Establishment And Application Of Denaturing High Performance Liquid Chromatography Technique Screening SLC26A4 Gene In Chinese Severe-Profound Hearing Impairment Population

GJB2 and SLC26A4 gene were the two major causes for non-syndromic hearing impairment, GJB2 gene mutation accounts for 50% autosomal recessive NSHI. . Ph.D YuFei had investigated the molecular epidemiology of GJB2 gene in Genetic Testing Center for Deafness of PLA General Hospital. GJB2 mutations causing hearing loss is of proportion 14.96% in Chinese NSHI cases , 6.05% proportion may be related to GJB2 mutations. (carried with identified pathogenic monoallelic ) and this study characterized the profile of the map of GJB2 genetic variations in Chinese NSHI cases, but there was no the molecular epidemiology of SLC26A4 gene in Chinese NSHI cases similar to GJB2. The screening of SLC26A4 gene mutation will assist us with genetic counseling, and it should be a rout detection to screen SLC26A4 gene mutation for severe or profound hearing loss. GJB2 gene has only one exon and is easily analysised by PCR and direct sequencing .SLC26A4 contains 21 exons with an open reading frame of 2343 base pairs. Up to date, more than 150 SLC26A4 mutations have been detected, including missense mutation, frame shift mutation, splice site mutation, nonsense mutation and small deletion mutation et al .These mutations covered all the 21 exons of SLC26A4 gene. Of all currently available methods, direct sequencing has the highest sensitivityand the gold standard but also the highest costs, obviously was not suitable for the highthroughput screening .The analysis of DNA sequence variation is offundamental importance in genetic studies[Lander, 1996], With the increasing availabilityof primary sequence from genomes of human andother organisms, the implementation of sensitive,efficient, and inexpensive technologies for detecting variation that can match the pace of primary sequencing becomes critical. Because of its size, high throughput mutation screening of SLC26A4 is challenging. The application of some techniques is unnecessarily laborious (SSCP, heteroduplexanalysis), expensive (direct sequencing) or insensitive (SSCP, heteroduplex analysis) for mutation screening. DHPLC is a recently developed method forDNA variationdetection that uses reversedphase ion pair liquid chromatography to detectDNA heteroduplexes,it represents an attractive consideration as an inexpensive option with a sensitivity that approaches that of direct sequencing [Liu et al., 1998;O'Donovan et al., 1998; Ellis et al., 2000].We used the plenty genetic resources established by the Otolaryngology Institute , Genetic Testing Center for Deafness of PLA General Hospital, to collect information of NSHI patients, and to investigate the molecular epidemiology of SLC26A4 gene , DHPLC analysis was carried out using WAVE-99-3500HT DHPLC device of our lab. in Chinese NSHI cases.Part one: collection of clinical data and establishing a novel method for the screening of SLC26A4 gene mutations based on DHPLCThe purpose of this study is to establishing a novel method for the screening of SLC26A4 gene mutations based on DHPLC. DHPLC primers were chosen to optimize mutation detection based on amplicon length and melting profile , 17pair primers provided by Bai-Lin Wu ,the director of Genetics Diagnostic Laboratory of Children's Hospital Boston affiliated Harvard university, primersof exon 7 and 8 were designed refered to the document of Coyle et al. Then definiting the condition of detection : using the DNA sample carried known mutation identified by direct sequcecing as positive control sample ,and wild-type DNA sample as negative control sample;Utilizing the recommended temperature according to Navigator software , the optima condition of detection were decided by experience eventually . The discrimination of chromatograms to the positive and negative sample were very direct-viewing.Part two: validating the DHPLC method of screening SLC26A4 gene mutations by blind methodThe purpose of this study is to investigate the sensitivity and specificity of DHPLC method for screening SLC26A4 gene mutations by blind method. In this section, 1552 patients with severe-profound hearing loss were enrolled fromdeafness school in 19 povince and cities . Exon7+8 of SLC26A4 were detected in these subjects by direct sequencing and identified 100 cases with mutational hot spot IVS7-2A>G heterozygous mutation, We present the application of the DHPLC protocol for 100 cases undergoing genetic testing for mutations were tested. Of the other exons fragments analyzed by DHPLC, simultaneously were also directly sequenced independently by two technicians. The comparison demonstrated that DHPLCdetected all alterations found by direct sequencing., no false-negative results were ever obtained. More importantly, under the same experimental conditions, it identified a further mutational event ,we obtained 25 novel mutations., the sensitivity is 100%, specificity is 89%, coincidence with the documented 96-100%. A rapid, simple, and low-cost assay for detecting both the known and new mutations occuring in the SLC26A4 gene was established.Part three: Incidence study of SLC26A4 mutation and Whole Spectrum Mutation Map Drawing among Patients with severe to profound Hearing LossThe purpose of this study is to detected SLC26A4 gene for mutations in 2217 severe-profound hearing loss cases by DHPLC. The general technique was DNA was prepared using standard conditions, exon sequences were amplified, Amplicons were checked by agarose gel electrophoresis before DHPLC analysis, to make sure that only the specific product was amplified and that no additional bands occurred thatcould lead to artificial heteroduplex conformation. Two samples mixed randomly were degenerated and annealed, subsequently analysised by DHPLC, positive or abnormal sampleswere selected to directedly sequence to assure mutation type. A total of 74 mutations were identified, including 54 novel mutations and 20 recurrent mutations. Of all these mutations, IVS7-2A>G is the commonest, which accounts for 75.9% of all mutant alleles(356/469). This mutation was detected in 307/2217 patients. Besides exon7+8 containing IVS7-2A>G, four exons (exon19, 10, 17 and 15 ) were found having more mutant alleles than others. These mutation sites spread all over 19 exons except exon1 , 20 and manifested widely the heterogenicity of SLC26A4 gene. The rateof mutation3.34%, 1.85%, 1.48%, 0.74%,respectively.In conclusion, our results show that various DHPLC systems are very effective, economical, fast, and sensitive equipment for the detection of mutations of SLC26A4 gene. This study characterized the profile of the map of SLC26A4 genetic variations in Chinese severe-profound hearing loss cases cases. These data provide basic information to foster genetic approaches to the early diagnosis and prediction of recurrent risks for hearing impairment in Chinese population.