全基因组外显子测序发现的垂体柄中断综合征致病基因的体内外功能研究

Pituitary stalk interruption syndrome (PSIS) is characterized by a thin or absent pituitary stalk with different degrees of anterior pituitary hormone deficiency, and is often associated with ectopic posterior pituitary, hypoplastic anterior pituitary and developmental malformations of structures or organs along the midline of central nervous system. PSIS is a rare disorder with an estimated incidence of about 0.5/100,000. Although congenital genetic defect was thought to be the cause, the reported known gene mutations only account for less than 5% of the patients. We still have a long way to go to fully understand the pathogenesis of PSIS. Here, at our center, we collected more than 100 Chinese PSIS patients samples with complete clinical data as well as the mutational status of genes currently known to be involved in pituitary development. Consistent with reports by others, we found that only a small number heterozygous mutations in coding sequences were found, while the intronic mutations or copy copy number variations were absent, suggesting the major PSIS-driving genetic defetcs are not yet to be identified. In this study, we firstly surveyed all coding genes systemically in 24 patient smaples to catalogue all PSIS related genetic defects by using whole exon sequencing, followed by validating the newly identified mutations in another cohort of 25 patient samples by targeted resequencing. Several mutations had been identified, such as NCOR2, NKD2 and ZIC2. Our preliminary study found that mutated NCOR2 could decrease the cell viability and migration of HepG cell and GH3 cell. Nextly, we are going to validate these newly identified mutations functionally through the moleucular and cell levels, and to discuss the functions of the genes in vitro and vivo using gene knockout animal models, with the purpose to define the roles of these genes on the pathogenesis and mechanism of PSIS. We believe our proposed study will unveil greatly the molecular mechanism of PSIS, and provide the foundation of better clinical management and genetic counseling for PSIS in future.

垂体柄中断综合征(PSIS)是指垂体柄缺如导致的系列严重临床症候群，大多患者终身不育、身材矮小、智力减退、甚至因垂体危象而危及生命。发病机制与先天基因缺陷有关，然而目前所发现的基因突变对该病的解释仍不到5%。我们前期工作收集了国内外最大样本量PSIS患者，仅发现2个文献报道的基因突变位点，提示其他重要致病基因尚未被发现。本研究应用新一代全基因组外显子测序分析了24例患者，发现多个候选高致病基因，如NCOR2、NKD2、ZIC2等，并在另25例患者中进行了验证。初步实验显示突变型NCOR2可降低HepG2和垂体GH3细胞迁移和增殖能力。本项目拟进一步对所上述致病基因从细胞、分子、动物模型层面进行体外、体内水平的功能研究，明确其在PSIS发病中的作用和机制。该研究的意义在于阐明通过全基因组外显子测序所发现的PSIS致病基因的致病作用和机制，为PSIS的临床分子诊断、遗传咨询及基因治疗提供依据。

垂体柄中断综合征(PSIS) 发病机制不详，临床漏诊误诊率高。本课题组通过系列研究，总结其临床特征和治疗经验，应用全外显子测序筛选高致病候选基因，并行功能验证。主要研究内容和结果：1 PSIS使用生长激素12月时显示可增加终身高。2运用全外显子测序24例PSIS，高频突变基因富集在垂体发育相关3条主要通路，包括Wnt/β-catenin、Notch以及Sonic Hedgehog通路。提示病因复杂，可能多基因、多调控通路参与。3进一步采用家庭成员(12岁男性家庭和8岁女性家庭)测序捕获具有潜在高致病基因，筛选出高致病候选基因NBPF9、MUC12、TYW1B，以及CDC27、NF1、USP9X等。4构建NBPF9、MUC12、TYW1B基因siRNA，分别转染进人胚胎干细胞，显示转染NBPF9 siRNA、MUC12 siRNA后细胞活性降低。5基因突变对垂体生长发育相关通路关键蛋白分子的影响分析。LHX3/LHX4是Wnt信号传导通路重要蛋白调控因子。PITX1参与脑垂体的衍生及发展。将NBPF9沉默表达后，PITX1表达未见明显改变，而LHX3蛋白表达下调。6通过转染siRNA-117与siRNA-1968使CDC27沉默后，GH3细胞迁移能力明显下降。7垂体特异性转录因子-1(POU1F1)和PROP1在垂体发育中非常重要。PROP1是Notch信号通路的关键因子。转染CDC27 siRNA后，GH3细胞POU1F1与PROP1表达均明显降低。8细胞转染CDC27siRNA后，分泌生长激素、泌乳素能力明显下降，促甲状腺激素无变化。本课题研究从临床和机制角度对PSIS进行深入研究，提供了我国患者的临床特征和治疗经验，并进行发病机制研究，是该病国内大规模的临床和基础系列研究，将为其临床诊治和遗传咨询提供有益的理论基础和临床经验。