Screening Of Gene Mutation Sites In Hereditary Dermatosis And In Vitro Splicing Assay To Predict The Effect Of Intron Mutation

Objectives:To determine the gene mutation sites of collected Chinese familis with porphyria,tuberous sclerosis,junctional epidermolysis bullosa,and hypohidrotic ectodermal dysplasia respectively through gene sequencing.And provide molecular diagnosis and genetic counselling for the four families.Another part,to analyze the genetic basis and pathogenicity of the mutation,c.925-14T>A in EDA gene,found in the family with hypohidrotic ectodermal dysplasia by ex vivo splicing assay.Methods:Peripheral blood samples were obtained from the patients,their parents and 200 unrelated healthy controls.Genomic DNA was extracted from these blood samples,polymerase chain reaction,high-throughput sequencing and Sanger sequencing were performed to identify mutations in aiming genes.Next,a pCAS2 minigene-based in vitro splicing assay was used to confirm the pathogenicity of the splicing variant resulting from,c.925-14T>A,an intron mutation of EDA.Results:Sanger sequencing identified three novel mutations,mosaic mutation c.5130-5131insT of TSC2 gene in the tuberous sclerosis patient,a large deletion approaching the LAMA3 amino acid terminal in the junctional epidermolysis bullosa family and c.925-14T>A in EDA gene of the hypohidrotic ectodermal dysplasia patient,which was heterozygous in his mother and aunt.And the following in vitro splicing assay showed that c.925-14T>A was pathogenic and led to a new dominant splicing variant with insertion of 4 amino-acids between Glu308 and Val309 of EDA.Conclusions:It is possible to identify gene mutation in genodermatosis through polymerase chain reaction,high-throughput sequencing and Sanger sequencing.Still more experiments are needed to verify the pathogenicity of the found mutation.The mosaic mutation of TSC2 c.5130-513 1insT is a novel frameshift mutation and may be responsible for TSC in the patient with milder manifestations.Somatic mosaicism may partially explain the low mutation-detecting rate in tuberous sclerosis.The intron mutation c.925-14T>A deprives EDA gene of proper splicing and the consequent four amino-acids insertion between Glu308 and Val309 dramatically alters the function of ectodysplasin-A.