基于基因捕获测序技术的生精障碍患者遗传学病因筛查方法的建立及其临床意义

The way of treatment we select for infertile patients could be greatly decided by genetic etiological test. For instance, better options such as ART would be suggested as early as possible to avoid unecessary drugs or operations. Besides, genetic couseling for patients undergoing ART could also gain benefits from the genetic etiological test. If decessive genetic defects were discovered by the test, freezing semen samples in an earlier stage would be recommended for the coming infertility in the future. Till now, karyotyping and Y microdeletion exam, by which only small parts of etiologies are able to be demonstrated, are still the predominant ways of genetic etiological test for infertile patients. In the present research, we tent to design a high throughput screen method based on gene trap and NGS technologies to detect quantities of infertility related gene mutations at the same time. Gene trap and NGS are less expensive and more specific comparing with other high throughput technologies, such as arrayCGH, arraySNP and NGS for whole genome sequencing. We believe that the technology of gene trap and NGS will dramatically improve the effeciency of clinical genetic etiological test for infertile patients. We wish that the method we design in our present research could be helpful in adopting better treatment options and couseuling for patients accepting ART.

生精障碍患者遗传学病因的检测有助于其治疗方式的选择，如进行辅助生殖技术助孕，避免不必要的药物或手术治疗。此外，亦有助于对行辅助生殖助孕的此类患者进行遗传咨询，如其男性后代也存在相应的基因缺陷，应及早就医或冷冻保存精液来解决生育问题。然而，目前临床工作中对于生精障碍患者的遗传学病因检测手段仍然停留于染色体核型分析和Y染色体微缺失检测，仅能阐明少部分患者的病因。为了让更多的生精障碍患者受益于其遗传学病因的明确，本研究拟采用基因捕获测序技术来建立一种同时检测大量生精障碍基因变异的筛查方法。和其他高通量检测技术如微阵列比较基因组杂交技术、微阵列单核苷酸多态技术及全基因组二代测序技术相比，基因捕获测序技术具有成本低、选择基因和疾病相关性高的特点，更利于其应用于临床生精障碍患者的遗传学病因筛查。希望本研究建立的方法可以用来指导生精障碍患者治疗方案的选择和患者行辅助生殖繁衍后代的遗传咨询。

本研究通过Pubmed文献和公共数据库检索，筛选出生精障碍相关的基因466个，构建生精障碍基因捕获测序平台。通过临床收集无精子症患者外周血样本、临床表型、临床精液分析和生殖激素等检测结果，共计完成100例无精子症患者和100例生育对照组外周血样本。应用构建的基因捕获测序平台进行高通量测序，对测序数据进行生物信息学分析，哈迪温伯格平衡检验、基因频率和基因型频率等统计学分析及case-control研究。获得主要结果包括筛选出包括21个SNV位点共12个有明确报道和致病机理研究相对明确的男性不育关联基因。通过构建针对非梗阻性无精子症（NOA）的基因捕获测序平台（gene panel），完成35例NOA外周血样本检测，为将来开发临床适用的生精障碍基因试剂盒提供了实验依据，未来将进一步扩大样本量和增加患者类型，如无精子症、少精子症、畸形精子症等，对此试剂盒进行验证，可用于指导临床病人治疗方案的选择和患者行辅助生殖繁衍后代的遗传咨询。

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