatgtgtg gcttgaatga acaagggact gttttgccaa cccattgtgc 80041 aagtattgag tccctgctta tggaggaggg aaactgcaga tcagagagcc agtgactggc 80101 ccgagctcga gggcggggct gggatctggc aaagcctcag tctctgacct caaggctcaa 80161 atagactgtg ggctcctctg cagcctcctc aatgcctggt ccaggtctgg gaccccatgg 80221 gaggtttgac gagggtacca gcgaatgagg aggtgagtgg tgagcaagcc tcgagatcca 80281 tagttacaag agtggccttg catgcatctg atagagggac agtgaggcct gaggctgcag 80341 agggattcaa atcccctggc caggtgtggt ggctcacgca tgtaatccca gcactttggg 80401 aggatcacct gaggccagga gttcaagact atcctggcca acatggcaaa accccatctc 80461 tactaaaaat ataaaaatta tccaggcatg ggggcggggc gcctataatc ccagctactc 80521 tggaggttaa ggcaagagaa ttgcttgaac caaagaggcg gaggttgcag tgaactgaga 80581 ttgtatcact gcactccagc atgggtgaca aagtgagact gtctcaaaaa aaaaaaagaa 80641 aaggcagatg actccaaaga ctctctcagg tgctccgggc ctggcacctg ctccccaatg 80701 ctctccacag ggctcacctg gcagaaggtg gggctgtgga cagctgacag cgcccggcac 80761 agagcgtcca catcgttgaa gccaggtggc aggcggtcca cacagaggca gcgggagtgg 80821 agaagggcag gcgtcagttg cccggcatcc gtccagtgca cgtagagggt gcgtggtccc 80881 agcggcttgc ccagcaggtc Ygacttggca cgggcagccg agtccttctt catgtactcR 80941 gcaaagccat agcccttgga ttggccagtg cgctcactgt agaccaggaa gcagcgctcc 81001 aggctgccga agggccgcac cagctcctcg aactgctgct gtgtgaggct ggggggcagg 81061 ttggccacac acagcagggc atccgtgggc tgcagctgca ccgacagttc Rcgctcccgc 81121 aggcggctct ggtggaaagc attgattgcg gcctcggcct gctccccatt cagcagggtc 81181 acgaaggctg cggtgggagg agggcagtgc gaggtcagcc gggccctgag ggcctgcccc 81241 tccaccccgc caccctgcaa accaggtggg acctccaaat gctgggcatc tcaggtgcct 81301 gctgatctga gcagttgcca gccttggcga ctcacacagg cgagagcgcc tgccattgac 81361 tctaggctcc acgaagccca ctccctctcc cagccctgcg tccacctgca cccacagcag 81421 cgctcaccaa ctgtgggcct ggctctgtcc taagaacgct gtgggtcact cgggtgatac 81481 caagggcagt ggttaagccc atgctcactg gggcccagat ttaagcccca agccccaggt 81541 ggctttgggg gtatcagttt cctcatctct caaatggata taacttaatc atcacaatca 81601 ccattttgcc agagaggaaa cgggcccaga gcagcacccc tgtgccccga gtaggggagc 81661 ctggacttga acccaggcag ccggactcca gaggcccact ctacacactg cccacccctg 81721 ccacctttcc aggccaccag tgcccctgaa ccctgctgcc acgtgctgcc ccagatttgc 81781 gatccaccca gcggccgcag gaagctttgt tcaaaacgat cacgccacat ccatcttgtg 81841 gcttctagct gcagggtgca gtgcagcctg tgcgggctcg ctgacctccc ctcccatctc 81901 accgctgcca cccggaccac gccaggcaca ctccagaggg gcctttgcag ctgctgtttc 81961 ttgagcatgg aacactgagc cccacctcct cacctggtta acctggaccc acccaggtag 82021 aaaccctgtc tctactaaaa atacaaaaat tagccgggca tggtggcaca tgcctgtaat 82081 cccagctact cgggaggctg aagcagggga atgggctgaa cccaggaggc ggaggttgca 82141 gcgagccgag ctcgcgccac tgcactccag cctggcgaca gagcaagact ccatctcaaa 82201 aaaaaaaaaa aaaagaccac acaagcaggg gtgactggct gagtgaggca agaggggtaa 82261 gaagagaatg aacccacggc caaacgttgt caagaattag ctcaatgcca agcgagtgga 82321 gtaggaagag aatcagcccg atgcccagtg ttctccaagg agaacatgaa ggctgtccca 82381 agatgacagg atgacaggca gattggggca acctgccatg ttggcctggg gggcaatact 82441 gggcctgaag ctcagagcag cagacagtga gccaggccct cccatcttca gggatggtca 82501 ctccctgccc tggcccRccc catggcccca caaacctgtc cctttgtatt tgtccacaaa 82561 acagtatttg agctcatagt cactgagcag gtcatgtact tcctgtggag atacaagaca 82621 aaagacaggg agttactgga gatggaaggg ggcaacccag tgccgcttca tagctgccac 82681 cagctatgaa ggcaggtcaa ttaccacccc ccattttacc aatgaggaaa ctgaggctcg 82741 atgcagtaat tttgtcaagg tcattctgct catacctggc agagatggga ttcagaacct 82801 agctgcagac tgtgctatta cccaggcaac atgctcttag agggtatacc tttagctggc 82861 tgggcggggt ggcYcacgct tataatccca gcattttggg aggccgaggc gggtgaatca 82921 cctgaggtca ggagttcgag accagcctgg ccaacatggt gaaaccttgt ctctactaaa 82981 aatacaaaaa ttagccagga gtggtggcgc acacctgtaa tcctagctac tcgggaggct 83041 gaggtaggag aactgcttgc acccaggagg cagaggttgc agtgtgccac tgcactccag 83101 cctgggtgac aaagggagac actgtcaaca acaataacaa caaggccggg cgcggtggct 83161 cacgcctgta atcccagcac tttgggaggc cgaggcgggt ggatcacgag gtcaggagat 83221 cgagaccatc ctggctaaca aggtgaaacc ccgtctctac taaaaataca aaaaaattag 83281 ccgggcgcgg tggcaggcgc ctgtattccc agctactcgg gaggctgagg caggagaatg 83341 gcgtgaaccc gggaagcaga gcttgcagtg agccgagatt gcgccactgc agtccgcagt 83401 ccagcctggg cgacagagcg agactccgtc tcaaaaaaaa aaaaaaaaaa aaaaaaaaat 83461 aacaacaaca aaaaaaaaaa aaaaaaaaga ggccgggctc acgcctgtaa ttccagcact 83521 ttggaggctg aggcaggcgg gtcacaagat caagagatcg agaccatcct ggccaggaag 83581 gtgaaacccc atctctacta aaaatacaaa aatcagctgg ctgtggtggc gtgcacctgt 83641 agtcctagct actcgggaga ctgaggcaag acaattgctt gaacccgagg ggtgaaggtt 83701 gcagtgagca gacatcaagc cactgcactc tagcctggcg acagagtgag actccgtctc 83761 aaaaaaaaaa aaaaaaaaaa aaagagtatg cctttaggga agccatttaa cctgcatgaa 83821 cctccttttg caagattcac tgtttccaac aactctgtaa gtgaagtatt aacaaactca 83881 tgttgtatat gagaaaactt aggctaggag aagtgaagag atctgctgga ggccatgcag 83941 ttgggcagcg cagccaccca tgggtacctg actggggagc ccaccttcta gtaagcccca 84001 cgttgtgtgg gttcacatgc ctcccaattt cctcctgctg ataaagctgc cctcttccaa 84061 gccagctcat ttatttttct tttttttaga gatagtgtct tgttctgtca cctaggctag 84121 agtgtagtag cacaatcata gctcactgca gcctcaacct cctaggctca agtgatcctc 84181 ccacctcagc ctcctgcgta gctgggacta caggcacgca ccaccatgcc tggctaattt 84241 ttaaaaattt tttgtagaga tgagggtttc actataccgc ccaggctagt ctcgaaccct 84301 tgggctcaat acagcctctt gcctcagcct ctcaaagtgc tgggattaaa gggatgagcc 84361 accatgccca gcctctcaag ccagctttga aggcaaagtg ctggcgcctg ctgggaccag 84421 acacactgtg catcttgcag gagagacaat gtgttcccag ctgagagact gcagacactg 84481 agaggaagac ccagaaagaa acctgaccat ccttaatgat ccctgtcccg atctgtcccc 84541 aaattcagcc cctcccaaca tccctggggt tccactagcc acgtgcccac ccggtgtgct 84601 ggtgggccgg agagcaggga atctcagcag caggaagcga gaacctattt caccgccata 84661 gggcagggca gtatagatgc catttccctg ggtccaaaga gcattgggaa gcgctgcccc 84721 atgctggccc catgagaagg gcactcctag caggaagatt agccacgagg ggctccatag 84781 aacacaggcc tcgcttcctt cctgtaggtc tgtaggctgg tggcagaaga ccccattttc 84841 ccaaattaat cctgaggttt tgggggctga gacccagggg ctgctgttaa cttccggatt 84901 tatctctgag agcagtgaat aaacgcgtga tatggagggg cgtactctcc gcagtcctga 84961 aacttctgga tgtgaggYcc cgccaccctg tggtagagtg ggtggagaaa agcatcatcc 85021 aagcaggggg caactggtgg tccaatgtac cagcccattc acaattcccc aacaggacca 85081 aagctgtccg cccccttcca ttccccgccc gcttccatca cttggtgtcg cccaagcctg 85141 tggtcgcccc tagcaacgcc ctccctcact tcgcagtcgg tttccccttt catcatcgcc 85201 cccccacgtc ccgctcttgg tctctcccga tcccgcgtgg atccggtgct tgggcgcccc 85261 cgccaacgac ccccgcgcac ctcagcgttg gtgtcgcccg gttccccccg cgcacgcgca 85321 ccgtggtatt tcccgccacg ctcctacacc gcccccccca atacctggtt ggtcacgtcc 85381 cccgggaggc cccggatcag tatcttgcgg cggttacgga actggcgctc ggtgtgttcc 85441 aggcgtttcc ggatctcttc tggatctaga ggcggcagct cttcttccgg cgcccggcgc 85501 tccgcggcat cgccggcttc gacttcggcc ccagacttag ggctcagcgg gggccggtga 85561 gtaacggaca cgtccgccgc catcttggga aacccggcgc cttctgggac cagcgagccg 85621 gggcggagcg gcatagagcg gcaacgaggg cgcgcccgtc gattggctgg gagaaacccc 85681 acctccttcc cgcccccctt gccccgttgg ctaagcccca gcccacctct caagagttca 85741 agctgcgtct gcgcgccaca ggccccgccc ccttccgtcg cagcgacgct actcagtgga 85801 tgtgcacctg ggtgggagag acttgcagcg cgggtggaat caaaccacag attagggagt 85861 ttgaaggctt tattggtgcg gaatctgagg gcacagccaa gcccccgcca actttgatcc 85921 cggatcccag cgtcactcag ctctggacgg ttcttccccc attgcttctg tcggctgcat 85981 agacgtgagg ggcagatagg tgStctcctc cctaacatgg taactgccgc tccgttggtg 86041 ctccctgaag acgtacatta aggccagtac gatagtcacc acgcccaggg tcagtaacac 86101 cgccacgaag acggggacaa agtgggagct cccagctgtg cagagaaagc gctaagtcaa 86161 tatgcgtccc ttctgtctcc aacccccccg ccccccggct taccggtcca gacccgcagc 86221 cccgtRttag ctcaccctca atgtccatca ccacgaccag ggtgtatttg cctcgtgagc 86281 tggacgcttg gcactgataa gtaccattat gtgttacgtt gacgaagaac gggatcccca 86341 ccggcacctc ccggctggag ccttccttca aacaccgcag ctcggggtac gggttgcccc 86401 tggcttggca ctgcaggacg tgtctcgttt tatctttcca tttcaagtgc tgggggcatg 86461 tggctcggtc aattttggga ccatctgtgg aaccaccatg tgtgatcaga cacccaacac 86521 acccgaggca cagtggtgca gaggagcgtc taatctttcc agggcagggg tggagggatt 86581 aaaggtcagg gtgaccgact cacacaggac tcgcagctgg acgctactgt tcctgtgcaa 86641 gaactcgccg tccacctcga gagtggcact gcagaagaag ctgcgtccgt cgtcactctc 86701 ggtagcattt agctgaagtt gagctggctg ccccggggcc gcggccggaa ctccgtccag 86761 cgtgacctgg actcgagccc cagccatgca actcacggtc actgtggacc cctcatgggc 86821 ggtgggctcg ctgaggttca caatgggtcc taggaagcct aaaggcgggg cattgcccag 86881 gagcttaatg aacaggacct tcctgtgggt caagccgctc cctccgccct cccctttcct 86941 ctcgggatat ccgggccacg ctttcggccg ttcaagcctc gccctctttc cgcgctgtgt 87001 ccagcttcgg gcactcaggc ccaacccacg ttgcaactgc tattggggca agccaggccc 87061 caccttttcg gctagtctcc gccccctctg ccacgccccc agactgctga ggccgcgccc 87121 ccttcccacg cctcctctta ctaaagaccg tcaagttctc ccgggcctcc cgtctctcgc 87181 cccctagggt cacgttgcag acgatYtccc gggcaccctc ctgatccgcg cgcgccgtgg 87241 ctgtggctgt ggccgttagc gtgtccccgt ggttcatgac tgtcgcattc agcatctggt 87301 cccccagcgc caggtagacc tgggcctctg aggctggaaa aagcccgtct agggtgcagt 87361 ccaccggcca cgacgtttcc acctccaaga accggggggc cacgaggcgc gggggggtca 87421 cgggcaggac tggggagaaa ggtgggcata gtacaacccc caggactgtg ccttccccag 87481 gacaccctca tcccccccag tcaggatatc ttgctgactg ggtcaccctt cttcRcagga 87541 cacacacaca gggccatgaa aacggccttc ttcaaagatc ccacggctcg ggcctccctt 87601 ccggggccac cttgcccagt ggccgggggc tttccttccc agaagcctgt gaggtccggg 87661 gtaccccact ctcaggaacc ccaaggtcag ggcaccgtct accctggctc agctcgtcac 87721 ccaccgtctg aagccccttc tctcaccaaa ggttcggagc tggcgggggg ctgaggtgtt 87781 cacgaacagt cccagcccct ggggctgcat gtccagttct gtgcggcatg agaaaggggc 87841 tccgtggtcg tctctgctgg ccagcacagt ggcagtgacc tccgctggct cctccactgc 87901 gggctgccgg ctcagctcct cctcccagcg aagcagcacc accgtgaggc tggtccgggg 87961 cgacccaYcc tccacttggc agcgcagggt gaagttctgg cccaccggct gccaaggagg 88021 caggggtgcc agctccacac gctccgggag ccctgagaga ggaggggagg atggcactta 88081 gcgggtcctg caaacccacc cactcacccc agggactggg gaggagacag ggtRgtcctg 88141 ccgagaactg tgagctttga gttaataaac ttagagggct tagagctggg ccagtcgaag 88201 cgtttgctat tatcattagc gcagtgatta tcatttcctg tgttgtcaga taccttgcaa 88261 ggcgctaaac aaaactttct gttctcaaag atggcaMaat aaaaaaaaat gaggatggaa 88321 gggatgaacg tttatgacta tgatatgaat attaaaaatt cctgtttatg gccagacgtg 88381 gtggctcacg cctgtaatcc cagagctttg ggaggccggg gtgggtggat caaRaggtca 88441 ggattattca gttctctcca atcaggttca gacactcagg gctttcctgg ttcaccagcc 88501 ctgtggcagc ctctcaagat actccggcac agtcacagga gacttgacac tcccaggctg 88561 ggtgccctcc ccagtacacc tacaaaacgc tgggcctcag gccgggcact gtggctcatg 88621 cctgtaatcc cagcactttg ggaggccaag gtggacggat cacctgagac caggagttca 88681 agaccagcct tgccaacatg gtgaaacccc atctctacta aaaatacaaa aattagccag 88741 gcatggtagc acgtgcctgt aatcccagct actcaggagg ctgaggcaca agaatcgctt 88801 gaacccagga ggcaaaattt gcagtgagct gagatagtgc cactgcactc cagcctggcg 88861 acagagagag actctatgtc aaaaaaaaaa aaaaaaaaaa aaaggctggg caaggtgact 88921 catgcctgta atcccaccac tttgggaggc cgagggaggc ggatcacttg aggtcaggag 88981 tttgagacca acctggccca tatggcaaaa ccctgtctct actaaaaata caaaaattag 89041 ccgggcatgg tgtcacacgc ctgtaatccc agctactaag gaggctaaga caggagaatc 89101 acttgaaccc aggaggcgga ggttgcaatg agctgacatc gcgccattgt actccagcat 89161 gggggacaat agcaagactg cgtctcaaag aaaagaaaag tcaaaaagta ggccaggcat 89221 ggtggctcac gcttataatc ccagcacttt gggaggccaa ggcgggcaga tcatttgagg 89281 ccaggagttt gagaccagcc tgggcaacat ggtgaaatcc tgtccctact aaaaatacaa 89341 aaattagctg ggcgtgttgg catgcgcctg taatcccagc tactcaggag gctgaggcag 89401 gagaatcgct tgaacccgga aggcagaggt tgcagtgagc caagattgcg ccactgcact 89461 ctagcctggg ggacagagtg agactctgtc tcaaaaaaaa aaaaaaagaa aagaaaagaa 89521 aaaaaaaagt agccaggcct gcaatcccag ctactggaga gactgaagtg ggaggattgc 89581 ctgagcccag gagtttgaga ccaacctagg caacataggg agatcctgtc tctaaaataa 89641 aggtataaaa aagtgtaaat gtaaaacctc catttgccta ctgctgactg aaaggggtac 89701 cccctttttt gcttaagaga caagggtctt gcttttttgc acaggctgga gtgcagtggt 89761 gcaattatag ctcactacag ccttgaactc ctgggctcaa gcgattctcc caccttggtc 89821 ccccaagtag ctgggacttc aggcatgtgc caccatgccc agctaatatt ttttatttat 89881 ttttgtagag acggggtgtc cctatattgc tctgcctggt cttaaatttc cggactcaag 89941 caatcctcct gccttggcct cccaaagtgt tgggattatt gttggggcca ggcgcagtgg 90001 ctcacacttg taatcccagc actttgagag gctgatgtgg gcggaacacc ccgtctctac 90061 taaaaataca aaacttagct gggcattagc tggacctggg aggcagaggt cgcagtgagc 90121 cgagattgca ccactgcact ccagcctggg ctacagagta agagacgcca tctcaaaaaa 90181 aaaaaaaagc ttgttggggc cgggcgcagt ggcccatgcc tgtaatccca gcactttggg 90241 aggctgaggt gggcggatcg cctgaggtca ggagttcaag accagactgg ccaacatggt 90301 gaaatgccat ctcaactaaa aatacaaaat ttgctgggtg tggtggcgtg catgtgtaat 90361 ctcagccact cgggaggctg aggcaggaga atcacttgaa cccaggaggc gaaggttgca 90421 gtgagccgac atcgtgccac tgcactccag cctaggcaaa aagagcgaaa ctctgtctca 90481 aaaaaaaaaa aaaaatgctt gttggggccc aagttaattg atttcacaac tgctgccttg 90541 gcatgagatt gagatgcgtc tctatatctc tgtctctgtc tctgtgtctc tgtctctgtc 90601 tRtctttgta tctcacacac aaatgtccag gtctgagctc tgggtcagaa ctgtggtcca 90661 ttctttgcaa actgagggtc cccactctcc tgagccattg gccacttgcc cacaccacta 90721 cccaagacca gtcccacctc cggacacgtY gactcactgt acacggtgat gttagaggag 90781 cctgttatct gRgagccatt gcagtacact gagcagagga tccgactgtt gccagtcacg 90841 ttgctgagat tgaaggctgc ccagcccatg ccactggcca ccagctcctt tgatagggac 90901 gtctccaagg cgattttctc agagctggga caatcagtac tgcagttcac aaacagggac 90961 cctccagcag agagcacagg gttctggggc tccacccgca aaaggaactc ctgcccctgg 91021 acaccttcag gaacatgaag aagtcctggt gtttgtttcc ttgtYcccca acccacgcat 91081 caacaggccc caccatttaa cacctctctc cttgtgccga gacagaaggg ttcctgcctt 91141 catggtccag Kgggaaaggt agaatccatc agagaccRgg gagaactcag aaggaggcca 91201 gaagaggctc tgatccagca taggaggagg tgagtcaggg aaggcttcct ggaagcgggg 91261 accattgcag cccgaaactg aaggctaagt aggagttagc aagtgaaagg gacaggtgat 91321 cctggcagag ggaagcacat acgcaaaggg caaatttcag gagttcagat ggagcaggaa 91381 gcatgagggg tgtgtgtgtg tgtgttaggg gagagggatg gcgtacaagc ctatgctggg 91441 gcctgaccac ctgaaggctg aactaaggaa ccagaacttt ctcccgagga tacagagtca 91501 gcggccaggt cgcagtgtgg caggatgtga acagcgttgc gttctattcc tctaccctct 91561 actgatcccc aaaacgcagc cctctccagc cctccccggc ttgactggtc tcacctgggg 91621 tcagcagaca gcagaccagc agagtccagc aggccctggg ccacaacacg gatggtacca 91681 tggtRgccat tctgacagag gaaggtgcct tcctgaggtg cccgaagggc aggcagggga 91741 aaaggcgggg cctcccactg cgacggaagc acagcggtta agattgcaga agtctgggga 91801 ctgcaccttg cccaggcctg tgggtggaag gggatgctgt ggcccttggg ggccagggtg 91861 ggggacccag acggggctct gcaaagtggc cagcactgct gagtctcccc cagggaatgg 91921 gctggtccca gttgcactgc tggcccccat ttgaattcac aggaaatgct aactaaactg 91981 gtttcttttg ttctcagcac tgggaagcct gggtgggggg gatgaggcat ctccttctgg 92041 ctgaacttag gtgggtaggg gaatattctg tccccagaga ggaagttaat ggggaaaact 92101 gtaggcacca ttgtgtagat gttaaggtgg gatttcgagg aaacattaaa aaacaaaaac 92161 aaaaacagaa acagaagcag agtcttgcta tgttgccccg actggtcttg aactcctggc 92221 cttaagatgg cctcctctca tctCgacctc ccaaagtgtt gcgattacag gcgtgagcca 92281 ccacgcctgg ccacctttga agaCcttgag gatacctccc tatctttgaa gatcttgggg 92341 atcccccatc ccctgcacaa agagctaagg taggtgattt ggggacagct gaKtccctga 92401 gccactgtct atccaggatt ctacttggtt tttgtttgtt tgttttgaga cagagtctca 92461 ctctgtcacc caggttggag tgcagtggcg ggatctcggc tcactgcagc ctctgcctcc 92521 cgggttcaaa cgattttcct gcctcagcct cccgagtagc tgggattaca ggcatgtgcc 92581 accacgcccg gctaattttt Following are ICAMl, ICAM4 and ICAM5 complementary DNA sequences (cDNAs; SEQ ID NOs: 2, 3 and 4, respectively). Following is an ICAMl cDNA sequence (SEQ ID NO: 2).

NM_000201 [gi:4557877] Homo sapiens intercellular adhesion molecule 1 (CD54) , human rhinovirus receptor (ICAMl) , mRNA gcgccccagtcgacgctgagctcctctgctactcagagttgcaacctcagcctcgctatggctcccagcagccccc ggcccgcgctgcccgcactcctggtcctgctcggggctctgttcccaggacctggcaatgcccagacatctgtgtc cccctcaaaagtcatcctgccccggggaggctccgtgctggtgacatgcagcacctcctgtgaccagcccaagttg ttgggcatagagaccccgttgcctaaaaaggagttgctcctgcctgggaacaaccggaaggtgtatgaactgagca atgtgcaagaagatagccaaccaatgtgctattcaaactgccctgatgggcagtcaacagctaaaaccttcctcac cgtgtactggactccagaacgggtggaactggcacccctcccctcttggcagccagtgggcaagaaccttacccta cgctgccaggtggagggtggggcaccccgggccaacctcaccgtggtgctgctccgtggggagaaggagctgaaac gggagccagctgtgggggagcccgctgaggtcacgaccacggtgctggtgaggagagatcaccatggagccaattt ctcgtgccgcactgaactggacctgcggccccaagggctggagctgtttgagaacacctcggccccctaccagctc cagacctttgtcctgccagcgactcccccacaacttgtcagcccccgggtcctagaggtggacacgcaggggaccg tggtctgttccctggacgggctgttcccagtctcggaggcccaggtccacctggcactgggggaccagaggttgaa ccccacagtcacctatggcaacgactccttctcggccaaggcctcagtcagtgtgaccgcagaggacgagggcacc cagcggctgacgtgtgcagtaatactggggaaccagagccaggagacactgcagacagtgaccatctacagctttc cggcgcccaacgtgattctgacgaagccagaggtctcagaagggaccgaggtgacagtgaagtgtgaggcccaccc tagagccaaggtgacgctgaatggggttccagcccagccactgggcccgagggcccagctcctgctgaaggccacc ccagaggacaacgggcgcagcttctcctgctctgcaaccctggaggtggccggccagcttatacacaagaaccaga cccgggagcttcgtgtcctgtatggcccccgactggacgagagggattgtccgggaaactggacgtggccagaaaa ttcccagcagactccaatgtgccaggcttgggggaacccattgcccgagctcaagtgtctaaaggatggcactttc ccactgcccatcggggaatcagtgactgtcactcgagatcttgagggcacctacctctgtcgggccaggagcactc aaggggaggtcacccgcgaggtgaccgtgaatgtgctctccccccggtatgagattgtcatcatcactgtggtagc agccgcagtcataatgggcactgcaggcctcagcacgtacctctataaccgccagcggaagatcaagaaatacaga ctacaacaggcccaaaaagggacccccatgaaaccgaacacacaagccacgcctccctgaacctatcccgggacag ggcctcttcctcggccttcccatattggtggcagtggtgccacactgaacagagtggaagacatatgccatgcagc tacacctaccggccctgggacgccggaggacagggcattgtcctcagtcagatacaacagcatttggggccatggt acctgcacacctaaaacactaggccacgcatctgatctgtagtcacatgactaagccaagaggaaggagcaagact caagacatgattgatggatgttaaagtctagcctgatgagaggggaagtggtgggggagacatagccccaccatga ggacatacaactgggaaatactgaaacttgctgcctattgggtatgctgaggcccacagacttacagaagaagtgg ccctccatagacatgtgtagcatcaaaacacaaaggcccacacttcctgacggatgccagcttgggcactgctgtc tactgaccccaacccttgatgatatgtatttattcatttgttattttaccagctatttattgagtgtcttttatgt aggctaaatgaacataggtctctggcctcacggagctcccagtccatgtcacattcaaggtcaccaggtacagttg tacaggttgtacactgcaggagagtgcctggcaaaaagatcaaatggggctgggacttctcattggccaacctgcc tttccccagaaggagtgatttttctatcggcacaaaagcactatatggactggtaatggttcacaggttcagagat tacccagtgaggccttattcctcccttccccccaaaactgacacctttgttagccacctccccacccacatacatt tctgccagtgttcacaatgacactcagcggtcatgtctggacatgagtgcccagggaatatgcccaagctatgcct tgtcctcttgtcctgtttgcatttcactgggagcttgcactattgcagctccagtttcctgcagtgatcagggtcc tgcaagcagtggggaagggggccaaggtattggaggactccctcccagctttggaagggtcatccgcgtgtgtgtg tgtgtgtatgtgtagacaagctctcgctctgtcacccaggctggagtgcagtggtgcaatcatggttcactgcagt cttgaccttttgggctcaagtgatcctcccacctcagcctcctgagtagctgggaccataggctcacaacaccaca cctggcaaatttgattttttttttttttttcagagacggggtctcgcaacattgcccagacttcctttgtgttagt taataaagctttctcaactgcc

Following isanICAM4cDNA sequence(SEQ IDNO: 3).

NM_001544 [gi:12545400] Homo sapiens intercellular adhesion molecule 4, Landsteiner-Wiener blood group (ICAM4) , transcript variant 1, mRNA. ctttttgccatggggtctctgttccctctgtcgctgctgttttttttggcggccgcctacccgggagttgggagcg cgctgggacgccggactaagcgggcgcaaagccccaagggtagccctctcgcgccctccgggacctcagtgccctt ctgggtgcgcatgagcccggagttcgtggctgtgcagccggggaagtcagtgcagctcaattgcagcaacagctgt ccccagccgcagaattccagcctccgcaccccgctgcggcaaggcaagacgctcagagggccgggttgggtgtctt accagctgctcgacgtgagggcctggagctccctcgcgcactgcctcgtgacctgcgcaggaaaaacacgctgggc cacctccaggatcaccgcctacaaaccgccccacagcgtgattttggagcctccggtcttaaagggcaggaaatac actttgcgctgccacgtgacgcaggtgttcccggtgggctacttggtggtgaccctgaggcatggaagccgggtca tctattccgaaagcctggagcgcttcaccggcctggatctggccaacgtgaccttgacctacgagtttgctgctgg accccgcgacttctggcagcccgtgatctgccacgcgcgcctcaatctcgacggcctggtggtccgcaacagctcg gcacccattacactgatgctcgcttggagccccgcgcccacagctttggcctccggttccatcgctgcccttgtag ggatcctcctcactgtgggcgctgcgtacctatgcaagtgcctagctatgaagtcccaggcgtaaagggggatgtt ctatgccggctgagcgagaaaaagaggaatatgaaacaatctggggaaatggccatacatggtggctgacgcctgt aatcccagcactttgggaggccgaggcaggagaatcgcttgagcccaggagttcgagaccagcctggacaacatag tgagaccccgtctatgcaaaaaatacacaaattagcctggtgtggtggcccgcacctgtggtcccagctacccggg aggctgagttgggaggatcctttgagccctgaaagtcgaggttgcagtgagccttgatcgtgccactgcactccag cctgggggacagagcacgaccctgtctccaaaaataaaataaaaataaaaataaatattggcgggggaaccctctg gaatcaataaaggcttccttaaccagc

Following is an ICAM5 cDNA sequence (SEQ ID NO: 4). NM_003259 [gi: 12545403] Homo sapiens intercellular adhesion molecule 5, telencephalin (ICAM5), mRNA

ccgtcctctagcccagctcctcggctcgcgctctcctcgcctcctgtgctttccccgccgcggcgatgccagggc cttcgccagggctgcgccgggcgctactcggcctctgggctgctctgggcctggggctcttcggcctctcagcgg tctcgcaggagcccttctgggcggacctgcagcctcgcgtggcgttcgtggagcgcgggggctcgctgtggctga attgcagcaccaactgccctcggccggagcgcggtggcctggagacctcgctgcgccgaaacgggacccagaggg gtttgcgttggttggcgcggcagctggtggacattcgcgagccggagactcagcccgtctgcttcttccgctgcg cgcggcgcacactacaggcgcgtgggctcattcgcactttccagcgaccagatcgcgtagagctgatgccgctgc ctccctggcagccggtgggcgagaacttcaccctgagctgtagggtccccggcgccgggccccgtgcgagcctca cgctgaccctgctgcggggcgcccaggagctgatccgccgcagcttcgccggtgaaccaccccgagcgcggggcg cggtgctcacagccacggtactggctcggagggaggaccatggagccaatttctcgtgtcgcgccgagctggacc tgcggccgcacggactgggactgtttgaaaacagctcggcccccagagagctccgaaccttctccctgtctccgg atgccccgcgcctcgctgctccccggctcttggaagttggctcggaaaggcccgtgagctgcactctggacggac tgtttccagcctcagaggccagggtctacctcgcactgggggaccagaatctgagtcctgatgtcaccctcgaag gggacgcattcgtggccactgccacagccacagctagcgcagagcaggagggtgccaggcagctgatctgcaacg tcaccctggggggcgaaaaccgggagacccgggagaacgtgaccatctacagcttcccggcaccactcctgaccc tgagcgaacccagcgtctccgaggggcagatggtgacagtaacctgcgcagctgggacccaagctctggtcacac tggagggagttccagccgcggtcccggggcagcccgcccagcttcagctaaatgccaccgagaacgacgacagac gcagcttcttctgcgacgccaccctcgatgtggacggggagaccctgatcaagaacaggagcgcagagcttcgtg tcctatacgctccccggctagacgattcggactgccccaggagttggacgtggcccgagggcccagagcagacgc tgcgctgcgaggcccgcgggaacccagaaccctcagtgcactgtgcgcgctccgacggcggggccgtgctggctc tgggcctgctgggtccagtcactcgggcgctctcaggcacttaccgctgcaaggcggccaatgatcaaggcgagg cggtcaaggacgtaacgctaacggtggagtacgcaccagcgctggacagcgtgggctgcccagaacgcattactt ggctggagggaacagaagcctcgctgagctgtgtggcgcacggggtaccgccgcctgatgtgatctgcgtgcgct ctggagaactcggggccgtcatcgaggggctgttgcgtgtggcccgggagcatgcgggcacttaccgctgcgaag ccaccaaccctcggggctctgcggccaaaaatgtggccgtcacggtggaatatggccccaggtttgaggagccga gctgccccagcaattggacatgggtggaaggatctgggcgcctgttttcctgtgaggtcgatgggaagccacagc caagcgtgaagtgcgtgggctccgggggcgccactgagggggtgctgctgccgctggcacccccagaccctagtc ccagagctcccagaatccctagagtcctggcacccggtatctacgtctgcaacgccaccaaccgccacggctccg tggccaaaacagtcgtcgtgagcgcggagtcgccaccggagatggatgaatctacctgcccaagtcaccagacgt ggctggaaggggctgaggcttccgcgctggcctgcgccgcccggggtcgcccttccccaggagtgcgctgctctc gggaaggcatcccatggcctgagcagcagcgcgtgtcccgagaggacgcgggcacttaccactgtgtggccacca atgcgcatggcacggactcccggaccgtcactgtgggcgtggaataccggccagtggtggccgaacttgctgcct cgccccctggaggcgtgcgcccaggaggaaacttcacgttgacctgccgcgcggaggcctggcctccagcccaga tcagctggcgcgcgcccccgggggccctcaacatcggcctgtcgagcaacaacagcacactgagcgtggcaggcg ccatgggaagccacggcggcgagtacgagtgcgcacgcaccaacgcgcacgggcgccacgcgcggcgcatcacgg tgcgcgtggccggtccgtggctatgggtcgccgtgggcggcgcggcggggggcgcggcgctgctggccgcggggg ccggcctggccttctacgtgcagtccaccgcctgcaagaagggcgagtacaacgtgcaggaggccgagagctcag gcgaggccgtgtgtctcaacggagcgggcggcggcgctggcggggcggcaggcgcggagggcggacccgaggcgg cggggggcgcggccgagtcgccggcggagggcgaggtcttcgccatacagctgacatcggcgtgagccgctcccc tctccccgcgggccgggggacgccccccagactcacacgggggcttatttattgctttatttatttacttattca tttatttatgtattcaactccaagggcgtcacccccattttctacccatcccctcaataaagtttttataaagga

Following are ICAMl, ICAM4 and ICAM5 amino acid sequences (SEQ ID NO: 5, 6 and 7, respectively). Following is an ICAMl cDNA sequence (SEQ ID NO: 5). NP_000192 [gi : 4557878 ] intercellular adhesion molecule 1 precursor; CD54 [Homo sapiens] MAPSSPRPALPALLVLLGALFPGPGNAQTSVSPSKVILPRGGSVLVTCSTSCDQPKLLGI ETPLPKKELLLPGNN RKVYELSNVQEDSQPMCYSNCPDGQSTAKTFLTVYWTPERVELAPLPSWQ PVGKNLTLRCQVEGGAPRANLTWL LRGEKELKREPA VGEPAEVTTTVL VRRDHHGANFSCRTELDLRPQGLELFENTSAPYQLQTFVLPATPPQLVSPR VLEVDTQGTWCSLDGLFPVSEAQVHLALGDQRLNPTVTYGNDSFSAKASVSVTAEDEGTQRLTCAVILGNQSQE TLQTVTI YSFPAPNVILTKPEVSEGTEVTVKCEAHPRAKVTLNGVPAQPLGPRAQLLLKATPEDNGRSFSCSATL EVAGQLIHKNQTRELRVLYGPRLDERDCPGNWTWPENSQQTPMCQAWGNPLPELKCLKDGTFPLPIGESVTVTRD LEGTYLCRARSTQGEVTREVTVNVLSPRYEIVI ITWAAAVIMGTAGLSTYLYNRQRKI KKYRLQQAQKGTPMKP NTQATPP

Following is an ICAM4 amino acid sequence (SEQ ID NO: 6).

NP_001535 [gi:4504561] intercellular adhesion molecule 4 isoform 1 precursor; Landsteiner-Wiener blood group protein [Homo sapiens].

MGSLFPLSLLFFLAAAYPGVGSALGRRTKRAQSPKGΞPLAPSGTSVPFWVRMSPEFVAVQPGKSVQLNCSNSCPQ PQNSSLRTPLRQGKTLRGPGWVSYQLLDVRAWSSLAHCLVTCAGKTRWATSRITAYKPPHSVILEPPVLKGRKYT LRCHVTQVFPVGYLVVTLRHGSRVIYSESLERFTGLDLANVTLTYEFAAGPRDFWQPVICHARLNLDGLWRNSS APITLMLAWSPAPTALASGSIAALVGILLTVGAAYLCKCLAMKSQA

Following is an ICAM5 amino acid sequence (SEQ ID NO: 7).

NP_003250 [gi: 12545404] intercellular adhesion molecule 5 precursor; telencephalin [Homo sapiens].

MPGPSPGLRRALLGLWAALGLGLFGLSAVSQEPFWADLQPRVAFVERGGSLWLNCSTNCPRPERGGLETSLRRNG TQRGLRWLARQLVDIREPETQPVCFFRCARRTLQARGLIRTFQRPDRVELMPLPPWQPVGENFTLSCRVPGAGPR ASLTLTLLRGAQELIRRSFAGEPPRARGAVLTATVLARREDHGANFSCRAELDLRPHGLGLFENSSAPRELRTFS LSPDAPRLAAPRLLEVGSERPVSCTLDGLFPASEARVYLALGDQNLSPDVTLEGDAFVATATATASAEQEGARQL ICNVTLGGENRETRENVTIYSFPAPLLTLSEPSVSEGQMVTVTCAAGTQALVTLEGVPAAVPGQPAQLQLNATEN DDRRSFFCDATLDVDGETLIKNRSAELRVLYAPRLDDSDCPRSWTWPEGPEQTLRCEARGNPEPSVHCARSDGGA VLALGLLGPVTRALSGTYRCKAANDQGEAVKDVTLTVEYAPALDSVGCPERITWLEGTEASLSCVAHGVPPPDVI

KPQPSVKCVGSGGATEGVLLPLAPPDPSPRAPRIPRVLAPGIYVCNATNRHGSVAKTWVSAESPPEMDESTCPS HQTWLEGAEASALACAARGRPSPGVRCSREGIPWPEQQRVSREDAGTYHCVATNAHGTDSRTVTVGVEYRPWAE LAASPPGGVRPGGNFTLTCRAEAWPPAQISWRAPPGALNIGLSSNNSTLSVAGAMGSHGGEYECARTNAHGRHAR RITVRVAGPWLWVAVGGAAGGAALLAAGAGLAFYVQSTACKKGEYNVQEAESSGEAVCLNGAGGGAGGAAGAEGG PEAAGGAAESPAEGEVFAIQLTSA

Modifications may be made to the foregoing without departing from the basic aspects of the invention. Although the invention has been described in substantial detail with reference to one or more specific embodiments, those of skill in the art will recognize that changes may be made to the embodiments specifically disclosed in this application, yet these modifications and improvements are within the scope and spirit of the invention, as set forth in the claims which follow.

Citation of the above publications or documents is not intended as an admission that any of the foregoing is pertinent prior art, nor does it constitute any admission as to the contents or date of these publications or documents. The content of each of the U.S. patents and other publications and documents referenced herein is hereby incorporated by reference in its entirety in jurisdictions allowing such incorporation.

Claims

What is claimed is:

1. A method for providing a prognosis for breast cancer in a subject, which comprises: detecting the presence or absence of one or more polymorphic variations associated with breast cancer severity in a nucleic acid sample from a subject, wherein the polymorphic variation is detected in a nucleotide sequence selected from the group consisting of:

(a) a nucleotide sequence in SEQ ID NOs: 1-4;

(b) a nucleotide sequence which encodes a polypeptide encoded by a nucleotide sequence in SEQ ID NOs: 1-4;

(c) a nucleotide sequence which encodes a polypeptide that is 90% or more identical to the amino acid sequence encoded by a nucleotide sequence in SEQ ID NOs: 1-4;

(d) a fragment of a nucleotide sequence of (a), (b), or (c) comprising the polymorphic variation; and administering a breast cancer preventative or detection procedure to a subject in need thereof based upon the presence or absence of the one or more polymorphic variations in the nucleic acid sample.

2. The method of claim 1 , wherein the one or more polymorphic variations are at one or more positions in SEQ ID NO: 1 is selected from the group consisting of 37083, 41510, and 44338.

3. The method of claim 1 , wherein the one or more polymorphic variations is at position 41510 of SEQ ID NO: 1.

4. The method of claim 1, wherein the one or more polymorphic variations are detected at one or more positions in linkage disequilibrium with one or more positions in SEQ ID NO: 1 selected from the group consisting of 37083, 41510, and 44338.

5. A method for identifying a subject at risk of prostate cancer, which comprises detecting the presence or absence of one or more polymorphic variations associated with prostate cancer in a nucleic acid sample from a subject, wherein the one or more polymorphic variations are detected in a nucleotide sequence selected from the group consisting of:

(a) a nucleotide sequence in SEQ ID NOs: 1-4;

(b) a nucleotide sequence which encodes a polypeptide encoded by a nucleotide sequence in SEQ ID NOs: 1-4;

(c) a nucleotide sequence which encodes a polypeptide that is 90% or more identical to the amino acid sequence encoded by a nucleotide sequence in SEQ ID NOs: 1-4;

(d) a fragment of a nucleotide sequence of (a), (b), or (c); whereby the presence of the polymorphic variation is indicative of the subject being at risk of prostate cancer.

6. The method of claim 5, wherein the one or more polymorphic variations are detected at one or more positions selected from the group consisting of 44338 and 44768.

7. The method of claim 5, wherein the one or more polymorphic variations are detected within a region spanning positions 44338 to 44768 in SEQ ID NO: 1.

8. The method of claim 7, wherein a polymorphic variation is detected at position 44338 in SEQ ID NO: 1.

9. The method of claim 7, wherein a polymorphic variation is detected at position 44768 in SEQ ID NO: 1.

10. The method of claim 5, wherein the one or more polymorphic variations are detected at one or more positions in linkage disequilibrium with one or more positions in SEQ ID NO: 1 selected from the group consisting of 44338 and 44768.

11. The method of claim 5, wherein detecting the presence or absence of the one or more polymorphic variations comprises: hybridizing an oligonucleotide to the nucleic acid sample, wherein the oligonucleotide is complementary to a nucleotide sequence in the nucleic acid and hybridizes to a region adjacent to the polymorphic variation; extending the oligonucleotide in the presence of one or more nucleotides, yielding extension products; and detecting the presence or absence of a polymorphic variation in the extension products.

12. A method for identifying a polymorphic variation associated with prostate cancer proximal to an incident polymorphic variation associated with prostate cancer, which comprises: identifying a polymorphic variation proximal to the incident polymorphic variation associated with prostate cancer, wherein the polymorphic variation is detected in a nucleotide sequence selected from the group consisting of:

(a) a nucleotide sequence in SEQ ID NOs: 1-4;

(b) a nucleotide sequence which encodes a polypeptide encoded by a nucleotide sequence in SEQ ID NOs: 1-4;

(c) a nucleotide sequence which encodes a polypeptide that is 90% or more identical to the amino acid sequence encoded by a nucleotide sequence in SEQ ID NOs: 1-4;

(d) a fragment of a nucleotide sequence of (a), (b), or (c) comprising the polymorphic variation; determining the presence or absence of an association of the proximal polymorphic variant with prostate cancer.

13. The method of claim 12, wherein the incident polymorphic variation is at a position in SEQ ID NO: 1 selected from the group consisting of 44338 and 44768.

14. The method of claim 12, wherein the incident polymorphic variation is within a region spanning positions 44338 to 44768 in SEQ ID NO: 1.

15. The method of claim 12, wherein the proximal polymorphic variation is within a region between about 5 kb 5' of the incident polymorphic variation and about 5 kb 3' of the incident polymorphic variation.

16. The method of claim 12, which further comprises determining whether the proximal polymorphic variation is in linkage disequilibrium with the incident polymorphic variation.

17. The method of claim 12, which further comprises identifying a second polymorphic variation proximal to the identified proximal polymorphic variation associated with prostate cancer and determining if the second proximal polymorphic variation is associated with prostate cancer.

18. The method of claim 17, wherein the second proximal polymorphic variant is within a region between about 5 kb 5' of the incident polymorphic variation and about 5 kb 3' of the proximal polymorphic variation associated with prostate cancer.

19. An isolated nucleic acid comprising a nucleotide sequence selected from the group consisting of:

(a) a nucleotide sequence in SEQ ID NOs: 1 -4;

(b) a nucleotide sequence which encodes a polypeptide encoded by a nucleotide sequence in SEQ ID NOs: 1-4;

(c) a nucleotide sequence which encodes a polypeptide that is 90% or more identical to the amino acid sequence encoded by a nucleotide sequence in SEQ ID NOs: 1-4;

(d) a fragment of a nucleotide sequence of (a), (b), or (c); and

(e) a nucleotide sequence complementary to the nucleotide sequences of (a), (b), (c), or (d); wherein the nucleotide sequence comprises a cytosine at position 44338 and/or a guanine at position 44768 of SEQ ID NO: 1.

20. An oligonucleotide comprising a nucleotide sequence complementary to a portion of the nucleotide sequence of (a), (b), (c), or (d) in claim 53, wherein the 3' end of the oligonucleotide is adjacent to a polymorphic variation associated with prostate cancer.

21. A microarray comprising an isolated nucleic acid of claim 20 linked to a solid support.

22. An isolated polypeptide encoded by the isolated nucleic acid sequence of claim 19.

23. A method for detecting or preventing prostate cancer in a subject, which comprises: detecting the presence or absence of one or more polymorphic variations associated with prostate cancer in a nucleic acid sample from a subject, wherein the polymorphic variation is detected in a nucleotide sequence selected from the group consisting of:

(a) a nucleotide sequence in SEQ ID NOs: 1-4;

(b) a nucleotide sequence which encodes a polypeptide encoded by a nucleotide sequence in SEQ ID NOs: 1-4; (c) a nucleotide sequence which encodes a polypeptide that is 90% or more identical to the amino acid sequence encoded by a nucleotide sequence in SEQ ID NOs: 1-4;

(d) a fragment of a nucleotide sequence of (a), (b), or (c) comprising the polymorphic variation; and administering a prostate cancer preventative or detection procedure to a subject in need thereof based upon the presence or absence of the one or more polymorphic variations in the nucleic acid sample.

24. The method of claim 23, wherein the one or more polymorphic variations are at one or more positions in SEQ ID NO: 1 is selected from the group consisting of 44338 and 44768.

25. The method of claim 24, wherein the one or more polymorphic variations are in a region spanning positions 44338 to 44768 in SEQ ID NO: 1.

26. The method of claim 23, wherein the prostate cancer detection procedure is selected from the group consisting of digital rectal examinations (DRE), prostate specific antigen (PSA) measurements, transrectal ultrasonography (TRUS), transrectal needle biopsy (TRNB), and a combination of the foregoing.

27. The method of claim 23, wherein the prostate cancer preventative procedure is selected from the group consisting of surgical prostatectomy, radiation therapy, hormone ablation therapy, and chemotherapy.

28. The method of claim 27, wherein the hormone therapy is selected from the group consisting of LHRH agonists and anti-androgens.

29. A method of targeting information for preventing or treating prostate cancer to a subject in need thereof, which comprises: detecting the presence or absence of one or more polymorphic variations associated with prostate cancer in a nucleic acid sample from a subject, wherein the polymorphic variation is detected in a nucleotide sequence selected from the group consisting of:

(a) a nucleotide sequence in SEQ ID NOs: 1-4;

(b) a nucleotide sequence which encodes a polypeptide encoded by a nucleotide sequence in SEQ ID NOs:l-4;

(c) a nucleotide sequence which encodes a polypeptide that is 90% or more identical to the amino acid sequence encoded by a nucleotide sequence in SEQ ID NOs: 1-4;

(d) a fragment of a nucleotide sequence of (a), (b), or (c) comprising the polymorphic variation; and directing information for preventing or treating prostate cancer to a subject in need thereof based upon the presence or absence of the one or more polymorphic variations in the nucleic acid sample.

30. The method of claim 29, wherein the one or more polymorphic variations are at one or more positions in SEQ ID NO: 1 is selected from the group consisting of 44338 and 44768.

31. The method of claim 29, wherein the one or more polymorphic variations are in a region spanning positions 44338 to 44768 in SEQ ID NO: 1.

32. The method of claim 29, wherein the information comprises a description of a prostate cancer detection procedure, a chemotherapeutic treatment, a surgical treatment, a radiation treatment, a preventative treatment of prostate cancer, and combinations of the foregoing.

33. A composition comprising a prostate cancer cell and an antibody that specifically binds to a protein, polypeptide or peptide encoded by a nucleotide sequence identical to or 90% or more identical to a nucleotide sequence in SEQ ID NOs: 1-4.

34. A composition comprising a prostate cancer cell and a RNA, DNA, PNA or ribozyme molecule comprising a nucleotide sequence identical to or 90% or more identical to a portion of a nucleotide sequence in SEQ ID NOs: 1-4.

35. The composition of claim 34, wherein the RNA molecule is a short inhibitory RNA molecule.

36. A composition comprising a prostate cancer cell and an agent that induces nucleic acid strand breakage.

37. A method of selecting a subject that will respond to a treatment of prostate cancer, which comprises: detecting the presence or absence of one or more polymorphic variations associated with prostate cancer in a nucleic acid sample from a subject, wherein the polymorphic variation is detected in a nucleotide sequence selected from the group consisting of:

(a) the nucleotide sequence of SEQ ID NOs: 1-4;

(b) a nucleotide sequence which encodes a polypeptide consisting of an amino acid sequence encoded by a nucleotide sequence in SEQ ID NOs: 1-4 ;

(c) a nucleotide sequence which encodes a polypeptide that is 90% or more identical to an amino acid sequence encoded by a nucleotide sequence in SEQ ID NOs: 1-4 ; and

(d) a fragment of a nucleotide sequence of (a), (b), or (c) comprising the polymorphic variation; and selecting a subject that will respond to the prostate cancer treatment based upon the presence or absence of the one or more polymorphic variations in the nucleic acid sample.

38. The method of claim 37, wherein the one or more polymorphic variations are detected at one or more positions in SEQ ID NO: 1 selected from the group consisting of 44338 and 44768.

39. The method of claim 37, wherein the one or more polymorphic variation are detected in a region spanning positions 44338 to 44768 in SEQ ID NO: 1.