Prestin, a Cochlear Motor Protein, Is Defective in Non-Syndromic Hearing Loss
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Human Molecular Genetics, 2003, Vol. 12, No. 10 1155–1162 DOI: 10.1093/hmg/ddg127 Prestin, a cochlear motor protein, is defective in non-syndromic hearing loss Xue Zhong Liu1,*, Xiao Mei Ouyang1, Xia Juan Xia2, Jing Zheng3, Arti Pandya2, Fang Li1, Li Lin Du1, Katherine O. Welch4, Christine Petit5, Richard J.H. Smith6, Bradley T. Webb2, Denise Yan1, Kathleen S. Arnos4, David Corey7, Peter Dallos3, Walter E. Nance2 and Zheng Yi Chen8 1Department of Otolaryngology, University of Miami, Miami, FL 33101, USA, 2Department of Human Genetics, Medical College of Virginia, Virginia Commonwealth University, Richmond, VA 23298-0033, USA, 3Department of Communication Sciences and Disorders, Auditory Physiology Laboratory (The Hugh Knowles Center), Northwestern University, Evanston, IL, USA, 4Department of Biology, Gallaudet University, Washington, DC 20002, USA, 5Unite´ de Ge´ne´tique des De´ficits Sensoriels, CNRS URA 1968, Institut Pasteur, Paris, France, 6Department of Otolaryngology University of Iowa, Iowa City, IA 52242, USA, 7Neurobiology Department, Harvard Medical School and Howard Hughes Medical Institute, Boston, MA, USA and 8Department of Neurology, Massachusetts General Hospital and Neurology Department, Harvard Medical School Boston, MA 02114, USA Received January 14, 2003; Revised and Accepted March 14, 2003 Prestin, a membrane protein that is highly and almost exclusively expressed in the outer hair cells (OHCs) of the cochlea, is a motor protein which senses membrane potential and drives rapid length changes in OHCs. Surprisingly, prestin is a member of a gene family, solute carrier (SLC) family 26, that encodes anion transporters and related proteins. Of nine known human genes in this family, three (SLC26A2, SLC26A3 and SLC26A4 ) are associated with different human hereditary diseases. The restricted expression of prestin in OHCs, and its proposed function as a mechanical amplifier, make it a strong candidate gene for human deaf- ness. Here we report the cloning and characterization of four splicing isoforms for the human prestin gene (SLC26A5a, b, c and d ). SLC26A5a is the predominant form of prestin whereas the others showed limited distribution associated with certain developmental stages. Based on the functional importance of prestin we screened for possible mutations involving the prestin gene in a group of deaf probands. We have identified a 50-UTR splice acceptor mutation (IVS2-2A>G) in exon 3 of the prestin gene, which is responsible for recessive non-syndromic deafness in two unrelated families. In addition, a high frequency of heterozygosity for the same mutation was observed in these subjects, suggesting the possibility of semi-dominant influence of the mutation in causing hearing loss. Finally, the observation of this mutation only in the Caucasian probands indicated an association with a specific ethnic background. This study thereby reveals an essential function of prestin in human auditory processing. INTRODUCTION passing to the brain. OHCs are responsible for amplification of cochlear vibrations, possibly through somatic length changes The mammalian inner ear consists of the cochlea (the sense driven by their membrane potentials (‘electromotility’). This organ of hearing) and vestibule (comprising organs responsible unique amplification is thought to significantly increase the for balance). Within the cochlea are two distinct receptor cell sensitivity and frequency selectivity of the cochlea (1). types: the inner hair cells (IHCs) and the outer hair cells (OHCs). A combination of subtractive PCR hybridization and IHCs are thought to be the true sensory receptors: they transduce differential screening from gerbil OHCs has recently identified vibration and are the source of essentially all auditory signals a protein, prestin, whose cellular distribution, subcellular *To whom correspondence should be addressed at: Department of Otolaryngology (D-48), University Of Miami, 1666 NW 12th Avenue, Miami, FL 33136, USA. Tel: þ1 3052435695; Fax: þ1 3052434925; Email: [email protected] Human Molecular Genetics, Vol. 12, No. 10 # Oxford University Press 2003; all rights reserved 1156 Human Molecular Genetics, 2003, Vol. 12, No. 10 localization and functional properties indicate that it is the Table 1. Primers used for RACE analysis of prestin electromotility motor of cochlear OHCs (2,3). Prestin is a isoforms member of a newly characterized gene family, solute carrier (SLC) family 26, which encodes anion-transporter-related Primers Sequences proteins. Like other members in this family, prestin has a Hu-pres1f AGTGACACTCAGGAAATGCTTGTC highly hydrophobic core of predicted 12 transmembrane Hu-pres2f AGGGGATAACGTCCACACTGTCAT Hu-pres3f TGTGGCCATATATCTCACAGAGCC domains with the N- and C-terminal located cytoplasmically Hu-pres4f CTCTTCCACCTTGTGTACCGTAGA (3). Although prestin has not been shown to have any anion Hu-pres5f TTTGAATCATTGCCCCAGGCTGTG transport capability, it does appear to share the overall structure Hu-pres6f AAAGACTGGAGTGAACCCAGCAGT and specific protein domains of the anion transporter family Hu-pres8f GACTTGGTCTCAGGCATAAGCACA SLC26 such as having a conserved sulfate transport motif and Hu-pres1r GGAGTACATGTGAATGCCTGTTTC Hu-pres2r TGAATTTGTATGCTGGCAGCCATT STAS (sulfate transporters and antisigma factor antagonists) Hu-pres3r TGTGCTTATGCCTGAGACCAAGTC domain. The nine family members cloned thus far in humans, Hu-pres4r AGTTGGCTGCCTAAAACTGCATCA SLC26A1-9, transport chloride, iodine, bicarbonate, oxalate Hu-pres6r CAGAGGCTCTGTGAGATATATGGC and hydroxyl anions, with different specificities (4). Mutations Hu-pres7r CCTAGAGGAAGTGTTCCAACGACA Hu-pres8r CACAGCCTGGGGCAATGATTCAAA in the SLC26A2, SLC26A3 and SLC26A4 genes are Hu-pres9r ACTGCTGGGTTCACTCCAGTCTTT responsible for three distinct recessive disorders: diastrophic Hu-pres17rev AGCTGGCATTCAAACCCTGTCCAC dysplasia, congenital chloride diarrhea and Pendred syndrome, Hu-pres18rev TAAGTGACTTGGCTGAGGTCACCC respectively (5). SLC26A4 encodes a chloride-iodide transport protein expressed in the thyroid, kidney and inner ear. Its different mutations can lead to either syndromic deafness (Pendred syndrome) (6) or non-syndromic recessive deafness from nested PCR with human ear cDNA, with the first round (DFNB4) (7). using primer pair Hu-pres1f/30 primer (Invitrogen) and the Dramatic progress has been made in the identification of more second round with Hu-pres3f/30-nest (Invitrogen). Both 50 and than 80 gene loci that can cause non-syndromic deafness, 30 RACE PCR products were gel purified and sequenced. The the most common form of genetic hearing loss (http:// sequencing data from overlapping PCR products was dnalab-www.uia.ac.be/dnalab/hhh/). Additional loci for non- assembled using Sequencher V4.0 (GeneCodes Corp., Ann syndromic deafness exist, but the lack of suitable families Arbor, MI, USA). for linkage analysis has made localization difficult (8). More- The single 50 RACE fragment indicated that there are no over, only nearly 30 of these genes have been cloned to date alternative splicing variants at the 50 portion of prestin, which (http://dnalab-www.uia.ac.be/dnalab/hhh/), owing to the absence was confirmed by the identical sequences of this segment from of distinguishing clinical signs in deaf individuals, the hetero- human ear cDNAs. Sequencing the four bands of 30 RACE geneity of genetic deafness and small size of affected human derived from primer pair Hu-pres3f/30-nest showed that they families. With the difficulty of positional cloning, a candidate were alternative splicing variants which differed at their 30 gene approach—based on the identification of genes uniquely portion. The assembly of the RT–PCR, 50- and 30-RACE or preferentially expressed in the inner ear—may produce products revealed four prestin cDNAs which were 2671, 2492, additional human deafness genes more efficiently (9–12). 1960 and 1416 bp in length and encoded predicted ORFs of Clearly, the unique function of prestin and its specific expres- 744, 685, 516 and 335 amino acids. We have designated them sion in OHCs make this gene an excellent candidate for a SLC26A5a, SLC26A5b, SLC26A5c and SLC26A5d, respec- human non-syndromic deafness. In the present study, we tively. SLC26A5b, SLC26A5c and SLC26A5d all share the same report the cloning and characterization of multiple isoforms terminal 30 exon, but differ in their intervening cDNA of human prestin gene and its genomic structure. Significantly, sequences (Fig. 1). SLC26A5a and SLC26A5b share the we identify a splicing junction mutation in the prestin gene, majority of the sequence, and differ only at the terminal 30 which leads to non-syndromic hearing loss. exon (see genomic structure, Fig. 1). SLC26A5a–c contain the complete set of predicted transmembrane domains, whereas SLC26A5d has only seven of the 12 predicted transmembrane RESULTS domains. All four prestin isoforms preserve the sulfate transport motif (seconds transmembrane domain), but not Cloning of human prestin gene and its splicing variants the STAS domain (sulfate transporters and antisigma-factor antagonists). A consensus polyadenylation signal (AATAAA) Standard RT–PCR amplified most of the coding region of the was present in the 30-UTR of SLC26A5b, SLC26A5c and human prestin gene from human fetal ear (at 10, 20 and 22 SLC26A5d, but not in the 30-UTR of SLC26A5a. weeks’ gestation) cDNA pools, using the oligonucleotides To further characterize the origin and distribution of the derived from sequence comparisons between the mouse prestin isoforms, RT–PCR was performed using human fetal prestin