一家族性单基因遗传自身免疫病致病基因SOX13的发现及功能分析

In the preliminary study, we collected several families and sporadic cases with Systemic lupus erythematosus, rheumatoid arthritis. We performed whole exome sequencing in one multi-generation family in which 8 family members affected with Systemic lupus erythematosus, rheumatoid arthritis. After filtering with ExAC, 1000 genomes,ESP6500 and gnomAD, then conducted prioritization analysis, a candidate variant p.S453I in SOX13 gene was identified in the family. After Sanger sequencing all the family members, we found that this missense variant co-segregate with the phenotype in the family and is conservative across different species. PolyPhen-2,SIFT and Mutationtaster were used to predict the possible pathogenic effect of the variant. The bioinformatic analysis results revealed the substitution p.S453I was predicted to be likely detrimental to protein function. All the results suggested that variant p.S453I in SOX13 might play an critical role in the pathogenesis of autoimmune disease. But the detailed mechanisms remained unclear..In this proposed project, we aimed to confirmed its pathogenic role by 1) screening more disease families and sporadic cases to find more mutations in the SOX13 gene to confirm its disease-causative role. 2) in vitro studies such as ChIP-Seq and CoIP to provide more functional supportive clues for the variant, 3) phenotype assay for the knock-in mouse model to investigate the pathophysiological effect of Sox13 on autoimmune diseases . In addition, in vivo and in vitro functional studies of identified disease-causing genes will be carried out to investigate their critical roles in the pathogenesis of rheumatic diseases. Identification of more genes implicated in the autoimmune diseases will provide us a better understanding of the molecular pathology and help find potential treatments.

自身免疫病具有较高的发生率，分离其致病基因对于了解疾病的发生机制及防治具有重要的意义。课题组前期利用全外显子组测序对一单基因遗传的自身免疫病大家系进行分析，过滤筛选后发现SOX13 p.S453I 错义突变在家系内与表型共分离且在进化中高度保守，生物信息学分析证实该变异影响了蛋白功能，提示SOX13基因突变导致了自身免疫病。为探索该变异的致病性及SOX13导致疾病发生的分子机制，课题组利用基因打靶技术构建Sox13 p.S453I 的基因敲入与敲除小鼠模型，通过对Knock in小鼠注射II型胶原（CII）诱导产生风湿性关节炎或注射pristane诱导产生系统性红斑狼疮等表型分析证实突变的致病性，分析敲除小鼠γδT等细胞的变化，通过ChIP-Seq、免疫荧光、流式细胞术等技术分析SOX13致病的分子机制，为揭示SOX13在自身免疫病发生的分子机制进行探索。

课题组拟对前期收集到的一个自身免疫病家系进行致病基因分析，明确导致该家系的基因突变，为家系提供基因诊断。同时明确突变的分子病理机制及表型效应，为自身免疫病的发生机制提供一个新的靶点。课题按研究计划顺利完成，利用全外显子组测序结合Sanger测序，发现SOX13基因存在c.G1358T，p.S453I错义突变，该变异在家系中与疾病表型共分离，在ExAC, 1000genome数据库中不存在。该变异在物种间高度保守，双荧光素酶报告基因实验表明SOX13基因S453I错义突变影响了SOX13转录因子的功能，体外实验证明该突变导致Wnt/β-catenin信号通路过度激活。由于SOX13基因能够调控γδT细胞分化，我们的实验证明变异影响了γδT细胞正常的分化进程。为深入明确变异的分子病理效应，课题组构建了S453I突变的knock-in小鼠模型。统计结果表明，与对照小鼠相比，KI小鼠的胸腺和外周血中，γδT细胞比例升高。对KI小鼠利用Pristane诱导后，与对照相比，KI小鼠脾脏明显肿大，尿蛋白含量明显升高。肾盂黏膜及肾小管周围炎细胞浸润，肾小球体积增大，基底膜增厚，血管内皮细胞及系膜细胞出现增生现象，同时伴有明显的单核细胞浸润。综上，课题利用体内外实验证明SOX13基因S453I错义突变通过影响γδT细胞的分化导致自身免疫病的发生。本项目培养博士研究生2名，硕士研究生4名，在Human molecular genetics, Journal of Immunology, Journal of Clinical Immunology等杂志发表论文8篇。