Whole Exome Sequencing Identifies The Causal Gene In A Six-generation Uygur Epidermolytic Palmoplantar Keratoderma Family And Its Mechanism Research

Objective:Epidermolytic palmoplantar keratoderma(EPPK,OMIM : 144200) is a rare autosomal dominant skin disorder characterized by diffuse hyperkeratosis on the palms and soles. EPPK is a genetic heterogeneity disease.There have some disease-causing genes reports in different ethnic,but it still no report on Uygur population. We collected a larger Uyghur EPPK family clinical symptoms, map the genealogy, analysis the Uyghur EPPK genetic characteristics. Whole exome sequencing combined the previous lingkage analysis identified the candidate causal gene,Sanger further validated the causal gene.To investigate the pathogenesis of EPPK, we used immunohistochemical method to search another keratins abnormal on EPPK,which can provide certain theoretical basis. Currently, EPPK paitients had few therapeutic options. RNA interference technology has been applied in single gene disease research. siRNA transfection into HaCaT cell to specific inhibit the pathogenic gene expression.This study preliminarily illustrate pathogensis of the EPPK,and provide certain theoretical basis and experimental basis of targeting therapy of EPPK.Methods:(1)Two affected individuals(iv56 and III31)were subjected toWhole exome sequencing,combined with previous linkage analysis to to identify the candidate genes.(2)To reduce the number of the candidate pathogenic genes,And then using the software that can predict the affection the mutations can make to the function of the protein, we further filter to identify the possible mutation/gene of EPPK.(3) Using PCR sequencing methods, we validate the pathogenic gene in the family and a wide range of normal population,collected a small EPPK family to further validation.(4)We designed and synthesized specific siRNA strands and plasmid, and then transfected the HaCaT cells by the lipofectamine 2000.(5)Expressions mRNA and protein were examined by semiquantitative reversepolymerase chain(RT-PCR) and Western blot in transfected siRNA into HaCaT cells respectively.(6) The mRNA levels of KRT1, KRT5, KRT6, KRT16, KRT10, KRT14 and KRT12 before and after siRNA transfection were detected by RT-PCR.(7) Confocal microscopy was used to detect the changes of cytoskeleton before and after siRNA transfection.(8)Immunohistochemistry was perfomed to determine the expression and location of keratins in epidermoly between the case and controls.Results:(1)KRT9 c.C487T(p.R163W)is the causal gene for this larger Uyghur EPPK family,using whole exome sequencing and pervious linkage analysis.(2)To HaCaT cells overexpression of K9,we co-transfected plasmid with KRT9 c.C487T(p.R163W)and siRNA into HaCaT cells,RT-PCR and weston bolt showed the mRNA of K9-R163 W and k9- R163 W +siRNA- R163 W relatively express 1.063±0.159,0.242±0.051;protein relatively express 1.1329±0.131,0.289±0.036;siRNA can siRNA can specificly inhibit mutant KRT9 expression(P <0.05 with 95% confidence limits).(3) The expression of KRT6, KRT16 and KRT10 mRNA was significantly lower than that of KRT9 c.C487T(p.R163W) in the high expression of KRT9 c.C487T(p.R163W)(P <0.05).(4) KRT9 c.C487T(p.R163W) high expression, keratinocyte skeleton structure was disorganized state, part of the cytoskeleton structure disappeared.(5)Immunohistochemistry revealed hyperproliferation, impaired terminal differentiation, and abnormal expression of keratins K5, K10. Furthermore, the mutation of K9 induces the stress-activated keratins K10,K6 and K 16.The normal of levels and distribution of Ki-67 and p63.Conculsion:(1)KRT9 c.C487T(p.R163W)is the causal gene for Uygur EPPK family,which can support the theoretical basis of pathogenesis.(2) RNA specific inhibit KRT9 c.C487T(p.R163W) gene and wild-type KRT9 expression, indicating that the high expression of pathogenic gene lesions caused by damage to the balance of keratin damage, affecting the cell skeletal structure, Low expression of the cell structure is not damaged, for the epidermolysis of palmoplantar keratosis pathogenesis and gene therapy to provide new ideas.(3)siRNA inhibition significantly down-regulated the KRT9 mRNA, it may provides a specific therapeutic strategy for EPPK.(4)KRT9 c.C487T(p.R163W)may disturb the balance of keratins,the basal cell over proliferation,which induce the EPPK.