Mutation Screening And Gene Diagnosis Of Rare Skin Diseases

Background A variety of rare skin diseases occur worldwide with low incidence but serious condition,such as xeroderma pigmentosum,hereditary palmoplantar keratoderma and pachyonychia congenita,which not only severely affect the physical and mental health of the patients,but also bring serious economic burdens to the families and the society.In some cases,it can also be life-threatening.No effective treatment is available so for.Early diagnosis and timely prevention will be of great significance.The phenotypes of some rare diseases are complex,which may be accompanied by multiple system damages,bringing challenges in the traditional clinical diagnosis.Broad application prospects have been seen in the mutation screening.In recent years,the technology of gene diagnosis has been developed rapidly and been widely used in many clinical fields,which can detect variants in gene structure and expression changes in patients at the DNA or RNA levels.High-throughput sequencing has played an important role in the detection of disease-causing genes and molecular diagnosis so far.In view of the complex phenotypic heterogeneity and genetic heterogeneity,the seletions of different methods for different genetic diseases have been currently accepted as the main idea in gene diagnosis.Object Based on the clinical symptoms and previous genetic studies,mutation screenings by whole-exome sequencing and direct sequencing were performed on the families clinically suspected with xeroderma pigmentosum or hereditary palmoplantar keratoderma so as to complete gene diagnosis and genetic consultation,which provides theoretical support for the pre-symptomatic genetic diagnosis such as prenatal diagnosis.Methods?1?Samples from a family clinically suspected with xeroderma pigmentosum and from five families with hereditary palmoplantar keratoderma were enrolled in.?2?In the xeroderma pigmentosum family,the proband and her parents were selected to conduct the whole-exome sequencing.Agilent Sure Select XT Library Prep Kit and Sure Select Human All Exon V6 kit were used for the library preparation and capture,followed by 150 bp pair-end sequencing on Illumina Hiseq XTen platform.After quality control of the rawdata,variants comparisons,mutation recognition and annotation,a list of suspected variants were obtained.Screening for disease-associated deleterious mutations was then made,with emphases on the reported xeroderma pigmentosum causative genes.The suspected mutations were then verified in other samples of the family.?3?Direct sequencing of gene KRT9 / KRT1 was performed in five pedigrees of hereditary palmoplantar keratoderma.Two cases and one control were selected in the family with negative results to perform whole-exome sequencing.The suspected disease-causing mutations were verified in the original family by Sanger sequencing to detect genotype-phenotype co-segregation.Results?1?A compound heterozygous mutation in the XPC gene,c.2218₂220del?p.Glu740₇40del?located in exon 12 and c.2257 dup C?p.Arg753fs?located in exon 13 was found in the xeroderma pigmentosum family,which has not been reported previously.Each parent of the proband carried one mutation.Genotype-phenotype co-segregating analysis was confirmed within the family.A conservative analysis revealed that the amino acid position on 740 and 753 were highly conserved across multiple species.According to the clinical manifestations and the result of gene detection,the diagnosis of xeroderma pigmentosum type C in the proband was made clearly.?2?Three different KRT9 mutations?c.482A>G,c.487C>T and c.488G>A?were detected in three palmoplantar keratoderma families,and mutation 482A>G was correlated with the knuckle pad.The diagnosis of epidermolysis palmoplantar keratoderma was clear.?3?No mutation in the gene KRT9 or KRT1 was found in a pedigree suspected with hereditary palmoplantar keratoderm.A mutation?c.T1262C?in the KRT16 gene was identified by whole-exome sequencing,which was previously found to cause the pachyonychia congenital.The diagnosis of type I pachyonychia congenita was made by the combination with clinical manifestations and the result of gene detection.?4?No mutation of the keratin gene was deteced in a pedigree with hereditary palmoplantar keratoderma,which led to a negative gene diagnosis,suggesting that there may be new mutant forms or pathogenic genes in this family.Conclusions For rare skin diseases with known causative genes,the combination of whole-exome sequencing and direct sequencing could identify the disease-causing mutations in a quick,economic and efficient way.Through the mutation screening in 6families with rare skin diseases,clear genetic diagnoses were made in 5 of them,emphasizing the important role of gene diagnosis as a reverse diagnosis method in the clinical work.At the same time,one novel mutation was identified in this study,which will enrich the mutation spectrum of xeroderma pigmentosum.We also report a family with negative gene detection,in which the relavant factors were analysed and the lessons were learned.