DNA Sequence Analysis of SLC26A5, Encoding Prestin, in a Patient-Control Cohort: Identification of Fourteen Novel DNA Sequence Variations

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.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.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

Heterozygosity of sequence variations in <i>SLC26A5.</i>

<p>Heterozygosity is provided for each DNA sequence variation. n = the number of individuals studied. Novel variants are shown in bold type.</p

Haplotype analysis of patients and controls compared to International HapMap Project database markers.

<p>Haplotype blocks as determined by HaploView 4.0 are shown as shaded bars and compared for patients, controls, patients and controls combined (Combined), and the International HapMap Project database markers (HapMap). The location of markers within the <i>SLC26A5</i> gene is shown at the top of the figure. DNA sequence variations are shown above the haplotype blocks. Note: The only marker observed in this study that is included in the International HapMap Project database is rs7779997 in IVS5. The genomic <i>SLC26A5</i> nucleotides spanned by the haplotype blocks derived from the International HapMap Project database markers are shown at the bottom of the figure. Abbreviations and symbols used: IVS, intervening sequence (intron); *formerly hCG1811409, Celera database <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005762#pone.0005762-Applied1" target="_blank">[17]</a>; #, C allele noted is variant allele, not reference sequence allele; −, Variant allele not detected; +, Reference allele not detected; 1, 2, 3, 4, represent haplotype block designations derived from and assigned by HaploView 4.0 software analysis of the International HapMap Project database markers. The reference sequence (undeleted) allele of variant rs66928926 (g.69917_69919delTCT) is designated as “T” and of variant rs72655394 (g.70078_70082delATATA) is designated as “A.” Novel variants are shown in bold type.</p

Other DNA sequence variations observed in <i>SLC26A5</i> IVS2-2A>G heterozygotes.

<p>Additional sequence variations observed in the patient and control samples heterozygous for the <i>SLC26A5</i> IVS2-2A>G variant are shown. All eight subjects were also homozygous for the variant alleles at g.56471G>A, g.67439T>C, and g.69036T>C (not shown). Het = heterozygous; Hom = homozygous; Ref = reference sequence allele; Var = variant allele.</p

Multiple sequence alignment for observed and previously reported [17], [18] amino acid sequence variations in prestin.

<p>Amino acid alignments of prestin orthologs from various species are shown using <i>H. sapiens</i> as the anchor sequence. Amino acid (aa) positions 67, 150, and 663 indicated in the top row of columns 3–5 refer to the amino acid positions in the human prestin amino acid sequence. Reference sequences used are derived from the NCBI protein database and aligned using the NCBI HomoloGene multiple sequence alignment tool (<a href="http://www.ncbi.nlm.nih.gov" target="_blank">http://www.ncbi.nlm.nih.gov</a>).</p

Reference sequence allele frequencies for variations in <i>SLC26A5</i>.

<p>Allele frequencies of reference sequence nucleotides are provided for each DNA sequence variant. n = the number of chromosomes studied. <i>P</i> values are calculated by comparing patients to the combined control group. Novel variants are shown in bold type.</p