伴有泌尿系统畸形的多器官出生缺陷的遗传基础及其致病机制的研究

Multiple organ congenital malformation is a significant cause of perinatal mortality and disability in children. It is known that genetic causes mainly account for multiple congenital malformations. However,there is limited research on the pathogenic mechanism of multiple congenital malformations associated with urinary system defects.Our pilot study on prenatal ultrasound of a monochorionic diamniotic twins showed discordant phenotype in which one fetus is normal and the other one with VACTERL syndrome,a severe multiple system congenital defect associated with kidney malformation.Pathogenic copy number change was only identified among the affected kidney by aCGH. Our data evidenced that the genetic difference of monozygotic twins may lead to the phenotypic difference in this case. It is hypothysized that both the germline variants and somatic variants could lead to the multiple congenital malformations including urinary system malformation. In this project, we will recruit the discordant MCDA twins with one normal fetus ,the other fetus showing multiple malformations including the urinary system; apply the massively paralell sequencing technology for high read depth whole genome sequence analysis to identify the pathogenic genomic variants and attempt to delineate the relationship between the genetic changes and congenital anomalies. To achieve this objective, we will establish the amniocyte and skin fibroblast-induced pluripotent stem cells with specific genetic defects identified in the affected twin to serve as in vitro disease model to study the differentiation of stem cells into different germ layers by investigating the differential potential and gene expression profile changes. This project may reveal the pathogenesis of this type of birth defect.It would significantly promote the prevention and control of birth defect in our country.

多发性先天畸形是围产儿死亡和儿童残疾的重要原因。目前认为遗传因素是多发性先天畸形的主要原因。然而对伴有泌尿系统畸形的多发先天畸形的致病机制研究甚少。我们在一例单绒毛膜双羊膜囊(MCDA)单卵双胞胎的产前超声检查发现仅一胎为VACTERL综合征，一种严重的伴有泌尿系统畸形的多发畸形，aCGH芯片检测出其受累组织中有致病性基因组拷贝数变异。单卵双胞胎遗传物质上不同程度的差异是导致其表型不一致的重要因素。由此推论生殖细胞的新发突变或受精后的体细胞突变可能导致伴有泌尿系统畸形的多发畸形。本课题选取仅一胎表现为该种畸形的MCDA单卵双胞胎，采用第二代测序技术进行全基因组高深度测序，定位致病性基因组变异，阐述变异类型与先天性畸形的相关性。同时构建羊水和皮肤纤维细胞来源的诱导性多能干细胞作为疾病模型，检测对干细胞分化的影响，基因表达谱的变化，该研究有望揭示先天性畸形的分子机制，促进我国对出生缺陷的防治。

先天性泌尿系统畸形在新生儿中的发病率约为3-6‰。产前超声常见的异常有：肾分离，肾发育不良，肾盂积水，巨膀胱或膀胱缺失等，占常规产前超声异常指标的1/3。文献显示先天泌尿系统畸形与遗传缺陷有关。利用Chromosomal Microarray Analysis(CMA)平台检出了多个与泌尿系统畸形相关的CNVs(copy number variations), 但在产前诊断中的研究有限，到目前为止仅有少量的病例报告。 本课题针对三个目标进行研究（1）收集产前诊断中超声发现为泌尿系统畸形的病例，研究致病CNV与疾病的关系；(2)也进行全基因组低深度测序 (low-pass whole genome sequencing) 全面检测染色体异常，特别是染色体结构异常；3）研究缺失型CNV与TAD的关系，探讨泌尿系统畸形表型的致病机制，筛选候选基因并验证。同时构建皮肤纤维细胞来源的诱导性多能干细胞 (iPSc) 作为疾病模型 一、 共收集207例产前超声指征提示泌尿系统畸形的病例，包括MCDA双胞胎5例有羊水，绒毛和脐带血标本，提取DNA后进行了CMA技术之一的aCGH（array comparative hybridization, aCGH ）检测，也进行全基因组低深度测序 (low-pass whole genome sequencing) 全面检测染色体异常，特别是染色体结构异常. CMA 结果如下：1.在186例产前诊断标本中，11.36%(19/186)的病例检测出致病性CNVs。2.将176例病例分为2组，第一组有85例为单发型泌尿系统畸形, 发现有3.5%（3/85）携带致病性CNV; 第二组有101例为综合征型伴泌尿系统畸形， 有15.8%（16/101）的致病性CNVs。 我们的结果显示泌尿系统畸形与遗传缺陷有关. CNV导致疾病有多种致病机制，包含（1）缺失型的CNV中包括关键基因导致单倍计量不足而致病；（2）CNV区域内包含调控因子，导致基因表达异常而致病。我们甚至发现打断拓扑相关区域（Topologically associated domain，TAD）会有新的拓扑区域（Topological domain）形成，调控因子或基因因“位置效应”出现错误调控或异常表达，导致多发畸形。