Genotyping with a 198 mutation arrayed primer extension array for hereditary hearing loss: assessment of its diagnostic value for medical practice

Abstract

Molecular diagnostic testing of individuals with congenital sensorineural hearing loss typically begins with DNA sequencing of the GJB2 gene. If the cause of the hearing loss is not identified in GJB2, additional testing can be ordered. However, the step-wise analysis of several genes often results in a protracted diagnostic process. The more comprehensive Hereditary Hearing Loss Arrayed Primer Extension microarray enables analysis of 198 mutations across eight genes (GJB2, GJB6, GJB3, GJA1, SLC26A4, SLC26A5, MTRNR1 and MTTS1) in a single test. To evaluate the added diagnostic value of this microarray for our ethnically diverse patient population, we tested 144 individuals with congenital sensorineural hearing loss who were negative for biallelic GJB2 or GJB6 mutations. The array successfully detected all GJB2 changes previously identified in the study group, confirming excellent assay performance. Additional mutations were identified in the SLC26A4, SLC26A5 and MTRNR1 genes of 12/144 individuals (8.3%), four of whom (2.8%) had genotypes consistent with pathogenicity. These results suggest that the current format of this microarray falls short of adding diagnostic value beyond the customary testing of GJB2, perhaps reflecting the array's limitations on the number of mutations included for each gene, but more likely resulting from unknown genetic contributors to this phenotype. We conclude that mutations in other hearing loss associated genes should be incorporated in the array as knowledge of the etiology of hearing loss evolves. Such future modification of the flexible configuration of the Hereditary Hearing Loss Arrayed Primer Extension microarray would improve its impact as a diagnostic tool.

Figure 1. APEX detection of the W24X (71G>A) mutation in GJB2 and the 1555A>G mutation in MTRNR1.

A) W24X in GJB2. Row 1. Wild-type genotype for nucleotide position 71 in the GJB2 gene. In the sense direction (S) the wild-type G allele is detected, and in the antisense (AS) direction the complementary C allele is identified. Row 2. Heterozygous for W24X. S: the wild-type G allele and the mutant A allele are both present. AS: the wild-type C allele and the mutant T allele are detected. Row 3. Negative control. B) 1555A>G in MTRNR1. Row 1. Wild-type genotype for nucleotide position 1555 in the MTRNR1 gene. The wild-type A allele is identified in S, and the complementary T allele is detected in AS. Row 2. Homoplasmic for 1555A>G. S: the mutant G allele is present. AS: the complementary mutant C allele is detected. Row 3. Negative control.