Diagnosis Of Variant-Type Xeroderma Pigmentosum In A Family By Whole-Exome Sequencing

BackgroundXeroderma pigmentosum(XP)is a rare autosomal recessive disorder resulting from deficiency in base excision repair caused by single-nucleotide mutations,especially in skin exposed to sunlight.Patients with XP show light sensitivity and skin pigmental changes in sun-exposed areas and have a higher incidence of neurological abnormalities,skin cancer,and other tumors.XP is classified into eight subtypes.Although the different XP subtypes present some specific clinical manifestations,the conditions are usually difficult to diagnose.The gene responsible for each type of XP has been identified.Most of the eight distinct genes encode proteins associated with the nucleotide excision repair(NER)function.The XP variant(XPV)type is the only variant that does not involve mutation in NER pathway components and instead results from mutations in the DNA polymerase eta(POLH)gene,which encodes the Y-DNA polymerase that participates in the repair of damaged DNA.POLH mutations weaken DNA of replication under exposure to ultraviolet light,and then lead to illness.ObjectiveWhole-exome sequencing as well as Sanger sequencing were performed in the family with pigment abnormality to clarify the diagnosis and analyze the relationship between clinical phenotype and genetic changes.Methods1.Case Collection:We collected a family,which include clinical data and whole blood sample of 2 patients and 7 normal people.2.DNA extraction and Sanger sequencing:To make diagnose,primers for ADAR gene were designed and sequenced using ABI3500 sequencer.3.Whole-exome sequencing:IlluminaTM HiSeq 2000 was used for whole-exome sequencing of DNA from 2 patients and 2 normal controls in this family,and mutation sites were identified by sequencing data analysis.4.Sanger sequencing verification:Sanger sequencing were performed on the whole family for the pathogenic mutations,using ABI3500 sequencer.5.Bioinformatics analysis(gene function analysis):Use the MutationTaster(http://www.mutationtaster.org)software to predict the functional change caused by potential pathogenic mutations.Use the Swiss-Model(https://swissmodel.expasy.org)software builds protein Model of POLH.Results1.Clinical features:The proband was a 36-year-old male with coarse skin on the face,chest and limbs.Large amounts of dark brown patches and papules,covered with a few scales,were fused into pieces partially.Histopathological examination of the skin lesions was performed and revealed melanin pigmentation in the basal layer and extension of the furcella into the dermis with a bud shape.Erythema and thick layer of scales were seen in both lower extremities.Upon histopathological examination of these skin lesions,the epidermal layer showed continuous parakeratosis and Munro’s microabscesses.The dermis layer exhibited dilated superficial vessels and infiltration of a few lymphocytes.The patient was diagnosed as DSH?psoriasis vulgaris,diabetes,renal dysfunction.2.Sanger sequencing:By Sanger sequencing of ADAR gene,no mutation was found and the diagnosis of DSH was excluded.3.Whole-exome sequencing:Ten possible mutation sites,including a novel homozygous nonsense mutation(c.353dupA,p.Y118 V119delinsX)in the POLH gene and a homozygous missense mutation(c.T214C,p.S72P)in the t-complexassociated-testis-expressed 1(TCTE1)gene were identified in the two patients.These 10 mutation sites were not present in the normal controls.4.Sanger sequencing verification:Based on Sanger sequencing for the 10 possible mutation sites in all 9 family members,eight sites not fitting the phenotype were excluded.The POLH variant(c.353dupA,p.Y118_V119delinsX)and the TCTE1 variant(c.T214C,p.S72P)were confirmed by Sanger sequencing and identified as being heterozygous in the patients’ normal parents and another normal family member.They were homozygous mutations in both the proband and another patient.These two mutations are co-isolated from the disease phenotype in the family.5.Bioinformatics analysis(gene function analysis):The homozygous POLH variant cosegregating with the disease phenotype in the family was predicted to lead to a change in amino acid sequence and a premature termination codon and to affect protein features and splice site changes,as assessed by MutationTaster;this may be a morbigenous variant in this pedigree.According to Swiss-Model,the protein model of POLH exhibits different lengths and configurations in the normal controls and patients with mutations in the POLH gene,suggesting significant functional deficiency.TCTE1 and POLH are both on 6p21.1,and the mutation in TCTE1 also co-segregated with the phenotype in the entire family.TCTE1 had no significant effect on the development of the disease.This POLH mutation site has not been reported before,and the above studies have proved its pathogenicity,which is the new pathogenic mutation site of XPV.ConclusionIn this study,we performed whole-exome sequencing of an XP family,which had been suspected as having DSH,and a novel XPV pathogenic homozygous nonsense mutation(c.353dupA,p.Y118_V119delinsX)was identified in the POLH gene.This patient was diagnosed as XPV.Our study proves that next-generation sequencing(NGS)is an effective method for the rapid diagnosis and determination of XP genetic etiology.