A Study Of Common Deafness Genes Using The Combination SNPscan And Sanger Sequencing In Nonsyndromic Hearing Loss Patients Of Minority In Northwest China And Mutation Analysis Of 9 Uncommon Deafness Genes Using SNPscan

Hearing impairment is the most common neurosensory disorder in humans,affecting approximately one to three of every 1000 newborns worldwide.In China,it has been estimated that there are approximately 0.8 million children（<7 years old）with hearing impairment,with an annual increase of 20,000 to 30,000 children.Approximately 50%of congenital hearing loss patients and 70%of childhood hearing loss patients are attributed to genetic causes.Nonsyndromic hearing impairment（NSHI）contribute 60%-70%of inherited hearing impairment and involves more than 100 different genes loci with autosomal dominant（20%）,autosomal recessive（75%）,X-linked（1%）,and maternal inheritance（at least 1%）.With the successful completion of Human Genome Project,tremendous progress has been made in genetic studies of hearing loss.To date,approximately 70 genes and more than 1000 mutations causing nonsyndromic deafness have been reported（http://deafness variation database.org）.More than 45 genes and 69 loci are associated with autosomal recessive nonsyndromic deafness（DFNB）.The GJB2 gene accounted for the etiology in approximately 4%-30.4%of the patients with hearing loss,SLC26A4 gene accounted for approximately 13.73%-15%,and mtDNAA1555G mutation accounted for 1.76%-8.56%.Thus,three genes have been conventionally screened in the Chinese deaf population.On the other side,molecular defects of majority of deaf individuals in China remain to be identified.The etiologic contribution of uncommon deafness genes,in particular,has yet to be investigated systematically.Due to the large number and presumably low mutation frequencies of those genes,it would be highly expensive and time-consuming to screen mutations for uncommon deafness genes by the conventional Sanger sequencing.This limitation,however,may be overcome by using patented SNP genotyping technology.The feasibility of this strategy has been confirmed in principle in several pilot studies.In this study,we performed a comprehensive mutations screening of 3 common deafness genes and 9 uncommon deafness genes in a large cohort of nonsyndromic deaf patients using SNPscan and Sanger sequencing.The aim of this study was to investigate the molecular etiology of NSHI patients from northwest China to provide effective risk assessment and genetic counseling for patients with hearing loss and their families.This study is divided into two parts:Part one:A study of common deafness genes using the combination SNPscan and Sanger sequencing in nonsyndromic hearing loss patients of minority in northwest ChinaThree genes of GJB2,mitochondrial DNA12SrRNA,and SLC26A4 in a total of 1120 nonsyndromic hearing impairment patients of minority were screened for mutations in our study cohort using the combination of SNPscan and Sanger sequencing in northwest China.（1）The carrier frequency of mtDNAA1555G mutation was 3.3%（37/1120）in this cohort.In 37 the homoplasmic mtDNAA1555G mutation carriers,18 cases were found a clear history of aminoglycoside use.The results suggested that administration of aminoglycoside antibiotics in this region was an important reason of higher incidence of the mtDNAA1555G mutation.Statistically significant difference in the prevalence of the mtDNAA1555G mutation was showed between different districts or different ethnicities.Three patients carried the homoplasmic mtDNAC1494T mutation,which gave an estimate of 0.26%.The frequencies of mitochondrial DNA12SrRNA mutation were identical by comparison with Sanger sequencing.（2）We detected complete sequence of GJB2 gene for mutation using SNPscan in 1120 NSHI cases.15.36%（172/1120）patients carried mutations of GJB2 gene,for which 71 patients were homozygous,43 were compound heterozygous,and 58 were heterozygous.10.18%（114/1120）patients carried pathogenic mutations of GJB2 gene.The allele frequency of GJB2 mutations in 1120 NSHI cases was 12.77%（286/2240）.The c.235delC showed the highest allele frequency,followed by c.35delG,and c.299₃00delAT.The c.504insAACG mutation wasn’t detected in 1120 NSHI cases.Statistically significant difference in the allele frequency of the GJB2 mutation was showed between different districts or different ethnicities.We detected complete sequence of GJB2 gene for mutation using Sanger sequencing in 1120 NSHI patients.13.84%（155/1120）patients carried mutations of GJB2 gene（66 homozygous,35compound heterozygous,and 54 heterozygous）.The allele frequency of GJB2 mutations was 11.43%（256/2240）.The most common mutant allele was c.235delC,followed by c.299₃00delAT,and c.35delG;The c.IVS1+1G>A mutation wasn’t detected in 1120 NSHI cases.No statistically significant difference in the allele frequencies of the GJB2 mutation was showed between different districts.Although statistically significant difference in the allele frequencies of GJB2 mutation was showed between different ethnicities.The order of allele frequency of GJB2 mutation was different between different ethnicities in comparison with SNPscan.In summary,the detection rate was relatively low by comparison with using SNPscan in mutation detection rate and allele frequency.However,polymorphisms and more nucleotide change were detected using Sanger sequencing.（3）We detected the mutation of SLC26A4 gene using SNPscan in 1120 NSHI patients from the northwest China.10.45%（117/1120）patients were found carrying SLC26A4 mutations,of which 69 patients carried diallelic SLC26A4 mutations（54 homozygotes,15compound heterozygotes）.48 cases carried monoallelic SLC26A4 mutations.The allele frequency of SLC26A4mutions was 8.3%（186/2240）.The c.IVS7-2A>G showed the highest allele frequency,followed by c.2027T>A,and c.2168A>G.We detected complete sequence of SLC26A4 gene for mutation using Sanger sequencing in 1120 NSHI patients,of which 41 patients carried diallelic SLC26A4 mutations（34 homozygotes,7compound heterozygotes）.37 cases carried monoallelic SLC26A4 mutations.6.96%（78/1120）patients were found carrying SLC26A4 mutations.3.66%（41/1120）patients carried pathogenic mutations of GJB2 gene.The allele frequency of SLC26A4 mutations was 5.31%（119/2240）.The most common mutant allele was c.IVS7-2A>G,followed by c.2168A>G.Although statistically significant difference in the allele frequencies of the SLC26A4 mutation was showed between different districts or different ethnicities.The order of allele frequencies of the SLC26A4 mutation was different between different districts or different ethnicities in comparison with SNPscan.In short,the detection rate was lower compared to using SNPscan in mutation detection rate,nucleotide change,and allele frequency.Moreover,Sanger sequencing is characteristic of high accuracy,the sequencing of long fragment,and low-throughput technologies.it fits the sequencing of specific regions;SNPscan has the advantage of high-throughput technologies and low cost,it is appropriate for sequencing of genome.Thus,we may choose different method according different need.Part two:mutation analysis of 9 uncommon deafness genes using SNPscan in nonsyndromic hearing loss patients in northwest ChinaTo investigate mutation frequencies of uncommon deafness genes in nonsyndromic hearing impairment（NSHI）patients in northwest China.We detected the mutations of CDH23,LRTOMT,MYO15A,OTOF,PCDH15,TMC1,TMIE,TMPPSS3,TRIOBP genes in 354 NSHI patients from the northwest China.Twenty-one of 354 NSHI patients were identified the mutations of 9 uncommon deafness genes,of which 15 patients was homozygous,1 were compound heterozygous,and 5 were heterozygous.The 22 identified mutations included 8 missense mutations,9 nonsense mutations,and 5 frame-shift mutations.22 genotypes of 9 uncommon deafness genes mutations in NSHL patients were identified in this study as the following:MYO15A—p.E2339X,p.Y1392X,p.Q3172X,c.10419-10423del,p.V2266M,p.D1451N;OTOF—c.907₉08del,p.P775fs,p.G79R,p.R568Q;PCDH15—p.Y394X,p.R245X,p.L777fs;TCM1—p.W557fs,p.G258D;TMIE—p.R96X;LRTOMT—p.C142X;TRIOBP—p.Q336X;TMPRSS3—c.346G>A;CDH23—p.P240L,p.A179P,p.I1657N.The most common mutation was MYO15A,followed by OTOF,CDH23,and TMC1,which accounted for 1.7%（6/354）,1.1%（4/354）,0.85%（3/354）,and 0.56%（2/354）,respectively.In short,5.93%of patients with nonsyndromic hearing loss in our study cohort were attributed to the mutations of 9 uncommon deafness genes.