两个隐性营养不良型大疱性表皮松解症家系COL7A1基因突变分析及产前诊断

Mutation analysis of the COL7A1 gene and prenatal diagnosis in two families with recessive dystrophic epidermolysis bullosa

目的 检测2个隐性营养不良型大疱性表皮松解症家系COL7A1基因突变情况，并在患儿母亲再次妊娠时进行胎儿产前诊断。方法 收集2例隐性营养不良型大疱性表皮松解症患儿临床资料，提取患儿及其父母的外周血DNA，应用PCR扩增COL7A1基因的全部118个外显子并测序；用100例无血缘关系的健康人外周血作对照。确定致病突变后，在患儿母亲再次妊娠时，羊膜腔穿刺术获取羊水细胞。分别自直接抽提的羊水细胞以及培养后的羊水细胞中提取基因组DNA进行扩增和测序，检测COL7A1基因突变，同患儿的检测结果进行比较，行产前诊断。胎儿娩出后抽取静脉血，检测COL7A1基因突变。所有测序结果都经过双向测序验证。结果 2例患儿均发现COL7A1基因复合杂合突变。例1 COL7A1基因存在c.5453G 〉 A及c.6781C 〉 T复合杂合突变，分别导致编码氨基酸发生p.G1818D突变和产生提前终止密码p.R2261Efs＊25，其中c.5453G 〉 A来自父亲，c.6781C 〉 T来自母亲。例2 COL7A1基因存在c.6205C 〉 T及c.8272_8272delG复合杂合突变，导致编码蛋白发生p.R2069C及p.V2758Sfs＊28复合杂合突变，其中c.6205C 〉 T来自父亲，c.8272_8272delG来自母亲。2例患儿的母亲再次怀孕时所取羊水细胞及羊水培养细胞均未测到患儿所携带的COL7A1基因突变。胎儿出生后皮肤黏膜正常，无水疱，基因检测亦未发现患儿所携带的COL7A1基因突变。结论 2例营养不良型大疱性表皮松解症患儿均存在COL7A1基因复合杂合突变，并成功在2例患儿的母亲再次妊娠时进行了产前诊断。

Objective To detect mutations of the COL7A1 gene in 2 families with recessive dystrophic epidermolysis bullosa（RDEB）, and to perform prenatal diagnosis during the pregnancy of patients′ mothers. Methods Clinical data were collected from 2 patients with RDEB. DNA was extracted from the peripheral blood samples from the patients, their parents and 100 unrelated healthy people who served as controls. PCR was performed to amplify all the 118 exons of the COL7A1 gene followed by DNA sequencing. After identification of pathogenic mutations, amniotic fluid cells were obtained by amniocentesis during the next pregnancy of the patients′ mothers, and genomic DNA was extracted from uncultured or cultured amniotic fluid cells followed by amplification and DNA sequencing to detect mutations in the COL7A1 gene. The results were compared with patients′ results for prenatal diagnosis. After delivery, venous blood samples were collected from the neonates to detect mutations in the COL7A1 gene. All the results were verified by bidirectional sequencing. Results Compound heterozygous mutations in the COL7A1 gene were identified in the 2 patients. Two heterozygous mutations （c.5453G 〉 A and c.6781C 〉 T） in the COL7A1 gene were found in case 1, which resulted in the p.G1818D mutation and the formation of a premature termination codon p.R2261Efs＊25. Additionally, the c.5453G 〉 A and c.6781C 〉 T mutations were inherited from his father and mother respectively. Another 2 heterozygous mutations （c.6205C 〉 T and c.8272_8272delG） in the COL7A1 gene were identified in case 2, which led to the p.R2069C and p.V2758Sfs＊28 mutations in encoded proteins, and the c.6205C 〉 T and c.8272_8272delG mutations were inherited from the patient′s father and mother respectively. None of the above mutations in the COL7A1 gene was found in the uncultured or cultured amniotic fluid cells, which were collected from the 2 patients′ mothers during the next pregnancy. After birth, the neonates showed normal skin and mucosa without blisters, and genetic testing showed none of the above mutations in the COL7A1 gene in the neonates. Conclusion Compound heterozygous mutations in the COL7A1 gene were found in the 2 patients with RDEB, and prenatal diagnosis was successfully performed in the 2 patients′ mothers during the next pregnancy.