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WO2015127853A1 - 家族特异性遗传病关联等位基因单体型变异标签确认方法 - Google Patents

家族特异性遗传病关联等位基因单体型变异标签确认方法 Download PDF

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WO2015127853A1
WO2015127853A1 PCT/CN2015/072589 CN2015072589W WO2015127853A1 WO 2015127853 A1 WO2015127853 A1 WO 2015127853A1 CN 2015072589 W CN2015072589 W CN 2015072589W WO 2015127853 A1 WO2015127853 A1 WO 2015127853A1
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disease
family
genomic dna
genetic
allele
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林巍
蔡长明
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林巍
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    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6809—Methods for determination or identification of nucleic acids involving differential detection
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

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  • the invention belongs to genetics and molecular biology, and particularly relates to a method for confirming a haplotype variation tag of a family-specific genetic disease-associated allele.
  • Preimplantation genetic diagnosis PGD is a generic method for determining whether a test tube baby embryo to be implanted has a disease risk or carries a disease allele.
  • Pregnancy fetal diagnosis of PD is a general term for determining whether a fetus after pregnancy has a disease risk or is carrying a disease allele.
  • PGD has become a very effective treatment for genetic diseases in the early detection of mutations in single genes.
  • the single cell or very small amount of DNA required for this method is required to be high.
  • PD can collect multi-cellular fetal samples, DNA manipulation is relatively easy.
  • PGD and PD are for whole chromosomes (euploidy abnormalities, such as children with Down syndrome), and others are for local DNA fragment range, even single nucleotide variation (base substitution, insertion, and Missing) to carry out.
  • the object of the present invention is to provide a family-specific genetic disease associated allele Body type variation label confirmation method.
  • the present invention provides a method for confirming a family-specific genetic disease-associated allele haplotype variation tag.
  • a family-specific genetic disease-associated allele haplotype variation tag confirmation method comprising the following steps,
  • the variant site is genetically analyzed in accordance with the disease trait of each genomic DNA sample to confirm the disease-associated allele haplotype, thereby confirming the allele haplotype variation tag associated with the disease.
  • the member A is a member of Mendelian genetic disease or a defective gene haplotype carrier and is about to undergo birth
  • member B is member A.
  • member C is the mother of member A
  • member D is a positive control, that is, suffering from Mendelian genetic disease
  • member E is a negative control, that is, without the Mendelian genetic disease trait.
  • member D and member E are selected from members of the paternal family; if member A has a disease risk from the maternal line, member D and member E are selected from members of the maternal family, among which members E has a direct blood relationship with other patients in the family, namely parents, children or siblings.
  • the whole genome DNA sample is derived from somatic cells and is not limited to peripheral blood, any somatic cells having allelic diploid information, such as oral epithelial cells, tissue culture, and even clinical samples such as hair.
  • the target gene information of step 2) determines the exon framework by literature and/or database.
  • the amplified primers in step 3 are designed on the intron and cover the exons, or multiple amplicon primers are designed to cover the longer exons.
  • the sequencing in step 3) is Sanger sequencing or high throughput sequencing.
  • sequencing includes Sanger monoclonal sequencing and high-throughput sequencing technology represented by Illumina HiSeq, Life Technologies Ion Torrent, ABI SOLiD or 454 pyrosequencing. (High-throughput sequencing) method. In high-throughput sequencing, it is also called Multiplex Amplicon Sequencing.
  • the mutation site in step 4) is caused by a deletion variation, a substitution variation or an insertion variation.
  • the genetic analysis described in step 6) adopts the following genotyping and disease state comparison table, and if a record is found, the rare allele haplotype a in the binary polymorphism is used as a haplotype variation tag, wherein
  • Aa indicates that the diploid allelic state is in a heterozygous state
  • AA or aa indicates that the diploid allele is in a homozygous state
  • "+" indicates that the disease trait is positive
  • "-" indicates that the disease trait is negative
  • the allelic haplotype variant tag is applied to a pre-implantation genetic diagnosis PGD of a IVF embryo or a fetal diagnosis PD during pregnancy to eliminate a high disease risk embryo or fetus containing the allele haplotype variation tag. .
  • the allele haplotype variation tag is searched and confirmed, and the DNA is translated into a protein, and the amino acid sequence of the protein is changed to further confirm the Reliability of allele haplotype variant tags associated with disease.
  • At least 5 samples from the family of Mendelian genetic diseases can ensure that the sample is sufficient to confirm the reliability of the haplotype variation label and save the detection cost;
  • the invention adopts an intuitive trait and an allelic type comparison table to facilitate the operation and understanding of the method implementer
  • a new upstream method for determining a family-specific genetic disease-associated allele haplotype variant tag in combination with PGD or PD according to which a standardized preimplantation genetic diagnosis PGD or a fetal diagnostic PD kit for a specific genetic disease is developed. And diagnostic methods.
  • Figure 1 is a flow chart for confirming the family-specific allele haplotype variation tag.
  • Figure 2 shows the principle of primer design for the exon interval in this method.
  • Figure 3 is a sequence of sequenced bases corresponding to deletion of the mutant site genotype AA in multiple osteophytes.
  • Figure 4 is a forward sequenced base peak map corresponding to the deleted mutation site genotype Aa in multiple osteophytes.
  • Figure 5 is a reverse-sequencing base peak map corresponding to the deleted mutation site genotype Aa in multiple osteophytes cases.
  • Figure 6 is a schematic representation of the alignment of the second generation sequencing reads corresponding to the deletion mutation genotype Aa in the colon polyp case and the reference genome sequence.
  • Figure 7 is a schematic representation of the alignment of the second generation sequencing reads corresponding to the deletion genomic AA in the colon polyp case and the reference genomic sequence.
  • a method for confirming a haplotype variation tag associated with a family of multiple hereditary osteophytes see Figure 1, including the following steps,
  • member A is about to have birth, and has family Mendel's genetic disease multiple osteophytes, males in both husband and wife; member B is member A's father, suffering from multiple osteophytes; members C is the mother of member A; member D is the cousin of member A, has multiple osteophytes disease, is the positive control of the disease family; member E is the cousin of member A, does not suffer from multiple osteophyte disease, is A negative control for the disease family.
  • member D and member E are from members of the paternal family who are at risk of multiple osteophyte diseases.
  • the exon 1 of EXT1 and the exon 15 of the EXT2 gene in Table 1 are 1735 bp and 1282 bp in the RefSeqGen database, respectively, and it is necessary to design at least 2 overlapping amplicons for full coverage.
  • the unidirectional sequencing length is currently limited to 400 bp.
  • the length of the amplicon should be less than 800 bp, that is, at least 3 amplicons are needed to cover. In this example, three amplicon coverage strategies were employed for these two exons.
  • the positive sequencing base peak corresponding to genotype Aa is shown in Figure 4 and the reverse-sequencing base peak is shown in Figure 5. Since one base deletion exists in one of the two alleles, the positive position at the mutation site is made. Both anti-two-way sequencing will result in an allelic misplacement downstream of the variant site, belonging to a typical allelic heterogeneity and containing a deletion mutation. Therefore, the genotyping and disease status of 5 samples are compared with Table 2, which is summarized as the genotype and phenotype of the corresponding 5 sample family members in Table 9 of Table 2.
  • Aa in the comparison table indicates that the diploid allelic state is in a heterozygous state, AA or aa indicates that the diploid allele is in a homozygous state; "+” indicates that the disease trait is positive, and "-” indicates disease trait. Negative; if Aa corresponds to disease traits, it indicates that the defective gene exhibits a dominant trait, and if Aa corresponds to a non-disease trait, it indicates that the defective gene exhibits a recessive trait.
  • the allele haplotype is defined as A. If the site is consistent with the reference genome sequence and belongs to the majority population, the allele haplotype is defined as A. If the site is mutated (deletion, insertion or substitution), the allele haplotype is defined as a. If both diploid A and a are present, the sample is genotyped here as Aa. In this embodiment, the Sanger sequencing peak map is shown in conjunction with the patterns of FIGS. 4 and 5. These two peak maps show the allelic diploid heterogeneity and are base deletion heterogeneity and are therefore classified as Aa. While Figure 3 shows the allelic diploid homology of the same site, and the sequence is identical to the reference genome sequence, it is classified as AA. The genotyping results of the five genomic DNA samples were genetically analyzed to confirm that a containing the deletion could be used as the family-specific Mendelian genetic disease-associated allele haplotype variation tag for future PGD and PD probe design. Or compare references.
  • the allele haplotype variation tag found in Example 1 was searched and confirmed, and the DNA was translated into protein. The amino acid sequence of the protein was changed, which facilitated further confirmation of the variation of the allele at the site. Reliability of disease association.
  • the present invention further investigated the deletion mutation and found that the deletion mutation was recorded in the literature (Seki H, Kubota T, Ikegawa S, Haga N, Fujioka F, Ohzeki S, et al. Mutation frequency of EXT1 and EXT2in 43 Japanese Families with hereditary multiple exostoses. American journal of medical genetics. 2001; 99(1): 59-62. Epub 2001/02/15. PubMed PMID: 11170095.).
  • the deletion mutation can be determined as a confirmation allele Monomer type label. If there is no documented after the search, it indicates that the mutation site has not been discovered, and it belongs to the new family genetic variation label, which deserves further study.
  • a method for confirming a haplotype variant tag associated with a family of hereditary colon polyp diseases is substantially the same as in Example 1.
  • member A is about to have birth, and has a family Mendelian hereditary colon polyps, which is a female patient in both spouses; member B is the father of member A, is a patient with colonic polyposis; member C is a member A's mother; member D is member A's abdomen with colon polyp disease, is a positive control patient of the disease family; member E is member A's cousin, also D's non-colon polyp disease brother, is the disease family Negative control. Others were treated as in Example 1.
  • the variation of the two genes is the main cause of the disease, taking into account the mutations on the APC Accounted for the vast majority of cases. Therefore, the gene is first fully sequenced in the exon region to find base variations that may affect protein coding.
  • the APC gene-specific sequence designed in Table 3 was used for the target gene APC interval, and the sample tag sequence and the Illumina sequencing universal Adapter sequence were added at the 5' end of the design sequence and synthesized.
  • Primer oligonucleotides, a total of 49 pairs of primers with a target amplicon length of 500-600 bp were designed. See Table 3 for 49 pairs of primer sequences. These primers were then used for PCR amplification of the template DNA of 5 samples.
  • the DNA fragments which were inserted at 500-600 bp were subjected to deep parallel sequencing.
  • the target amplicon length limit is consistent with the optimized sequencing lengths mentioned in the methods of the invention, as the Illumina MiSeq 2 x 300 bp platform is used for sequencing.
  • Primer design principles are the same as in Example 1.
  • the DNA sequence of the disease in 5 members of the multiple colon polyp disease family is 8687 bp in length on the APC gene, and the 22nd in Table 3
  • a deletion mutation site of 5 bp in length was found, and the upstream and downstream sequences were: CACATAATAGAAGATGAAAT[AAAAC]AAAGTGAGCAAAGACAAT (the starting point of the universal annotation of the bp exon is the 1221th base, the end point is 1226 bases). This mutation caused an overall offset of the reading frame.
  • the deletion of the polybase is a genetic marker of a family of multiple colon polyps, and can be applied to the diagnosis of PGD of the family.
  • the invention adopts a sample of at least 5 persons in the family of Mendelian genetic diseases, and only adopts an intuitive trait and an allelic type comparison table for the method of sequencing the PCR amplicon of the exon of a specific disease-related gene, and is convenient for the implementer.
  • the operation the target area is small, the sequencing cost is low, and the implementation period is short; the sample can be ensured to confirm the reliability of the haplotype variation tag, and the detection cost is saved; the PGD or PD provided by the present invention determines the family-specific inheritance.
  • the method of disease-associated allele haplotype variation tags can be used to develop standardized IVD preimplantation genetic diagnosis PGD or pregnancy fetal diagnostic PD kits and diagnostic methods for specific genetic diseases.

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Abstract

本发明提供了一种家族特异性遗传病关联等位基因单体型变异标签确认方法。从孟德尔遗传疾病的家系中至少选取5人提取基因组DNA；获取与疾病相关的目标基因信息；对每个基因组DNA中的目标基因区间的DNA片段进行扩增、测序；分别挑选每个基因组DNA在目标基因区间存在的所有变异位点；获取每个变异位点在每个基因组DNA的基因型；变异位点在每个基因组DNA的分型结果结合疾病性状进行遗传分析，从而确认该疾病关联的等位基因单体型变异标签。

Description

家族特异性遗传病关联等位基因单体型变异标签确认方法 技术领域
本发明属于遗传学、分子生物学，具体涉及一种家族特异性遗传病关联等位基因单体型变异标签确认方法。
背景技术
胚胎植入前遗传诊断PGD是一种用于判断待植入试管婴儿胚胎是否具有疾病风险或者是携带疾病等位基因的方法统称。孕期胎儿诊断PD则是一种判断怀孕后的胎儿是否具有疾病风险或者是携带疾病等位基因的方法统称。随着细胞DNA序列特异性荧光标记以及单细胞测序方法的出现，PGD成为在胚胎早期检测单基因上所发生变异而导致遗传疾病的一种非常有效的处理手段。但是该方法所要求的单细胞或极少量DNA操作要求较高。而PD则可以采集多细胞的胎儿样本，DNA操作就相对容易。PGD和PD有的是针对整条染色体的(整倍体异常、例如唐氏综合症患儿)，有的则是针对局部DNA片段范围，甚至单个核苷酸的变异(含碱基的替换、插入以及缺失)来进行的。通过这种诊断，可以通过胚胎植入干预或者中止妊娠的方式在家族后代有效地减小疾病的风险，达到优生目的。
为了达到诊断局部DNA片段变异的目的，首先需要在某一家族里确认针对其遗传性疾病的特定DNA区域的变异标签，即在某一基因区域的找到局部替换、插入或缺失。这是进一步进行PGD和PD的重要前提。在此，为了确认针对遗传性疾病在特定家族里的特定参考物信息标签，即以较为经济且可靠的方式在特定DNA区域的找到局部替换、插入或缺失，并确认其与该家族的遗传疾病的关联性，是操作PGD和PD的重要前提。到目前为止，虽然有报道某些疾病关联点突变的某些个例，并可用于相应的PGD和PD诊断，但是针对不同疾病甚至同一症状疾病在不同家族里个性化PGD标签，一直都没有出现过统一的标准化方法。不同的机构或研究人员根据各自偏好，采用不同的采样标准和技术路线，获取各自认为可靠的PGD或PD诊断标签。因此，发明了一种方法，寻找针对不同单基因变异疾病关联的一个或多个家族特异性变异位点，并以其为目标设计用于PGD或者PD胚胎诊断的参考物。
发明内容
针对现有技术不足，本发明的目的是提供一种家族特异性遗传病关联等位基因单 体型变异标签确认方法。
为实现上述目的，本发明提供了一种家族特异性遗传病关联等位基因单体型变异标签确认方法。
一种家族特异性遗传病关联等位基因单体型变异标签确认方法，包括以下步骤，
1)从单个位点基因变异导致的遗传性疾病即孟德尔遗传疾病的家系中至少选取5人提取基因组DNA样本；
2)获取与所述疾病相关的目标基因信息；
3)对步骤1)提取的每个基因组DNA样本中的所述目标基因区间的DNA片段进行扩增、测序；
4)分别挑选每个基因组DNA样本在所述目标基因区间存在的所有变异位点；
5)获取每个所述变异位点在每个基因组DNA样本的纯合或杂合状态基因型AA、Aa或aa；
6)所述变异位点在每个基因组DNA样本的分型结果结合疾病性状进行遗传分析以确认疾病关联等位基因单体型，从而确认该疾病关联的等位基因单体型变异标签。
优选的，所述步骤1)，所选孟德尔遗传疾病的家系成员中，包括成员A为作为孟德尔遗传疾病患者或者缺陷基因单体型携带者并即将进行生育者，成员B为成员A的父亲，成员C为成员A的母亲，成员D为阳性对照，即患孟德尔遗传疾病，成员E为阴性对照，即无所述孟德尔遗传疾病性状。
进一步地，如果成员A的患疾病风险来自父系，则成员D和成员E从父系家族成员中选择；如果成员A的疾病风险来自母系，则成员D和成员E从母系家族成员中选择，其中成员E与家族中的其他疾病患者有直接的血缘关系，即父母、子女或者兄弟姐妹。
优选的，全基因组DNA样本来源于体细胞，并不局限于外周血，任何具有等位基因二倍体信息的体细胞，例如口腔上皮细胞、组织培养物、甚至毛发等临床样本。
优选的，步骤2)的目标基因信息通过文献和/或数据库确定外显子框架。
进一步地，步骤3)中所述扩增的引物设计在内含子上并覆盖外显子，或者设计多个扩增子引物而全面覆盖较长外显子。
优选的，步骤3)中所述测序为Sanger法测序或高通量测序。
进一步地，所述测序包括Sanger单克隆测序以及Illumina HiSeq、Life Technologies Ion Torrent、ABI SOLiD或454 pyrosequencing等为代表的高通量测序技术 (High-throughput sequencing)方法。在高通量测序中，又被称之为多重扩增子测序(Multiplex Amplicon Sequencing)。
进一步地，步骤4)中所述变异位点由缺失变异、替换变异或插入变异导致。
优选的，步骤6)所述的遗传分析采用如下基因分型和疾病状态对照表，如果找到记录，则二元多态性中罕见的等位基因单体型a作为单体型变异标签，其中，该对照表中Aa表示二倍体等位基因状态处于杂合子状态，AA或aa表示二倍体等位基因处于纯合子状态，“+”表示疾病性状为阳性，“-”表示疾病性状为阴性，
记录 本人 父亲 母亲 阳性对照 阴性对照
1 Aa|+ AA|- Aa|+ Aa|+ AA|-
2 Aa|+ AA|- Aa|+ aa|+ AA|-
3 Aa|- AA|- Aa|- aa|+ AA|-
4 Aa|- AA|- Aa|- aa|+ Aa|-
5 Aa|+ AA|- aa|+ Aa|+ AA|-
6 Aa|+ AA|- aa|+ aa|+ AA|-
7 Aa|- AA|- aa|+ aa|+ AA|-
8 Aa|- AA|- aa|+ aa|+ Aa|-
9 Aa|+ Aa|+ AA|- Aa|+ AA|-
10 Aa|+ Aa|+ AA|- aa|+ AA|-
11 Aa|- Aa|- AA|- aa|+ AA|-
12 Aa|- Aa|- AA|- aa|+ Aa|-
13 Aa|+ Aa|+ Aa|+ Aa|+ AA|-
14 Aa|+ Aa|+ Aa|+ aa|+ AA|-
15 Aa|- Aa|- Aa|- aa|+ AA|-
16 Aa|- Aa|- Aa|- aa|+ Aa|-
17 Aa|+ Aa|+ aa|+ Aa|+ AA|-
18 Aa|+ Aa|+ aa|+ aa|+ AA|-
19 Aa|- Aa|- aa|+ aa|+ AA|-
20 Aa|- Aa|- aa|+ aa|+ Aa|-
21 Aa|+ aa|+ AA|- Aa|+ AA|-
22 Aa|+ aa|+ AA|- aa|+ AA|-
23 Aa|- aa|+ AA|- aa|+ AA|-
24 Aa|- aa|+ AA|- aa|+ Aa|-
25 Aa|+ aa|+ Aa|+ Aa|+ AA|-
26 Aa|+ aa|+ Aa|+ aa|+ AA|-
27 Aa|- aa|+ Aa|- aa|+ AA|-
28 Aa|- aa|+ Aa|- aa|+ Aa|-
29 aa|+ Aa|+ Aa|+ Aa|+ AA|-
30 aa|+ Aa|- Aa|- aa|+ AA|-
31 aa|+ Aa|- Aa|- aa|+ Aa|-
32 aa|+ Aa|+ aa|+ Aa|+ AA|-
33 aa|+ Aa|- aa|+ aa|+ AA|-
34 aa|+ Aa|- aa|+ aa|+ Aa|-
35 aa|+ aa|+ Aa|+ Aa|+ AA|-
36 aa|+ aa|+ Aa|- aa|+ AA|-
37 aa|+ aa|+ Aa|- aa|+ Aa|-
38 aa|+ aa|+ aa|+ Aa|+ AA|-
39 aa|+ aa|+ aa|+ aa|+ AA|-
40 aa|+ aa|+ aa|+ aa|+ Aa|-。
优选的，所述等位基因单体型变异标签应用于试管婴儿胚胎植入前遗传诊断PGD或孕期胎儿诊断PD，以淘汰含有该等位基因单体型变异标签的高疾病风险的胚胎或胎儿。
本发明的另一方案，上述步骤6)后还包括将等位基因单体型变异标签进行检索确认有记载，并将DNA翻译成蛋白质，所述蛋白质的氨基酸序列发生改变，则进一步确认所述等位基因单体型变异标签与疾病关联的可靠性。
本发明的有益效果：
1、采用孟德尔遗传疾病的家系中至少5人样本，既可以保证样本足以确认单体型变异标签的可靠性，又节省了检测成本；
2、利用一部分孟德尔遗传疾病的关联基因区域已经非常清楚，只是不同家族有不 同的变异位点导致相似的疾病性状，根据文献和数据库对已有遗传疾病症状和特定基因的关联知识，采用只针对特定疾病关联基因外显子的PCR扩增子测序的方式，目标区域小，测序成本较低，实施周期短；
3、本发明采用直观的性状和等位基因型对照表的方式，便于该方法实施者的操作和理解；
4、配合PGD或PD确定家族特异性遗传病关联等位基因单体型变异标签的上游新方法，根据该方法开发针对特定遗传疾病的标准化胚胎植入前遗传诊断PGD或孕期胎儿诊断PD试剂盒以及诊断方法。
附图说明
图1家族特异性等位基因单体型变异标签的确认流程图。
图2本方法中外显子区间的引物设计原则。
图3在多发性骨疣病例中缺失突变位点基因型AA对应的测序碱基峰图。
图4在多发性骨疣病例中缺失突变位点基因型Aa对应的正向测序碱基峰图。
图5在多发性骨疣病例中缺失突变位点基因型Aa对应的反向测序碱基峰图。
图6在结肠息肉病例中缺失突变位点基因型Aa对应的二代测序读取与参考基因组序列的比对示意图。
图7在结肠息肉病例中缺失突变位点基因型AA对应的二代测序读取与参考基因组序列的比对示意图。
具体实施方式
以下实施例用于说明本发明，但不用来限制本发明的范围。
实施例1
用于某一家族多发性骨疣疾病(Hereditary Multiple Exostoses)关联等位基因单体型变异标签的确认方法，参见图1，包括以下步骤，
1)从该孟德尔遗传疾病家系中选取5人提取基因组DNA样本。选取5个家庭成员，成员A为即将进行生育，并患有家族孟德尔遗传疾病多发性骨疣，为夫妻双方中的男性；成员B为成员A的父亲，患有多发性骨疣疾病；成员C为成员A的母亲；成员D为成员A的堂姐，患有多发性骨疣疾病，是疾病家系的阳性对照；成员E为成员A的姑表弟，未患有多发性骨疣疾病，是疾病家系的阴性对照。其中，成员D和成员E来自有多发性骨疣疾病风险的父系家系成员。分别采集上述5个成员的外周静脉血5ml，并采用TaKaRa  MiniBEST全血DNA提取试剂盒提取DNA，具体细节可参考该试剂盒的操作手册说明。5个成员提取基因组DNA样本分别为样本A、样本B、样本C、样本D、样本E。
2)获取与疾病相关的目标基因信息。检索了在线人类孟德尔遗传学数据库(http://www.omim.org/)，并且根据相关文献(Cook A,Raskind W,Blanton SH,Pauli RM,Gregg RG,Francomano CA,et al.Genetic heterogeneity in families with hereditary multiple exostoses.American journal of human genetics.1993；53(1):71-9.Epub 1993/07/01.PubMed PMID:8317501；PubMed Central PMCID:PMC1682231和Wu YQ,Heutink P,de Vries BB,Sandkuijl LA,van den Ouweland AM,Niermeijer MF,et al.Assignment of a second locus for multiple exostoses to the pericentromeric region of chromosome 11.Human molecular genetics.1994；3(1):167-71.Epub 1994/01/01.PubMed PMID:8162019)了解到在8号染色体的EXT1与11号染色体EXT2以及在19号染色体EXT3(Le Merrer M,Legeai-Mallet L,Jeannin PM,Horsthemke B,Schinzel A,Plauchu H,et al.A gene for hereditary multiple exostoses maps to chromosome 19p.Human molecular genetics.1994；3(5):717-22.Epub 1994/05/01.PubMed PMID:8081357.)，三个基因的变异为该疾病发生的主要原因，考虑到EXT1和EXT2上的突变占了绝大多数的病例。因此先对这两个基因进行外显子区域的全面测序，从而寻找可能影响蛋白质编码的碱基变异。
3)对步骤1)提取的5个基因组DNA样本中的目标基因区间的DNA片段进行扩增，测序。在获取5个全基因组DNA以后，以5个全基因组DNA为模版，针对EXT1和EXT2两个基因在NCBI RefSeqGen最新的基因注释信息中总共26个外显子(EXT111个，EXT215个)，每个外显子都设计了一对或多对引物，参见表1，对模版DNA进行PCR扩增。
引物设计原则：参见图2，针对常规Sanger测序仪的片段长度400－900bp限制，并考虑到其双向测序的可能，对外显子设计PCR反应的扩增子长度限制在2倍测序仪单个反应所能够检测到的序列平均长度以内。以ABI 3730XL全自动DNA测序仪为例，单个反应可以获取平均长度为700bp的序列，那么定义扩增子最大长度为2×700bp＝1400bp。如果出现外显子大于该长度，则需要设计多于1个并且彼此有叠加的扩增子，以确保对整个外显子区间的100％覆盖率，从而不至于错过可能的突变。例如，表1中EXT1的第1号外显子和EXT2基因的第15号外显子，在RefSeqGen数据库注释长度分别为1735bp和1282bp，要全面覆盖需要设计至少2个重叠的扩增子。又以Life Technologies Ion PGMTM深度测序平台为例，目前其单向测序长度限制为400bp，则设计引物时应保证 扩增子长度应小于800bp，即需要至少3个扩增子来覆盖。在此实施例中，对这两个外显子采用了3个扩增子覆盖策略。
表1针对常规Sanger测序仪器的通用引物设计列表
Figure PCTCN2015072589-appb-000001
Figure PCTCN2015072589-appb-000002
4)分别挑选5个基因组DNA样本在目标基因区间存在的所有变异位点。经过多组Sanger单克隆测序，在该多发性骨疣疾病家族中的5个成员中，在EXT2基因上长度为567bp的第3号外显子的第258个碱基上，找到一个缺失突变位点[C]，其上下游序列为：CCGACAGTCC[C]ATCCCAGAGC(处于该567bp外显子通用注释的起点为第248个碱基，终点为第268个碱基)。本发明还采用Ion PGMTM对5个样本进行了多重扩增子深度测序(Multiplex Amplicon Sequencing)，获得同样的结果支持。
5)获取每个变异位点在5个基因组DNA样本的纯合或杂合状态基因型AA、Aa或aa。在5个基因组DNA样本中只表现为两种等位基因状态，分别为AA和Aa。而Aa出现在具有多发性骨疣疾病性状的样本A、样本B、样本D。因此为显性基因表型，体现在测序碱基峰图上，基因型AA对应的测序碱基峰如图3所示，即上述第3号外显子的第258个碱基位点，上下游序列与参考基因组序列完全一致，两个等位基因序列也一致。而基因型Aa对应的正向测序碱基峰如图4和反向测序碱基峰图5所示，由于两个等位基因中的一条存在一个碱基缺失，使得在该变异位点的正反两向测序都会在该变异位点的下游出现等位基因读框错位，属于典型等位基因异质并且包含缺失突变。因此，5个样本的基因分型和疾病状态对照表2，总结为表2中记录9中对应的5位采样家族成员的的基因型和表型情况。
表2基因分型和疾病状态对照表
Figure PCTCN2015072589-appb-000003
Figure PCTCN2015072589-appb-000004
Figure PCTCN2015072589-appb-000005
注：该对照表中Aa表示二倍体等位基因状态处于杂合子状态，AA或aa表示二倍体等位基因处于纯合子状态；“+”表示疾病性状为阳性，“-”表示疾病性状为阴性；如果Aa对应疾病性状则表示该缺陷基因呈现显性性状，如果Aa对应非疾病性状则表示该缺陷基因呈现隐性性状。
6)变异位点在5个基因组DNA样本的分型结果结合疾病性状进行遗传分析以确认多发性骨疣疾病疾病关联等位基因单体型，从而确认该疾病关联的等位基因单体型变异标签。
该位点如果和参考基因组序列一致且属于多数人群的情况，则等位基因单体型定义为A，如果该位点发生变异(缺失、插入或替换)，则等位基因单体型定义为a。如果二倍体A和a都存在，则该样本在此处基因分型为Aa。在本实施例中，体现在Sanger测序峰图则须结合图4和图5的模式来表示。这两个峰图显示等位基因二倍体异质，而且是碱基缺失异质，因此被分型为Aa情况。而图3显示同样位点的等位基因二倍体同质，而且序列和参考基因组序列一致，因而被分型为AA。在5个基因组DNA样本的分型结果进行遗传分析，确认包含该缺失的a可以作为该家族特异性孟德尔遗传疾病关联等位基因单体型变异标签，用于今后PGD和PD的探针设计或比对参考。
实施例2
将实施例1中找到等位基因单体型变异标签进行检索，确认有记载，并将DNA翻译成蛋白质，蛋白质的氨基酸序列发生改变，则有利于进一步确认等位基因在该位点的变异与疾病关联的可靠性。本发明进一步对该缺失突变进行了文献调研，发现该缺失突变曾经在文献中有记载(Seki H,Kubota T,Ikegawa S,Haga N,Fujioka F,Ohzeki S,et al.Mutation frequencies of EXT1and EXT2in 43Japanese families with hereditary multiple exostoses.American journal of medical genetics.2001；99(1):59-62.Epub 2001/02/15.PubMed PMID:11170095.)。由此，确定该缺失变异可以作为确认等位基因 单体型标签。如果检索后，没有文献记载，则说明该变异位点尚未被发现，则属于新的家族遗传性变异标签，值得进一步进行研究。
实施例3应用
在成功认定实施例1确认的等位基因单体型序列标签以后，申请人和第三方机构(北京大学生物动态光学成像中心和北医三院生殖中心)通过胚胎单细胞操作以及MALBAC扩增方法(Zong C,Lu S,Chapman AR,Xie XS.Genome-wide detection of single-nucleotide and copy-number variations of a single human cell.Science.2012；338(6114):1622-6.Epub 2012/12/22.doi:10.1126/science.1229164.PubMed PMID:23258894；PubMed Central PMCID:PMC3600412.)对EXT2的目标外显子片段进行测序，从而从18个体外授精的胚胎中确认了4个不包含该缺失突变的，等位基因状态表现为AA的优质体外授精的胚胎用于植入。而其它二倍体等位基因分型表示为Aa的胚胎或者某些整倍体异常的被淘汰。
实施例4应用
某一家族遗传性结肠息肉疾病关联等位基因单体型变异标签的确认方法，步骤基本同实施例1。
1)从该孟德尔遗传疾病家系中选取5人提取基因组DNA样本。选取5个家庭成员，成员A为即将进行生育，并患有家族孟德尔遗传疾病结肠息肉，为夫妻双方中的女性患者；成员B为成员A的父亲，是结肠息肉疾病患者；成员C为成员A的母亲；成员D为成员A的患有结肠息肉疾病的姑表姐，是疾病家系的阳性对照患者；成员E为成员A的姑表弟，也是D的无结肠息肉疾病亲兄弟，是疾病家系的阴性对照。其它同实施例1处理。
2)获取与疾病相关的目标基因信息。检索在线人类孟德尔遗传学数据库(http://www.omim.org/)，并且根据相关文献(Soravia C1,Berk T,Madlensky L,Mitri A,Cheng H,Gallinger S,Cohen Z,Bapat B.,Genotype-phenotype correlations in attenuated adenomatous polyposis coli.Americal Journal of Human Genetics,1998:Vol.62:6,P2190-1301)了解到在5号染色体的APC与1号染色体MUTYH(Sampson JR,Dolwani S,Jones S,Eccles D,Ellis A,Evans DG,Frayling I,Jordan S,Maher ER,Mak T,Maynard J,Pigatto F,Shaw J,Cheadle JP(2003)."Autosomal recessive colorectal adenomatous polyposis due to inherited mutations of MYH".Lancet 362(9377):39-41.doi:10.1016/S0140-6736(03)13805-6.PMID 12853198)，两个基因的变异为该疾病 发生的主要原因，考虑到APC上的突变占了绝大多数的病例。因此先对这个基因进行外显子区域的全面测序，从而寻找可能影响蛋白质编码的碱基变异。
3)对步骤1)提取的5个全基因组DNA样本，针对目标基因APC区间利用表3设计的APC基因特异性序列并在设计序列5‘端添加样本标签序列和Illumina测序通用Adapter序列并合成的引物寡核苷酸，总共设计了49对目标扩增子长度为500－600bp的引物。49对引物序列参见表3。然后用这些引物对5个样本的模版DNA进行PCR扩增。对产生插入为500-600bp的DNA片段进行深度并行测序。该目标扩增子长度限制符合在本发明方法中提到的优化测序长度，因为此处采用的是Illumina MiSeq 2×300bp的平台进行测序。引物设计原则同实施例1。
表3针对常规Illumina MiSeq 2×300bp测序平台的APC特异引物设计列表
Figure PCTCN2015072589-appb-000006
Figure PCTCN2015072589-appb-000007
Figure PCTCN2015072589-appb-000008
4)在Illumina MiSeq测序仪上完成深度并行测序以后，该多发性结肠息肉疾病家族中的5个成员中的疾病患者的DNA序列上，在APC基因上长度为8687bp，表3中第22号外显子的第1221碱基上，找到一个长度为5bp的缺失突变位点，其上下游序列为：CACATAATAGAAGATGAAAT[AAAAC]AAAGTGAGCAAAGACAAT(处于该bp外显子通用注释的起点为第1221个碱基，终点为第1226个碱基)。该突变造成了读码框的整体偏移。
5)同实施例1中描述情况，有该缺失突变等位基因的标记为a，没有该等位基因缺失突变的标记为A，结果见图6、7。5个家族成员样本的基因型和疾病表型状态对照表2，也总结为记录9的情况。从而确认该多碱基的缺失为家族多发性结肠息肉的遗传标签，可以应用于该家族的PGD诊断。
工业实用性
本发明采用孟德尔遗传疾病的家系中至少5人样本，只针对特定疾病关联基因外显子的PCR扩增子测序的方式，采用直观的性状和等位基因型对照表的方式，便于实施者的操作，目标区域小，测序成本较低，实施周期短；既可以保证样本足以确认单体型变异标签的可靠性，又节省了检测成本；本发明提供的配合PGD或PD确定家族特异性遗传病关联等位基因单体型变异标签的方法可用于开发针对特定遗传疾病的标准化试管婴儿胚胎植入前遗传诊断PGD或孕期胎儿诊断PD试剂盒以及诊断方法。
虽然，上文中已经用一般性说明、具体实施方式及试验，对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。
Figure PCTCN2015072589-appb-000009
Figure PCTCN2015072589-appb-000010
Figure PCTCN2015072589-appb-000011
Figure PCTCN2015072589-appb-000012
Figure PCTCN2015072589-appb-000013
Figure PCTCN2015072589-appb-000014
Figure PCTCN2015072589-appb-000015
Figure PCTCN2015072589-appb-000016
Figure PCTCN2015072589-appb-000017
Figure PCTCN2015072589-appb-000018
Figure PCTCN2015072589-appb-000019
Figure PCTCN2015072589-appb-000020
Figure PCTCN2015072589-appb-000021
Figure PCTCN2015072589-appb-000022
Figure PCTCN2015072589-appb-000023
Figure PCTCN2015072589-appb-000024
Figure PCTCN2015072589-appb-000025
Figure PCTCN2015072589-appb-000026
Figure PCTCN2015072589-appb-000027
Figure PCTCN2015072589-appb-000028
Figure PCTCN2015072589-appb-000029
Figure PCTCN2015072589-appb-000030

Claims (10)

  1. 一种家族特异性遗传病关联等位基因单体型变异标签确认方法，其特征在于，包括以下步骤，
    1)从单个位点基因变异导致的遗传性疾病即孟德尔遗传疾病的家系中至少选取5人提取基因组DNA样本；
    2)获取与所述疾病相关的目标基因信息；
    3)对步骤1)提取的每个基因组DNA样本中的所述目标基因区间的DNA片段进行扩增、测序；
    4)分别挑选每个基因组DNA样本在所述目标基因区间存在的所有变异位点；
    5)获取每个所述变异位点在每个基因组DNA样本的纯合或杂合状态基因型AA、Aa或aa；
    6)所述变异位点在每个基因组DNA样本的分型结果结合疾病性状进行遗传分析以确认疾病关联等位基因单体型，从而确认该疾病关联的等位基因单体型变异标签。
  2. 根据权利要求1所述的方法，其特征在于，所述步骤1)，所选孟德尔遗传疾病的家系成员中，包括成员A为作为孟德尔遗传疾病患者或者缺陷基因单体型携带者并即将进行生育者，成员B为成员A的父亲，成员C为成员A的母亲，成员D为阳性对照，即患孟德尔遗传疾病，成员E为阴性对照，即无所述孟德尔遗传疾病性状。
  3. 根据权利要求2所述的方法，其特征在于，如果成员A的患疾病风险来自父系，则成员D和成员E从父系家族成员中选择；如果成员A的疾病风险来自母系，则成员D和成员E从母系家族成员中选择，其中成员E与家族中的其他疾病患者有直接的血缘关系，即父母、子女或者兄弟姐妹。
  4. 根据权利要求1所述的方法，其特征在于，步骤2)的目标基因信息通过文献和/或数据库确定外显子框架。
  5. 根据权利要求4所述的方法，其特征在于，步骤3)中所述扩增的引物设计在内含子上并覆盖外显子，或者设计多个扩增子引物而全面覆盖较长外显子。
  6. 根据权利要求1所述的方法，其特征在于，步骤3)中所述测序为Sanger法测序或高通量测序。
  7. 根据权利要求6所述的方法，其特征在于，步骤4)中所述变异位点由缺失变异、替换变异或插入变异导致。
  8. 根据权利要求1所述的方法，其特征在于，步骤6)所述的遗传分析采用如下基因分型和疾病状态对照表，如果找到记录，则二元多态性中罕见的等位基因单体型a作为单体型变异标签，其中，该对照表中Aa表示二倍体等位基因状态处于杂合子状态，AA或aa表示二倍体等位基因处于纯合子状态，“+”表示疾病性状为阳性，“-”表示疾病性状为阴性，
    Figure PCTCN2015072589-appb-100001
    Figure PCTCN2015072589-appb-100002
  9. 根据权利要求1-8任一所述的方法，其特征在于，步骤6)后还包括将等位基因单体型变异标签进行检索确认有记载，并将DNA翻译成蛋白质，所述蛋白质的氨基酸序列发生改变，则进一步确认所述等位基因单体型变异标签与疾病关联的可靠性。
  10. 根据权利要求1-9任一所述的方法，其特征在于，所述等位基因单体型变异标签应用于试管婴儿胚胎植入前遗传诊断PGD或孕期胎儿诊断PD，以淘汰含有该等位基因单体型变异标签的高疾病风险的胚胎或胎儿。
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