Human Cochlear Expressed Sequence Tags Provide Insight into Cochlear Gene Expression and Identify Candidate Genes for Deafness

To identify candidate genes for human hearing dis-orders and to understand better human hearing at themolecular level, we constructed a human cochlearcDNA library. An aliquot of the unsubtracted cochlearlibrary was contributed to the IMAGE Consortium atLawrence Livermore National Laboratory for the gen-eration of expressed sequence tags (ESTs) by theMerck/WashU EST project. Over 4000 ESTs were devel-oped from the cochlear cDNA library and deposited inthe GenBank EST database. Sequence clusteringshows that the majority of clones are in low copynumbers, demonstrating the high complexity of the li-brary. The sequences of 1388 cochlear ESTs (33%)match 517 known human genes. Among these aregenes previously shown to cause both syndromic andnon-syndromic hearing loss. A number of the cochlearESTs show high homology to non-human genes,suggesting new gene family members or human ho-mologs of animal genes. We also report the chromoso-mal map positions of 437 cochlear ESTs. These pro-vide positional candidate genes for 18 different non-syndromic hearing disorders. A Human Cochlear ESTDatabase web site (http://www.bwh.partners.org/pathology ) has been created to provide access to thecochlear clone data for gene discovery investigations.INTRODUCTIONHearing loss is our most common sensory disorder. One in 1000children is born deaf, and an equal number lose their hearing byadulthood (1). Additionally, half the population experiencesignificant hearing impairment by age 65 years ( 1). Approximate-ly 50% of deafness has a genetic etiology with autosomaldominant, autosomal recessive, X-linked or mitochondrial pat-terns of inheritance. Hundreds of syndromes have been identifiedin which hearing loss is a component (2); however, the majorityof hearing loss is non-syndromic (i.e. associated with no otherclinical findings).Rapid progress is underway in studies of hearing and deafnessat the molecular level. To date, >40 chromosomal loci containinggenes involved in non-syndromic hearing loss (NSHL) have beenidentified by linkage analysis. Loci are designatedDFNA1–DFNA15 for autosomal dominant forms of NSHL,DFNB1–DFNB20 for autosomal recessive NSHL, andDFN2–DFN8 for the X-linked forms of NSHL (3). Thechromosomal locus for each disorder, linked markers andappropriate references can be found on the Hereditary HearingLoss Home Page (4).Within the past year, genes responsible for several humanhearing disorders have been identified: the human homolog of theDrosophila diaphanous gene (DIAPH) in DFNA1 (5), the gapjunction protein connexin 26 (GJB2) in DFNB1 (6,7) andDFNA3 ( 8), the POU4F3 transcription factor in DFNA15 ( 9), theputative sulfate transporter PDS in both Pendred syndrome andDFNB4 (10,11), the tectorial membrane protein α-tectorin(TECTA) in DFNA8/12 ( 12), the unconventional myosin MYO15in human DFNB3 and mouse shaker-2 (4,13), a gene (USH2A)with homologies to laminin epidermal growth factor andfibronectin in Usher syndrome type IIa (15), the novel cochleargene COCH in DFNA9 ( 16) and a gene with very little homologyto any known protein, DFNA5 ( 17 ). Each of these genes has beenshown to be expressed in the cochlea, demonstrating howknowledge of gene expression in the membranous labyrinth iscritical to our further understanding of hearing and deafness.Despite these recent successes in discovering hearing lossgenes, the vast majority of NSHL genes remain to be identified.This is partially due to the fact that many families in which NSHLsegregates are small, with an insufficient number of informativerecombination events to allow narrowing of the genetic intervalto which an NSHL gene maps. Thus, in many cases, severalmegabases of genomic DNA must be analyzed to identifycandidate genes for each of the NSHL loci.Analysis of expressed sequence tags (ESTs) has proven usefulrecently in identifying positional candidate genes for humandisease. ESTs provide short nucleotide sequences that act as