| Background: Hearing loss is one of the most common distressing disorders,which affects speech development and language acquisition in children and,as a resμlt,often leads to isolating themselves from society.The incidence of congenital hearing loss is estimated to be 1 to 3 in 1000 newborns worldwide,and about 30000 deafen children are born every year in China,and most of them are severe or profound sensorineural hearing loss.The etiology of hearing loss is mμltifactorial.Hearing loss can be a resμlt of environmental causes;it can also be the resμlts of a mutation in a single gene or a combination of mutations of different genes,or the resμlt of an association between environmental factors and genetics.According to studies,about 60% of the congenital hearing loss cases are caused by genetic factors.Many delayed hearing loss is related to gene defect or gene polymorphism.Approximately 70% of cases of hereditary hearing loss are non-syndromic,with the remaining 30% being syndromic.Non-syndromic hereditary hearing loss exhibits extremely heterogeneous disease traits.Up to now,More than 140 loci have been reported to be responsible for non-syndromic hearing loss,and 88deafnesss-causing genes have been cloned.The molecμlar epidemiology investigation of deafness in China showed that the common pathogenic genes were GJB2,SLC26A4,GJB3 and mitochondrial DNA 12 Sr RNA gene and so on.Genetic testing is the main method for the diagnosis of non-syndromic hearing loss.Recently,many studies have reported that suspension array technology is a high throughput,sensitive and specific detection system.In addition,next generation sequencing has been introduced as an alternative approach for hereditary deafness.Targeted genome enrichment and massively parallel sequencing(TGE+MPS)can be used to detect nearly all deafnesss-causing genes and identify genetic variation at a single base-pair resolution.In this study,based on suspension array technique,a one-off detection platform for 20 loci of four common genes GJB2,GJB3,SLC26A4,and mitochondrial DNA 12 Sr RNA gene was developed in order to detect hot spot mutations in patients with non-syndromic hearing loss.And,we explored the possibility of incorporating suspension array technique and TGE+MPS into clinical deafness genes diagnosis,in an attempt to develop a sensitive and specific diagnostic test for non-syndromic hearing loss and provide better genetic counselling and fertility guidance for deaf patients.Objective:To develop a set of suspension array technology detection system to simμltaneously detect 20 loci of the 4 common deafness-causing genes in the patients with non-syndromic hereditary deafness.And,we explored the possibility of incorporating suspension array technique and TGE+MPS into clinical deafness genes diagnosis,in an attempt to develop a sensitive and specific diagnostic test for non-syndromic hearing loss and provide better genetic counselling and fertility guidance for deaf patients.Methods:1.We selected one person without mutation of hearing loss gene as negative control and thirty genomic DNA of the patients with Sanger sequencing-confirmed known genotypes,and established the suspension array technology detection system for simμltaneously detecting 20 loci of the 4 common deafness-causing genes in the patients with non-syndromic hereditary deafness,including GJB2:c.235 del C,c.299-300 del AT,c.109A>G,c.176-191del16,c.35 del G,and c.512 ins AAGG;GJB3:c.538C>T;SLC26A4:IVS7-2A>G,c.2168C>T,c.1229C>T,c.1975G>C,C.1226G>A,c.2086C>T,c.754T>C,c.1174A>T,IVS15+5G>A,c.1693 ins A,and c.2027T>A;and mitochondrial DNA 12 Sr RNA:m.1555A>G and m.1494C>T.Using the suspension array technology,a number of 160 samples were analyzed,Nine Deafness Gene Mutation Detection Kit and Sanger sequencing were simμltaneously used to compare this test.2.A cohort of 316 patients initially diagnosed as non-syndromic hereditary hearing loss were collected at the Prenatal Diagnosis Centre and the Otolaryngology Department of Guangdong Women and Children Hospital from November 2011 to June 2016.The DNA genomes of 316 specimens were first detected by the established suspension array technique;second,patients carrying heterozygous variant of GJB2 or SLC26A4 gene were detected by Sanger sequencing.And TGE+MPS was used to detect the patients who were still undiagnosed by suspension array technology and Sanger sequencing.First,the non-syndromic hereditary hearing loss gene panel 1(including 82 known mutation sites of18 non-syndromic hereditary deafness genes)was applied in all cases who were undiagnosed;15 patients were randomly selected for genetic deafness gene panel 2(including 215 known hereditary deafness genes,of them including 88 non-syndromic hereditary deafness genes and 127 syndromic deafness genes).Resμlts:1.A method which coμld simμltaneously detecting 20 mutation spots of the 4common deafness-causing genes was established.2.Of 316 patients,a total of 161 cases were found out to be carrier of mutation by suspension array technology,including 65 cases carried homozygous mutations or compound heterozygous mutations,the mutation positive ratio and diagnostic ratio were 50.9% and 21.2%,respectively.3.Eighteen patients with single heterozygosity of GJB2 or SLC26A4 gene detected by suspension array technique were sequenced by Sanger sequencing.The diagnostic rate was increased to26.9%.4.Two hundred and twenty-nine patients who were still undiagnosed by suspension array technology and Sanger sequencing,were tested 82 known mutation sites of 18non-syndromic hereditary deafness genes by TGE+MPS,and ten mutations in five genes were detected in these patients,and two patients were diagnosed.Fifteen of 229 patients were randomly selected to test 215 known hereditary deafness genes,and eighteen suspicious pathogenic mutations in twelve genes were detected in fourteen patients.Conclusion:1.A suspension array technique was successfμlly established for detecting 20 hot spots in four common deafness-causing genes in Chinese popμlation.The technique has the advantages of high throughput,high efficiency,low cost and easy to operate.It is suitable for wide application in clinical practice.It is expected to be a potential gene detection tool for deafness.2.TGE+MPS has the advantages of high coverage and can make up for the shortcomings of traditional genetic detection techniques for deafness.It is a powerfμl tool for the detection of rare mutations and new pathogenic mutations.3.The combination of various detection techniques not only preserves the advantages of high efficiency,high speed,low cost and accuracy of hot spot mutation screening method,but also makes up for the shortcoming of non-mutation hot spot being easily missed in deafness popμlation,and can find new pathogenic mutation.It provides a set of optional technical methods for clinical diagnosis of deafness,better prevention and treatment of hereditary deafness and the work of eugenics.It also lays a foundation for the functional study of the gene causing deafness. |
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