Identifying The Causative Gene Of An Olmsted Syndrome Kindred By Whole Exome Sequencing,and KRT9 Gene Mutation Analysis And Prenatal DNA Diagnosis Of Four Epidermolytic Palmoplantar Keratoderma Pedigrees

Background:(1)Olmsted syndrome is a rare congenital skin disorder characterized by two main clinical features:mutilating palmoplantar keratoderma and periorificial hyperkeratotic plaques.Other clinical manifestations of the disease include diffuse alopecia,onychodystrophy,leucokeratosis of the oral mucosa,constriction of the digits.TRPV3 and MBTPS2 are two disease-causing genes of Olmsted syndrome.(2)Epidermolytic palmoplantar keratoderma(EPPK)is a relative common autosomal dominant genodermatoses by clinical manifestations of diffuse hyperkeratosis of the palms and soles.EPPK is mainly caused by mutations in KRT9 or KRT1.Objectives:(1)To identify the causative gene of a suspected Olmsted syndrome pedigree by whole-exome sequencing(WES).The results could reveal the pathogenic gene and confirm the clinical diagnosis of the disease and provide support for the further prenatal DNA diagnosis,or preimplantation genetic diagnosis(PGD)service.(2)To screen the gene mutation spectrums of four EPPK pedigrees.After the possible disease-causing mutations are demonstrated,to provide the DNA-based prenatal diagnosis for three families who strongly required it.Methods:(1)A suspected Olmsted syndrome kindred including 9 family members at Chengdu city was collected.The previous studies using PCR-Sanger sequencing failed to find any KRT9 or KRT1 gene mutations.In order to elucidate the disease-causing gene mutations,WES was carried out.Sanger sequencing were performed to verify the results.(2)Four EPPK families were enrolled and their peripheral anticoagμlant blood samples were obtained.PCR-Sanger sequencing were performed to identify the causive mutations,and the variants were confirmed by AS-PCR.Prenatal DNA diagnosis was performed on three families at high risk of EPPK.Resμlts:(1)Either KRT9 or KRT1 mutations did not exist in this suspected Olmsted syndrome pedigree.WES study revealed a heterozygous missense mutation of TRPV3,c.1703G>A(p.Gly568Asp).Sanger DNA sequencing verified the resμlts of WES.(2)The four EPPK families all had KRT9 heterozygous missense mutations.Among them,c.487C>T(p.Arg163Trp),the most common mutation of KRT9,was found in 2 pedigrees,and c.482A>G(p.Asn161Ser)in 1 pedigree,and c.566A>G(p.Tyr 189Cys)in 1 pedigree.Fetal genomic DNA was extracted from amniotic fluid to detect the known mutations existed in the 3 families.Unfortunately,the results showed that all fetuses from the three families carried the disease-causing mutations.Conclusions:(1)Combined with the clinical manefestations,TRPV3/c.1703G>A(p.Gly568Asp)mutation gave the precisive diagnosis that the pedigree affected Olmsted syndrome.So further possible prenatal DNA diagnosis or PGD could be provided for the kindred.The results also fulfilled the TRPV3 gene mutation database.(2)Rapid screening KRT9 gene mutation is the first choice for those clinically suspected EPPK families.Once the disease-causing gene mutation was determined,the DNA-based prenatal diagnosis or PGD could be carried out to prevent the birth of affected offspring.