Objective To Investigate The Pathogenic Mutations Of The Osteogenic System In The Osteogenic System

Epidermolytic palmoplantar keratoderma (EPPK) is an autosomal dominant inherited skin disease clinically characterized by epidermolyic hyperkeratosis of the palms and soles as well as histologically by suprabasal epidermolysis. Recent molecular biological studies have showed that most cases of EPPK are caused by mutations of keratin 9 gene (KRT9) and these mutations usually occur in the highly conserved rod domains.ObjectiveTo analyse genetic pattern of three EPPK families, determine the candidate gene of mutation detection and find the mutation sites.MethodsIn this study, three EPPK families were collected.Family 1 was carried out microsatellite linkage analysis. PCR and Sanger DNA sequencing were used to analyze the causative mutation in the proband. Next, mutation identification was performed in these three families.Results(1) Family 1 was located in 17q21 by microsatellite linkage analysis(2) A novel mutation of c.491T>C (p.L164P) in KRT9 was found in the proband of Family 1 and the restriction enzyme reaction identified that the mutation was co-segregated with the EPPK in all family members and 50 normal samples.(3) Patients of family 2 and 3 respectively contained c.488 G>A (p.Rl 63Q) and c.482 A>G (p.N161S)Conclusionsa novel mutation of c.491T>C (p.L164P) in KRT9 was co-segregated with the EPPK in all members of family 1and 50 normal samples. The causative mutations of Family 2 and 3 were respectively c.488 G>A (p.R163Q) and c.482 A>G (p.N161S). This study further confirmed that the 1A domain was the mutational hotspots regain of EPPK.ObjectiveTo analyse genetic pattern of three OI families, determine the candidate gene of mutation detection and find the mutation sites.MethodsGenomic DNA was extracted from peripheral blood and the fetal villi tissue by standard phenol/chloroform method. PCR amplified the coding regions of COL1 A1 and COL1A2 and Sanger DNA sequencing were used to analyze the causative mutation in the probands of three families; Mutation identification was performed in familial members of three families by PCR-HRM. DNA sequencing was used to validate the genotype of the samples in different HRM-curves.ResultsHomozygotes of the missense mutations c.2560G>A (p.G854S), c.358C>T (p.R120*) and c.814G>A (p.G272S) c.2333G>T(p. R778L) were separately found in probands of three OI families, and the latter two mutations were first found. All were located in gene COL1A1. HRM analysis displayed the affecteds of each family presented the same curve, which was different from the one of the unaffected and normal controls. These curves were consistent with genetic sequencing.ConclusionsWe found two new mutations in gene COL1A1 and established a technology platform of diagnosing osteogenesis imperfecta based on PCR-HRM to perform the validation of mutations andprenatal genetic diagnosis.ObjectiveTo identify the ATP7B mutation and perform the prenatal gene diagnosis in a family with Wilson’s disease(WD)MethodsGenomic DNA was extracted from peripheral blood and the fetal villi tissue by standard phenol/chloroform method. PCR and Sanger DNA sequencing were used to analyze the causative mutation in the proband; Mutation identification was performed in other 4 familial members including a fetus with high risk of WD by PCR-HRM. In the same way, a prenatal gene diagnosis was completed successfully in the mother of the proband. DNA sequencing was used to validate the genotype of the samples in different HRM-curves.ResultsThe proband was a homozygote of the missense mutation c.2333G>T(p. R778L), located in the exon 8 of gene ATP7B. HRM analysis displayed three different curves, representing three different genotypes. The curve types for the 5 familial members and 4 normal control samples were consistent with genetic sequencing:the proband himself was a mutant homozygote, all the other family members were the heterozygote of the mutation, and the 4 normalcontrols were wild type.ConclusionsThe authors found a known mutation of p.R778L in a WD family, and the prenatal gene diagnosis was achieved in a fetus with high risk by PCR-HRM.