Mapping And Positional Cloning The Causative Genes In Chinese Pedigrees With Non-syndromic Hereditary Hearing Impairment

The mapping and cloning of deafness genes is a hot focus that geneticists and otolaryngologists.The great progress of the research on hereditary non-syndromic deafness has been made since the first non-syndromic deafness locus was mapped in 1988.Until May 2008,144 loci are located,from which and 49 genes are cloned.The mapping and cloning on of deafness genes grew rapidly since the first gene of non-syndromic deafness was cloned in 1995.On average, 8.3 loci and 4 genes were identified every year,from 1995 to 2006.Chinese scholars achieved great success during the period.In 1999,Academician Xia Jia-hui et al from Chinese Academy of Science cloned the underling gene GJB3 for DFNA2,which was the only gene cloned by Chinese scientist.Up to now, Chinese scientist have discovered 13 loci(13/144,9%),7 of which were the same as the previous identified,and the other 6 were novel(6/144,4.2%).The future for positional cloning of deafness gene is bright.The underling genes in nearly 2/3 of the identified loci remain to be cloned,in addition to continuously identified genes/loci.However,those opportunities parallel with challises.The year of 2007 was a bottle-neck in the history of genetic studies of hearing loss.The speed for mapping and cloning of deafness genes was dramatically reduced,indicated by less successful attempts of positional cloning annually discovered.Only 4 loci and 2 genes were discovered during 2007 and 2008.In this study,we worked on the research of three non-syndromic hereditary deafness pedigrees by the strategy of positional cloning and screening the candidate genes based on the deaf people in China.We succeeded in locating 2 loci in two pedigrees and identifying a novel deafness locus named DFNA61 and attempted to predict causative genes in a deafness family mapped before.The research included three parts. Part one:mapping of the novel locus DFNA61 for late-onset mid to high frequency autosomal dominant hereditary hearing lossIn this study,mapping and cloning of the underlying gene for a scarce frait of hereditary deafness in a Chinese family suffering from middle and high-frequency hearing loss was performed.The phenotypic characteristics included autosomal dominant inheritance,late-onset occurence,progressivity and the hearing loss mainly in middle frequencies hearing.The family included 21 members in 3 generations,of which 10 members were deaf.We got the maximum LOD value of 3.45 on chromosome 17 with peak microsatellite marker D17S1852 by using genome-wide genesan and linkage analysis,and the linked region, defined by markers D17S804 and D17S969,was 6.74 cM long.No known hearing loss locus/gene was reported previously in this region,and thus,this locus was named as DFNA61.It contains 14 protein-coding genes,and further effort will be directed to identification of the causative genes in the locus.Part two:fine mapping of the linkage for hereditary late-onset hearing loss(W727 family)Almost all the DFNA loci have been linked to late-onset hearing loss phenotypes.Studies of hereditary late-onset hearing loss might give insights for early aging in hearing or age-related hearing loss.Consequently,our group have worked in this area for many years.In this study,we fine-mapped the locus for a Chinese late-onset hearing loss family named W727,which was previously mapped on chromosome 9(LOD=2.06,D9S157) in 2007.This family was extended by adding 15 new members including 6 deaf and 9 normal subjects.We got the maximum LOD value of 3.75 on Maker D1S2797 on chromosome 1,the interval of 18.9cM distance was defined by markers D1S255 and D1S2890.This interval overlapped with DFNA2 overlaps in part,we screened the mutations of GJB3 gene,one of responsible genes for DFNA2,but no mutation was found.In the next step,we will screen the other gene(KCNQ4).If no causative mutataion was found either,the underlying gene for this family might be novel. Part three:Network-based global inference and identification of human deafness genes by using modularization methodMapping deafness gene was just the first step in genetic research,it followed by difficult task finding the causative gene in the locus.Network-based global inference was a newly development bioinfornatics method to predict causative genes via the gene network.In order to verify the potential of this method for prioritizing the genes to be cloned,we applied this method tp predict the putative for causative gene for autonomic dominant deafness in family 686,which had been mapped on to a Chromosome 9 interval of 4.12 cM,flanked by markers D9S165 and D9S1874.Two different modular methods,CIPher model and Endeavour online tools were implemented,leading to identification of 7 genes were chosen,including TLN1,STOML,AQP3,DNAI1,C9ORF24,CCIN, and GALT.We first selected AQP3 gene to screen 25 family members including 8 deafness patients.Two polymorphisms were found:390C＞T/390C＞T and 394G＞A/WT.The latter can change the 132nd amino acid,from Asp to Asn, possibly leading to changes both in functionalities and spatial structure of the resulted protein.Reasonably,the 4 members with this functional polymorphism night be affected,soon or later.However,none of the healthy members in this family had this mutation.This study demonstrate that in-silico bioinformatics methods might provide effective alternative or pfiofitizing the hearing loss gene to be loned.