KR100647304B1 - 2 A polynucleotide associated with a type II diabetes mellitus comprising single nucleotide polymorphism microarray and diagnostic kit comprising the same and method for analyzing polynucleotide using the same - Google Patents

Abstract

The present invention provides a polynucleotide comprising 10 or more consecutive nucleotides derived from the group consisting of SEQ ID NOs: 1 to 80, and a polynucleotide for diagnosis or treatment of type 2 diabetes comprising the 101st base or a complementary polynucleotide thereof. .

Polynucleotide, Type 2 Diabetes, Monobasic, Polymorphic, SNP

Description

A polynucleotide associated with a type II diabetes mellitus comprising single nucleotide polymorphism, microarray and diagnostic kit comprising the polynucleotide associated with type 2 diabetes including a monobasic polymorph, and a method for analyzing polynucleotides using the same same and method for analyzing polynucleotide using the same}

The present invention relates to polynucleotides for use in connection with type 2 diabetes, microarrays comprising the same, diagnostic kits and methods of analyzing polynucleotides associated with type 2 diabetes.

The genomes of all organisms undergo spontaneous mutations that result in variants in the sequence of progeny during evolution (Gusella, Ann. Rev. Biochem. 55, 831-854 (1986)). Variants may be evolutionarily beneficial or disadvantageous or neutral compared to progeny forms. In some cases, variants may be fatally penalized and not passed on to their offspring. In other cases, it gives evolutionary benefit to the species, and eventually DNA is inserted into most of the species, effectively forming a ancestral form. In many cases, these ancestral forms and variants survive and coexist among species. By coexistence of plural forms of sequence, polymorphism occurs.

Such polymorphisms are known as restriction fragment length polymorphism (RFLP), short tandem repeats (STR), variable number tandem repeats (VNTR), and single nucleotide polymorphisms (SNPs). Dual SNPs are monobasic polymorphs and take the form of single nucleotide variations between individuals of the same species. When a monobasic polymorph occurs in a coding region sequence, a defective or mutated protein may be expressed by either of the polymorphic forms. In other cases, single base polymorphisms can occur in non-coding region sequences. Some of these may result in the expression of defective or mutated proteins (eg, as a result of defective splicing). Some monobasic polymorphs may have no effect on the phenotype.

Single base polymorphisms are known to occur once in about 1,000bp in humans. Where these monobasic polymorphs affect phenotypes such as diseases, polynucleotides comprising such single base polymorphs can be used as primers or probes to diagnose such diseases. Monoclonal antibodies that specifically bind the monobasic polymorphs can also be used for the diagnosis of diseases. Currently, several research institutes are conducting research on single base analysis and its functional analysis. The nucleotide sequence of the single base polymorphism and other experimental results thus found are made into a database so that anyone can access and use it for research.

However, these monobasic polymorphisms only found the presence of monobasic polymorphisms on the human genome or cDNA, and did not reveal their effect on the phenotype. Some of these functions are known, but most are unknown.

Type 2 Diabetes mellitus is known to account for 90 to 95% of all diabetics. Type 2 diabetes occurs in people who produce insulin in the body but have abnormal amounts of insulin or low sensitivity to insulin, which is characterized by large variations in blood glucose levels. This is known to cause difficulty in obtaining energy from food because glucose in the blood cannot move into the cells due to the abnormality of insulin. It is known that there is a genetic factor in the development of type 2 diabetes, and other risk factors include the age of 45 years or older, family history of diabetes, overweight, high blood pressure and cholesterol levels. Currently, the diagnosis of diabetes is mainly performed by measuring fasting blood glucose (FSB) test and oral glucose tolerance test (OGTT) to change the phenotype due to disease, that is, blood glucose level. (National Institute of Diabetes and Digestive and Kidney Diseases of the National Institutes of Health, http://www.niddk.nih.gov, 2003). Type 2 diabetes can be said to be a disease with a high need for early diagnosis because it can slow down the progress of treatment or diabetes through changes in exercise and dietary habits, weight control, and various medications. Millennium Pharmaceuticals has announced that genotype mutations in the HNF1 gene can be used to diagnose and predict type 2 diabetes (PR Newswire, Sept 1, 1998). Sequenom has reported that the FOXA2 (HNF3β) gene is highly associated with the development of type 2 diabetes (PR Newswire, Oct 28, 2003). Although several genes have been reported to be associated with the development of type 2 diabetes, research has focused on only a few specific genes on some chromosomes and has been tested on specific populations. As a result, there may be different results depending on the race, and not all the causative genes of type 2 diabetes have been identified, and there are not many cases where the type 2 diabetes is diagnosed using these molecular biological methods. to be. In addition, early diagnosis before onset has not been made. Therefore, the necessity of finding new SNPs and related genes highly related to type 2 diabetes in the entire human genome and using them for early diagnosis.

It is an object of the present invention to provide a polynucleotide comprising a monobasic polymorph associated with type 2 diabetes.

Still another object of the present invention is to provide a microarray or type 2 diagnosing kit comprising a polynucleotide comprising a single base polymorph related to type 2 diabetes.

Another object of the present invention is to provide a method for analyzing polynucleotides associated with type 2 diabetes of the present invention.

The present invention relates to a polynucleotide comprising 10 or more consecutive nucleotides derived from the group consisting of SEQ ID NOs: 1 to 80, comprising a polymorphic site (position 101), a polynucleotide for diagnosing or treating type 2 diabetes or its complement Provided are polynucleotides.

The polynucleotide has a length of 10 or more nucleotides including the polymorphic sites of SEQ ID NOs: 1 to 80. The length is 10 to 400 nucleotides, preferably 10 to 100 nucleotides, more preferably 10 to 50 nucleotides. Wherein the polymorphic site of each sequence is at position 101.

The polynucleotide having the nucleotide sequence of SEQ ID NO: 1 to 80 is a polymorphic sequence. A polymorphic sequence refers to a sequence comprising a polymorphic site representing a single base polymorph in a nucleotide sequence. A polymorphic site is a site where a single base polymorphism occurs in the polymorphic sequence. The polynucleotide can be DNA or RNA.

In the present invention, the polymorphic site (position 101) of the polymorphic sequences of SEQ ID NOs: 1 to 80 is associated with type 2 diabetes. This was confirmed through sequence analysis of DNA obtained from blood samples derived from type 2 diabetic patients and normal persons. Tables 1-1, 1-2, 2-1 and 2-2 show the association between the polymorphic sequences of SEQ ID NOs: 1 to 80 with type 2 diabetes and the characteristics of the polymorphic sequences.

In Table 1-1 and 1-2, what each column means is as follows.

Assay_ID indicates the name of the marker.

SNP represents a base observed at a single nucleotide polymorphism site, where A1 and A2 are small masses during sequencing by a homogeneous MassExtension (hME) technique. The allele is named A1 and the large allele is named A2 for convenience of experiment.

The sequence containing SNP shows the sequence number of the sequence containing each SNP site | part, and shows the sequence containing A1 and A2 allele in 101st position, respectively.

In the allele frequency column, cas-A2, con_A2 and Delta are the frequency of the A2 allele in the case sample, the frequency of the A2 allele in the normal sample, and the cas-A2, respectively. The absolute value of the difference between and con_A2. Where cas-A2 is (frequency of A2A2 genotype x2 + frequency of A1A2 genotype) / (number of samples in disease group x2) and con-A2 is (frequency of A2A2 genotype x2 + frequency of A1A2 genotype) / ( Number of samples in the normal group x2).

Genotype frequency represents the frequency of each genotype, and cas_A1A1, cas_A1A2 and cas_A2A2 and con_A1A1, con_A1A2 and con_A2A2 represent the number of humans with A1A1, A1A2 and A2A2 genotypes in the disease group and normal group, respectively.

Chi-square (df = 2) represents the chi-square value with a degree of freedom of 2. Chi-value is the chi-squared value and is a value for p-value calculation. The chi-exact-p-value represents the p-value of Fisher's exact test of chi-square test. Fisher's exact because the result of the general chi-square test may be inaccurate if the value of the genotype number is less than 5 Test is a variable used to test for more accurate statistical significance (p-value). In the present invention, when the p-value ≤ 0.05, it was determined that the genotypes of the disease group and the normal group were not the same.

The risk allele is the reference allele as A2. If the frequency of A2 in the disease group is greater than the frequency of A2 in the normal group (ie cas_A2> con-A2), then A2 as the risk allele. On the contrary, A1 was used as the risk allele.

Odds ratio represents the ratio of the probability of having a risk allele among disease groups to the probability of having a risk allele among normal groups. In the present invention, the Mantel-Haenszel odds ratio method was used. CI represents the 95% confidence interval of the odds ratio and represents (lower confidence interval, upper confidence interval). Confidence intervals with a value of 1 cannot be considered significant for association with disease.

The HWE state represents the state of Hardy-Weinberg Equilibrium, and con_HWE and cas_HWE represent the state of Hardy-Wineberg equilibrium in normal and diseased groups. For chi-value = 6.63 (p-value = 0.01, df = 1) in the chi-square (df = 1) test, Hardy-Weinberg Disequilibrium (HWD) if greater than 6.63, greater than 6.63 In small cases, it was judged by Hardy-Weinberg Equilibrium (HWE).

ㆍ Call ratio indicates the number of samples tested successfully genotype for the total number of samples put into the original experiment, the cas_call_rate and con_call_rate is the genotype of the total sample (300 people) used in the disease group and normal group experiment, respectively To represent the analyzed ratio.

Tables 2-1 and 2-2 describe the properties associated with each SNP marker based on NCBI build 123.

As shown in Tables 1-1, 1-2, 2-1 and 2-2, the polymorphic markers of SEQ ID NOs: 1-80 of the present invention showed 95% squared analysis between the expectation and observation of allele frequency. The reliability of Chi-exact-p-value ranged from 4.54x10 ^{-4 to} 0.0104, which was significantly different from the control group, and the odds ratio ranged from 1.34 to 2.43, which is associated with type 2 diabetes. Can be.

The monobasic polymorphisms of SEQ ID NOS: 1 to 80 of the present invention showed a significant frequency of appearance in the type 2 diabetic patients and normal subjects. Thus, the polynucleotides of the present invention can be effectively used for diagnosis, genetic fingerprinting analysis or treatment associated with type 2 diabetes. Specifically, it may be used as a primer or probe for the diagnosis of type 2 diabetes. It can also be used as antisense DNA or compositions for treating type 2 diabetes.

The present invention also provides an allele specific polynucleotide for diagnosing type 2 diabetes that hybridizes to a polynucleotide comprising 10 or more consecutive nucleotides derived from SEQ ID NOs: 1 to 80 including a polymorphic site or its complement.

The allele-specific polynucleotide means to hybridize specifically to each allele. That is, it means hybridizing so that the base of the polymorphic site in each polymorphic sequence of SEQ ID NO: 1-80 can be distinguished specifically. Here, hybridization can usually be carried out under stringent conditions, for example salt concentrations of 1 M or less and temperatures of 25 ° C. or higher. For example, conditions of 5 × SSPE (750 mM NaCl, 50 mM Na Phosphate, 5 mM EDTA, pH 7.4) and 25-30 ° C. may be suitable for allele specific probe hybridization.

In the present invention, the allele specific polynucleotide may be a primer. “Primer” herein refers to template-directed DNA synthesis under appropriate conditions (eg, four different nucleoside triphosphates and polymerizers such as DNA, RNA polymerase or reverse transcriptase) in appropriate buffers and at appropriate temperatures. It refers to a single stranded oligonucleotide that can act as a starting point. The appropriate length of the primer may vary depending on the purpose of use, but is usually 15 to 30 nucleotides. Short primer molecules generally require lower temperatures to form stable hybrids with the template. The primer sequence need not be completely complementary to the template, but should be sufficiently complementary to hybridize with the template. Preferably, the primer 3 'end is aligned with the polymorphic site (base 101 position) of SEQ ID NO: 1 to 80. The primer hybridizes to the target DNA comprising the polymorphic site and initiates amplification of an allelic form in which the primer shows complete homology. This primer is used in pairs with a second primer that hybridizes to the other side. By amplification the product is amplified from two primers, which means that certain allelic forms are present. Primers of the invention include polynucleotide fragments used in ligase chain reaction (LCR).

In the present invention, the allele specific polynucleotide may be a probe. As used herein, the term "probe" refers to a hybridization probe, and refers to an oligonucleotide capable of sequence-specific binding to the complementary strand of a nucleic acid. Such probes include peptide nucleic acids described in Nielsen et al., Science 254, 1497-1500 (1991). The probe of the present invention is an allele-specific probe, in which polymorphic sites exist in nucleic acid fragments derived from two members of the same species, and hybridize to DNA fragments derived from one member, but not to fragments derived from other members. . Hybridization conditions in this case show significant differences in hybridization strength between alleles and should be sufficiently stringent to hybridize to only one of the alleles. In the probe of the present invention, it is preferable that the center portion (ie, position 7 when the probe is 15 nucleotides and position 8 or 9 when the probe is 16 nucleotides) is aligned with the polymorphic site of the sequence. This can lead to good hybridization differences between different allelic forms. The probe of the present invention can be used for diagnostic methods for detecting alleles. The diagnostic method includes detection methods based on hybridization of nucleic acids such as Southern blot and the like, and may be provided in a form that is pre-bound to the substrate of the DNA chip in a method using a DNA chip.

The invention also includes a type 2 diabetes diagnostic microarray comprising the polynucleotide of the invention or its complementary polynucleotide. The microarray may include DNA or RNA polynucleotides. The microarray consists of conventional microarrays, except that they contain the polynucleotides of the present invention.

The present invention also provides a kit for diagnosing type 2 diabetes comprising the polynucleotide of the present invention. The kit may include not only the polynucleotide of the present invention but also reagents required for the polymerization reaction, such as dNTP, polymerases of various kinds, and colorants.

In addition, the present invention comprises the steps of obtaining a nucleic acid from a sample; And

A method for diagnosing type 2 diabetes comprising determining the nucleotide sequence of one or more polymorphic sites (101) of the polynucleotides of SEQ ID NOs: 1 to 80 or complementary polynucleotides thereof. Here, the nucleotide sequence of the polymorphic site is shown in Table 1-1. If one or more of the risk alleles as shown in 1-2, 2-1, 2-2, 3, 4, and 5 are the same, determining that the risk of type 2 diabetes is high Can be.

The first step of obtaining a nucleic acid from a sample may be performed by a conventional DNA separation method. For example, the target nucleic acid can be amplified by PCR and purified. Ligase chain reaction (LCR) (Wu and Wallace, Genomics 4, 560 (1989), Landegren et al., Science 241, 1077 (1988)), transcription amplification (Kwoh et al . , Proc. Natl. Acad. Sci. USA 86, 1173 (1989)) and self-sustaining sequence replication (Guatelli et al., Proc. Natl. Acad. Sci. USA 87, 1874 (1990)) and nucleic acid based sequence amplification (NASBA). The last two methods involve isothermal reactions based on isothermal transcription, producing 30 or 100-fold single-stranded RNA and double-stranded DNA as amplification products.

In one embodiment of the method of the present invention, the step of determining the nucleotide sequence of the polymorphic site is a polynucleotide comprising at least 10 consecutive nucleotides derived from the group consisting of SEQ ID NOS: 1 to 80 of the present invention, the 101 st base Hybridizing the nucleic acid sample to a microarray in which a polynucleotide for diagnosis or treatment of type 2 diabetes or a complementary polynucleotide thereof is immobilized; And detecting the hybridization result.

Methods of preparing microarrays by immobilizing microarrays and probe polynucleotides on substrates are well known in the art. The immobilization of the probe polynucleotides associated with type 2 diabetes of the present invention can also be readily prepared using this prior art. In addition, detection of hybridization and hybridization results of nucleic acids on microarrays is well known in the art. Such detection may, for example, detect a nucleic acid sample by detecting a detectable signal comprising a fluorescent material such as Cy3 and Cy5. The hybridization results can be detected by labeling with a labelable substance that can be generated and then hybridizing on a microarray and detecting a signal from the labeling substance.

In another embodiment of the method of the present invention, SEQ ID NO: 2,4,5,8,9,11,13,16,18,20 having a nucleotide corresponding to a risk allele as a result of determining the nucleotide sequence of the polymorphic site , 21,23,25,27,30,32,33,36,38,40,42,44,46,47,49,51,53,5 5,57,59,62,63,65,67, When the sequence of 69,71,74,75,77 or 80 is present, the method may further include determining that the sample belongs to a high risk group of type 2 diabetes. The more the nucleic acid sequence having the risk allele is detected in one sample, the higher the probability of belonging to the risk group.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

Example

Example 1

In this example, DNA was isolated from blood of a group of Koreans (300 patients) who were found to be type 2 diabetic patients and were treated with the same age group as those of the patient group, but did not yet have symptoms of type 2 diabetes, The frequency of appearance of specific monobasic polymorphs was analyzed. The single base polymorphs selected in this example are published databases (NCBI dbSNP: http://www.ncbi.nlm.nih.gov/SNP/) or realsnp.com (http: //: //www.realsnp.com) from Sequenom. /), And the single base sequence in the sample was analyzed using primers using the sequence around the selected single base polymorphism.

1. Preparation of DNA Samples

DNA was extracted from blood collected from type 2 diabetic patients and normal individuals. DNA extraction was performed according to known extraction methods (Molecular cloning: A Laboratory Manual, p 392, Sambrook, Fritsch and Maniatis, 2nd edition, Cold Spring Harbor Press, 1989) and the instructions of the commercial kit (Gentra system). Among the extracted DNAs, only those having a purity of at least 1.7 or more UV measurement (260/280 nm) were used.

2. Amplification of Target DNA

Target DNA, which is a constant DNA region containing a single nucleotide polymorphism to be analyzed, was amplified by PCR. PCR method proceeded in a conventional manner, the conditions were as follows. First, target genomic DNA was prepared at 2.5 ng / ml. Next, the following PCR reaction solution was prepared.

2.24 µl of water (HPLC grade)

0.5 μl 10x buffer (with 15 mM MgCl ₂ and 25 mM MgCl ₂ )

0.04 μl dNTP Mix (GIBCO) (25 mM / each)

Taq pol (HotStar) (5U / μl) 0.02μl

0.02 μl of potential / back primer mix (1 μM / each)

1.00 μl DNA

5.00 μl total reaction volume

Here, the forward and reverse primers were selected at appropriate positions from upstream and downstream of a single base polymorph of known day database. The primers are summarized in Table 3.

The thermal cycling reaction was maintained at 95 ° C. for 15 minutes, repeated for 30 seconds at 95 ° C., 30 seconds at 56 ° C., 1 minute at 72 ° C., and maintained at 72 ° C. for 3 minutes, and then at 4 ° C. Archived. As a result, a target DNA fragment having a length of 200 nucleotides or less was obtained.

3. Analysis of Monobasic Polymorphs in Amplified Target DNA

The analysis of single nucleotide polymorphisms in the target DNA fragment was carried out using a homogeneous MassExtension (hME) technique. The principle of the MassExtension technique is as follows. First, a primer (also referred to as an extension primer) that is complementary to the base immediately before the single base polymorphism in the target DNA fragment is prepared. Next, the primer is hybridized to the target DNA fragment, and a DNA polymerization reaction is caused. In this case, a reagent (eg, ddTTP) which stops the reaction after the base complementary to the first allele base (for example, the A allele) of the target monobasic polymorph allele is added to the reaction solution. Let's do it. As a result, when a first allele (eg, A allele) is present in the target DNA fragment, a product is obtained in which only one base (eg, T) complementary to the first allele is added. . On the other hand, if a second allele (eg, G allele) is present in the target DNA fragment, the closest first allele with the base (eg, C) complementary to the second allele added A product extending to the base (eg A) is obtained. By determining the length of the product extended from the primer thus obtained by mass spectrometry, it is possible to determine the type of allele present in the target DNA. Specific experimental conditions were as follows.

First, unbound dNTPs were removed from the PCR product. To this end, 1.53 μl of pure water, 0.17 μl of HME buffer, and 0.30 μl of SAP (shrimp alkaline phosphatase) were added and mixed in a 1.5 ml tube to prepare an SAP enzyme solution. The tube was centrifuged at 5,000 rpm for 10 seconds. The PCR product was then placed in the SAP solution tube, sealed and held at 37 ° C. for 20 minutes, 85 ° C. for 5 minutes, and then stored at 4 ° C.

Next, a homogeneous extension reaction was performed using the target DNA product as a template. The reaction solution was as follows.

1.728 μl of water (nano-grade pure water)

0.200 μl hME extension mix (10 × buffer with 2.25 mM d / ddNTPs)

0.054 μL extension primer (100 μM each)

0.018 μl Thermosequenase (32U / μl)

Total volume 2.00 μl

The reaction solution was mixed well, followed by spin down centrifugation. Seal the tube or plate containing the reaction solution well, hold for 2 minutes at 94 ℃, then repeat 5 seconds at 94 ℃, 5 seconds at 52 ℃, 5 seconds at 72 ℃ 40 times, and then 4 ℃ Stored in. The homogeneous extension reaction product thus obtained was washed using resin (SpectroCLEAN). The extension primers used for the extension reaction are shown in Table 3.

Table 3. Primers used for target DNA amplification and extension primers used for homogeneous extension reaction

The obtained extended reaction product was analyzed by the sequence Assisted Laser Desorption and Ionization-Time of Flight (MALDI-TOF) in the mass spectrometry. The MALDI-TOF operates on the principle of mass analysis by calculating the time taken for the material to be analyzed to fly with an ionized matrix to fly from the vacuum to the opposite detector. The smaller mass arrives at the detector quickly, and the single nucleotide polymorphic sequence of the target DNA can be determined based on the difference in mass and the known single nucleotide polymorphic sequence.

Determining the sequence of the polymorphic site of the target DNA using the MALDI-TOF as described above Table 1-1. It is shown to 1-2, 2-1, and 2-2. At this time, each allele may exist in the form of a homozygote or heterozygote in each individual. However, at the group level, the composition between the heterozygote frequency and the homozygote frequency is not statistically significant. Mendel's genetic law and Hardy-Weinberg law show that the composition of the alleles that make up a group is maintained at a constant frequency and, if statistically significant, can give a biological function meaning. Monobasic polymorphs of the present invention are shown in Table 1-1. As shown in 1-2, 2-1, and 2-2, it was found that the patient appeared in a statistically significant level in type 2 diabetes patients and could be used for diagnosis of type 2 diabetes.

The polynucleotides of the present invention can be used for applications related to type 2 diabetes, such as diagnosis, treatment and genetic fingerprint analysis of type 2 diabetes.

In addition, the microarray and the kit containing the polynucleotide of the present invention can effectively diagnose type 2 diabetes.

According to the method for analyzing polynucleotides related to type 2 diabetes of the present invention, it is possible to effectively diagnose the presence or risk of type 2 diabetes.

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Claims (9)

A polynucleotide consisting of 10 or more contiguous nucleotides selected from a polynucleotide selected from the group consisting of SEQ ID NOs: 1 to 80, and comprising a 101 st base, a polynucleotide for diagnosis or treatment of type 2 diabetes or a complementary polynucleotide thereof. A polynucleotide for diagnosing or treating type 2 diabetes, which hybridizes with the polynucleotide of claim 1 or a complementary polynucleotide thereof. The polynucleotide of claim 1 or 2, or a complementary polynucleotide thereof, having a length of 10 to 100 nucleotides. The polynucleotide of claim 1 which is a primer or probe. A microarray for diagnosing type 2 diabetes comprising the polynucleotide of claim 1 or a complementary polynucleotide thereof. A diagnostic kit for type 2 diabetes comprising the polynucleotide of claim 1 or a complementary polynucleotide thereof. delete delete delete