| Background Epidermolysis bullosa simplex(EBS) comprises a group of inherited skin diseases characterized by intraepidermal blisters caused by little or no trauma.The exact prevalence of EBS is uncertain;It was estimated about from 1:30,000 to 1:50,000. Traditionally,four clinical subtypes of EBS range from relatively mild blistering of the hands and feet(Weber-Cockayne type,EBS-WC,OMIM 131800) to more generalized blistering(Koebner type,EBS-K,QMIM 131900;Dowling-Meara type,EBS-DM, OMIM 131760;EBS with mottled pigmentation,EBS-MP,OMIM 131960).EBS-WC is the mildest variant with blistering restricted to the hands and feet;EBS-K is associated with generalized blistering;EBS-DM is the most severe with clustered blisters at any body site following mild trauma.EBS-MP associated with exceptional clinical as well as genetic features.Skin fragility is evident at birth,progressive brown pigmentation interspersed with depigmented spots,developing on the trunk and then on the extremities.EBS is usually inherited in an autosomal dominant manner,that results from mutations in the genes that encode keratin 5(K5) and keratin 14(K14).These 2 keratin molecules form the bulk of the protein contents of basal keratinocytes and participate as obligatory heterodimers in the formation of the keratin intermediate filament(KIF) cytoskeleton in these cells.Mutatins in either 1 of 2 basal keratin genes consequently impair keratinocyte capacity to resist mechanical stress,leading to cytolysis and blister formation upon exposure of the skin to friction.It has previously been described that there is a correlation between the respective EBS subtypes and the location of the K5 and KI4 gene mutations.The extension of the catalogue of mutations in EBS further advances our understanding of the molecular pathophysiology and provides the basis for appropriate diagnosis and genetic counselling of this patient group..Objective To identify K5 and K14 genes mutations in 3 sporadic Chinese patients and 3 pedigrees with EBS.This study will contribute to expand database of K5 and K14 genes and further illustrate the extensive diversity of mutational events that led to the different phenotypes of EBS.Methods Genomic DNA was isolated from blood samples using salt chloroform extraction method.Using genomic DNA as a template,exons 1-9 of K5 gene were amplified by polymerase chain reaction(PCR).Before exons.1-8 of K14 gene were amplified by PCR,a Long-Range Polymerase Chain Reaction(LR-PCR) was used to avoid amplification of the K14 pseudogene,which shows a 93%-95%homology to.the functional K14 gene.PCR products were directly sequenced with the same primers as used for PCR amplification by CEQ8800 DNA sequencer after purified by Exonuclease I-Shrimp Alkaline Phosphatase before sequencing and subjected to eleetrophoresis in 1%agarose gel.Results In this study,we described 3 pedigrees and 3 sporadic EBS patients.Direct sequencing of the entire coding sequence of K5 and K14 gene in these patients revealed five mutations,two of which were novel.For all of these mutations,sequence analysis of unaffected family members and 100 unrelated control individuals was performed to exclude polymorphisms.The analysis of the K5 gene exon 9 in the patient 1 and affected family members revealed a c.1649delG(p.Gly550AlafsX77) mutation; c.508G＞A(p..Glul70Lys) mutation was detected in the exon 1 of K5 in the patient 2 and the affected family members;It reveals an heterozygote G to A transition at position 280(c.280G＞A,p.Ala94Thr) of the K14 gene in the patient 3 and the affected family members,in the patient 4;an heterozygote T to A transition at position 1730 (c.1730T＞A,p.Phe577Tyr) of the K5 gene was detected;mutation(c.1237G＞A, p.Ala413Thr)of K14 gene exon 6 in patient 5.We failed to detected any mutation of K5 and K14 gene in patient 6 with EBS-WC.Conclusions These mutations in keratin genes are confirmed as the major cause of EBS in the Chinese patients.The cases reported supplement the available clinical and genetic data for EBS worldwide.
|
PDF Full Text Request