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

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.

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

本项研究通过对CLCN7基因R767W突变导致常染色体显性遗传性骨硬化症2型（OPTA2）患者及家系成员中的健康突变携带者进行全基因组重测序，并对测序结果进行分析，发现与表型可能潜在相关的基因多达173个，因而无法实现对与疾病表型有关的候选修饰基因的鉴定；为此本项目组又尝试进一步对OPTA2患者及家系中健康致病突变携带者进行甲基化分析，虽然仍未有阳性发现，但综合分析考虑可能与骨髓细胞增殖分化、成骨-破骨偶联及溶酶体有关。在细胞水平，我们发现与家系中携带致病突变的健康人相比，来源于OPTA2患者的外周血单核细胞经诱导分化生成的破骨细胞，虽然细胞数量无明显减少但其基本功能丧失，不能形成正常的吸收陷窝；为了能够充分的了解和阐明CLCN7基因R767W突变导致OPTA2的病理生理变化及外显不全的机制，本研究团队成功地构建了此致病突变条件性敲入小鼠模型（Clcn7-G763R），并成功地与破骨细胞特异Ctsk-Cre小鼠进行繁配，获得了可以用于骨硬化症研究的动物模型。在本项目进行的过程中，我们建立了骨硬化症样本库，样本包括血清、DNA、影像学资料、羊水和脐带等。通过对表型和基因型进行的分析，我们发现CLCN7基因突变导致的骨硬化症，患者的临床症状多变，严重程度不一，存在着较为严重的异质性，为临床确诊带来一定的困难。通过家系研究，我们总结了目前国人CLCN7基因突变导致OPTA2所有的临床表现，同时通过对CLCN7基因全外显子及外显子－内含子交界区的测序，明确致病突变，从而构成了国人骨硬化症的突变谱，指出对临床表现不典型或较轻的患者特别需要注意检测内含子区域的突变。本项研究为阐明OPTA2的病理生理变化以及外显不全的机制进行了探讨，虽然最终没能确定与表型有关的修饰基因，但是获得了机制研究所必需的重要工具－OPTA2小鼠模型，为后续研究奠定了重要基础。

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