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Mutations Of COL7Al Gene Of Dystrophic Epidermolysis Bullosa Pruriginosa

Posted on:2012-04-16Degree:DoctorType:Dissertation
Country:ChinaCandidate:Z L TangFull Text:PDF
GTID:1114330335485135Subject:Dermatology and Venereology
Abstract/Summary:PDF Full Text Request
BackgroundEpidermolysis bullosa (EB) is a group of disease with dermal-epidermal separation in the different level of basement membrane zone. According to the ultrastructure and the precise split location, EB can be divided into four main types: epidermolysis bullosa simplex (EBS), junctional epidermolysis bullosa (JEB), dystrophic epidermolysis bullosa (DEB) and Kindler syndrome. EBS, with cleft within basal keratinocytes, is caused by mutations in KRT5, KRT14 or plectin protein. JEB, of which the separation occurs predominantly in the lamina lucida, can be due to the defects of lamina-332, integrin╬▒6╬▓4 and collagen XVII. DEB, with separation under the lamina densa, is caused by the defect in collagen VII. Finally, Kindler syndrome, without definitely locations of the cleft, is caused by mutation in Fermitin family homologous protein 1.Dystrophic epidermolysis bullosa (DEB) is a subtype of inherited epidermolysis bullosa characterized by clefts under the lamina densa. DEB can be inherited by autosomal dominant or autosomal recessive pattern, based on the mutation mode. DEB is caused by mutations in type VII collagen gene which encodes one of the important components of the anchoring fibrils.Dystrophic epidermolysis bullosa pruriginosa (DEBP) is a rare type of dystrophic epidermolysis bullosa (DEB). Patients with DEBP may show clinical features of DEB early after birth, with vesicles and blisters, which were diminished with age. Gradually patients developed pruriginosa-like lesions, mainly on the shins. Patients can also have no clinical manifestations until adult age, when pruriginosa lesions gradually appeared on the shins, with slightly nail destrophy. Albopapuloid lesions may be found in some patients with DEBP.Although DEBP can be inherited either in an autosomal dominant or in an autosomal recessive pattern, most of them are autosomal dominant. Disease-causing mutations in COL7A1 gene of DEBP includes missense mutations, frameshift mutations and splice mutation. Glycine substitution missense mutations in COL7A1 is the most common one in DEBP, which affects the characterized structural Gly-Xaa-Yaa in collagen proteins. Because glycine is the smallest amino acid and plays an important role in the function and structure of VII collagen. The Tri-helix structural formation of VII collagen was interfered as Glycine substituted by other amino acids, affecting its stability and integrity. As a result, functions of anchoring fibrils under lamina densa in bsasement, which is mainly composed by collagen VII, may be altered. The mechanism of severe pruritus in DEBP and the cause of the prurigo-like lesions are still unclear. Our research focused on the relationship between clinical phenotypes and genotypes in three cases with DEBP. It may further rich DEBP mutations database and prepare for the prenatal diagnosis in the families of these three cases.ObjectiveTo determine mutations of COL7A1 gene in three cases with dystrophic epidermolysis bullosa pruriginosa.Methods1. The first patient was a 17 y old male who developed vesicle and bulla on the friction site of hand and foot after brith.The frictional vesicle and bulla diminished gradully with age, but papules and nodules were noted on his shins,dorsa,and extremities with severe pruritus.The same condition was formed in this mother and relatives.with denial parent consanguineous marriage. The second and third cases were adult female. The first onset was occurred after juvenile and manifested by gradually increased nodule on the shins and dorsa with severe pruritus.The second case has family history and the third was sporadic.Physical examination revealed multiple purple red or black papules and nodula,with epidermal excoriations, hyperkeratosis on shins and trunk.Nail involvement including dystrophy,atrophy was found in three cases.The lesion in first case were severe,with multiple papule and scare on dorsa. The pathology revealed hyperkeration,acanthosis,fissure formation under epidermis, and lymphocyte infiltration around blood vessel in upper dermis.2. Electromicroscope examination:The skin lesion was taken from the shins of the patient 1 and patient 3, fixed in 5% glutaraldehyde solutions, and observed by transmission electromicroscope.3. Genomic DAN extraction:5ml peripheral blood was taken from the patients and their relatives. DNA was isolated by phenol-chloroform.4. PCR amplification and DNA sequencing:72 pairs of primers were designed according to COL7A1 gene sequence. PCR were peformed and all the amplicons were subjected to the direct sequencing.150 normal individuals were also sequenced as controls. The purified PCR products were sequenced by Beijing Tianyihuyuan company.ResultsPatient 1 and 2 are familial cases, whereas patient 3 is sporadic. Transmission electron microscopy examination showed cleft under lamina densa and slightly decreased anchoring fibrils in part of the regions. Genomic DNA was subjected to mutation detection by PCR methods. All the exons, including the flanking intronic sequences, were amplified and the products were purified and send for direct sequencing. By comparison with COL7A1 sequences published by Ensembl, we detect a heterozygous G-T substitution affecting nucleotide 6734, exons of 85 leading to Gly2245Val heterozygous mutation in case 1. In case 2, sequencing of the PCR product revealed a heterozygous G-A mutation at the nucleotide 6859, resulting in Gly2287Arg. These mutations were also found in their affected relatives. In case 3, heterozygous G-T mutation at the nucleotide 5318, leading to Gly1773Val was detected, but the same mutation was not found in her unaffected parents. All mutations was confirmed by reverse sequencing. The novel mutations, Gly2245Val and Gly1773Val, were not detected in 150 normal controls. ConclusionMutations of p.G2245V,p.G2287R and p.G1773V of COL7Al may be the causes of the clinical phenotype of the three patients. Further proved glycine replace mutation the important pathogenic significance in DEBP.Among them,2245V p.G p.G 1773V for two not reported new mutations. This riched DEBP genotype phenotype and the clinical research, and had laid the foundation of prenatal diagnosis.
Keywords/Search Tags:Dystrophic Epidermolysis Bullosa, Pruriginosa, COL7Al, Gene, Mutation
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