Recent advances in molecular genetics have enabled to determine the genetic causes of non-syndromic hearing loss, and more than 100 genes have been related to the phenotype. Due to this extraordinary genetic heterogeneity, a large percentage of patients remain without any molecular diagnosis. This condition imply the need for new methodological strategies in order to detect a greater number of mutations in multiple genes. In this work, we optimized and tested a panel of 86 mutations in 17 different genes screened using a high-throughput genotyping technology to determine the molecular etiology of hearing loss. The technology used in this work was the MassARRAY iPLEX® platform. This technology uses silicon chips and DNA amplification products for accurate genotyping by mass spectrometry of previous reported mutations. The generated results were validated using conventional techniques, as direct sequencing, multiplex PCR and RFLP-PCR. An initial genotyping of control subjects, showed failures in 20 % of the selected alterations. To optimize these results, the failed tests were re-designed and new primers were synthesized. Then, the specificity and sensitivity of the panel demonstrated values above 97 %. Additionally, a group of 180 individuals with NSHL without a molecular diagnosis was screened to test the diagnostic value of our panel, and mutations were identified in 30 % of the cases. In 20 % of the individuals, it was possible to explain the etiology of the HL. Mutations in GJB2 gene were the most prevalent, followed by other mutations in in SLC26A4, CDH23, MT-RNR1, MYO15A, and OTOF genes. The MassARRAY technology has the potential for high-throughput identification of genetic variations. However, we demonstrated that optimization is required to increase the genotyping success and accuracy. The developed panel proved to be efficient and cost-effective, being suitable for applications involving the molecular diagnosis of hearing loss.

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.

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 ∼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).

Mutations in the mitochondrial DNA (mtDNA) have been associated with aminoglycoside-induced and nonsyndromic deafness in different populations. In the present study, we investigated the contribution of mutations in mitochondrial genes to the etiology of hearing loss in a Brazilian sample. Using mass spectrometry genotyping technology, combined with direct sequencing, 50 alterations previously described in 14 mitochondrial genes were screened in 152 patients with sensorineural hearing loss and in104 normal hearing controls. Fifteen known mitochondrial alterations were detected (G709A, A735G, A827G, G988A, A1555G, T4363C, T5628C, T5655C, G5821A, C7462T, G8363A, T10454C, G12236A, T1291C, G15927A). Pathogenic mutations in MT-RNR1 and MT-TK genes were detected in 3 % (5/152) of the patients with hearing loss. This study contributed to show the spectrum of mitochondrial variants in Brazilian patients with hearing loss. Frequency of A1555G was relatively high (2.6 %), indicating that this mutation is an important cause of hearing loss in our population. This work reports for the first time the investigation and the detection of the tRNALys G8363A mutation in Brazilian patients with maternally inherited sensorineural hearing loss.