WO2024027569A1 - 一种不依赖于先证者的单体型构建方法 - Google Patents

一种不依赖于先证者的单体型构建方法 Download PDF

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WO2024027569A1
WO2024027569A1 PCT/CN2023/109731 CN2023109731W WO2024027569A1 WO 2024027569 A1 WO2024027569 A1 WO 2024027569A1 CN 2023109731 W CN2023109731 W CN 2023109731W WO 2024027569 A1 WO2024027569 A1 WO 2024027569A1
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pathogenic
haplotype
parents
embryos
carrier
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French (fr)
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许瑞霞
顾梦南
单文琪
杨玉妍
孔令印
梁波
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苏州贝康医疗器械有限公司
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    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B20/00—ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
    • G16B20/30—Detection of binding sites or motifs
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B20/00—ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
    • G16B20/50—Mutagenesis
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A90/00—Technologies having an indirect contribution to adaptation to climate change
    • Y02A90/10—Information and communication technologies [ICT] supporting adaptation to climate change, e.g. for weather forecasting or climate simulation

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  • This application belongs to the field of genetic testing technology and relates to a haplotype construction method that does not depend on the proband.
  • Monogenic abnormalities are a type of disease caused by mutations in a single gene.
  • the transmission of these diseases between parents and children follows Mendel's laws, so they are also called Mendelian genetic diseases.
  • Traditional prenatal diagnosis during pregnancy can prevent the birth of sick children, but termination of pregnancy brings great pain and mental burden to pregnant women and their families. Therefore, fundamentally blocking the transmission of genes responsible for genetic diseases is of great significance and clinical value.
  • Chromosomal structural variations include chromosomal translocations and inversions. Chromosomal translocations are divided into reciprocal translocations and Robertsonian translocations. The incidence of chromosomally balanced translocations in the population is 0.19%, and its carriers usually There are no obvious phenotypic abnormalities, but it is closely related to adverse pregnancy outcomes such as recurrent miscarriage, stillbirth, stillbirth, neonatal mental retardation and other congenital malformations.
  • PTT-M monogenic diseases
  • PTT-SR chromosomal structural variations
  • FISH FISH
  • arrayCGH microarray-comparative genomic hybridization
  • SNP array single nucleotide polymorphism microarray
  • NGS next-generation sequencing
  • the pathogenic chain of the father or mother is used to determine the pathogenicity of the embryo/fetus.
  • the above method cannot be used to determine whether the embryo/fetus carries a pathogenic variant in the gene.
  • This application provides a haplotype construction method that does not rely on the proband.
  • sequencing only the chromosomal abnormality carrier the pathogenic site and its upstream and downstream sequence information can be directly obtained, which will help solve clinical problems. It is difficult to detect PGT-M/PGT-SR in non-proband/imbalanced embryo samples, thus expanding the scope of application of PGT-M/PGT-SR.
  • this application provides a method for constructing haplotypes that does not depend on the proband, and the method includes:
  • a haplotype construction method is designed that does not depend on the proband. Only long-segment sequencing of the parents of the chromosome pathogenic variant carriers, combined with SNPs site screening, can be independent of the proband or reference. , to obtain the pathogenic/normal haplotype of chromosomal abnormality carriers, which can then be used to determine whether the embryo has inherited the pathogenic variant, thereby assisting in screening normal embryos for implantation and avoiding the birth of children carrying the pathogenic variant.
  • long fragment sequencing can be a single-molecule long-read sequencing method, or it can be a method that indirectly implements long fragment sequencing by introducing sequence identifiers into large fragments.
  • the genotype information of the pathogenic variant includes structural variation, point mutation or indel, and duplication.
  • the method of marking the haplotype of a parent includes marking the haplotype of a parent as a specific color.
  • the haplotype color of the parent can be marked according to requirements.
  • the chromosome chain carrying the pathogenic variant is marked in blue.
  • the method further includes the step of extracting genomic DNA from both spouses.
  • the proband-independent haplotype construction method includes the following steps:
  • the present application provides a device for screening embryos for genetic diseases and chromosomal abnormalities.
  • the device includes a haplotype building unit and a screening unit.
  • the haplotype building unit is used to perform the first step.
  • the method for constructing a haplotype that does not depend on the proband described in the aspect; the screening unit is used to perform the following steps: perform whole-genome SNP analysis on the whole-genome amplification product of the offspring embryo sample; and conduct a whole-genome SNP analysis based on the pathogenic variant carriers.
  • the haplotype results of the parents were used to perform SNP linkage analysis on the homozygous genotypes of the offspring embryos, and the typing results of a single chromosome chain of the offspring embryos were obtained.
  • the pathogenic/normal haplotypes of chromosomal abnormality carriers are constructed, combined with SNP linkage analysis correction of embryos , can achieve accurate haplotype typing of the entire chromosome, and then determine whether the embryo carries a pathogenic mutation, which can help such situations where proband samples cannot be obtained, new mutations or consanguineous marriages have a large number of homologous regions and cannot construct haplotypes.
  • the whole genome amplification (WGA) method can be a whole genome amplification method based on PCR, a whole genome amplification method based on isothermal amplification, or amplification based on multiple annealing circular cycles. technology for whole-genome amplification.
  • the screening unit is further configured to perform a step including whole genome amplification of embryo samples, the embryo samples including embryo biopsy samples.
  • the embryo biopsy sample includes a blastomere biopsy sample or a blastocyst trophectoderm biopsy sample.
  • the screening unit is further configured to perform steps including correcting incorrect assembly.
  • a haplotype unit is used to perform the following steps:
  • the screening unit is used to perform the following steps:
  • proband or family-linked haplotype is required: This application does not rely on the haplotype construction method of the proband. It only performs long-segment sequencing on carriers of chromosomal pathogenic variants to construct the carrier's haplotype. Type, can be applied to genetic screening, combined with SNP linkage analysis and correction of embryos, to achieve accurate haplotype typing of the entire chromosome, and then determine whether the embryo carries a pathogenic mutation;
  • High-precision, long-fragment sequencing can easily obtain balanced translocation breakpoints across the entire genome, including complex regions of the genome, and can locate breakpoints to single bases level, significantly improving the sensitivity and accuracy of structural variation (SV) detection; and
  • Figure 1 shows the detection results of the analysis method used in the haplotype construction method of the present application that does not rely on the proband in Example 1.
  • Figure 2 shows the detection results of the conventional PGH family linkage analysis method in Comparative Example 1.
  • Figure 3 shows the detection results of the analysis method used in the haplotype construction method of the present application that is not dependent on the proband in Example 2.
  • Figure 4 shows the detection results of the conventional PGH family linkage analysis method in Comparative Example 2.
  • This embodiment provides a haplotype construction method that does not rely on balanced translocation in the proband or reference, and combines it with SNP linkage analysis of embryos to detect preimplantation chromosome structural variation.
  • genomic DNA samples of CYJ a balanced translocation carrier, were constructed and sequenced according to the library construction and sequencing instructions of the long fragment sequencing platform.
  • PGH chips for SNP genotype detection.
  • Each PGH chip contains 690,000 SNPs, which can fully cover 23 pairs of human chromosomes.
  • the PGH chip SNP genotypes were performed on the WGA amplification products of embryonic trophectoderm biopsy cells of the patient's two offspring. detection. The specific experimental methods are carried out according to the instructions and will not be repeated here.
  • Genotyping of balanced translocation carriers Perform whole-genome analysis on the long-fragment sequencing raw data of balanced translocation carriers to obtain genotype information of their pathogenic variants (including structural variations, point mutations, indels, and duplications) );
  • step (3) Construct the haplotype of the balanced translocation carrier: assemble the heterozygous SNPs determined in step (2) for whole-genome assembly and typing; obtain the translocation/normal haplotype of the parents of the balanced translocation carrier; according to flat The typing results of balanced translocation mark the haplotype color of the parents (blue indicates the chromosome chain carrying the balanced translocation);
  • This embodiment provides a method for constructing haplotypes of single-gene diseases that are independent of the proband or reference and combines them with SNP linkage analysis of embryos to perform pre-implantation single-gene genetic disease detection.
  • Genomic DNA samples were constructed and sequenced according to the library construction and sequencing instructions of the long fragment sequencing platform.
  • PGH chip SNP genotype detection was performed on the WGA amplification products of the patient's two offspring embryonic trophectoderm biopsy cells.
  • Genotyping of single-gene disease carriers Perform whole-genome analysis on the long-segment sequencing raw data of single-gene disease carriers to obtain genotype information of their pathogenic variants (including point mutations and indels, Repeat, etc.);
  • step (3) Construct the haplotypes of single gene disease carriers: assemble the heterozygous SNPs determined in step (2) for whole genome assembly and typing; obtain the pathogenic/normal haplotypes of the parents of single gene disease carriers; according to The typing results of single-gene disease pathogenic variants mark the haplotype color of the parents (blue indicates the carrying chromosome chain);
  • Sample genotyping SNP genotype detection was performed on the WGA amplification products of the embryo biopsy samples of the male (normal), female (balanced translocation), female mother (reference), and two offspring of the couple in the family. Specifically, The experimental method was carried out according to the instructions and will not be repeated here;
  • step (3) Construct family-linked haplotypes: The effective SNPs sites determined in step (2) are collected and used to obtain monomers covering the two break areas, the entire chromosome of the translocated chromosome and the entire chromosome of the homologous chromosome through family linkage analysis. Type, the collection of different chromosome haplotypes is called family haplotype.
  • the haplotype information of the breakpoint region in the embryo (blastocyst) cells is compared with the chromosome haplotype information of the reference sample to distinguish whether the embryo carries a balanced translocation.
  • Sample genotyping Conduct large-scale SNP on samples from couples and probands of single-gene disease patients genotype testing;
  • step (3) Construct the reference sample haplotype in the family haplotype: Collect the effective SNPs sites determined in step (2), obtain the haplotype covering the region of the pathogenic variant through family linkage analysis, and obtain the reference haplotype in the family haplotype Sample haplotype.
  • the haplotype information of the pathogenic mutation region in the embryo (blastocyst) cells is compared with the haplotype information of the reference sample to distinguish whether the embryo carries the pathogenic mutation.
  • Example 1 the typing results of balanced translocation family samples detected by the method of the present application are shown in Figure 1.
  • the blank area of the long arm of the CYJ01E chromosome is the unbalanced translocation area, and the start and end sites of the area are the break points.
  • the long arm of the CYJ02E chromosome is yellow.
  • the block crosses the break point and is a balanced translocation carrier;
  • the results of the conventional PGH family linkage analysis method in Comparative Example 1 are shown in Figure 2.
  • the results of the application method in Example 1 and the conventional PGH family linkage analysis method in Comparative Example 1 For comparison, the results are shown in Table 3.
  • the karyotype results of the woman and her mother in the balanced translocation family are balanced translocation carriers; the detection results of the method of this application are consistent with the karyotype and PGH results of the embryo, and the breakpoint of the carrier is The results are consistent with those of embryonic PGH, indicating that this application can construct haplotypes independently of the proband or reference, and can be effectively used in genetic screening.
  • the typing results of single gene disease family samples detected by the method of the present application in Example 2 are shown in Figure 3.
  • the results of the conventional PGH family linkage analysis method in Comparative Example 2 are shown in Figure 4.
  • the method of the present application in Example 1 and the Comparative Example are shown in Figure 4. 1 Comparing the results of conventional PGH family linkage analysis methods (Table 4), the single sample haplotype typing results of the method of this application are consistent with the PGH typing results, and the offspring all carry the ⁇ -thalassaemia mutation.
  • this application provides a haplotype construction method that does not rely on the proband or reference. It only performs long-segment sequencing on carriers of chromosomal pathogenic variants to construct the haplotype of the carrier, which can be effectively used in Genetic screening, combined with SNP linkage analysis and correction of embryos, can achieve accurate haplotype typing of the entire chromosome, and then determine whether the embryo carries a pathogenic mutation, helping to obtain proband samples, new mutations, or the existence of consanguineous marriages. It is difficult to screen normal embryos for patients undergoing traditional PGT-M/PGT-SR treatment due to factors such as a large number of homologous regions and the inability to construct haplotypes.

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Abstract

公开了一种不依赖于先证者的单体型构建方法，其包括对夫妻双方中致病变异携带者亲本一方的DNA样本进行长片段测序；对致病变异携带者亲本的测序数据进行全基因组分析，获得其致病变异的基因型信息；筛选在致病变异携带者亲本中的杂合型SNPs；集合杂合型SNPs进行全基因组组装与分型；根据致病变异所在的长片段组装信息，获得致病变异位点的分型结果；根据致病位点的分型结果标记致病变异携带者亲本的单体型，构建致病变异携带者亲本的致病/正常单体型。本申请仅对染色体致病变异携带者进行长片段测序，构建携带者的单体型，可有效应用于遗传筛查，结合胚胎的SNP连锁分析校正，可实现整条染色体的单体型准确分型。

Description

一种不依赖于先证者的单体型构建方法 技术领域
本申请属于基因检测技术领域，涉及一种不依赖于先证者的单体型构建方法。
背景技术
单基因异常是一类由单个基因突变引起的疾病，这类疾病在亲子之间传递规律遵循孟德尔定律，因此也被称为孟德尔遗传病。传统的孕期产前诊断可以避免患病儿的出生，但是终止妊娠给孕妇及其家庭带来较大痛苦和精神负担。因此，从根本上阻断遗传病致病基因的传递具有重大的意义和临床价值。
染色体结构变异包括染色体易位和倒位，染色体易位分为相互易位(reciprocal translocation)和罗伯逊易位(Robertsonian translocation)，染色平衡易位在人群中的发病率为0.19％，其携带者通常没有明显的表型异常，但与反复流产、死胎、死产、新生儿精神发育迟缓及其他先天畸形等不良妊娠结局密切相关。目前报道用于单基因病胚胎植入前遗传学检测(PGT-M)和植入前染色体结构变异遗传学检测(PGT-SR)的检测技术相似，包括FISH，微阵列-比较基因组杂交技术(arrayCGH)，单核苷酸多态性微阵列芯片(SNP array)和二代测序(NGS)。
由于PGT-M和PGT-SR检测依赖于胚胎微量样本的扩增，扩增过程中可能发生等位基因脱扣(Allele drop-out，ADO)，即其中一个等位基因优势扩增或完全扩增失败，造成检测结果的假阳性或假阴性。因此，临床上常利用家系连锁单体型分析来提高胚胎植入前遗传学检测(PGT检测)的准确性。首先对夫妻双方、先证者或参照者，即与先证者具有血缘关系的相关亲属的样本来构建正常/致病的单体型，基于先证者(或参照者)区分胚胎/胎儿的父亲或母亲的致病链，进而判断胚胎/胎儿的致病性。但是，对于特殊家系，如在无先证者或参照者的情况下，通过上述方法就不能对胚胎/胎儿的基因中是否携带致病变异进行判断。
综上所述，在临床实践中，部分单基因病患者/结构变异携带者出于种种原因无法获得先证者和参照者的基因组样本，或者夫妻携带的突变属于新发突变， 无法通过家系连锁分析构建单体型，还有少量患者因近亲婚配存在大量同源区域而无法构建单体型，限制了PGT检测技术的应用，因此，开发不依赖于先证者的单体型构建方法，对于基因检测技术领域具有重要意义。
发明内容
本申请提供一种不依赖于先证者的单体型构建方法，仅通过对染色体异常携带者一方进行测序，即可直接获得致病位点及其上下游序列信息，将有助于解决临床无先证者/不平衡胚胎样本难以进行PGT-M/PGT-SR检测的难题，扩大PGT-M/PGT-SR的适用范围。
第一方面，本申请提供一种不依赖于先证者的单体型构建方法，所述方法包括：
对夫妻双方中致病变异携带者亲本一方的DNA样本进行长片段测序；
对致病变异携带者亲本的测序数据进行全基因组分析，获得其致病变异的基因型信息；
筛选在致病变异携带者亲本中的杂合型SNPs(单核苷酸多态性)；以及
集合所述杂合型SNPs进行全基因组组装与分型；根据致病变异所在的长片段组装信息，获得致病变异位点的分型结果；根据致病位点的分型结果标记致病变异携带者亲本的单体型，构建致病变异携带者亲本的致病/正常单体型。
本申请中，设计一种不依赖于先证者的单体型构建方法，仅对染色体致病变异携带者亲本进行长片段测序，结合SNPs位点筛选，能够不依赖于先证者或者参照者，获得染色体异常携带者致病/正常单体型，进而可应用于判断胚胎是否遗传了致病变异，从而辅助筛选正常的胚胎进行植入，避免携带致病变异的患儿出生。
本申请中，长片段测序可以是单分子长读长测序方法，也可以是通过对大片段引入序列标识间接实现长片段测序的方法。
优选地，所述致病变异的基因型信息包括结构变异、点突变或插入缺失、重复。
优选地，所述标记亲本的单体型的方法包括标记亲本的单体型为特定颜色。
本申请中，可根据需求标记亲本的单体型颜色，如本申请具体实施例中将致病变异携带染色体链标记为蓝色。
优选地，所述方法还包括提取夫妻双方基因组DNA的步骤。
作为优选的技术方案，所述不依赖于先证者的单体型构建方法包括以下步骤：
(1)对夫妻双方中致病变异携带者一方的DNA样本进行长片段测序；
(2)对致病变异携带者亲本的测序数据进行全基因组分析，获得其致病变异的基因型信息；
(3)筛选在致病变异携带者亲本中的杂合型SNPs；以及
(4)集合所述杂合型SNPs进行全基因组组装与分型；根据致病变异所在的长片段组装信息，获得致病变异位点的分型结果；根据致病位点的分型结果标记致病变异携带者亲本的单体型，构建致病变异携带者亲本的致病/正常单体型。
第二方面，本申请提供一种对胚胎进行遗传病筛查和染色体异常筛查的装置，所述装置包括构建单体型单元和筛查单元，所述构建单体型单元用于执行第一方面所述的不依赖于先证者的单体型构建方法；所述筛查单元用于执行包括：对子代胚胎样本的全基因组扩增产物进行全基因组SNP分析；根据致病变异携带者亲本的单体型结果对子代胚胎的纯合基因型进行SNP连锁分析，得到子代胚胎单条染色体链的分型结果。
本申请中，基于所述不依赖于先证者的单体型构建方法，不依赖于先证者或者参照者，构建染色体异常携带者致病/正常单体型，结合胚胎的SNP连锁分析校正，可实现整条染色体的单体型准确分型，进而判断胚胎是否携带了致病突变，帮助无法获得先证者样本、新发突变或近亲婚配存在大量同源区域而无法构建单体型等因素难以进行传统PGT-M/PGT-SR治疗的患者筛选染色体正常的胚胎。
本申请中，全基因组扩增(WGA)方法可以是以PCR为基础的全基因组扩增方法，也可以是以等温扩增为基础的全基因组扩增方法、基于多次退火环状循环扩增技术的全基因组扩增方法。
优选地，所述筛查单元还用于执行包括对胚胎样本进行全基因组扩增的步骤，所述胚胎样本包括胚胎活检样本。
优选地，所述胚胎活检样本包括卵裂球活检样本或囊胚滋养外胚层活检样本。
优选地，所述筛查单元还用于执行包括对错误组装进行校正的步骤。
本申请中，由于亲本存在错误组装的block，会在胚胎之间的单体型结果中有相同的断点，对上述的错误组装情况进行校正，得到准确的分型结果。
作为优选的技术方案，所述构建单体型单元用于执行包括以下步骤：
(1)对夫妻双方中致病变异携带者一方的DNA样本进行长片段测序；
(2)对致病变异携带者亲本的测序数据进行全基因组分析，获得其致病变异的基因型信息；
(3)筛选在致病变异携带者亲本中的杂合型SNPs；以及
(4)集合所述杂合型SNPs进行全基因组组装与分型；根据致病变异所在的长片段组装信息，获得致病变异位点的分型结果；根据致病位点的分型结果标记致病变异携带者亲本的单体型，构建致病变异携带者亲本的致病/正常单体型；
所述筛查单元用于执行包括以下步骤：
(1)对子代胚胎样本的全基因组扩增产物进行全基因组SNP分析；
(2)根据致病变异携带者亲本的单体型结果对子代胚胎的纯合基因型进行SNP连锁分析，对错误组装进行校正，得到子代胚胎单条染色体链的分型结果。
与现有技术相比，本申请具有以下有益效果：
(1)无需先证者，不需要家系连锁单体型：本申请不依赖于先证者的单体型构建方法，仅对染色体致病变异携带者进行长片段测序，构建携带者的单体型，可应用于遗传筛查，结合胚胎的SNP连锁分析校正，实现整条染色体的单体型准确分型，进而判断胚胎是否携带了致病突变；
(2)通用性强：对样本进行一次测序，即可获得样本全基因组范围内的各种变异信息，应用于不同的分析流程同时获得PGT-A、PGT-M、PGT-SR和其他的胚胎异常状态结果，无需针对不同的染色体异常进行个体化设计；
(3)检测全面：通过高精度长片段测序，一次测序获得高准确度的SNP/InDel/CNV/SV变异检测结果，以及单体型分型结果，检测更加精准，范围更广，适用于各种易位、倒位、染色体非整倍体、缺失、重复、单基因病、CNV变异来源分析等；
(4)高灵敏度和精确度：高精度、长片段测序，轻松获取包括基因组复杂区域在内的全基因组范围内的平衡易位断裂点，且能够将断裂点定位至单碱基 水平，显著提高结构变异(SV)检出的灵敏度和精确度；以及
(5)不需要分析子代的CNV和结构变异等，简化分析步骤，减少人工判读。
附图说明
图1为实施例1中应用于本申请不依赖于先证者的单体型构建方法的分析方法的检测结果。
图2为对比例1中常规PGH家系连锁分析方法的检测结果。
图3为实施例2中应用于本申请不依赖于先证者的单体型构建方法的分析方法的检测结果。
图4为对比例2中常规PGH家系连锁分析方法的检测结果。
具体实施方式
为进一步阐述本申请所采取的技术手段及其效果，以下结合实施例和附图对本申请作进一步地说明。可以理解的是，此处所描述的具体实施方式仅仅用于解释本申请，而非对本申请的限定。
实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件，或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可通过正规渠道商购获得的常规产品。
实施例1
本实施例提供不依赖于先证者或参照者的平衡易位的单体型构建方法并结合胚胎的SNP连锁分析进行胚胎植入前染色体结构变异检测。
接受辅助生殖的染色体平衡易位携带者家庭(已有染色体核型检测结果)，患者家系染色体核型如表1所示，抽取染色体平衡易位携带者及其配偶和患者直系亲属的外周血5mL，于EDTA抗凝采血管中保存，同时获取两对夫妻各自的胚胎活检样本经过全基因组扩增后的产物，家系外周血样本按照本领域高分子量DNA提取方法提取基因组DNA。
表1
1、平衡易位携带者单样本长片段建库测序
平衡易位携带者CYJ基因组DNA样本参照长片段测序平台建库测序说明书进行建库测序。
2、胚胎PGH芯片SNP基因型检测
使用PGH芯片进行SNP基因型检测，每个PGH芯片包含69万个SNPs，可全面覆盖人23对染色体，对患者的两个子代胚胎滋养外胚层活检细胞的WGA扩增产物进行PGH芯片SNP基因型检测。具体实验方法参照说明书进行，在此不再赘述。
3、平衡易位携带者单样本单体型构建
(1)平衡易位携带者基因分型：对平衡易位携带者的长片段测序原始数据进行全基因组分析，获得其致病变异的基因型信息(包括结构变异、点突变和插入缺失、重复)；
(2)确定SNPs位点：筛选在染色体平衡易位携带者中为杂合型的SNPs；
(3)构建平衡易位携带者的单体型：集合步骤(2)确定的杂合型SNPs进行全基因组组装与分型；获得平衡易位携带者亲本的易位/正常单体型；根据平 衡易位的分型结果标记亲本的单体型颜色(蓝色为平衡易位携带染色体链)；
4、构建子代胚胎的单体型：根据平衡易位携带者亲本的单体型结果对胚胎的纯合基因型进行SNP连锁分析，得到胚胎单条染色体链的分型结果；由于亲本存在错误组装的block，会在胚胎之间的单体型结果中有相同的断点，对上述的错误组装情况进行校正，得到准确的分型结果。
实施例2
本实施例提供不依赖于先证者或参照者的单基因病的单体型构建方法并结合胚胎的SNP连锁分析进行胚胎植入前单基因遗传病检测。
接受辅助生殖的单基因病患者家庭(已有基因检测结果)，患者家系基因检测结果如表2所示，抽取单基因病患者及其配偶和患者直系亲属的外周血5mL，于EDTA抗凝采血管中保存，同时获取两对夫妻各自的胚胎活检样本经过全基因组扩增后的产物。家系外周血样本按照本领域高分子量DNA提取方法提取基因组DNA。
表2
1、单基因病患者单样本长片段建库测序
基因组DNA样本参照长片段测序平台建库测序说明书进行建库测序。
2、胚胎PGH芯片SNP基因型检测
对患者的两个子代胚胎滋养外胚层活检细胞的WGA扩增产物进行PGH芯片SNP基因型检测。
3、单基因病携带者单样本单体型构建
(1)单基因病携带者基因分型：对单基因病携带者的长片段测序原始数据进行全基因组分析，获得其致病变异的基因型信息(包括点突变和插入缺失、 重复等)；
(2)确定SNPs位点：筛选在单基因病携带者中为杂合型的SNPs；
(3)构建单基因病携带者的单体型：集合步骤(2)确定的杂合型SNPs进行全基因组组装与分型；获得单基因病携带者亲本的致病/正常单体型；根据单基因病致病变异的分型结果标记亲本的单体型颜色(蓝色为携带染色体链)；
4、构建子代胚胎的单体型：根据单基因病携带者亲本的单体型结果对胚胎的纯合基因型进行SNP连锁分析，得到胚胎单条染色体链的分型结果；由于亲本存在错误组装的block，会在胚胎之间的单体型结果中有相同的断点，对上述的错误组装情况进行校正，得到准确的分型结果。
对比例1
本对比例进行平衡易位家系样本进行常规PGH检测。
1、家系单体型构建：
(1)样本基因分型：对家系的男方(正常)、女方(平衡易位)、女方母亲(参照者)以及该夫妻的2个子代胚胎活检样本WGA扩增产物进行SNP基因型检测，具体实验方法参照说明书进行，在此不再赘述；
(2)确定有效SNPs位点：选择在染色体平衡易位携带者中为杂合型，在其配偶中为纯合型，并且在参照者中也是纯合型的SNP位点；选择覆盖染色体易位断裂点、易位染色体和与其相对应的正常的同源染色体上的有效SNPs，染色体每Mb范围内至少选择1个有效SNP；在覆盖断裂点区域，有效SNPs从断裂点上下游2Mb范围内选择；
(3)构建家系连锁单体型：集合步骤(2)确定的有效SNPs位点通过家系连锁分析得到覆盖两处断裂区域、易位染色体整条染色体及其同源染色体的整条染色体的单体型，不同染色体单体型的集合称为家系单体型。
2、区分携带平衡易位或正常染色体的胚胎：
将胚胎(囊胚)细胞中断裂点区域的单体型信息和参照者样本的染色体单体型信息进行比对，从而区分胚胎是否携带平衡易位。
对比例2
本对比例对单基因病患者家系进行常规PGH检测。
1、家系单体型构建：
(1)样本基因分型：将单基因病患者夫妇双方、先证者样本进行大规模SNP 基因型检测；
(2)确定有效SNPs位点：选择在单基因病携带者中为杂合型，在其配偶中为纯合型，并且在先证者中也是纯合型的SNPs位点；选择覆盖致病突变上下游2Mb范围内的有效SNPs；
(3)构建家系单体型中参照样本单体型：集合步骤(2)确定的有效SNPs位点，通过家系连锁分析得到覆盖致病变异的区域的单体型，获得家系单体型中参照样本单体型。
2、区分携带单基因病致病变异或正常的胚胎：
将胚胎(囊胚)细胞中致病突变区域的单体型信息和参照者样本的单体型信息进行比对，从而区分胚胎是否携带致病突变。
结果对比
实施例1中本申请方法检测平衡易位家系样本分型结果如图1所示，CYJ01E染色体长臂的空白区域为不平衡易位区域，区域起止位点即为断裂点，CYJ02E染色体长臂黄色block跨过断裂点，为平衡易位携带者；对比例1中常规PGH家系连锁分析方法结果如图2所示，将实施例1中本申请方法与对比例1中常规PGH家系连锁分析方法结果进行对比，结果如表3所示，该平衡易位家系女方和其母亲核型结果为平衡易位携带者；本申请方法检测结果与核型及胚胎的PGH结果一致，且携带者断裂点的结果与胚胎PGH的一致，表明本申请可不依赖于先证者或参照者构建单体型，并能有效应用于遗传筛查。
表3
实施例2中本申请方法检测单基因病家系样本分型结果如图3所示，对比例2中常规PGH家系连锁分析方法结果如图4所示，将实施例1中本申请方法与对比例1常规PGH家系连锁分析方法结果进行对比(表4)，本申请方法单样本单倍型分型结果与PGH分型结果一致，子代均携带了α地贫突变。
表4
综上所述，本申请提供了不依赖于先证者或参照者的单体型构建方法，仅对染色体致病变异携带者进行长片段测序，构建携带者的单体型，可有效应用于遗传筛查，结合胚胎的SNP连锁分析校正，可实现整条染色体的单体型准确分型，进而判断胚胎是否携带了致病突变，帮助无法获得先证者样本、新发突变或近亲婚配存在大量同源区域而无法构建单体型等因素难以进行传统PGT-M/PGT-SR治疗的患者筛选正常的胚胎。
申请人声明，本申请通过上述实施例来说明本申请的详细方法，但本申请并不局限于上述详细方法，即不意味着本申请必须依赖上述详细方法才能实施。所属技术领域的技术人员应该明了，对本申请的任何改进，对本申请产品各原料的等效替换及辅助成分的添加、具体方式的选择等，均落在本申请的保护范围和公开范围之内。

Claims (5)

  1. 一种不依赖于先证者的单体型构建方法，其包括以下步骤：
    (1)对夫妻双方中致病变异携带者一方的DNA样本进行长片段测序；
    (2)对致病变异携带者亲本的测序数据进行全基因组分析，获得其致病变异的基因型信息；
    (3)筛选在致病变异携带者亲本中的杂合型SNPs；以及
    (4)集合所述杂合型SNPs进行全基因组组装与分型；根据致病变异所在的长片段组装信息，获得致病变异位点的分型结果；根据致病位点的分型结果标记致病变异携带者亲本的单体型，构建致病变异携带者亲本的致病/正常单体型；
    其中，步骤(1)中仅对致病变异携带者一方进行长片段测序，不检测夫妻中正常一方、携带致病基因的子代、携带者亲属或染色体异常的胚胎。
  2. 一种对胚胎进行遗传病筛查和染色体异常筛查的装置，其包括构建单体型单元和筛查单元；
    所述构建单体型单元用于执行权利要求1所述的不依赖于先证者的单体型构建方法；
    所述筛查单元用于执行包括：
    对子代胚胎样本的全基因组扩增产物进行全基因组SNP分析；以及
    根据致病变异携带者亲本的单体型结果对子代胚胎的纯合基因型进行SNP连锁分析，得到子代胚胎单条染色体链的分型结果。
  3. 根据权利要求2所述的对胚胎进行遗传病筛查和染色体异常筛查的装置，其中，所述筛查单元还用于执行包括对胚胎样本进行全基因组扩增的步骤，所述胚胎样本包括胚胎活检样本；
    其中，所述胚胎活检样本包括卵裂球活检样本或囊胚滋养外胚层活检样本。
  4. 根据权利要求2所述的对胚胎进行遗传病筛查和染色体异常筛查的装置，其中，所述筛查单元还用于执行包括对错误组装进行校正的步骤。
  5. 根据权利要求2-4任一项所述的对胚胎进行遗传病筛查和染色体异常筛查的装置，其中，所述构建单体型单元用于执行包括以下步骤：
    (1)对夫妻双方中致病变异携带者一方的DNA样本进行长片段测序；
    (2)对致病变异携带者亲本的测序数据进行全基因组分析，获得其致病变异的基因型信息；
    (3)筛选在致病变异携带者亲本中为杂合型的SNPs；以及
    (4)集合所述杂合型SNPs进行全基因组组装与分型；根据致病变异所在的长片段组装信息，获得致病变异位点的分型结果；根据致病位点的分型结果标记致病变异携带者亲本的单体型，构建致病变异携带者亲本的致病/正常单体型；
    其中，步骤(1)中仅对致病变异携带者一方进行长片段测序，不检测夫妻中正常一方、携带致病基因的子代、携带者亲属或染色体异常的胚胎；
    所述筛查单元用于执行包括以下步骤：
    (1)对子代胚胎样本的全基因组扩增产物进行全基因组SNP分析；以及
    (2)根据致病变异携带者亲本的单体型结果对子代胚胎的纯合基因型进行SNP连锁分析，对错误组装进行校正，得到子代胚胎单条染色体链的分型结果。
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