CLCN7基因R767W突变导致常染色体显性遗传骨硬化症2型外显不全修饰基因的鉴定和功能研究

CLCN7基因突变导致常染色体显性遗传骨硬化症2型（OPTA2）是汉族人群中最常见骨硬化类型，仅60%的外显率使临床诊断和干预困难巨大，修饰基因可能是关键因素但机制不明。本课题拟选择前期研究发现的15个外显不全CLCN7基因R767W突变OPTA2家系，首先对3个家系4例患者和4例未发病突变携带者进行全基因组重测序，通过生物信息分析对两者基因间的差异进行注释以发现与基因显性有关的位点；利用上述受检者外周血单核细胞诱导生成破骨细胞进行数字表达谱分析，观察与破骨细胞相关的信号通路中差异基因表达；综合上述基因组和转录组两个层面的分析，在相互交集区域中确定候选修基因并在其余12个家系中验证，获得与外显不全有关的修饰基因；构建同时携带R767W突变和修饰基因的RAW264.7细胞进行功能研究，了解突变和修饰基因对破骨细胞的作用。不仅有望阐明外显不全发生机制，发现治疗靶点，且为遗传咨询产前诊断奠定基础

Autosomal dominant osteopetrosis type II (OPTA2), caused by CLCN7 gene mutations, is the main type of osteopetrosis in Chinese population. The penetrance is circa 60%. Because of the incomplete penetrance, it is very difficult to establish clear diagnoses and effective intervention. So far the mechanism of incomplete penertrance is still unknown. Modifier genes and environmental factors may be the major influence factors. In order to identify modifier genes and explore the mechanism of incomplete penetrance, fifteen OPTA2 families caused by CLCN7 gene R767W mutation with incomplete penetrance from our previous study will be recruited in this study. Firstly, the whole genome-wide resequencing and digital expression profiling will be performed in four patients and four unaffected mutation carriers. The genetic difference between patients and carriers will be annotation by bioinformatic analysis. We will focus on the differential gene expression in the pathways involved in the osteoclast formation and function. The candidate modifier genes will be identified according to the above DNA and RNA studies. Secondly, the following replication study will be conducted in another 19 patients and 11 unaffected mutation carriers to find out the true-positive modifier genes as being associated with incomplete penetrance. Thirdly, in order to investigate the combined effect of CLCN7 gene R767W mutation and modifier genes on the number and activity of osteoclast, the functional study will be conducted by using the RAW264.7 cell line transfected with the R767W mutations and modifier genes simultaneously. We believe that this study will be very important for the exploration of the mechanism of incomplete penetrance in OPTA2 and the discovery of new treatment targets. It is hoped that the results from this study will lay the groundwork for the studies of antenatal diagnosis and genetic counseling.