反向型营养不良性大疱性表皮松解症COL7A1基因的突变

Analysis of COL7A1 gene mutations in a family with recessive dystrophic epidermolysis bullosa inversa

目的报告1例反向型营养不良性大疱性表皮松解症，并进行组织学超微结构分析和分子遗传学诊断。方法患者男，24岁。主诉反复皮肤水疱24年。出生后全身反复出现泛发水疱和大疱，以摩擦部位为主，夏重冬轻，伴有明显的瘙痒感，无光敏感现象。3～4岁后水疱、大疱减少，仅躯干、腹部余留水疱，症状逐年减轻。5岁时患肾炎，15岁时发展为肾衰竭，进行了肾移植手术，长期口服他克莫司和吗替麦考酚酯治疗，服药后皮损减轻。父母非近亲结婚，无相关家族史。皮肤科检查：全身未见水疱，腰背腹部可见大片边缘不规则色素减退萎缩性瘢痕，伴多个甲营养不良。收集先证者临床资料，通过免疫荧光抗原检测、透射电镜检查对患者进行分型，应用PCR扩增COL7A1基因并测序寻找致病突变，通过软件预测和逆转录PCR对突变结果在RNA水平进行验证。结果患者皮损部皮肤Ⅶ型胶原表达量减少；皮肤裂隙位于真皮侧，致密板下锚纤维数量减少。COL7A1基因上存在同义杂合的c．C5499T剪接突变和c．C6205T（p．Arg2069Cys）错义突变，两突变分别来自父母。150例无关健康人对照中未发现相应突变。结论c．C5499T的剪接突变和c．C6205T（p．Arg2069Cys）错义突变可能是导致患者反向型营养不良性大疱性表皮松解症的致病突变。

Objective To observe the ultrastructural features of recessive dystrophic epidermolysis bullosa inversa （RDEB-I） and to detect the mutations of COLTA1 gene in a family with RDEB-I. Methods A 24-year-old male patient complained of recurrent vesicles in the skin for 24 years. The lesions began as generalized pruritic vesicles and bullae soon after birth, with a predilection for areas subject to friction, and showed a trend to be worse in summer but mild in winter. No photosensitivity was observed. When he was 3 to 4 years old, the lesions were decreased in number, with the only involvement of the trunk and abdomen; thereafter, the lesions were improved year by year. The patient suffered from nephritis at the age of 5 years, which progressed into renal failure at the age of 15 years. He received renal transplantation and was given long-term oral tacrolimus and mycophenolate mofetil, which leaded to an improvement in the lesions. The family history was unremarkahle, and the marriage between her parents was not consanguineous. Dermatological examination revealed large areas of irregularly-marginated, hypopigmented, atrophic scar on the waist, back and abdomen with onychodystrophy involving multiple nails. No vesicles were observed. Immunofluorescence antigen mapping and transmission electron microscopy were conducted to observe the expression of type VII collagen in and ultrastructure of cutaneous lesions from the patient. Venous blood samples were obtained from the patient as well as his parents and 3 sisters, and drill biopsy specimens were obtained from the margin of vesicular lesions and unaffected anterior tibial skin of the patient. DNA specimens were obtained from the blood samples of the family members and 150 unrelated healthy controls, and RNA was extracted from the biopsy samples of the patient. PCR and direct sequencing were carried out to detect mutations in COLTA1 gene, and reverse transcription-PCR was conducted to confirm the mutation at mRNA level. Results Skin cleavage was observed under lamina densa in the dermis, with a decrease in anchoring fibrils and expression of type VII collagen in the lesions of the patient. A heterozygous synonymous mutation c.C5499T which created a new splicing site and leaded to a premature terminal codon, as well as a heterozygous missense mutation c.C6205T （C-T transition at codon 2069： CGT to TGT）which leaded to the substitution of arginine by eysteine, were identified in the COL7A1 gene of the proband and all of his sisters, but not in any of the unrelated controls. The c.C5499T and c.C6205T mutations were inherited from the patient＇s father and mother respectively. Conclusion The compound heterozygous mutations c.C6205T and c.C5499T may be responsible for RDEB-I in this patient.