全基因组连锁分析结合新一代靶向捕获测序技术定位杯状耳畸形罕见大家系致病突变

Approximately 5% of the population has some sort of congenital ear malformation which affect position, shape and even function of the ear. The cupped or constricted ear deformity occurs as a result of hooding of the superior helix of the ear which account for 10% among all kinds of ear malformation. Different from other ear deformities, cupped ear is characterized by bilateral occurrence and potential genetic predisposition. Literatures showed that most of the studies focused on the surgical treatment rather than the etiology, inculding it genetic mechanism, specificity and susceptibility gene of the disease. In this study, we aim to identify the susceptibility gene using targeted next-generation sequencing combined with the linkage region based on the cupped ear families which follow Mendelian genetic mode. The candidate mutations or genes obtained from next-generation sequencing will be further subjected to Sanger sequencing with additional pedigrees and sporadic cases/controls. Relationship between genotype and phenotype will be further elucidated by the prediction of susceptibility gene and its ecoding protein through analyzing the biological pathways, cellular localization, protein interaction networks and tissue specificity. Next-generation sequencing is a powerful new technology which is frequently used in mapping the causal genes for common and rare diseases. Its high efficiency has been proved nowadays. The identification of the susceptibility gene for congenital cupped ear will not only improve the understanding of the genetic mechanism, but also facilitate relevant genetic counseling and early diagnosis, targeted intervention, and has an important social value as well.

先天性耳部畸形是影响耳廓位置和形态的常见出生缺陷，人群中约有5%的样本有不同程度的耳部发育畸形。杯状耳是其中的一种，约占各种先天性耳部畸形的10%；区别于其他常见的耳部畸形，杯状耳多为双侧，具有明显的遗传倾向。国内外针对杯状耳畸形的报道多侧重于其治疗方法的改进，缺乏针对发病机制的深入探讨。本课题组将选择遗传模式符合孟德尔规律的杯状耳大家系，采用经典的连锁分析结合当前的二代测序技术鉴定易感基因的策略查找致病突变；进一步在杯状耳散发病例及正常对照人群中进行验证，确定致病基因对群体发病风险的贡献度。结合生物学通路、细胞定位、蛋白作用网络和组织表达特异性等多种分析，预测易感基因编码蛋白结构和功能，进一步阐明基因型和表型关系。该策略具有所需样本量小，简便快捷，可重复性强的优点；将为耳部畸形的遗传咨询、早期诊断和临床早期干预、以及靶向治疗奠定遗传学基础，具有重要的经济价值和社会意义。

先天性耳部畸形是影响耳廓位置和形态的常见出生缺陷，人群中约有5%的样本有不同程度的耳部发育畸形。杯状耳是其中的一种，约占各种先天性耳部畸形的10%；区别于其他常见的耳部畸形，杯状耳多为双侧，具有明显的遗传倾向。国内外针对杯状耳畸形的报道多侧重于其治疗方法的改进，缺乏针对发病机制的深入探讨。.本课题组对一个杯状耳大家系进行了连锁定位研究，在4号染色体上找到了一个连锁区域4p15.32-4p16.2，其LOD值为2.7，NPL值为 12.28。对于单纯外耳畸形而言，4p15.32-4p16.2是一新发现的连锁候选区域。我们首先对该易感位点内的基因HMX1、NKX3-2、EVC、EVC2测序后并未找到共分离的突变位点，而后开展了全外显子组研究，依旧未在该易感位点内甚至全基因组范围内发现编码的功能性变异位点。我们先后又收集了2个单纯型外耳畸形家系并进行了连锁定位后，发现4p15.32-4p16.2区域的LOD = 7，因此推测导致该畸形的突变一定位于该易感区域，且极有可能位于基因间区。因此，我们对这3个家系进行了目标区域捕获研究，完成了32个样本的二代测序工作，最终在该区域找到了一个和所有家系患者共分离的变异。.本研究不仅找到了我国一个杯状耳大家系的染色体连锁区域，同时还找到了一个和其发病相关的致病突变位点。本研究是国内外首个针对杯状耳的研究，该结果将推动杯状耳致病突变的定位工作，为该疾病的病因学研究提供遗传基础，为该病的产前诊断提供重要的科学依据。