利用目标基因靶向测序技术研究血友病A新的分子致病机制

血友病A（HA）是临床最常见的遗传性出血性疾病，主要是由F8基因突变造成凝血因子Ⅷ（FⅧ）蛋白生成异常或功能障碍所致。目前绝大多数HA患者的发病机制已明确，但仍有2%~5%患者的分子致病机制尚未明了。本课题组前期在对450个HA家系进行研究时发现8例先证者的发病机制未明。本项目拟以此8例HA先证者为研究对象，采用Agilent HaloPlex靶向捕获系统和Illumina Miseq技术平台，对12个可能导致FⅧ异常的目标基因进行高通量靶向基因组测序，结合生物信息学分析，并经Sanger测序法验证和家系分析，发现新的HA相关基因突变；随后，通过构建相关基因突变表达质粒，研究相关基因突变对F8 mRNA水平、蛋白质水平及FⅧ活性等的影响，探讨相关基因突变在HA发病中的作用。本研究有望发现新的HA分子致病机制，为进一步完善HA的基因诊断、产前诊断和遗传咨询提供理论依据。

Hemophilia A (HA) is the most common inherited bleeding disorder and is mainly caused by mutations in the F8 gene, resulting in a quantitative and qualitative abnormality of coagulation factor VIII (FVIII). At present, the pathogenic mechanisms for most HA patients have been identified; however, in 2%~5% of the HA patients, the pathogenesis is still not yet clear. Our previous studies have shown that the causal mutations couldn't be identified in 8 of 450 HA pedigrees by routine molecular diagnostics. This study is designed to capture and sequence 12 target genes that are associated with synthesis and functioning of FVIII protein in samples from the eight HA proband with Agilent HaloPlex Target Enrichment System and Illumina Miseq paltform. Sequencing data will be processed and analyzed by a variety of bioinformatic tools, and the candidate causal variants will be identified through stringent filtering procedures. Sanger sequencing and co-segregation analysis will be undertaken to validate the candidate causal variants. Furthermore, the functional impacts of the candidate causal variants will be assessed in vitro through constructing of mutant plasmids on levels of mRNA, protein and bio-activity of FVIII. This study may help further elucidating the new molecular mechanism for FVIII deficiency in HA cases without detectable mutations, and provide the theoretical basis for prenatal diagnosis and genetic counseling.