The Genetic Analysis Based On High-throughput Sequencing In A Non-syndromic Hearing Loss Family

Background and objectiveHearing loss,namely deafness,is one of the most common sensory defect diseases in clinic treatment,and is also one of the most important problems that affects human life and leads to permanent disabilities.Nowadays,there are 0.278 billion people who suffering different degrees of hearing loss,and in China,the number of deafness patients totals nearly 30 million,topping all kinds of disabilities.There are one-three newborns with hearing loss per thousand people,and also approximately one in thousand to three in thousand would be deaf during growth and development.So many people suffering deafness,this would seriously affects patients' recognition and communication and burdens themselves,families and society.The pathogenic factors of deafness vary,which can be roughly categorized into two kinds: environmental and genetic factors.Among deafness patients,more than 60% suffers congenital sensorineural deafness due to genetic factors,and the other 40% may be caused by environmental factors.Deafness can be divided into syndromic hearing loss(SHL)and non-syndromic hearing loss(NSHL),according to whether patients suffering outer ear organs' developmental anomalies and dysfunction or not.In terms of genetic ways,generic deafness can be divided into four kinds: autosomal dominant deafness(DFNA),autosomal recessive deafness(DFNB),sex chromosome link(X-linked deafness,DFNX,Y-linked deafness,DNFY)and mutation of mitochondria matrilineal inheritance.The statistics suggest that over 70% of the deafness patients suffer NSHL.The number of already found disease-causing genes totally over 200 kinds,and are extensively spread on each chromosome.So,it is hard to study how to determine the disease-causing gene of deafness patients.So far,the common gene testing methods include Sanger sequencing,gene chip sequencing and high through-put sequencing.Sanger sequencing is deemed as the gold standard of diagnosing genetic mutations with high accuracy,and it can provide the tested gene's whole sequencing information,but costs a lot and is time-consuming;gene chip sequencing can test several genes at the same time,but the so far developed generic deafness gene diagnosing chip can only test some common deafness genes' mutation all at once,not including rare or unknown mutations,it may miss some of the gene mutations;while high through-put sequencing is rapid,accurate,sensitive,effective and wide-spread,and it can test several genes and locus at the same time.Compared with other methods,high through-put sequencing has big advantages.Therefore,we chose high through-put sequencing to rest the disease-causing gene of a non-syndromic hearing loss family,in hope to find its generic disease-causing basis and offer references to the NSHL family's deafness gene diagnosis,generic consult and deafness prevention and treatment.MethodsMaterials are collected from a non-syndromic hearing loss family,Nanyang,Henan province,China,with 3 deafness patients out of 26 persons from 3 generation.Register all people's basic information,and investigate the deafness patients' age of onset,inducing factors,concomitant symptoms,deafness progressing,the history of ototoxicity drug use,the history of head or ear trauma,the history of exposure to noise,mother's incubation situation,the history of newborn jaundice.All the family number should have tests,including pure tone test,acoustic immittance measurement,otoacoustic emission,auditory evoked potential,high-resolution computed tomography(CT),magnetic resonance imaging(MRI),inner acoustic duct water imaging in order to exclude that they all have no acoustic neuroma and other craniocerebral space-occupying lesions and dysgnosia.Collect 5ml peripheral venous blood of the family numbers,using EDTA-K2 to anti-freezing and extract DNA with DNA extraction kit and sequencing genes by the method of high through-put sequencing.Compare the gained gene sequencing with reference sequence on the UCSC website,version GRch37/hg19(http://genome.ucsc.edu),in order to get mutant genes of this NSHL family numbers.ResultsThree patients carry GJB3 c.375C>T heterozygous mutation,KCNQ4 c.777T>C heterozygous mutation,ILDR1 c.1545T>G homozygous mutation,GJB2 c.1277C>T?c.1152G>A?c.1067G>T homozygous mutation,and mt DNA 12 S rRNA m.1121T>C mutation.Other family numbers,one carries ILDR1 c.1545T>G heterozygous mutation,GJB2 c.1277C>T?c.1152G>A?c.1067G>T homozygous mutation,one carries GJB2 c.1277C>T homozygous mutation,three carry GJB2 c.1277C>T heterozygous mutation.Conclusions1.We didn't find any clear genes lead this Non-syndromic hearing loss family's deafness,but we doubt this NSHL family suffers deafness because of mtDNA 12 S rRNA m.1121T>C mutation.2.The gene mutation of mtDNA 12 S rRNA m.1121T>C which we suspect can lead Non-syndromic hearing loss needs some more research.3.The high through-put sequencing has the characteristics of speediness,accuracy,delicacy,efficient,wide coverage in the screening of deafness genes.4.The high through-put sequencing can promote the discovery of new virulence gene.