COL7A1基因复合杂合突变致隐性营养不良型大疱性表皮松解症1家系研究

Recessive dystrophic epidermolysis bullosa caused by COL7A1 gene mutation in a pedigree

目的 分析1例常染色体隐性营养不良型大疱性表皮松解症患者临床表型,探讨该家系COL7A1基因突变情况。方法 收集1例常染色体隐性营养不良型大疱性表皮松解症患者(先证者)及其家系临床资料。采集先证者及其母亲外周血(先证者父亲病逝),提取基因组DNA,采用高通量测序法对先证者皮肤病相关基因各外显子编码区进行测序,确定基因变异位点,采用PCR-Sanger测序法验证致病性突变情况。应用软件分析剪接突变、错义突变位点保守性及致病性。结果 先证者临床表现为双手背、腹部及双足多发红色斑块、结痂,瘙痒明显,指/趾甲营养不良及脱落,口腔舌体黏膜白斑;血清总蛋白、球蛋白、IgG水平升高,自然杀伤细胞绝对数、自然杀伤细胞百分比、淋巴细胞绝对数降低;心电图示多导联ST-T改变;家系3代仅先证者1人患病。先证者存在COL7A1基因c.841_846+1dup和c.5560G>A(p.Gly1854Arg)复合杂合突变;母亲存在c.5560G>A(p.Gly1854Arg)突变;c.841_846+1dup为剪接突变,突变导致6号外显子3′端正常供体剪接位点丢失,并在下游7 bp处激活形成新的供体剪接位点,使7号外显子发生提早终止密码子,此突变在人类基因突变数据库中未收录,为新突变。c.5560G>A(p.Gly1854Arg)为错义突变,65号外显子编码区第5 560位核苷酸由鸟嘌呤突变为腺嘌呤,编码蛋白第1 854位甘氨酸突变为精氨酸,COL7A1编码蛋白在多个物种第1 854位甘氨酸序列中呈完全保守,该突变具有致病性。根据先证者临床表现、辅助检查、基因检测及家系特征,诊断为常染色体隐性遗传营养不良型大疱性表皮松解症。结论 COL7A1基因c.841_846+1dup和c.5560G>A(p.Gly1854Arg)复合杂合突变是该先证者临床表型的可能原因。

Objective To analyze the clinical phenotype of a case of autosomal recessive dystrophic epidermolysis bullosa,and to investigate COL7A1 gene mutation in a pedigree.Methods The clinical data were collected from the proband and her family.The peripheral blood samples were collected from the proband and her mother(her father died),the genomic DNA was extracted,and high throughput sequencing was used to detect the coding regions of exons of the skin disease-related genes to define the gene mutation sites,and the pathogenic mutation was verified by PCR-Sanger sequencing.The conservation and pathogenicity of gene mutation sites were predicted by bioinformatics software.Results The proband was clinically shown as multiple red plaques and scabs with marked itching on the back of hands,abdomen and feet,dystrophic and absent nails,leukoplakia of oral tongue mucosa,elevated serum total protein,globulin and IgG levels,and decreased absolute natural killer cell count,natural killer cell percentage and absolute lymphocyte count;electrocardiogram showed multi-lead ST-T changes;only the proband in three generations of the family was affected.The proband carried compound heterozygous mutations of c.841_846+1 dup and c.5560G>A(p.Gly1854Arg)in COL7A1 gene;her mother carried a c.5560G>A(p.Gly1854Arg)mutation;c.841_846+1 dup was a splice mutation that caused the loss of the normal donor splice site at the 3′end of exon 6 and activated at 7bp downstream to form a new donor splice site,causing an early termination codon in exon 7.This mutation was not included in the Human Mutation Database and it was a new mutation.c.5560G>A (p.Gly1854Arg)was a missense mutation in which the nucleotide at position 5 560 in the coding region of exon 65 was mutated from guanine to adenine,the protein coding for position 1 854 was mutated from glycine to arginine,the protein encoded by COL7A1 was fully conserved in the glycine sequence at position 1 854 in several species,and the mutation was pathogenic.By comprehensive consideration of the clinical manifestations,auxiliary examination results,genetic testing results and family characteristics,the patient was diagnosed with recessive dystrophic epidermolysis bullosa.Conclusion The compound heterozygous mutation of COL7A1c.841_846+1 dup and c.5560G>A (p.Gly1854Arg)may be the cause of the clinical phenotype of the proband.