Genetic Variation Analysis And Prenatal Diagnosis Of Hereditary Deafness In Henan Province

BackgroundDeafness is one of the most common birth defects in human beings.Every year,about 30000 deaf children are born in China,and 60000-80000 children with delayed hearing loss are found.Most of them are severe to profound sensorineural hearing loss,which seriously affects the communication and cognitive ability of them.The causes of deafness are complex,more than 50%are associated with genetic factors,with high genetic heterogeneity and phenotypic similarities.So far,we have identified 123 genes related with nonsyndromic hearing loss,53 genes related with syndromic hearing loss(http://hereditaryhearingloss.org),so it is difficult for clinicians directly determing their molecular causes through clinical phenotypes.According to epidemiological survey data of deafness patients in China,the most common hereditary deafness gene are GJB2,SLC26A4,12SrRNA,accounting for 70%of genetic deafness patients.Screening of the three major genes has been a routine screening project for deafness patients at present,but there are still some patients with unknown etiology who need to be further screened for rare deafness genes.Due to the large number of rare deafness genes and low mutation frequency,it is expensive and time-consuming to solve this problem through traditional gene-by-gene Sanger sequencing.The next-generation sequencing technology developed in recent years is often used in the diagnosis of genetic disorders with obvious heterogeneity.This technology can be used to find mutations of known pathogenic genes as well as find new genes.It becomes an effective tool for molecular research of genetic diseases.Waardenburg syndrome(WS)is the most common syndromic deafness inherited in autosomal dominant mode.Its main clinical symptoms are sensorineurotic deafness,hypopigmentation of skin,white forelock or premature white hair,and heterochromia iris,accounting for 2%of congenital deafness,and the prevalence is about.1/42000.WS is generally considered to be a related syndrome caused by the lack of neural crest cell-derived melanocytes.WS is divided into four subtypes(WS1-4)based on the presence or absence of additional symptoms.WS1 is characterized by congenital sensoriniural hearing loss,heterochromatic or blue eyes of the iris,and depigmentation plaques of the skin and hair;The difference between WS2 and WS1 lies in the absence of inner canthus displacement;Limb abnormalities were found in WS3 patients;WS4 is characterized by congenital megacolon on the basis of WS2 phenotype.At present,six genes(PAX3,SOX10,MITF,EDN3,EDNRB,SNAI2)have been found to be related to WS.The researchers proposed that almost all WS1 and WS3 patients have heterozygous variation in PAX3,and 15%of WS2 patients have heterozygous variation of MITF.SOX10 accounted for about 15%of WS2 cases and 50%of WS4 cases,while SNAI2 and EDN3/EDNRB variation accounted for a small part of WS2 and WS4 patients.Due to the typical symptoms of WS patients,Sanger sequencing of corresponding genes was usually carried out after clinical classification to confirm the diagnosis.At the same time,there is the possibility that the direction of genetic testing is wrong and even false negative due to the wrong clinical typing.Permanent hearing loss of children leads to problems related to speech development,mental health,cognitive development and social survival,in addition to hearing AIDS and cochlear implants,there are no effective treatments to restore hearing,therefore preventing hearing and speech impairments is important for families at high risk of deafness.However,fetal hearing abnormalities during pregnancy can not be evaluated by ultrasound or enzymology examination,only DNA sequence analysis can be used to predict fetal hearing phenotype.Thus,genetic prenatal diagnosis of deafness could provide detailed information to help these families make reproductive decisions.In the near future,timely interventions,such as gene therapy may be used for causing mutations detected in fetuses.ObjectiveTo study the application value of variation analysis by targeted genomic capture and massively parallel sequencing technology in nonsyndromic deafness and Waardenburg syndrome from Henan province,analyze the hotspot mutation gene and hotspot structural domain,and to explore the potential relationship between clinical phenotype and genotype,to investigate the value of interventional prenatal diagnosis combined with DNA analysis in the prevention of birth defects caused by hereditary deafness.Methods1.Research objectPart Ⅰ:A total of 125 Henan native deafness patients from the First Affiliated Hospital of Zhengzhou University were collected.They were diagnosed as nonsyntactic bilateral sensorinural deafness,aged from 1 month to 67 years.The screening results of GJB2,SLC26A4 and 12SrRNA were negative in the past.Part Ⅱ:11 Waardenburg syndrome patients and their family members from the First Affiliated Hospital of Zhengzhou University were collected.Part Ⅲ:197 families with deafness for prenatal diagnosis from Genetic and Prenatal Diagnosis Center of the First Affiliated Hospital of Zhengzhou University were collected.2.Targeted genomic capture and massively parallel sequencing168 deafness genes were screened.The process was as follows:quality detection of DNA samples→preparation of whole gene DNA library→ capture of target deafness genes→high-throughput sequencing→off machine→data analysis.3.Multiplex ligation-dependent probe amplification and fluorescence quantitative PCRMultiplex ligation-dependent probe amplification and fluorescence quantitative PCR were used to verify the deletion or duplication of the deafness gene detected by high-throughput sequencing.4.Primer design and Sanger sequencingGene Tool software was used to design PCR amplification and sequencing primers for the mutation sites of deafness genes according to Ensembl database.The PCR products were directly sequenced by Sanger sequencing.5.Bioinformatics analysis5.1 Variation screening and pathogenicity analysisRetention mutation criterion:The sequencing depth was greater than 5,the mutation frequency was greater than 30%,and there were no or less than 0.05 loci in the six normal human mutant databases(1000 Genome,ESP6500SI,INHOUSE,EXAC_ALL,GNOMAD,EXAC_EAS).Synonyms mutation sites(previously reported not removed),intron variation,UTR region variation were removed.The GERP++software was used to analyze the conservation of variants in species,and the SIFT,Ployphen2,MutationTaster software were used to predict pathogenicity.The SPIDEX software was used to predict RNA splicing and subsequent protein functional changes after site mutation.5.2 Variation pathogenicity assessmentCombined with clinical phenotypes of patients,types of gene variation,prediction results of biological software,co-separation of family variation,literature reports,and research on variation function,etc.,a comprehensive analysis was made.According to the variation classification standard developed by the American College of Medical Genetics and Genomic,the variation was classified into five categories,benign,likely benign,uncertain significance,likely pathogenic,pathogenic.6.Prenatal diagnosisAccording to the gestational age of the pregnant woman,an appropriate invasive prenatal diagnosis method was selected.At 10-14 weeks of pregnancy,10-20mg of villus tissue was extracted by ultrasound-guided transperitoneal chorionic puncture sampling.10ml of amniotic fluid was extracted by ultrasoundguided transabdominal amniocentesis at 18-24 weeks of gestation.After the samples were collected,the maternal contamination was eliminated by STR method,and the deafness gene variation was analyzed.Results1.Analysis of rare deafness gene variation in 125 patients with non-syndromic deafness23 patients were diagnosed,and the pathogenic grade of genetic variation was possibly pathogenic variation or pathogenic variation.The diagnostic rate was 18.4%(23/125),and 11 deafness genes were involved,including MYO15A(6),TMPRSS3(3),MYO7A(3),POU3F4(3),ACTG1(2),CDH23(1),OTOF(1),and OTOGL(1),KCNQ4(1),TECTA(1)and MT-TS1(1).33 pathogenic or possibly pathogenic variants were detected,among which 16 variants were found for the first time in this study.7 patients were detected with one pathogenic or possible pathogenic variant and one clinically unknown variant in the same deafness gene,and 4 deafness genes were involved,including MYO15A(4),MYO7A(1),LOXHD1(1),and TBC1D24(1).10 patients were detected with MYO15A compound heterozygous or homozygous variants,6 patients carried double pathogenic variants,4 patients carried one pathogenic variant and one clinically unknown variant,and a total of 17 variants were detected,of which 16 variants were located in the main structural domain of myosin XVa,accounting for 94.1%(16/17).The Motor domain is the hotspot mutation domain of MYO15A.All the MYO15A variants detected in this study lied in non-N-terminal region,and all patients showed severe hearing loss.We summarized 50 variants with autosomal dominant modes,these variants’frequency are less than 0.001,and they were predicted to be harmful by at least one software,but these variants derived from parents with normal phenotype,including 47 missense mutations,accounted for 94%(47/50),three splic mutations,accounted for 6%(3/50),this 50 variants are evaluated as uncertain significance according to the ACMG.The genes involved including TNC,DIAPH1,DIAPH3,MYH14,CHD7,WFS1,TJP2 and COL1A1 et al.2.Analysis clinical characteristics and gene variation in 11 Waardenburg syndrome predigreesAmong the 11 predigrees with Waardenburg syndrome,5 predigrees had clear family history,and 6 predigrees were sporadic cases,with a total of 22 patients.Among them,3 probands were clinically diagnosed as non-syndromatic deafness at initial diagnosis,and the diagnosis were corrected to Waardenburg syndrome after the genetic diagnosis were confirmed.In 11 WS pedigrees,8 pedigrees were determined as WS2,3 pedigrees were determined as WS1,involving three genes(SOX10,PAX,MITF)with eleven variants,eight of which had not been reported in the HGMD professional database.The most common causative gene of WS in our study was SOX10,accounted for 54.5%(6/11).Through family verification,SOX10 given priority to form with spontaneous mutation,accounting for 83.3%(5/6),while MITF variation was common in parental inheritance(2/2).The hotspots of SOX10,PAX3 and MITF mutation were exon 2(4/6),the exon 5(3/3)and the exon 9(2/2),respectively.Sensorineural hearing loss and heterochromia iris were the most common symptoms of Waardenburg syndrome patients,accounting for 68.2%(15/22)and 63.6%(14/22)of all patients,respectively.Both of them were mainly bilateral,accounting for 93.3%(14/15)and 64.3%(9/14),respectively.The differences of clinical phenotypes were great among different families or even different patients in the same family.The results of genotype and phenotype analysis by Fisher’s exact probability method showed that PAX3,MITF and SOX10 had significant differences in the incidence of deafness,heterochromia iris and freckles(P<0.05),but there was no significant difference in the incidence of forehead white hair(P>0.05),Bonferroni correction method was used for pair comparison,and it was found that WS patients with PAX3 and SOX10 mutation had a higher probability of heterochromia of iris than those with MITF mutation group.However,WS patients with MITF variation had the highest incidence of facial freckle-like pigment changes.3.Analysis of prenatal diagnosis in 197 families with hereditary deafnessAmong 197 families,13 probands carried rare deafness genes causing variants,and the inheritance mode was autosomal recessive,including MYO15A(5),TMC1(2),TMPRSS3(2),PCDH15(2),USH2A(1),CDH23(1).GJB2 or SLC26A4 were the pathogenic genes of deafness in 184 families.The hearing phenotype of couples were normal in 182 families.In 2 families,both couple were deaf patients.In one family,the husband carried c.235delC homozygous variation of GJB2,and the wife carried c.223C>T(p.R75W)of GJB2 with autosomal dominant inheritance pattern.In another family,the husband had no mutation in GJB2,and the wife had detected the dominant pathogenicity c.551 G>A(p.R1 84Q)heterozygous variation of GJB2.We performed 208 prenatal diagnosis,involved with 212 fetuses,51 fetuses(24.1%)did not carry pathogenic mutation,124 fetuses(58.5%)were carriers of monoheterozygous variation,and 37 fetuses(17.4%)carried double pathogenic variation.37 fetuses were identified as deaf patients according to genetic diagnosis,their parents chose to laboration after consideration.175 fetuses were carriers or not carrying any pathogenic mutations,fetal parents all choose continue to pregnancy,up to December 2020,all reserved children has found no abnormal condition.Conclusion1.The combination of targeted gene capture and large-scale parallel sequencing technology established in this study can greatly improve the clinical diagnosis rate and detection rate of patients with non-syndromic deafness and Waardenburg syndrome,which has important application value in molecular diagnosis of hereditary deafness.2.MYO15A is the hot spot mutation gene of the rare deafness gene in Henan,and the hot spot mutation domain is the Motor domain,which is mainly in the form of compound heterozygous mutation.3.WS2 is common in WS patients in Henan.SOX10 is the main pathogenic gene,and it is mainly in the form of spontaneous,while MITF mutation is mainly inherited by parental generation.Sensorineural deafness and heterochromia iris are the most common symptoms of WS patients,and both of them are mostly bilateral.WS patients with PAX3 and SOX10 variation have a higher probability of heterochromia iris than the MITF variation group,while WS patients with MITF variation have the highest incidence of facial freckle pigmentation change.4.The 24 pathogenic variants found in this study for the first time enrich the gene mutation spectrum of hereditary deafness in Chinese population,and provide theoretical support for the study on the molecular pathogenic mechanism of hereditary deafness.5.The 50 low-frequency autosomal dominant variants inherited from parents with normal hearing phenotype in our study provide data reference for the accumulation of pathogenic evidence in the deafness gene mutation database.6.The prenatal diagnosis and genetic counseling of deafness have realized the strategy of "early screening,early diagnosis and early intervention",which is of great value in the prevention of birth defects of deafness.