导致Schmid型干骺端软骨发育不良COL10A1新发突变的功能变化及基因型-表型关系  

Functional changes and genotypic-phenotypic relationship of new COL10A1 mutations leading to Schmid type metaphyseal chondrodysplasia

作　　者：吴慧潇 王姗姗 姚阳阳 方丽 袁嘉欣 夏维波[2] 赵家军 高聆 王延宙 徐潮 WU Hui-xiao;WANG Shan-shan;YAO Yang-yang;FANG Li;YUAN Jia-xin;XIA Wei-bo;ZHAO Jia-jun;GAO Ling;WANG Yan-zhou;XU Chao(Department of Endocrinology and Metabolism,Shandong Provincial Hospital Affiliated to Shandong First Medical University,Institute of Endocrinology,Shandong Academy of Clinical Medicine,Shandong Provincial Key Laboratory of Endocrinology and Lipid Metabolism,Jinan 250021,China;Department of Endocrinology,Key Laboratory of Endocrinology,National Health Commission,Peking Union Medical College Hospital,Chinese Academy of Medical Science&Peking Union Medical College,Beijing 100730,China)

机构地区：[1]山东第一医科大学附属省立医院内分泌科,山东省内分泌代谢病临床医学中心,山东省临床医学研究院内分泌代谢研究所,济南250021 [2]中国医学科学院北京协和医学院北京协和医院内分泌科,卫生部内分泌重点实验室,北京100730

出　　处：《中华骨质疏松和骨矿盐疾病杂志》2022年第4期327-335,共9页Chinese Journal Of Osteoporosis And Bone Mineral Research

基　　金：国家自然科学基金(81974124);泰山学者计划(tsqn20161071);国家重点研发计划(2016YFC0901503)。

摘　　要：目的对一个Schmid型干骺端软骨发育不良(Schmid type metaphyseal chondrodysplasia,SMCD)三代家系进行致病基因分析并探讨该突变在SMCD发病机制中的潜在作用,并探索SMCD基因型与表型的关系。方法回顾性分析1例SMCD患儿的临床资料并定期随访,抽取患儿及家系成员的外周血进行全外显子组测序分析,针对可疑变异进行Sanger测序验证和生物信息学预测突变的致病性,并对突变蛋白质的三维结构进行建模,分析突变蛋白三级结构的变化。通过构建野生型和突变型COL10A1质粒,进行体外功能研究。通过PubMed数据库查询1997年1月至2021年8月既往报道对SMCD所有病例进行的文献综述和基因型-表型分析,总结COL10A1突变基因型-表型关系。结果经全外显子测序检测到患儿COL10A1基因存在杂合突变(c.1863_1866delAATG,p.M622Tfs*54),为未报道过的新变异。经家系验证分析,家系成员均未检测出此突变。生物信息学软件预测可能为有害变异。SWISS-MODEL同源建模显示该突变位于NC1结构域,预测突变蛋白产生截短的Ⅹ型胶原α-1(X)链,引起Ⅹ型胶原蛋白α-1(X)亚基的错误折叠和三聚体不正确组装。体外功能实验发现突变体α-1(X)链表达降低,α-1(X)链三聚体降低,而α-1(X)链二聚体水平明显升高。基因型-表型分析结果显示,携带截短突变或NC1结构域突变的患者比携带非截短突变或非NC1结构域突变的患者发病年龄更早,临床表型更严重。结论COL10A1基因新发突变(p.M622Tfs*54)引起X胶原α-1(X)链三聚化及组装异常,导致SMCD。COL10A1蛋白NC1结构域是SMCD的热点突变区域,携带NC1结构域突变患者发病年龄较早且临床症状更严重。Objective To analyze the pathogenic gene of a three-generation family with Schmid type metaphyseal chondrodysplasia(SMCD),and to explore the potential roles of this mutation in the pathogenesis of SMCD and the relationship between genotype and phenotype of SMCD.Methods The clinical data of one child suffered by SMCD were retrospectively analyzed and we conducted a follow-up study regularly.The peripheral blood of the case and family members was extracted for whole-exome sequencing analysis.Sanger sequencing was performed for suspicious variants,and the pathogenicity of the mutation was predicted by bioinformatics.The three-dimensional structure of the mutant protein was modeled to analyze changes in tertiary structure of the mutant protein.In vitro functional studies were performed by constructing wild-type and mutant COL10A1 plasmids.In addition,all previously reported SMCD cases from January 1997 to August 2021 were queried through the PubMed database for literature review and genotype-phenotype analysis.Results A novel heterozygous variant(c.1863_1866delAATG,p.M622Tfs*54)was identified in COL10A1 gene in the affected child.And it was predicted to be pathogenic by bioinformatics software.SWISS-MODEL homology modeling revealed that the mutation was located in the NC1 domain,which was predicted to produce truncated collagen and impair the trimerization of alpha 1(X)chains and combination with molecules in the matrix.Consistently,in vitro functional studies detected that alpha-1(X)chain and trimerization of alpha-1(X)chains were significantly decreased while the dimer level of alpha-1(X)chain was obviously increased.Moreover,genotype-phenotype correlation analysis demonstrated that patients with truncating mutations or mutations in NC1 domain often presented earlier onset and severer symptom compared with those with non-truncating mutations or mutations in non-NC1 domains.Conclusion A de novo mutation in COL10A1 gene(p.M622Tfs*54)causes abnormal trimerization and assembly of X-collagenαlpha-1(X)chain,resulting in

关 键 词：遗传性骨骼发育不良 COL10A1基因突变 生物信息学分析 功能研究 

分 类 号：R681[医药卫生—骨科学]