基于NGS技术的遗传性多囊肾病植入前遗传学诊断研究

Polycystic kidney disease (PKD) is one of the most common life-threatening genetic diseases affecting an estimated 1.5 million people in China with an incidence of 1 in 400 to 1 in 1,000. However, molecular diagnostics by direct mutation screening has proved difficult in this disorder due to genetic and allelic heterogeneity and complexity of the major locus, PKD1. In this study, we try to quickly identify disease-causing mutations in the entire coding regions of PKD1 and PKD2 with the use of next generation sequencing (NGS) combined with target enrichment method and bioinformatics, which are capable of overcoming the limitations or bottlenecks of PKD genetic analysis. To obviates the risk of misdiagnosis due to allele drop out(ADO) and the inefficiency of whole genome amplification of singe cell, a NGS based haplotype-linkage analysis using SNP markers within the 1M bp upstream and downstream range of PKD1/PKD2 will be carried out. All approaches will be validated using a cohort of 42 patients with ADPKD previously analyzed by Sanger sequencing in our hospital. After finishing the feasibility study on the application of target NGS base technology in testing mutation or informative SNP of PKD in single cell, patients of childbearing age will be chosen to undergo in vitro fertilization(IVF) followed by target NGS based preimplantation genetic diagnosis. Preimplantation genetic haplotyping using informative SNP markers on either side of the mutation will also be performed using NGS based technology. We hope the technology established by us will widen the scope and availability of preimplantation genetic testing for polycystic kidney disease, and will ultimately reduce the incident of PKD in China.

遗传性多囊肾病（PKD）是我国最常见、危害巨大的单基因遗传性肾病，发病率1‰-2.5‰，我国患者人数高达150万。PKD1基因结构复杂，PKD的临床基因诊断和植入前遗传诊断存在瓶颈和盲区。本项目率先运用新一代测序（NGS）技术，结合芯片捕获及生物信息学的研究方法，快速有效地检出PKD1/PKD2基因中的突变位点，突破PKD的检测瓶颈；同时在基因上下游1M范围内筛选可提供家系致病基因连锁信息的SNP位点，规模性开发PKD的单细胞SNP单体型分析体系，克服单细胞全基因组扩增后存在的等位基因丢失（ADO）和由此引起的误诊问题。在我们已完成基因突变谱绘制的42个PKD家系中选取育龄患者，进行体外受精（IVF）和植入前遗传学诊断（PGD），研究基于NGS技术的胚胎植入突变点检测和单体型分析（PGH）的可行性。最终建立可适合规模化开展遗传性多囊肾病的PGD标准体系，为降低人群中的发病率做贡献。

本研究使用直接PCR测序和多重连接依赖性探针扩增（MLPA）对49名无亲缘关系的ADPKD患者进行了PKD1和PKD2基因的突变分析。为了进一步克服PKD突变基因检测瓶颈，我们开发了一套基于芯片捕获及长链多聚酶链式反应（long-range polymerase chain reaction, LRPCR）的下一代靶向测序的方法，建立了一套稳定应用于ADPKD家系突变基因检测和靶向SNP单体型分析的高通量基因测序检测方案,并利用候选标记SNP位点的sanger测序结果对靶向NGS单倍型分析结果进行评估；我们完成了8个有强烈生育愿望的ADPKD家庭的植入前遗传学诊断，共获得24个胚胎，其中23个胚胎获得了PGD结果，诊断率为95.8%。其中3对夫妻胚胎的检测结果都是异常，未获得可移植的胚胎， 2对夫妻仍在等待移植，2对夫妻移植后没有妊娠，1对夫妻移植后临床妊娠，虽然该对夫妻在孕早期发生了流产。但本研究证明靶向NGS结合靶向单倍型分析可应用于胚胎植入前胚胎PKD基因突变的鉴定。因为我们的捕获定制阵列包含数千个在基因组中具有广泛分布的SNP，所以它非常适合于PKD基因的单倍型分析。不仅如此，靶向NGS与靶向单倍型分析相结合可用于PGD的其他单基因疾病，如DMD或SMA,而无需在不同的人中筛选遗传标记，具有敏感、快速、准确和高通量的优点。