Genetic diagnosis in two families with dystrophic epidermolysis bullosa / 中华皮肤科杂志

Objective:To analyze clinical characteristics of and causative genes in two families with dystrophic epidermolysis bullosa, and to reveal the pathogenesis of the disease and mechanisms underlying phenotypic differences between patients.Methods:DNA was extracted from peripheral blood samples of members from two families with dystrophic epidermolysis bullosa, and subjected to high-throughput sequencing and Sanger sequencing.Results:The clinical manifestations of the 2 probands in the 2 families were consistent with the diagnosis of dystrophic epidermolysis bullosa, and the symptoms of the proband in family 1 were more serious than those of other patients in the family. Genetic testing showed that all patients in family 1 carried a mutation c.6082G>C (p.G2028R) in the COL7A1 gene, and the proband and her phenotypically normal mother and uncle also carried a splice-site mutation c.7068+2 (IVS91) T>G in the COL7A1 gene, both of which were first reported. The proband in family 2 carried the mutations c.6081_6082 ins C (p.G2028Rfs*71) and c.1892G>A (p.W631X, first reported) in the COL7A1 gene, which were inherited from her father and mother, respectively.Conclusion:The two pathogenic mutations may be the molecular mechanism underlying the severe clinical phenotype in the proband in family 1; the first reported mutations enriched the mutation spectrum of the COL7A1 gene.

Mutation analysis of the COL7A1 gene in 4 cases of recessive dystrophic epidermolysis bullosa / 中华皮肤科杂志

Objective:To analyze gene mutations in and clinical phenotypes of 4 children with recessive dystrophic epidermolysis bullosa (RDEB) .Methods:Clinical data were collected from 4 children with RDEB, and DNA was extracted from peripheral blood samples of the children and their parents. A multi-gene panel targeting congenital epidermolysis bullosa was used for high-throughput sequencing. After identification of causative gene mutations, Sanger sequencing was performed to bidirectionally verify the mutations in the patients and their parents.Results:Genetic testing showed 8 compound heterozygous mutations in the COL7A1 gene in the 4 cases. Case 1 was diagnosed with moderate generalized RDEB, and inherited a paternal mutation c.7828C>T and a maternal mutation c.448G> A; case 2 was also diagnosed with moderate generalized RDEB, and inherited a paternal mutation c.3625_3635del and a maternal mutation c.2726_2728del; case 3 was diagnosed with localized RDEB, carrying a paternal mutation c.682+1G>A and an allelic mutation c.5532+1G>A, but the mutation c.5532+1G>A was not identified in the DNA extracted from the parental peripheral blood samples; case 4 was diagnosed with severe generalized RDEB, and inherited a paternal mutation c.5196delC and a maternal mutation c.500_501insAGGG. Among these mutations, c.2726_2728del and c.5196delC had not been reported previously.Conclusions:All the 4 children with RDEB carried compound heterozygous mutations in the COL7A1 gene, which may be the cause of RDEB. The phenotypes of biallelic frameshift mutation carriers appearred more severe than those of carriers of compound heterozygous mutations composed of biallelic splice site, missense and nonsense, frameshift and amino acid deletion or insertion mutations.

Gene mutation analysis of congenital skin defects: a case report / 中华皮肤科杂志

A female infant presented with skin defects and blisters for 2 hours after birth.Physical examination showed asymmetric skin defects on both lower extremities and left wrist,a thin-walled blister on the dorsal side of the right hand,and partial loss of the oral mucosa.No other abnormal signs were found.Genetic testing showed a heterozygous pathogenic mutation c.481C ＞ T (p.Gln161*) in exon 4 of the COL7A1 (NM-000094) gene and a heterozygous pathogenic mutation c.1837C ＞ T (p.Arg613*) in exon 14 of the COL7A1 (NM-000094) gene,which were also identified in the patient's father and mother respectively.The infant was diagnosed with congenital skin defects.The patient received protective isolation,focal washing with 0.9％ sodium chloride physiological solution,topical epidermal growth factor and comprehensive treatment for infection prevention.After 6-day treatment,the patient was discharged with dry and non-exudative skin lesions.This case prompted that abnormal heterozygosis mutation at C.481 and C.1837 sites on the COL7A1 (NM-000094) gene could form compound heterozygote,acting as pathogenic mutation.

Mutation analysis of the COL7A1 gene and prenatal diagnosis in two families with recessive dystrophic epidermolysis bullosa / 中华皮肤科杂志

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.

Mutation analysis of the COL7A1 gene in pretibial dominant dystrophic epidermolysis bullosa / 中华皮肤科杂志

Objective To explore the role of type Ⅶ collagen (COL7A1) gene in the pathogenesis of pretibial dominant dystrophic epidermolysis bullosa (DDEB-Pt).Methods Peripheral blood samples were obtained from a sporadic Chinese patient of Han nationality with DDEB-Pt,his parents and 100 healthy human controls.A modified salting-out method was used to extract genomic DNA from the blood samples,and PCR was performed to amplify 118 exons of the COL7A1 gene followed by DNA sequencing.Results A G→A mutation was identified at position 6109 (G6109A) in exon 78 of the COL7A1 gene in this patient,which caused a change from GCT to ACT at codon 2037 in the triple helix region,and resulted in the substitution of glycine (Gly) by arginine (Arg) (p.Gly2037Arg).Conclusion A novel glycine substitution mutation was identified in the COL7A1 gene in the patient with DDEB-Pt,which may be a pathogenic mutation.

Application of next generation sequencing technology for genetic diagnosis of a neonate and the family with heredi-tary dystrophic epidermolysis bullosa / 临床儿科杂志

Objectives To detect genetic causes of dystrophic epidermolysis bullosa (DEB). Methods Next-generation sequencing was used to detect a neonate with DEB. Sanger sequencing was used to confirm the results and detect his parents and grandmother on his mother side from the family. Results The neonate was found to have heterozygous mutation c.6781C>T of exon 86 in COL7A1 gene.This mutation results in R2261X nonsense mutation in typeⅦcollagen. His mother and grand-mother on his mother side have the same mutation. Conclusion Next-generation sequencing technology is a useful tool for the detection of mutations of COL7A1 gene, which is valuable for clinical application.

Epidermolysis bullosa pruriginosa: A rare presentation with asymptomatic lesions.

Epidermolysis bullosa pruriginosa (EBP) is a subtype of dominant dystrophic epidermolysis bullosa (DDEB) and is clinically characterized by pruritic lichenified plaques or prurigo-like lesions with violaceous linear scarring. Pruritus has always been described as one of the most striking features in EBP. Mutations in COL7A gene, especially in the glycine residue, have been shown to cause this form of DDEB. In this report, we describe a north Indian familial clustering of three cases of EBP, spread across two generations, presenting with hypertrophic lichenoid cutaneous lesions, which were completely asymptomatic. Clinical and histopathological analysis favored the diagnosis of EBP in all three cases. They are being reported for their unusual asymptomatic presentation.

Analysis of COL7A1 gene mutations in a family with recessive dystrophic epidermolysis bullosa inversa / 中华皮肤科杂志

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.

Mutations of COL7A1 gene in three cases of dystrophic epidermolysis bullosa pruriginosa / 中华皮肤科杂志

Objective To detect the mutations of COL7A1 gene in three cases of dystrophic epidermolysis bullosa pruriginosa (DEBP). Methods Clinical data were collected from 3 patients with DEBP. Skin lesions were obtained from these patients and subjected to transmission electron microscopy. DNA was extracted from the peripheral blood of the 3 patients, their 16 relatives, and 150 unrelated normal human controls, and PCR was performed to amplify all the exons and flanking sequences of COL7A1 gene followed by sequencing.Results The patient 1 and 2 had family history, whereas the case 3 was sporadic. Transmission electron microscopy showed tissue cleavage beneath lamina densa in case 1 and slightly decreased anchoring fibrils in some areas of the lesions in case 1 and 3. Three heterozygous mutations of COL7A1 gene, i.e., c. G6734T, c.G6859A and c. G5318T, which leaded to three amino acid mutations, i.e., p. G2245V, p. G1773V and p. G2287R, were found in patient 1, 2 and 3 respectively. Of them, p. G2245V and p. G1773V were novel mutations. The mutations strictly cosegregated with the phenotype in the patients of family 1 and 2. No mutation was detected in the unaffected parents of patient 3 or the 150 unrelated healthy controls. Conclusions The p. G2245V, p. G2287Rand p. G1773V mutations of COL7A1 gene may be responsible for the phenotype of DEBP in the three cases,and of them, p. G2245V and p. G1773V have never been reported.

Type VII Collagen Gene Mutations (c.8569G>T and c.4879G>A) Result in the Moderately Severe Phenotype of Recessive Dystrophic Epidermolysis Bullosa in a Korean Patient

Dystrophic epidermolysis bullosa (DEB) are caused by mutations in the COL7A1 gene, which encodes type VII collagen. Even though more than 500 different COL7A1 mutations have been identified in DEB, it still remains to be under-investigated. To investigate the mutation of COL7A1 in moderately severe phenotype of recessive DEB (RDEB) in a Korean patient, the mutation detection strategy was consisted of polymerase chain reaction (PCR) amplification of genomic DNA, followed by heteroduplex analysis, nucleotide sequencing of the PCR products demonstrating altered mobility. In this study, we found that one mutation (c.8569G>T) was detected within exon 116. The mutation of c.8569G>T in exon 116 changed the GAG (Glu) to TAG, eventually resulted in premature termination of type VII collagen polypeptide. Furthermore the mother did not have the mutation c.8569G>T in exon 116. The other novel mutation (c.4879G>A) was detected within exon 51 of both patient and mother, thereby resulting in changing valine (Val) to isoleucine (Ile) in type VII collagen polypeptide. Taken together, in this study we identified compound heterozygosity for COL7A1 mutations (c.8569G>T and c.4879G>A) in moderately severe RDEB in a Korean patient. We hope that this data contribute to the expanding database on COL7A1 mutations in DEB.

Three Cases of Recessive Dystrophic Epidermolysis Bullosa-mitis / 대한피부과학회지

Recessive dystrophica epidermolysis bullosa(RDEB) is rare, chronic non - inflammatory bullous disease of hereditary trait, which produces blister formation in early childhood. A localized, less severe form, often called RDEB-mitis, occurs at birth and usually involves the acral areas, with atrophic scarring of the joint surfaces and nail dystrophy but little mucosal involvement. Severe RDEB is a multilating disease, known also as the Hallopeau-Siemens (HS) varient. We report three cases which developed at birth and infancy, respectively. Bullae, atrophic scars were noted on the knees and ankles, and milia on the both hands and feet. Histologically, sections from the old blister lesion showed characteristic separation of the epidermis from the dermis forming bulla. Identification of the mutations COL7A1 gene was revealed by direct sequencing of each exon. We present three cases of RDEB-mitis considering the abscence of family history with clinical, histological and molecular analysis findings.