| Deafness is one of the most common birth defects.It was estimated that about 60%of deafness was caused by genetic factors.Genetic deafness was characterized by a high degree of genetic heterogeneity and ethic differences.According to the molecular epidemiological survey data of deafness in China,GJB2,SLC26A4 and mitochondrial 12S rRNA are the main pathogenic genes.It provided the molecular basis for diagnosis of deaf gene.In order to meet the needs for clinical diagnosis of deaf gene in China,in this dissertation we established a high-throughput efficient diagnostic system for genetic deafness,including hot spot mutations screening and rare mutations identifying for deaf gene.Using a robotic liquid handler and MALDI-TOF-MS technology,a high-throughput detection system for Chinese common deaf genes was established in this dissertation,including automatic sample preparation for dried blood spots and high-throughput deaf gene testing.The accuracy and validity of the detection system were verified by clinical samples of deafness patients.Using targeted gene capture sequencing technology,we also established an efficient identification system for the novel mutations in patients with non-syndromic deafness.The above two detection systems constituted a complete solution to non-syndromic deaf gene testing,and it was great significant to improved the capability for diagnosis of deaf gene.In this dissertation,30 hot spot mutations of Chinese common deaf gene(GJB2,SLC26A4,mitochondrial 12S rRNA)were selected to design the testing panel based on MALDI-TOF-MS technology.The panel was designed successfully to detect the 30 mutations in the single reaction.Using this panel,we detected 327 non-syndromic deafness patients with clear clinical genetic diagnosis.It was shown that all 30 mutations were detected by this panel and genotyping result was consistent with clinical genetic diagnosis.Further,373 non-syndromic deafness patients were tested using the method.The results showed that 210 patients(56.30%,210/373)carried at least one mutation,and 155 patients(41.55%,155/373)were diagnosed,including 63 cases induced by GJB2(16.89%,30/373),88 cases induced by SLC26A4(23.59%,88/373)and 4 cases induced by mitochondrial 12S rRNA(1.07%,4/373).The genotyping results of 373 patients were consistent with Sanger sequencing.Automatic sample preparation method for dried blood spots was also established by Bravo liquid handler.64 newborns' dried blood spots DNA were extracted using this method and 4 different magnetic beads kits.Average concentration of DNA was 10.76ng/?l to 21.88ng/?l and average value of A260/A280 was 1.84 to 1.99.5 newborns carring one mutation of deaf gene were found by MALDI-TOF-MS in these samples.Variations in 200 deafness-related genes were screened by targeted gene capture sequencing in 92 non-syndromic deafness patients undiagnosed by MALDI-TOF-MS technology established in this study.These patients included 90 probands and 2 affected sibs.All identified variations were performed bioinformatic analysis and verified by Sanger sequencing.The results showed that 20 deaf probands were diagnosed and the diagnostic rate of the panel of 200 deafness-related genes was 22.2%.There were 38 mutations located in 15 deafness-related genes were identified in the 20 deaf probands,among those 23 mutations were novel.This study based on a robotic liquid handler and MALDI-TOF-MS technology.We established a high-throughput,low cost and accurate testing system to diagnose and screen deafness-related gene mutations in Chinese.The establishment of the system provided technical support for large-scale prevention of deafness.We also diagnosed 22.2%deaf probands and reported 23 novel mutations by targeted gene capture sequencing.These enriched the deaf gene mutation database and improved the ability to explain the underlying etiology in more families. |
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