DNA sequence analysis and genotype-phenotype assessment in 71 patients with syndromic hearing loss or auditory neuropathy.

OBJECTIVES: Aetiological assessment of 71 probands whose clinical presentation suggested a genetic syndrome or auditory neuropathy. METHODS: Sanger sequencing was performed on DNA isolated from peripheral blood or lymphoblastoid cell lines. Genes were selected for sequencing based on each patient's clinical presentation and suspected diagnosis. Observed DNA sequence variations were assessed for pathogenicity by review of the scientific literature, and mutation and polymorphism databases, through the use of in silico tools including sorting intolerant from tolerant (SIFT) and polymorphism phenotyping (PolyPhen), and according to the recommendations of the American College of Medical Genetics and Genomics for the interpretation of DNA sequence variations. Novel DNA sequence variations were sought in controls. RESULTS: DNA sequencing of the coding and near-coding regions of genes relevant to each patient's clinical presentation revealed 37 sequence variations of known or uncertain pathogenicity in 9 genes from 25 patients. 14 novel sequence variations were discovered. Assessment of phenotypes revealed notable findings in 9 patients. CONCLUSIONS: DNA sequencing in patients whose clinical presentation suggested a genetic syndrome or auditory neuropathy provided opportunities for aetiological assessment and more precise genetic counselling of patients and families. The failure to identify a genetic aetiology in many patients in this study highlights the extreme heterogeneity of genetic hearing loss, the incompleteness of current knowledge of aetiologies of hearing loss, and the limitations of conventional DNA sequencing strategies that evaluate only coding and near-coding segments of genes.

DNA sequence analysis of SLC26A5, encoding prestin, in a patient-control cohort: identification of fourteen novel DNA sequence variations.

BACKGROUND: Prestin, encoded by the gene SLC26A5, is a transmembrane protein of the cochlear outer hair cell (OHC). Prestin is required for the somatic electromotile activity of OHCs, which is absent in OHCs and causes severe hearing impairment in mice lacking prestin. In humans, the role of sequence variations in SLC26A5 in hearing loss is less clear. Although prestin is expected to be required for functional human OHCs, the clinical significance of reported putative mutant alleles in humans is uncertain. METHODOLOGY/PRINCIPAL FINDINGS: To explore the hypothesis that SLC26A5 may act as a modifier gene, affecting the severity of hearing loss caused by an independent etiology, a patient-control cohort was screened for DNA sequence variations in SLC26A5 using sequencing and allele specific methods. Patients in this study carried known pathogenic or controversial sequence variations in GJB2, encoding Connexin 26, or confirmed or suspected sequence variations in SLC26A5; controls included four ethnic populations. Twenty-three different DNA sequence variations in SLC26A5, 14 of which are novel, were observed: 4 novel sequence variations were found exclusively among patients; 7 novel sequence variations were found exclusively among controls; and, 12 sequence variations, 3 of which are novel, were found in both patients and controls. Twenty-one of the 23 DNA sequence variations were located in non-coding regions of SLC26A5. Two coding sequence variations, both novel, were observed only in patients and predict a silent change, p.S434S, and an amino acid substitution, p.I663V. In silico analysis of the p.I663V amino acid variation suggested this variant might be benign. Using Fisher's exact test, no statistically significant difference was observed between patients and controls in the frequency of the identified DNA sequence variations. Haplotype analysis using HaploView 4.0 software revealed the same predominant haplotype in patients and controls and derived haplotype blocks in the patient-control cohort similar to those generated from the International HapMap Project. CONCLUSIONS/SIGNIFICANCE: Although these data fail to support a hypothesis that SLC26A5 acts as a modifier gene of GJB2-related hearing loss, the sample size is small and investigation of a larger population might be more informative. The 14 novel DNA sequence variations in SLC26A5 reported here will serve as useful research tools for future studies of prestin.

DNA sequence analysis of GJB2, encoding connexin 26: observations from a population of hearing impaired cases and variable carrier rates, complex genotypes, and ethnic stratification of alleles among controls.

Mutations in GJB2 are associated with hereditary hearing loss. DNA sequencing of GJB2 in a cohort of hearing impaired patients and a multi-ethnic control group is reported. Among 610 hearing impaired cases, 43 DNA sequence variations were identified in the coding region of GJB2 including 24 mutations, 8 polymorphisms, 3 unclassified variants (G4D, R127C, M163V), 1 controversial variant (V37I), and 7 novel variants (G12C, N14D, V63A, T86M, L132V, D159, 592_600delinsCAGTGTTCATGACATTC). Sixteen non-coding sequence variations were also identified among cases including the IVS1+1A>G mutation, 2 polymorphisms, and 13 novel variants. A diagnosis of GJB2-associated hearing loss was confirmed for 63 cases (10.3%). Heterozygous mutations were found in 39 cases (6.4%). Eleven cases carrying novel or unclassified variants (1.8 %) and 18 cases carrying the controversial V37I variant were identified (3%). In addition, 294 control subjects from 4 ethnic groups were sequenced for GJB2. Thirteen sequence variations in the coding region of GJB2 were identified among controls including 2 mutations, 6 polymorphisms, 2 unclassified variants (G4D, T123N), 1 controversial variant (V37I), and 2 novel variants (R127L, V207L). Nine sequence variations were identified among controls in the non-coding regions in and around GJB2 exon 2. Of particular interest among controls were the variability in carrier rates and ethnic stratification of alleles, and the complex genotypes among Asians, 47% of whom carried two to four sequence variations in the coding region of GJB2. These data provide new information about carrier rates for GJB2-based hearing loss in various ethnic groups and contribute to evaluation of the pathogenicity of the controversial V37I variant.

High frequency of the IVS2-2A>G DNA sequence variation in SLC26A5, encoding the cochlear motor protein prestin, precludes its involvement in hereditary hearing loss.

BACKGROUND: Cochlear outer hair cells change their length in response to variations in membrane potential. This capability, called electromotility, is believed to enable the sensitivity and frequency selectivity of the mammalian cochlea. Prestin is a transmembrane protein required for electromotility. Homozygous prestin knockout mice are profoundly hearing impaired. In humans, a single nucleotide change in SLC26A5, encoding prestin, has been reported in association with hearing loss. This DNA sequence variation, IVS2-2A>G, occurs in the exon 3 splice acceptor site and is expected to abolish splicing of exon 3. METHODS: To further explore the relationship between hearing loss and the IVS2-2A>G transition, and assess allele frequency, genomic DNA from hearing impaired and control subjects was analyzed by DNA sequencing. SLC26A5 genomic DNA sequences from human, chimp, rat, mouse, zebrafish and fruit fly were aligned and compared for evolutionary conservation of the exon 3 splice acceptor site. Alternative splice acceptor sites within intron 2 of human SLC26A5 were sought using a splice site prediction program from the Berkeley Drosophila Genome Project. RESULTS: The IVS2-2A>G variant was found in a heterozygous state in 4 of 74 hearing impaired subjects of Hispanic, Caucasian or uncertain ethnicity and 4 of 150 Hispanic or Caucasian controls (p = 0.45). The IVS2-2A>G variant was not found in 106 subjects of Asian or African American descent. No homozygous subjects were identified (n = 330). Sequence alignment of SLC26A5 orthologs demonstrated that the A nucleotide at position IVS2-2 is invariant among several eukaryotic species. Sequence analysis also revealed five potential alternative splice acceptor sites in intron 2 of human SLC26A5. CONCLUSION: These data suggest that the IVS2-2A>G variant may not occur more frequently in hearing impaired subjects than in controls. The identification of five potential alternative splice acceptor sites in intron 2 of human SLC26A5 suggests a potential mechanism by which expression of prestin might be maintained in cells carrying the SLC26A5 IVS2-2A>G DNA sequence variation. Additional studies are needed to evaluate the effect of the IVS2-2A>G transition on splicing of SLC26A5 transcripts and characterize the hearing status of individuals homozygous for the IVS2-2A>G variant.

Genetic susceptibility to aminoglycoside ototoxicity: how many are at risk?

PURPOSE: To assess the occurrence of two mutations associated with susceptibility to aminoglycoside ototoxicity. METHODS: Genetic analysis of anonymized, residual diagnostic specimens. RESULTS: One occurrence of the A1555G mutation and seven occurrences of the 961delT + C(n) nucleotide change were found. Two previously unreported sequence changes, T961G and 956-960insC, were also found in six and five specimens, respectively. CONCLUSIONS: Genetic susceptibility to aminoglycoside ototoxicity may be more common than previously suspected. Further study of the 961delT + C(n) mutation is recommended to confirm its role in aminoglycoside ototoxicity and assess penetrance and variability with and without exposure to aminoglycoside antibiotics.