Genetic Study Of Congenital Painless And Anhidrosis

Background:Congenital insensitivity to pain with anhidrosis?CIPA;MIM 256800?,also known as hereditary sensory and autonomic neuropathy type IV?HSAN-IV?,is a rare autosomal recessive disorders.CIPA is characterized by the absence of normal responses to painful stimuli,anhidrosis?inability to sweat?,recurrent episodic hyperthermia,self-mutilating behavior,as well as mild-to-severe intellectal disabilities.All cases borned with a developmental defect,and recurrent episodes of unexplained fever,which result in a high fatality rate.The incidence of the disease is extremely low and it is easily misdiagnosed.In recent years,with the development of clinical diagnosis and molecular testing,there have been a number of cases and genetic studies being reported at home and abroad.However,there has not been a representative genetic study of a large sample of CIPA in China.Objective:In this study,a mature gene detection platform was used to study mutations of causative genes in the CIPA family in Chinese population.The mutation spectrum of CIPA patients in Chinese population was explored to provide evidence for the diagnosis and genetic counseling of the disease.Methods:In this study,Sanger sequencing and Next Generation Sequencing were combined to conduct genetic research in the patients of CIPA.We collected the clinical data and peripheral blood samples from these CIPA probands and their available family members.Genomic DNA was extracted from blood samples using the standard sodium dodecyl sulfate-proteinase K-phenol/chloroform extraction method.Sanger sequencing was performed to screen the mutations in coding regions and exon-intron boundaries of NTRK1 in the probands.Long-range PCR and real-time PCR were used to detect the large fragment deletions of the NTRK1 gene.For families with no pathogenic mutations detected by Sanger sequencing,the whole genome sequencing method was used to further screen for mutations in the deep intron regions,and family validations were performed for candidate mutations.For the intronic mutation at non-canonical splice sites,Minigene and mRNA analysis were used to study the function of the splice site mutations.Results:In this study,34 families were subjected to genetic analysis,and we identified 27 mutations in NTRK1 gene from this cohort of patients,including 15 novel mutations and 12 known.The most common mutations were c.851-33T>A and c.287+2dupT,and a total of 24?35%?mutant alleles were identified.In addition,we identified a recurrent Alu element-mediated intrachromosomalnon-allelic homologous recombination that resulted in large fragment deletions?c.428-374₇18+485de1?.Through whole-genome sequencing,we identified a deep intronic mutation?c.[851-794C>G;851-798C>T]?for the first time in four unrelated families,which resulted in abnormal mRNA splicing.Conclusions:In conclusion,we performed a genetic analysis of NTRKl gene in the largest cohort of Chinese CIPA patients and found 15 novel mutations.We show for the first time that gross deletion and deep intronic mutation may be two novel causes of CIPA in these patients.Current findings expand the NTRK1 mutation spectrum responsible for CIPA,which would improve precise diagnosis of CIPA.