Objective: To analyze two mutations in voltage gated chloride channel 7 gene (CLCN7) of two osteopetrosis patients using bioinformatic methods, and compare the effects of mutations on protein structure and physicochemical characteristics, so as to predict the damaging effect of the mutations on the disease development.Methods: Genomic DNA were collected from peripheral blood of two osteopetrosis patients and their family members.CLCN7 gene was PCR amplified and sequenced.NCBI website, PolyPhen-2, DNAStar, SWISS MODEL were used to analyze the effect of the mutated genes.Results: Case 1 was diagnosed as intermediate autosomal recessive osteopetrosis (IARO), and his CLCN7 gene showed a de novo homozygous mutation c.1409 C>T in exon 16, which resulted in a residue substitution from proline to leucine (c.1409 C>T;p.Pro470Leu).Case 2 was diagnosed as autosomal dominant osteopetrosis type Ⅱ (ADOⅡ).The sequence analysis revealed a heterozygous mutation c.856 C>T in exon 10 of CLCN7 gene, which resulted in arginine to tryptophan residue substitution (c.856 C>T;p.Arg286Trp).Both of the mutations are located in highly conserved domain among different species.The mutations cause the changes in the secondary structure, tertiary structure and physicochemical properties and so on.Conclusions: Multiple bioinformatic analyses indicate that the mutations in CLCN7 gene (c.1409 C>T and c.856 C>T) can change the molecule structure and affect biological function of ClC-7, which shows a damaging effect and might be the key of disease development in the patients.%目的:采用生物信息学方法对两名骨硬化症患者的电压门控氯通道7基因(CLCN7)突变位点进行分析,比较突变对蛋白结构、理化性质等方面的影响,预测突变可能对疾病发生产生的作用.方法:提取两例骨硬化症患者及家庭成员外周血基因组DNA,PCR扩增CLCN7基因并进行序列分析;采用NCBI在线搜索、PolyPhen-2数据库、DNAStar软件、SWISS MODEL等多种手段对突变基因编码的蛋白质进行多方面预测.结果:病例1为中间型常染色体隐性遗传骨硬化症,基因测序表明其CLCN7基因16号外显子产生了c.1409 C>T纯合突变,所编码氨基酸由脯氨酸变为亮氨酸;病例2为Ⅱ型常染色体显性遗传骨硬化症,其10号外显子发生了c.856 C>T杂合突变,精氨酸变为色氨酸.两个突变位点在多个物种均表现出高度同源性,突变可导致CLC-7蛋白二级结构发生变化,从而改变蛋白质立体结构、理化特性等方面的特征.结论:多种生物信息学分析表明CLCN7基因的c.856 C>T和c.1409 C>T可改变蛋白的分子结构和功能,表现为有害类型的突变,可能导致骨硬化症的发生.
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