目的基因捕获结合二代测序技术诊断遗传性大疱性表皮松解症及COL7A1基因新突变分析

Diagnosis of hereditary epidermolysis bullosa and identification of a novel COL7A1 mutation with target next generation sequencing technique

目的:应用目的基因捕获技术诊断遗传性大疱性表皮松解症,探讨其价值及发现致病突变。方法:收集复旦大学附属儿科医院皮肤科临床诊断为遗传性大疱性表皮松解症的患儿临床资料。提取患儿及其父母外周血DNA,采用目的基因捕获结合二代测序技术,并与人类参考基因组hg19进行比对。Sanger测序进行位点及突变来源验证。1000genomes数据库查询变异频率,对于新发变异应用软件预测序列保守性和致病意义。PhastCons软件在物种间比对核苷酸序列保守性,取值范围0～1,接近1提示高度保守。MutationTaster2软件预测突变致病意义。结果:患儿出生后左侧小腿伸侧至足背皮肤缺损。测序发现与临床表型符合的靶基因COL7A1的2个变异位点,c.6900+2T>G和c.8819-3C>T,Sanger测序验证存在上述变异,先证者父亲在2个位点均未发生变异,母亲存在c.8819-3C>T变异。其中c.6900+2T>G为新发突变,位于经典的前体m RNA剪接区。c.8819-3C>T在正常人群频率0.04%,为低频突变位点。PhastCons软件计算c.6900+2T序列保守性得分为1,提示高度保守性序列,c.8819-3C得分为0.485,提示保守性较低。MutationTaster2软件预测c.6900+2T>G和c.8819-3C>T均为致病性突变。结论:目的基因捕获结合二代测序技术可以对遗传性大疱性表皮松解症进行诊断和分型,发现COL7A1 c.6900+2T>G新发突变。

Objective： Using targeted DNA capture and massively parallel sequencing technique to diagnose epidermolysis bullosa （EB） and to identify pathogenic mutation. Method： Genomic DNA was extracted from peripheral blood leukocytes of a patient and her parents. Targeted DNA capture was performed, followed by massively parallel sequencing of 17 EB genes. The gene sequences were mapped to the reference gene of human hg19. Sanger sequencing was used to confirm the potential pathogenic mutation. The mutation source was verified by analyzing parents＇DNA. The frequency of variations was originated from 1000 genomes. PhastCons software was used to determine the grade of conservation of a given nucleotide （values vary between 0 and 1, the closer the value is to 1, the more probable the nucleotide is conserved）. MutationTaster2 was used to evaluate the pathogenic potential of DNA sequence alteration. Results： A neonatal girl suffered fi＇om congenital localized absence of skin on her left lower leg without family history. Targeted DNA sequencing showed COL7A1 c.6900＋2T〉G and COL7AI c.8819- 3C〉T. Sanger sequencing confirmed two variations. Her father had no mutation while her mother carries COLTA1 c.8819-3C〉 T. The allele frequency of c.8819-3C〉T was 0.04%, a low frequency of mutation, in normal humans. Conservation scores of c.6900＋2T〉G and c.8819-3C〉T were 1 and 0.485, respectively. MutationTaster2 indicated that both COL7A1 c.6900＋2T〉G and COL7AI c.8819-3C〉T were pathogenic mutations. Conclusions： Target next generation sequencing can be used to diagnose EB. A novel COL7A1 mutation of c.6900＋2T〉G is identified.