一个先天性指间关节粘连家系致病基因鉴定  

Identification a pathogenic gene in a pedigree with symphalangism

作　　者：崔恒庆 孙滨 方霞 周晟博 杨皓然 戴心怡 韩刚 王斌[1] Cui Hengqing;Sun Bin;Fang Xia;Zhou Shengbo;Yang Haoran;Dai Xinyi;Han Gang;Wang Bin(Department of Plastic and Reconstructive Surgery,Shanghai Ninth People’s Hospital,Shanghai Jiao Tong University School of Medicine,Shanghai 200011,China)

机构地区：[1]上海交通大学医学院附属第九人民医院整复外科,200011

出　　处：《中华整形外科杂志》2020年第5期499-506,共8页Chinese Journal of Plastic Surgery

基　　金：国家自然科学基金(81571930,81772115);国家重点研发计划(2018YFC1105801)。

摘　　要：目的鉴定1个先天性指间关节粘连家系的致病基因,体外验证致病位点并分析其功能意义。方法收集1个2017年9月就诊于上海交通大学医学院附属第九人民医院患有指间关节粘连的家系临床资料,采集其外周血并提取基因组DNA,PCR扩增NOG、FGF9、GDF5外显子区段,使用一代测序技术测定外显子区段基因突变。计算机模拟noggin-GDF5蛋白复合体空间结构。体外COS-7细胞转染5μg空载质粒、野生型noggin质粒及V202G突变型质粒,每组设置3个复孔,实验重复3次,并以蛋白质印迹法检测noggin蛋白表达量。C2C12细胞体外同样转染上述质粒,进行成骨诱导分化,通过碱性磷酸酶染色、定量实验,并用qRT-PCR对成骨相关基因Col1α1、ALP及Runx2 mRNA进行定量分析,每组设置3个复孔,实验重复3次。采用Graphpad Prism 6软件对数据进行分析,组间比较采用非配对t检验,P<0.05表示差异有统计学意义。结果先证者(1岁男性患儿)及其母亲均患有指间关节粘连。一代测序结果显示,该家系中患者均存在NOG基因(c.605T>G p.V202G)杂合错义突变,FGF9、GDF5未测及与疾病相关的突变。计算机三维结构模拟显示该位点位于noggin蛋白α螺旋处。蛋白质印迹法结果显示突变型蛋白表达量显著低于野生型。体外成骨诱导分化显示,V202G突变型蛋白抑制成骨分化作用下降。碱性磷酸酶染色定量结果显示,空白载体组碱性磷酸酶活性为(12.3±0.8)U/L,野生型质粒组为(2.6±0.3)U/L,与空白载体组相比其碱性磷酸酶活性显著降低(t=11.550,P<0.001),突变型质粒组碱性磷酸酶活性为(10.8±0.3)U/L,与空白载体组相比差异无统计学意义(t=1.830,P=0.141)。成骨相关基因mRNA表达水平定量分析,与前述结果一致,与空白载体组相比,野生型noggin组成骨相关基因ALP、Col1α1及Runx2 mRNA表达量显著降低(t=5.987、4.498、4.170,P=0.004、0.011、0.014),突变型质粒组与空白载体组相比差异�Objective To identify the pathogenic gene of a pedigree with symphalangism and to prove the pathogenicity of this locus in vitro.Methods The clinical data of patients’families were collected at Shanghai Ninth People’s Hospital,Shanghai Jiao Tong University School of Medicine,peripheral blood was collected and genomic DNA was extracted and NOG,FGF9,GDF5 exon regions were amplified by PCR,and the exon gene mutations were detected by first-generation sequencing technique.The structure of noggin-GDF5 protein complex was simulated in silicon.COS-7 cells were transfected with 5μg empty plasmid,wild type plasmid and V202G mutant plasmid in vitro.Each group of plasmids was transfected into 3 well cells.The experiment was repeated for 3 times,and the expression of noggin protein was detected by Western blotting.C2C12 cells were also transfected with the above plasmids in vitro for osteogenic differentiation.By applying alkaline phosphatase staining and quantitative assay.Relative expression level of osteoblast-related genes Col1α1,ALP and Runx2 were detected by qRT-PCR.Each group of plasmids was transfected into 3 well cells,and the experiment was repeated for 3 times.All statistical analysis were performed by Prism 6 software.The result were shown as mean±standard deviation,and the comparison between groups was done by unpaired t-test.Data were considered statistically significant when P value is less than 0.05.Results Both the proband and his mother suffered from symphalangism.The result of Sanger sequencing showed that there was a heterozygous missense mutation of NOG gene(p.V202G)in all patients in this pedigree.No disease-related mutations were detected in FGF9 and GDF5.Computer three-dimensional mechanism simulation showed that the site was located at theαhelix.The result of Western blotting showed that the expression of mutant protein was significantly lower than that of wild type.Osteogenic differentiation in vitro showed that the inhibitory effect of V202G mutant protein on osteogenic differentiation decr

关 键 词：关节粘连 基因突变 遗传家系 关节强直 

分 类 号：R682[医药卫生—骨科学] R440[医药卫生—外科学]