RNA解旋酶DHX8基因突变导致新型矮小综合征的致病机制研究

RNA helicases, a large evolutionary conserved family of genes, are important players in all types of processes involving RNA. Yet little is known about their association with human diseases. In our initial stages of the research, our team identified a new candidate disease gene DHX8 by exome sequencing in four individuals with the symptoms of extremely short, hypoplastic pituitary, skeletal and gonadal dysplasia, which constitute a novel short stature syndrome. DHX8 encodes a RNA helicase. It is an important component of spliceosome, which is responsible for mRNA splicing. Based on the findings from clinical phenotyping of the patients and in vitro experiments which demonstrated that DHX8 mutations lead to RNA splicing defects in key genes responsible for pituitary development, we propose that DHX8 mutations cause this novel short stature syndrome via its effect on RNA splicing on pituitary developmental genes, which in turn lead to the failure of normal pituitary gland development. This project intends to further elucidate the mechanism by a multiple-level approach involving examining the effects of de novo DHX8 mutations on helicase activities, RNA splicing and protein expression, as well as on organ development and animal growth in in vivo animal models. This study will elucidate the roles of DHX8 gene in the pathways that regulate the pituitary development and understand a new pathogenic mechanism of short stature. The finding will reveal new biological significance of RNA helicase and provide new ideas for the treatment of related diseases.

RNA解旋酶在进化上高度保守，参与几乎所有与RNA相关的生物学过程，其基因家族庞大，但对其家族成员与疾病的关系仍知之甚少。课题组前期在4例具有极度矮小、垂体发育缺陷、骨骼发育和性腺发育不良等症状的新型矮小综合征患者中，通过家系全外显子组测序鉴定到新的候选致病基因DHX8。该基因编码的RNA解旋酶是剪接体的重要组分，与mRNA剪接密切相关。结合患者临床表型及前期实验研究推测DHX8突变通过影响垂体发育关键基因的表达，进而影响垂体的发育及功能，是导致该罕见综合征的主要原因。本项目拟通过细胞实验和构建DHX8突变小鼠模型等，从解旋酶活性、RNA剪接、蛋白表达、器官发育、动物生长等层面展开研究。探讨DHX8在垂体发育环节及相关信号通路中的作用，以期阐明DHX8基因突变导致该新型矮小综合征的发病机制，为进一步了解RNA解旋酶的生物学意义奠定基础，并为相关疾病的治疗研究提供理论依据。

RNA解旋酶基因家族参与真核细胞内多种与RNA相关的生物学过程，目前其家族成员与疾病的关系及其致病机理尚未阐明。本项目前期利用家系全外显子组测序在4例以极重度矮小、垂体发育缺陷、骨骼发育不良等为表型的新型矮小综合征患者中，鉴定到新的致病基因DEAH盒解旋酶8（DEAH-BOX HELICASE 8, DHX8）。DHX8基因编码的RNA解旋酶是剪接体的重要组分，与mRNA的剪接密切相关。本项目通过构建DHX8基因野生型和突变型表达载体，进行体外功能研究，分析DHX8基因突变对DHX8蛋白表达、定位和功能的影响；通过CRISPR/Cas9 基因编辑技术建立Dhx8基因敲除和错义突变（p.H712R）敲入小鼠模型，研究Dhx8基因对生长发育、垂体发育和骨骼表型的影响。体外功能研究发现，DHX8基因发生突变后，DHX8蛋白的ATP酶和RNA解旋酶活性下降，垂体发育和RNA剪接相关基因的表达下调，导致细胞增殖能力下降、细胞周期阻滞，以及细胞凋亡增加。小鼠模型研究发现，p.H712R突变小鼠表现为体长缩短，再现了患者表型；而全身性敲除则导致纯合小鼠胚胎期致死。通过本项目研究，阐明了DHX8突变导致矮小综合征的分子致病机制，即DHX8基因发生功能丧失型突变后，其ATP酶和RNA解旋酶活性下降，导致垂体发育相关基因的RNA剪接出现异常，阻碍了细胞正常的增殖，细胞周期阻滞于S和G2期，并引起细胞凋亡增加，导致垂体发育缺陷及矮小症表型。本项目研究结果加深了我们对剪接体生物学过程的理解，拓宽了RNA解旋酶对个体生长发育重要作用的认识；本项目中建立的Dhx8 p.H712R小鼠模型可进一步用于矮小综合征治疗的相关研究，为治疗药物的研发提供了非常有价值的线索。

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