Genetic Mapping And Gene Diagnosis For Inherited Deafness Pedigrees

Chapter 1:Analysis of the clinical features and family-related gene mutations for two pedigrees of typeⅣWaardenburg syndromeObjective:Analysis of the clinical features and family-related gene mutation for the two pedigrees of typeⅣWaardenburg syndrome （WSIV） which were collected （WSO1 and WS02） in the last year.Methods:After History taking, clinical analysis, haring test and physical examination for the two pedigrees, EDNRB、EDN3、SOX1O sequencing was taken for the two pedigrees to identify the Pathogenic mutation.Results:There were 5 persons from a 2 generations pedigree of WS01.The proband was a WSIV patient and his eldest sister was WSII. Both of them showed heterochromia iridis of left eye. Hirschsprung disease was found in the proband of WS01. Proband of WS01, a 1 year old girl, is a WSIV patient showed heterochromia iridis of eyes, hirschsprung disease. All of the patients presented bilateral profound hearing loss, a generalized decrease in retinal pigment with a focal hypopigmented lesion in the eyes with blue irides, without dystopia canthorum and pigmentary abnormalities of hair and skin. Heterozygous mutation Trp85X（c.254G>A） was detected in the proband, eldest sister and mother of WS01 pedigree. But the height of mutation peak was One third lower than that of normal one in the mother who was a healthy subject; Heterozygous mutation c.698-2A>T was detected in the proband of WS02 pedigree. However the mutation was not detected in the parents of the proband. All the mutations identified in these patients were not seen in any other unaffected family members and 100 unrelated control subjects.Conclusions:1.This was the first report of WSIV in Chinese patients.2. P.Trp85X （c.254G>A） and c.698-2A>T were the novel mutation for SOX 10 gene.3. SOX 10 gene was suspected of having mosaicism.Chapter 2:Localization for an autosomal dominant non-syndromic deafness family of ChinaObjective:We collected a four-generation family from the southern part of China with autosomal dominant sensorineural hearing impairment. In order to identify the responsible pathogenic mutations of the family, we set out to identify the locus and to sequentially analyze the candidate genes in the identified region.Methods:After family ascertainment and clinical analysis, exclusive analysis was performed. And then a Genome-wide scan was performed by using Illumina Linkage-12 DNA Analysis Kit （average spacing 0.58 cM）. Fine-mapping markers were genotyped to identify the locus. Finally, we performed haplotype analyses, and candidate gene DNA sequencing for the family.Results:The known genetic loci and genes were not associated with our family. The genome-wide scan and haplotype analyses traced the disease to chromosome 1p34.2-p34.3 with maximum multi-point LOD score of 3.2 which overlaps with DFNA2. We failed to identify any of the known or novel variants within KCNQ4, a voltage-gated potassium channel gene, and GJB3, a gene that encodes the gap junction protein connexin 31, which were the cloned deafness gene in DFNA2. Conclusions:There could be another candidate gene in DFNA2 which be responsible for hearing loss phenotype.Chapter 3:Designing and development of Illumina Goldengate 384k high-throughput BeadArrey for deafness gene mutations screening, and screening for patients in deafness pedigreesObjective:Designing and development of Illumina Goldengate 384k high-throughput BeadArrey. Screen for patients in deafness pedigrees and exclude locus for large deafness pedigrees by use of the BeadArrey.Methods:Design principles:Interval of 60bp for primer probe was needed to ensure accurate cross over on BeadArrey. So we selected mutations which have been reported twice, and then put the other mutations on the BeadArrey according the design priciple. Totally 240 mutations were selected, including 77 dominant mutations and 163 recessive mutations. On the other side, we selected 144 SNPs in deafness genes which were reported in Asian populations. Therefore, the BeadArrey we designed could screen for the deafness gene mutations and exclude locus for large deafness pedigrees. We conducted a call rate statistics and accurate verification for the BeadArrey. An allele frequency analysis of the 144 SNPs and exclusive location analysis were carried on. Finally, we analyzed the test results of BeadArrey for 135 DNA samples of 90 deafness pedigrees which were collected by ENT clinic of Xiangya Hospital and State Key Laboratory of Medical Genetics in 1997-2010.Results:1. Total Call rate of the BeadArrey was 96.32%,110 of 384 test points’ Call rate were 100%.2. False negative rate was 3/97 （3.1%） and false positive rate was 0 for test point GJB2₂35delC.3. Minimum allele frequencies of 19 SNPs in 8 genes were less than 0.1 （10%）.4. The region located by BeadArrey was similar to which was located by traditional micro-satellite scan.5. We detected 31 mutations of 12 genes in the BeadArrey screening for 135 patients.189 mutations which might be the rare mutations in Chinese deafness were not detected. Detection rate of GJB2 1 BP DEL 235C and SLC26A4 IVS7-2A>G were the same as previous reports. But mutations like PJVK（DFNB59）_1BP_DEL₉88G （57.36%） and SLC26A4_Gly497Ser （7.14%） which were not the hot mutations had a high detection rate.Conclusions:Illumina Goldengate 384k high-throughput BeadArrey could test more mutations for the patients and exclude locus for large deafness pedigrees. PJVK（DFNB59）_1BP_DEL₉88G （57.36%） and SLC26A4_Gly497Ser （7.14%） et al which were not the hot mutations had a high detection rate. To find the reason whether they are snps or the low accuracy, in the next step, we will conduct a accurate verification for more test point and remove the points with low call rate, mutation frequency and accuracy, add in the other mutation points which did not add in the BeadArrey as a result of the principle of 60bp Interval. We will remove the SNPs which allele frequencies are less than 10% and add in the other SNPs in the similar position.