复杂基因组变异导致Y连锁遗传性耳聋的机制研究

Y-linked hearing loss is the only one Mendelian trait linked to the human Y chromosome so far. It is based on the extensive investigation of a large hearing loss pedigree which had a family history of more than 1000 years and was ascertained by the applicant in 2000. In 2004, the applicant reported the Y-linked inheritance pattern of hereditary hearing loss in this Chinese family for the first time and denominated it as DFNY1 locus. In Feb. 2013, the applicant co-operating with the British scientist found that the Y chromosomes of all the male patients in DFNY1 family carried the same duplicated region arising from a complex rearrangement and including three sections of Y chromosome and 160kb sequence from chromosome 1. The 160kb sequence carried five RefSeq genes and was completely cosegregated with the phenotype of the family. All the breakpoint sequences revealed microhomology, a pattern indicative of fork stalling and template switching (FoSTeS). Our previous study is the first report of an interchromosomal rearrangement resulting from FoSTeS. So, this project is focusing on the following questions: How to identify the exact breakpoints of the complex rearrangement in DFNY1? How the copy number variation caused by FoSTeS in DFNY1 affect the hearing function? How about the pathophysiological change in the related animal model? In this project, the TAIL-PCR, qPCR, CRISPR/Cas9 making transgenic mouse models, and auditory electrophysiological and pathological methods will be used to discover the detailed structure of the complex duplication segment, to identify the causative gene for DFNY1 family, and to explain the molecular mechanisms of Y-linked hereditary hearing loss. This project will disclose the novel genetic and genomic mechanism of hereditary hearing loss caused by FoSTeS-mediated complex duplication. This study will help to improve the classic genetic theory and have theoretical innovation.

Y连锁遗传性耳聋是申请人于2004年在国际上首次发现的一种新型遗传性耳聋方式，定位和命名了Y染色体上的第一个耳聋DFNY1基因。2013年，申请人与英国小组合作，发现Y连锁遗传性耳聋家系中所有男性患者Y染色体上由于复制叉停滞与模板转换（FoSTeS）而产生了160kb基因组的复杂重组，并与耳聋表型共分离，文章发表在美国人类遗传学杂志上，并被称为国际遗传学领域唯一被证实的Y连锁遗传孟德尔疾病。那么，这种基因组复杂重组的断点在何处？复制重组的第三个基因拷贝如何影响听觉功能？如何导致耳聋发生的？本项目应用Tail PCR、Split reads技术分析FoSTeS突变断点，明确Y染色体短臂复杂扩增的精细结构；利用CRISPR/Cas9技术构建候选基因小鼠模型，从听力学、内耳形态、离子通道等系统观察候选基因在内耳中的作用，阐明DFNY1分子病理机制。这将对耳聋遗传学及基因组变异研究产生重大新突破。

Y连锁遗传性耳聋是项目负责人于2004年在国际上首次发现的一种新型遗传性耳聋方式，定位和命名了Y染色体上的第一个耳聋DFNY1基因。2013年项目负责人与英国小组合作，发现Y连锁遗传性耳聋家系中所有男性患者Y染色体上由于复制叉停滞与模板转换（FoSTeS）而产生了160kb基因组的复杂重组，文章发表在美国人类遗传学杂志上，并被称为国际遗传学领域唯一被证实的Y连锁遗传孟德尔疾病。为了分析这种基因组复杂重组的断点在何处？这个全新不同染色体之间FoSTeS的复杂重组的结构如何？本项目应用Nanopore全基因组测序，对该家系1例男性患者进行结构变异检测, 发现患者Y染色体上存在9个拷贝数增加的片段, 在插入区域INS1、INS7、INS9及横跨断点的Seg Dup区域均有基因分布。而在101例汉族正常男性Y染色体上，未检测到与患者插入序列区域特征相同的结构变异。同时对比上述正常样本TSPY1基因及受检者中4个较大插入区域的拷贝数分布情况，结果表明研究区域在DFNY1患者中拷贝数高于正常样本（82%-100%）；而在28个与DFNY1表型相似的常染色体显性遗传迟发性耳聋家系CNV分析中未发现基因组大片段DNA拷贝数变异。我们联合利用stLFR（单管长片段测序）和基因组组装、三代数据的处理和组装及混合组装方式，尝试全基因组组装、1号染色体和Y染色体局部组装。发现Y染色体上来自1号染色体的2个易位变异，初步明确了Y染色体断点位置chrY:9,520,205。同时应用Cas9技术构建了2款候选基因过表达转基因小鼠。本项目的研究成果初步阐明DFNY1分子机制，以全新的视角揭示基因组变异对男性听力特征、迟发性听力损失甚至于听觉老化的分子作用机理，丰富了耳聋遗传学及基因组变异研究的理论内容。