ABSTRACT
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.
ABSTRACT
Objective To observe the ultrastructural features of recessive dystrophic epidermolysis bullosa inversa(RDEB-Ⅰ)and to detect the mutations of COL7A1 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 unremarkable,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 COL7A1 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 cysteine,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.