Searching for Digenic Inheritance in Deaf Brazilian Individuals Using the Multiplex Ligation-Dependent Probe Amplification Technique

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Mutations in the genes coding for connexin 26 (Cx26), connexin 30 (Cx30), and connexin 31 (Cx31) are the main cause of autosomal recessive nonsyndromic sensorineural hearing loss (AR-NSNHL). The 35delG mutation is the most frequent in the majority of Caucasian populations and may account for up to 70% of all GJB2 mutations. As a large number of affected individuals (10%-40%) with GJB2 mutations carry only one mutant allele, it has been postulated that the presence of additional mutations in the GJB6 gene (Cx30) explains the deafness condition found in these patients. In the present study, we screened the c. 35delG mutation in similar to 600 unrelated Brazilian patients, with moderate to profound AR-NSNHL. Other point mutations in the coding region of the GJB2 gene were screened by sequencing analysis as well as the IVS 1 + 1 G> A splice site mutation in the same gene. Digenic mutations including large deletions and duplications were investigated in the Cx26, 30, and 31 genes in monoallelic individuals for mutations in the GJB2 gene. Large deletions and duplications were assessed by multiplex ligation-dependent probe amplification. We found 46 patients with mutations in only one GJB2 allele. Different pathogenic mutations associated with c. 35delG were found in 13 patients. Two patients were identified with digenic heterozygous mutations. Our findings contributed to more accurate diagnosis and more appropriate genetic counseling in 28% of patients studied (13/46).1512849853Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Optimization of simultaneous screening of the main mutations involved in non-syndromic deafness using the TaqMan (R) OpenArray (TM) Genotyping Platform

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Background: Hearing loss is the most common sensory deficit in humans, affecting approximately 10% of the global population. In developed countries, one in every 500 individuals suffers from severe to profound bilateral sensorineural hearing loss. For those up to 5 years old, the proportion is higher, at 2.7 in 1000 individuals, and for adolescents the average is 3.5 in 1000. Among the causes of hearing loss, more than 50% are related to genetic factors. To date, nearly 150 loci and 64 genes have been associated with hearing loss. Mutations in the GJB2 gene, which encodes connexin 26, constitute the main genetic cause. So far, more than 300 variations have been described in this gene. As a response to the clinical and genetic heterogeneity of hearing loss and the importance of correct molecular diagnosis of individuals with hereditary hearing loss, this study worked in the optimization for a diagnostic protocol employing a high-throughput genotyping technology. Methods: For this work, was used the TaqMan (R) OpenArray (TM) Genotyping platform. This is a high performance, high-throughput technology based on real-time PCR, which enables the evaluation of up to 3072 SNPs (Single Nucleotide Polymorphisms), point mutations, small deletions, and insertions, using a single genotyping plate. For the study, were selected the layout allowing to analyze 32 alterations in 96 individuals simultaneously. In the end, the generated results were validated by conventional techniques, as direct sequencing, Multiplex PCR and RFLP-PCR. Results: A total of 376 individuals were analyzed, of which 94 were healthy controls, totaling 4 plates in duplicate. All 31 of the changes analyzed were present in the nuclear genes GJB2, GJB6, CRYL1, TMC1, SLC26A4, miR-96, and OTOF, and in the mitochondrial genes MT-RNR1 and MT-TS1. The reactions were subsequently validated by established techniques (direct sequencing, multiplex PCR, and RFLP-PCR) that had previously been used to perform molecular screening of hearing loss at the Human Genetics Laboratory of the Center for Molecular Biology and Genetic Engineering (CBMEG), at the State University of Campinas (UNICAMP). In total, 11,656 genotyping reactions were performed. Of these, only 351 reactions failed, representing approximately 3.01% of the total. The average accuracy of genotyping using the OpenArray (TM) plates was 96.99%. Conclusions: The results demonstrated the accuracy, low cost, and good reproducibility of the technique, indicating that the TaqMan (R) OpenArray (TM) Genotyping Platform is a useful and reliable tool for application in molecular diagnostic testing of hearing loss.14Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES