Skip Navigation

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (49)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Skvorak, A. B.
Right arrow Articles by Morton, C. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Skvorak, A. B.
Right arrow Articles by Morton, C. C.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Human Molecular Genetics Pages 439-452  

Human cochlear expressed sequence tags provide insight into cochlear gene expression and identify candidate genes for deafness
Introduction
Results
   Cochlear EST clustering
   Known human genes and hearing disorder genes detected among the cochlear ESTs
   Mapped cochlear ESTs for positional candidate genes
Discussion
   Known genes expressed in cochlea
   Mapped cochlear ESTs
Materials And Methods
Acknowledgements
References

Human cochlear expressed sequence tags provide insight into cochlear gene expression and identify candidate genes for deafness

Human cochlear expressed sequence tags provide insight into cochlear gene expression and identify candidate genes for deafness

Anne B. Skvorak1,3, Zhiping Weng4, Andrew J. Yee3, Nahid G. Robertson1 and Cynthia C. Morton1,2,3,*

Departments of 1Pathology and 2Obstetrics, Gynecology and Reproductive Biology, Brigham and Women’s Hospital, 75 Francis Street, 3Harvard Medical School and 4Department of Biomedical Engineering, Boston University, Boston, MA 02215, USA

Received September 15, 1998; Revised and Accepted November 30, 1998

To identify candidate genes for human hearing disorders and to understand better human hearing at the molecular level, we constructed a human cochlear cDNA library. An aliquot of the unsubtracted cochlear library was contributed to the IMAGE Consortium at Lawrence Livermore National Laboratory for the generation of expressed sequence tags (ESTs) by the Merck/WashU EST project. Over 4000 ESTs were developed from the cochlear cDNA library and deposited in the GenBank EST database. Sequence clustering shows that the majority of clones are in low copy numbers, demonstrating the high complexity of the library. The sequences of 1388 cochlear ESTs (33%) match 517 known human genes. Among these are genes previously shown to cause both syndromic and non-syndromic hearing loss. A number of the cochlear ESTs show high homology to non-human genes, suggesting new gene family members or human homologs of animal genes. We also report the chromosomal map positions of 437 cochlear ESTs. These provide positional candidate genes for 18 different non-syndromic hearing disorders. A Human Cochlear EST Database web site (http://www.bwh.partners.org/pathology ) has been created to provide access to the cochlear clone data for gene discovery investigations.

INTRODUCTION

Hearing loss is our most common sensory disorder. One in 1000 children is born deaf, and an equal number lose their hearing by adulthood (1). Additionally, half the population experience significant hearing impairment by age 65 years (1). Approximately 50% of deafness has a genetic etiology with autosomal dominant, autosomal recessive, X-linked or mitochondrial patterns of inheritance. Hundreds of syndromes have been identified in which hearing loss is a component (2); however, the majority of hearing loss is non-syndromic (i.e. associated with no other clinical findings).

Rapid progress is underway in studies of hearing and deafness at the molecular level. To date, >40 chromosomal loci containing genes involved in non-syndromic hearing loss (NSHL) have been identified by linkage analysis. Loci are designated DFNA1-DFNA15 for autosomal dominant forms of NSHL, DFNB1-DFNB20 for autosomal recessive NSHL, and DFN2-DFN8 for the X-linked forms of NSHL (3). The chromosomal locus for each disorder, linked markers and appropriate references can be found on the Hereditary Hearing Loss Home Page (4).

Within the past year, genes responsible for several human hearing disorders have been identified: the human homolog of the Drosophila diaphanous gene (DIAPH) in DFNA1 (5), the gap junction protein connexin 26 (GJB2) in DFNB1 (6,7) and DFNA3 (8), the POU4F3 transcription factor in DFNA15 (9), the putative sulfate transporter PDS in both Pendred syndrome and DFNB4 (10,11), the tectorial membrane protein [alpha]-tectorin (TECTA) in DFNA8/12 (12), the unconventional myosin MYO15 in human DFNB3 and mouse shaker-2 (4,13), a gene (USH2A) with homologies to laminin epidermal growth factor and fibronectin in Usher syndrome type IIa (15), the novel cochlear gene COCH in DFNA9 (16) and a gene with very little homology to any known protein, DFNA5 (17). Each of these genes has been shown to be expressed in the cochlea, demonstrating how knowledge of gene expression in the membranous labyrinth is critical to our further understanding of hearing and deafness.

Despite these recent successes in discovering hearing loss genes, the vast majority of NSHL genes remain to be identified. This is partially due to the fact that many families in which NSHL segregates are small, with an insufficient number of informative recombination events to allow narrowing of the genetic interval to which an NSHL gene maps. Thus, in many cases, several megabases of genomic DNA must be analyzed to identify candidate genes for each of the NSHL loci.

Analysis of expressed sequence tags (ESTs) has proven useful recently in identifying positional candidate genes for human disease. ESTs provide short nucleotide sequences that act as unique identifiers of both novel and known genes, and can be used as probes to clone genes from appropriate cDNA libraries. As of December 1997, 91% of positionally cloned genes mutated in human diseases were represented by exact sequence matches to ESTs in the EST division of GenBank (http://www.ncbi.nlm.nih.gov/Bassett/dbEST/PosiClonNew.html ), indicating that the vast majority of disease genes are already present within the GenBank EST collection. A large-scale effort to map ESTs by use of radiation hybrid panels is being carried out by genome centers around the world. Currently (December 31, 1998), >49 000 human sequence-tagged sites (STSs), many of them derived from ESTs, have been assigned a chromosomal locus (http://www.ncbi.nlm.nih.gov/dbSTS ). Map positions of ESTs from dozens of cDNA libraries are a crucial resource for determining positional candidates for disease genes.

To identify candidate genes for human hearing disorders and to gain a more fundamental understanding of human hearing at the molecular level, we constructed a human cochlear cDNA library (18). The cochlear library is a resource for researchers studying human hearing and deafness, and facilitates identification of genes expressed in the human membranous labyrinth. An aliquot of the unsubtracted cochlear library was contributed to the IMAGE Consortium at Lawrence Livermore National Laboratory for gridding, and then sequencing by the Washington University Genome Sequencing Center. Over 4000 ESTs were created from the cochlear cDNA library and deposited in the GenBank EST database. All of the cochlear clones and the gridded microtiter plates are commercially available.

This report describes our analysis of the human cochlear ESTs. We present a listing of the known human genes represented by ESTs derived from the cochlear library. Mutations in several of these have already been demonstrated as the cause of both syndromic and non-syndromic hearing disorders. We also report the chromosomal map positions of 437 cochlear ESTs. This information identifies positional candidate genes for many hearing disorders mapped by linkage analysis. In addition, a listing of cochlear ESTs that have significant sequence homology to genes of non-human organisms suggests the possibility of new gene family members, or human homologs of animal genes. A complete listing of the data abstracted here as well as any future additions and updates can be found on the Human Cochlear EST Database website (19).

RESULTS

A total of 4304 human cochlear ESTs were generated from the unsubtracted, non-normalized Morton fetal cochlear cDNA library by the IMAGE Consortium and deposited in GenBank. Sequences were generated from 3373 independent clones, with 1207 5[prime] reads and 3097 3[prime] reads. Of the total cochlear ESTs, nucleotide sequences from 110 were either too short or of insufficient sequence complexity (i.e. contained excessive repetitive elements) to yield useful data for this study. Thus, 4194 cochlear ESTs (3263 clones) were of adequate length and complexity for further analysis (Table 1).


Figure 1. Northern blot of panel of human fetal RNAs, probed with cochlear cDNA clone from a ‘unique’ cluster, demonstrating high expression in cochlea and validating the ‘clustering’ technique. Ten micrograms of total RNA from each of the tissues listed was loaded in each lane. A longer exposure shows slight expression in testis and kidney. The transcript is ~3.5 kb in size.

Each of the 4194 cochlear ESTs was compared by WU-BLAST 2.0 analysis [Washington University BLAST (20,21)] with GenBank release 105.0 to identify ESTs derived from known human genes. The sequence of 1388 cochlear ESTs (33%) match 517 known human genes (Table 1). Of the remaining 2806 ESTs, BLAST analysis shows that 2265 (54% of the total) have significant matches to ESTs from other cDNA libraries. The remaining 541 ESTs (13% of the total) show no significant sequence homology to any nucleotide sequence deposited in GenBank to date, suggesting that they may be unique to the cochlear library, and may represent genes that are expressed exclusively in the human membranous labyrinth.

Table 1. Summary of human cochlear ESTs
  n (%)
Total ESTs generated from 3373 independent cochlear clones 4304
ESTs selected for further study 4194 (97)
Cochlear ESTs matching known human genes 1388 (33)
Cochlear ESTs matching other ESTs in GenBank 2265 (54)
ESTs unique to the cochlea 541 (13)

Cochlear EST clustering

‘Clustering’ cochlear clones, by grouping those with overlapping DNA sequence and presuming that the transcripts derive from the same gene, yields information on the abundance of a transcript and the complexity of the library. Of the 3263 independent clones that have had 5[prime], 3[prime] or both ends sequenced, 1137 (35%) are ‘singletons’: the sequence does not match the sequence of any other clone in the library. Clones for which both ends were sequenced are considered singletons if neither end matches any other sequence in the library other than its opposite end. The remaining 2126 clones fall into 414 clusters, the majority of which (80%) consist of only two or three clones. Only four clusters consist of 20 or more clones. The large percentage of singletons demonstrates the high complexity of the library, with many low copy number clones present, and suggests that sequencing of additional cochlear ESTs would identify other novel sequences.

Because the cochlear library was not subtracted or normalized in any way, the number of clones derived from an individual gene approximates the expression level of that gene in the membranous labyrinth. The gene most frequently found among the cochlear clones was collagen type I [alpha]2 (COL1A2). Eighty six (2.6%) of the cochlear clones represented COL1A2. The next most frequently expressed genes were COL3A1 (38 clones), translation elongation factor 1[alpha] (30 clones) and osteonectin/SPARC (16 clones).

Table 2. Known human genes among cochlear ESTs

Three of these four genes, COL1A2, COL3A1 and SPARC, were identified previously by subtractive hybridization and differential screening to be among the genes most highly expressed in human cochlea (18), confirming that EST sequencing and clustering yield results similar to alternative methods of gene expression analysis. Other genes represented by 10 or more cochlear clones include translationally controlled tumor protein, vimentin, COL1A1, myelin proteolipid protein and osteopontin.

Clustering just the clones that are ‘unique’ to cochlea, we found that the largest cluster consists of six independent clones, suggesting that this ‘cochlea-specific’ gene may be relatively highly expressed in human membranous labyrinth. To confirm this, one of the cochlear cDNAs in this cluster was used to probe a northern blot of human tissues (Fig. 1). This cochlear clone is indeed highly expressed in human cochlea (lane 3), and not at all or at comparatively low levels in other tissues. Overexposure of the blot in Figure 1 shows very low level expression in testis and kidney, and no expression in any other tissue. This demonstrates that transcription of this gene is not ‘unique’ to cochlea, but much more prominent in this organ compared with others.

Known human genes and hearing disorder genes detected among the cochlear ESTs

The known human genes that are identical to at least one cochlear EST are listed in Table 2, grouped into functional categories. GenBank accession numbers for the cochlear ESTs, the accession numbers of the corresponding known genes and their map locations, if determined, can be found on the Human Cochlear EST Database website (19).

Several of the cochlear ESTs match genes that have been shown previously to be mutated in both syndromic and non-syndromic human hearing disorders. An example of such an EST is that for GJB2 (connexin 26). Mutations in GJB2 were identified recently as the cause of the most common form of autosomal recessive NSHL, DFNB1 (6,7,22-24), and the dominant hearing loss DFNA3 (6,8), although there remains some controversy over the autosomal dominant forms. It has been speculated that GJB2 mutations may account for 20% of all cases of childhood deafness in the populations studied (7,25).

Ten different collagen genes are represented among the cochlear ESTs including COL1A1, COL1A2, COL2A1, COL3A1, COL4A1, COL4A2, COL5A1, COL6A1, COL9A3 and COL11A1. Mutations in four are pathogenic in syndromes in which hearing loss is a component. COL1A1 and COL1A2 mutations are responsible for osteogenesis imperfecta (OI) type 1, a disorder chiefly characterized by multiple bone fractures. About half of OI patients have conductive hearing loss that begins in the second decade (26). COL2A1 (27) and COL11A1 (28) mutations cause Stickler syndrome. The progressive pathobiology of this autosomal dominant disorder includes myopia, retinal detachment, joint and bone involvement and sensorineural hearing loss.

Branchio-oto-renal (BOR) syndrome is an autosomal dominant disorder with renal anomalies, hearing loss, pre-auricular pits and branchial clefts. The disease-causing gene is EYA1, the human homolog of the Drosophila eyes absent gene (29). EYA1 must play a critical developmental role in the inner ear and kidney.

The autosomal dominant hearing loss and vestibular defects in DFNA9 are caused by mutations in the novel gene COCH (16). COCH is highly expressed in human cochlea and vestibule, and is likely to be a secreted protein. Mutations in COCH result in acidophilic deposits in cochlear and vestibular labyrinths.

Mapped cochlear ESTs for positional candidate genes

Of the 40 non-syndromic hearing disorders that have been mapped by linkage analysis, the responsible gene has been identified in only 10 (4). The remaining disorders have been localized to various genetic intervals, but the gene causing hearing loss remains to be identified. Factors hindering efforts to identify these genes are large chromosomal regions and a lack of candidate genes. Because many of the hearing loss loci were mapped in small kindreds with few informative recombination events to narrow the genetic interval of the disease locus, hearing loss loci are assigned frequently to intervals spanning several centiMorgans, which may encompass hundreds of genes. Indeed, DFNB15 maps to two chromosomal loci, 3q and 19p, with equal LOD scores (30).

To identify candidate genes for hearing loss loci, each of the cochlear ESTs that did not match a known human gene was BLASTed against the database of sequence tagged sites (dbSTS). Genome centers including the Whitehead Institute Center for Genome Research (http://www.genome.wi.mit.edu/ ), the Sanger Center (http://www.sanger.ac.uk/ ) and the Stanford Human Genome Center (http://www-shgc.stanford.edu/ ) are using radiation hybrid mapping to develop STSs from ESTs and place them on genetic framework maps. When a cochlear EST sequence significantly matched an STS sequence, the cochlear transcript was considered ‘mapped’. A total of 872 cochlear ESTs match STSs in dbSTS. These represent 437 independent chromosomal loci. A list of the cochlear EST map positions is provided in Table 2 and more detailed information regarding the map positions is given on the website (19). The cochlear ESTs map to every chromosome except Y, and are fairly evenly distributed throughout the genome. Thirty five of the cochlear ESTs whose sequence matches no other nucleotide sequences in GenBank were made into STSs and mapped by the Whitehead Institute Center for Genome Research using the GeneBridge 4 radiation hybrid panel. These ESTs are underlined in Table 3.

Among cochlear ESTs that match STSs, 57 map to the intervals of 18 NSHL loci and four Usher syndrome subtypes (Table 3), providing immediate positional candidates for these disorders. These 22 disorders are DFNA2, -4, -7, -10, -13 and -18, DFNB5, -6, -7, -8, -12, -13, -15, -16, -17 and -19, DFN2 and -6, and USH1D, -1E, -1F and -3.

Table 3. Chromosomal map positions of cochlear ESTs and deafness loci
Underlined numbers indicate ESTs that are ‘unique’ to the cochlear library (i.e. the nucleotide sequence of the cochlear EST has not been deposited in any of the GenBank databases to date from any other resource). The second column is the approximate map position in centiMorgans (cM) of the EST. The third column lists any hearing loss loci that have been mapped by linkage analysis to the same chromosomal segment. More complete information, including STS accession numbers and map positions of hearing loss loci, can be found in the Human Cochlear EST Database (19).

Cochlear ESTs with homology to non-human genes

Two human homologs of genes from other species were shown recently to cause human hearing loss. Mutations in the human homolog of the Drosophila diaphanous gene are etiologic in DFNA1 (5), and mutations in human TECTA, previously identified in the mouse, result in DFNA8/DFNA12 (12). Seventy four of the cochlear ESTs have the highest sequence homology to 41 non-human genes (Table 4). These clones may identify novel human homologs of animal genes. The ESTs match genes previously identified in mouse, rat, cow, guinea pig, pig, dog, rabbit and yeast, with sequence identity ranging from 78 to 98%. Among these ESTs are transcription factors, signal transduction proteins, trafficking proteins and extracellular matrix-type proteins.

Table 4. Non-human genes with significant homology to human cochlear ESTs
Cochlear EST Gene namea GenBank Species % Identityb
accession no.   accession no.
N63142 [beta]-1,4-galactosyltransferase D37790 mouse 85
H89125 E25 (integral membrane protein) L38971 mouse 86
H88505 fat facets homolog U67874 mouse 81
H88006 FT1 (fused toes) Z67963 mouse 89
N66426 helix-loop-helix transcription factor M97636 mouse 95
H89345 interleukin 10 M84340 mouse 90
N67138 mrg1 Y15163 mouse 98
H88648 PG-M core protein D45889 mouse 78
N67393 quaking type 1 U44940 mouse 96
N66759 ras-related YPT1 Y00094 mouse 96
N71911 SCID complementing gene 2 D78188 mouse 94
N66904 serum inducible kinase M96163 mouse 87
N22583 spindlin U48972 mouse 92
N22165 Sycp3 gene Y08486 mouse 86
N66880 tetracycline transporter-like protein D88315 mouse 96
N67149 transcription factor PEBP2a1 D14636 mouse 92
N22163 ubiquitin-conjugating enzyme M3 X92665 mouse 84
N71196 X16 (DNA-binding protein) X53824 mouse 89
H88563 14-3-3 protein [gamma] subtype S55305 rat 89
H88637 acidic calponin U06755 rat 78
N66965 clathrin heavy chain J03583 rat 93
N64047 collagen type XII [alpha]1 U57362 rat 91
H88970 DNA-binding protein URE-B1 U08214 rat 90
H88289 dynein light intermediate chain LIC2 U15138 rat 87
N69774 glycoprotein 65 X99338 rat 86
N66389 guanine expression factor 2 (GEF2) AB003515 rat 85
H88361 phospholipase C-[beta]1b L14323 rat 89
N75864 Rap1B U07795 rat 95
H89079 SCIP (transcriptional repressor) M72711 rat 88
N21993 transcription factor Maf1 U56241 rat 90
N22596 zinc finger protein L03386 rat 97
H88072 [gamma] COP X70019 cow 85
N63362 guanine nucleotide exchange protein ARF-GEP AF023451 cow 97
N64163 NADH ubiquinone oxoreductase complex B15 X64898 cow 82
H89271 unr (upstream of N-ras) X71978 guinea pig 88
N75887 zinc finger protein L26335 guinea pig 92
H89106 non-histone protein HMG1 M21683 pig 90
H88060 succinyl-CoA synthetase [alpha] subunit AF008589 pig 81
N63219 non-erythroid [beta]-spectrin L02897 dog 95
N66989 mannosyl-oligosaccharide [alpha]1,2 mannosidase U04301 rabbit 85
N22012 MALR X15241 yeast 98
aGene which the cochlear sequence most closely matches.
bPercentage identity of the cochlear EST sequence to the gene sequence in the aligned region.

Several genes could prove particularly interesting in terms of human hearing and hearing disorders. For example, EST N64047 may represent a novel collagen because it is highly similar to rat collagen type 12 [alpha]1. At least 10 collagens are expressed in the human membranous labyrinth, and mutations in several contribute to hearing loss as described above. EST H89079 has highest homology to the rat transcription factor SCIP. SCIP is a POU domain protein expressed during Schwann cell differentiation (31). Two POU domain genes have been shown to be involved in human hearing loss: POU3F4 in X-linked mixed deafness (32) and POU4F3 in DFNA15 (9). In addition, homozygous deletion of Pou4f3 (Brn3c) also causes hearing loss and vestibular defects in mice (33).

DISCUSSION

This study describes a reverse molecular genetic approach to identify genes involved in human cochlear anatomy and physiology. Investigation of the mammalian cochlea at the molecular level will be crucial to an understanding of the auditory system. Sequencing a large number of human cochlear cDNAs to create ESTs has identified known and novel genes that are expressed in this complex and delicate organ. Positional candidate genes for several non-syndromic hearing disorders have been identified.

Known genes expressed in cochlea

Thousands of genes are expected to be expressed in an organ as intricate as the human cochlea. Partial sequencing of >3300 cochlear-derived clones revealed >1500 gene clusters and 500 known genes. Not surprisingly, many of the genes are ‘housekeeping’ genes encoding proteins that are part of the metabolism or structure of every living cell. Also among the cochlear ESTs are genes that are probably ‘contaminants’ from tissue collection. For example, the serum/blood cell proteins such as hemoglobins and complement factors are more likely to originate from contaminating blood than from tissue of the membranous labyrinth. Likewise, not every gene that is actually expressed in the cochlea will be among the cochlear ESTs; only one histone gene was found and only a subset of the ribosomal proteins, reflecting the incomplete sequencing and the developmental timing of transcripts. Undoubtedly, generating additional cochlear ESTs would create an increasingly more accurate picture of gene expression in this organ.

Among the known genes represented by cochlear ESTs are several that are the cause of human hearing loss: GJB2, COL1A1, COL1A2, COL2A1, COL11A1, EYA1 and COCH. There are several other hearing loss genes that have been shown independently to be expressed in cochlea but are not in the cochlear EST collection. For instance, we have shown that DIAPH, POU4F3, MYO15 and USH2A, genes mutated in DFNA1, DFNA15, DFNB3 and Usher syndrome type 2A, respectively (5,9,13,15), are all expressed in the membranous labyrinth by RT-PCR of human cochlear mRNA. This may reflect the low level expression of these genes in the cochlea. Coupled with the gene clustering data, this argues strongly that continued creation of cochlear ESTs would be useful in identifying novel hearing loss genes.

Mapped cochlear ESTs

Fifty seven of the cochlear ESTs fall within the genetic interval of 17 different non-syndromic hearing disorders and four Usher syndrome subtypes (Table 4). Because STSs and ESTs are mapped in radiation hybrid panels relative to framework markers, locus assignment is generally within an interval of several centiMorgans. When a cochlear EST is denoted as a positional candidate for a hearing loss gene because it maps to a given interval, it is done so with this caveat of the localization. However, when faced with an interval for a hearing disorder spanning hundreds or thousands of kilobases, potentially containing hundreds of genes, an obvious first choice for careful consideration are genes and ESTs expressed in the cochlea. Indeed, our laboratory has shown recently that radiation hybrid mapping of a gene found among the cochlear ESTs, COCH, placed it wholly within the interval of the DFNA9 locus (34), and subsequent investigation revealed missense mutations in three DFNA9 kindreds (16). Thus, positional candidate cochlear ESTs are now a proven and successful approach to disease gene identification.

Clearly the majority of mapped cochlear ESTs (Table 4) are not within the genetic interval of a known human hearing disorder. Over 40 deafness loci have been identified to date, and certainly more will be determined as additional families are recruited and linkage mapping techniques are improved. Having cochlear ESTs already mapped to future hearing loss loci will provide a starting point for deafness gene discovery. In addition, there are many mouse mutants that have hearing and vestibular defects (35). Regions of conserved synteny between human and mouse allow cross-referencing between the genomes of these two organisms. Maps of human cochlear ESTs potentially could identify mouse deafness genes, providing invaluable model systems for studying human genetic hearing loss.

MATERIALS AND METHODS

Construction of the human fetal cochlear cDNA library in the UniZap vector (Stratagene) is described by Robertson et al. (18). The directionally cloned library was not subtracted or normalized for this analysis. Mass in vivo excision of the library was performed according to the manufacturer’s protocol to remove phage sequences before being donated to the IMAGE Consortium. Gridding of clones and DNA preparation and sequencing for generation of ESTs has been described (36,37).

Northern blots were performed as described (38) using 10 µg of total RNA extracted from each of 11 human fetal tissues.

To obtain the data described here, the nucleotide sequence of each cochlear EST was used to conduct a WU-BLAST 2.0 search (20,21) against release 105.0 of the GenBank primate database. A cochlear EST nucleotide sequence was considered to be a significant match to a gene sequence when P < 1E-35 and percent identity was >80% over the entire length of the EST. P is the smallest sum probability; the lower the P-value is, the more identical are the two sequences being compared. The majority of cochlear EST sequences were >95% identical to the query sequence, disregarding 1 bp frameshifts and ambiguous (N) nucleotides. When a BLAST search revealed a significant match to more than one known gene, only the highest scoring hit was included in the data set.

Cochlear ESTs that did not match a known gene subsequently were BLASTed against the GenBank EST, STS and non-human (i.e. rodent, insect, microbial, etc.) databases. A cochlear EST nucleotide sequence was considered to be a match to an EST, STS or non-human sequence when P < 1E-30, the aligned region was >50 nucleotides and the identity was >85% over the aligned region, or P < 1E-20, the aligned region was >50 nucleotides and the identity was >90% over the aligned region. Any cochlear ESTs that did not match human, non-human or EST sequences other than self in the GenBank databases were considered ‘cochlear specific’ or ‘unique’ to cochlea.

The 110 cochlear ESTs that were excluded from analysis in the data set had no significant matches to any of the GenBank databases and self-hit values greater than P = 1E-20. Visual inspection of these ESTs revealed either very short clone inserts (<100 bp of sequence), a large percentage of N nucleotides or a large degree of repetitive elements.

A cochlear EST was considered ‘mapped’ when the EST matched an STS with P < 1E-30, the aligned region was >50 nucleotides and the identity was >85% over the aligned region, or P < 1E-20, the aligned region was >50 nucleotides and the identity was >90% over the aligned region. In the majority of cases, the nucleotide sequence was a sequence match of 96% identity or better to an STS. Map positions listed for the cochlear ESTs are those for the corresponding STSs that can be found on genetic maps at the Whitehead Institute Center for Genome Research (http://www.genome.wi.mit.edu/ ), the Stanford Human Genome Center (http://www-shgc.stanford.edu/ ) or the NCBI human gene mapping web page (http://www.ncbi.nlm.nih.gov/genemap/ ). The STS corresponding to each cochlear EST can be found in the Human Cochlear EST Database (19). All map distances are reported as centiMorgans (cM) from the telomere of the p arm of each chromosome. When these data were not provided directly for an STS at one of the above web sites, the centiMorgan distance was estimated by the position of the STS relative to flanking framework markers. CentiMorgan assignment of deafness loci was determined in the same manner, using informative markers for each locus provided in the Hereditary Hearing Loss Homepage (4). An explanation of the methodology for radiation hybrid mapping can be found in Cox et al. (39).

ACKNOWLEDGEMENTS

We thank Drs Greg Lennon and LaDeanna Hillier for providing helpful information regarding data derived from gridding and sequencing the cochlear cDNA library. We thank Dr James Battey for helpful discussion. We thank Juan Small for assistance with gene map assignments. This work was supported by NIH grants DC00038 (to A.B.S.) and DC03402 (to C.C.M.).

REFERENCES

1. Morton, N.E. (1991) Genetic epidemiology of hearing impairment. Ann. NY Acad. Sci., 630, 16-31. MEDLINE Abstract

2. Online Mendelian Inheritence in Man (OMIM) (1997) http://www3.ncbi.nlm.nih.gov/omim/ . Center for Medical Genetics, Johns Hopkins University, Baltimore, MD, and National Center for Biotechnology Information, National Library of Medicine, Bethesda, MD.

3. Van Camp, G., Willems, P.J. and Smith, R.J.H. (1997) Nonsyndromic hearing impairment: unparalleled heterogeneity. Am. J. Hum. Genet., 60, 758-764. MEDLINE Abstract

4. Van Camp, G. and Smith, R.J.H. (1998) Hereditary Hearing Loss Homepage (http://dnalab-www.uia.ac.be/dnalab/hhh/ ).

5. Lynch, E.D., Lee, M.K., Morrow, J.E., Welcsh, P.L., Leon, P.E. and King, M.C. (1997) Nonsyndromic deafness DFNA1 associated with mutation of a human homolog of the Drosophila gene diaphanous. Science, 278, 1315-1318. MEDLINE Abstract

6. Kelsell, D.P., Dunlop, J., Stevens, H.P., Lench, N.J., Liang, J.N., Parry, G., Mueller, R.F. and Leigh, I.M. (1997) Connexin 26 mutations in hereditary nonsyndromic sensorineural deafness. Nature, 387, 80-83. MEDLINE Abstract

7. Kelley, P.M., Harris, D.J., Comer, B.C., Askew, J.W., Fowler, T., Smith, S.D. and Kimberling, W.J. (1998) Novel mutations in the connexin 26 gene (GJB2) that cause autosomal recessive (DFNB1) hearing loss. Am. J. Hum. Genet., 62, 792-799. MEDLINE Abstract

8. Denoyelle, F., Lina-Granade, G., Plauchu, H., Bruzzone, R., Chaib, H., Levi-Acobas, F., Weil, D. and Petit, C. (1998) Connexin 26 gene linked to a dominant deafness. Nature, 393, 319-320. MEDLINE Abstract

9. Vahava, O., Morrell, R., Lynch, E.D., Weiss, S., Kagan, M.E., Ahituv, N., Morrow, J.E., Lee, M.K., Skvorak, A.B., Morton, C.C., Blumenfeld, A., Frydman, M., Friedman, T.B., King, M.C. and Avraham, K.B. (1998) Mutations in transcription factor POU4F3 cause inherited progressive hearing loss in humans. Science, 279, 1950-1954. MEDLINE Abstract

10. Everett, L.A., Benjamin, G., Beck, J.C., Idol, J.R., Buchs, A., Heyman, M., Adawi, F., Hazani, E., Nassir, E., Baxevanis, A.D., Sheffield, V.C. and Green, E.D. (1997) Pendred syndrome is caused by mutations in a putative sulfate transporter gene (PDS). Nature Genet., 17, 411-422.

11. Li, X.C., Everett, L.A., Lalwani, A.K., Desmukh, D., Friedman, T.B., Green, E.D. and Wilcox, E.R. (1998) A mutation in PDS, the gene responsible for Pendred syndrome, also causes nonsyndromic recessive deafness. Nature Genet., 18, 215-217.

12. Verhoeven, K., Van Laer, L., Kirschhofer, K., Legan, P.K., Hughes, D.C., Schattman, I., Verstreken, M., Van Hauwe, P., Coucke, P., Chen, A., Smith, R.J.H., Somers, T., Officiers, F.E., Van de Heyning, P., Richardson, G.P., Wachtler, F., Kimberling, W.J., Willems, P.J., Govaerts, P.J. and Van Camp, G. (1998) Mutations in the human [alpha]-tectorin gene cause autosomal dominant nonsyndromic hearing impairment. Nature Genet., 19, 60-62. MEDLINE Abstract

13. Wang, A., Liang, Y., Friddell, R.A., Probst, F.J., Wilcox, E.R., Touchman, J.W., Morton, C.C., Morell, R.J., Noben-Trauth, K., Camper, S.A. and Friedman, T.B. (1998) Association of uncoventional myosin MYO15 mutations with human nonsyndromic deafness DFNB3. Science, 280, 1447-1451. MEDLINE Abstract

14. Probst, F.J., Fridell, R.A., Raphael, Y., Saunders, T.L., Wang, A., Liang, Y., Morell, R.J., Touchman, J.W., Lyons, R.H., Noben-Trauth, K., Friedman, T.B. and Camper, S.A. (1998) Correction of deafness in shaker-2 mice by an unconventional myosin in a BAC transgene. Science, 280, 1444-1447. MEDLINE Abstract

15. Eudy, J.D., Weston, M.D., Yao, S., Hoover, D.M., Rehm, H.L., Ma-Edmonds, M., Yan, D., Ahmad, I., Cheng, J.J., Ayuso, C., Cremers, C., Davenport, S., Moller, C., Talmadge, C.B., Beisel, K.W., Tamayo, M., Morton, C.C., Swaroop, A., Kimberling, W.J. and Sumegi, J. (1998) Mutation of a gene encoding a protein with extracellular matrix motifs in Usher syndrome type IIa. Science, 280, 1753-1757. MEDLINE Abstract

16. Robertson, N.G., Lu, L., Heller, S., Merchant, S.N., McKenna, M., Eavy, R.D., Nadol, J.B., Myamoto, R.T., Lubianca Neto, J.F., Hudspeth, A.J., Seidman, C.E., Morton, C.C. and Seidman, J.G. (1998) Mutations in a novel cochlear gene cause DFNA9, a human nonsyndromic deafness with vestibular dysfunction. Nature Genet., 20, 299-303.

17. Van Laer, L., Huizing, E.H., Verstreken, M., van Zuijlen, D., Wauters, J.G., Bossuyt, P.J., Van de Heyning, P., McGuirt, W.T., Smith, R.J.H., Willems, P.J., Legan, P.K., Richardson, G.P. and Van Camp, G. (1998) Nonsyndromic hearing impairment is associated with a mutation in DFNA5. Nature Genet., 20, 194-197.

18. Robertson, N.G., Khetarpal, U., Gutierrez-Espelata, G.A., Bieber, F.R. and Morton, C.C. (1994) Isolation of novel and known genes from a human fetal cochlear cDNA library using subtractive hybridization and differential screening. Genomics, 23, 42-50. MEDLINE Abstract

19. Skvorak, A.B. and Morton, C.C. (1998) The Human Cochlear EST Database (http://www.bwh.partners.org/pathology ).

20. Altschul, S.F. and Gish, W. (1996) Local alignment statistics. Methods Enzymol., 266, 460-480.

21. Karlin, S. and Altschul, S.F. (1993) Applications and statistics for multiple high-scoring segments in molecular sequences. Proc. Natl Acad. Sci. USA, 90, 5873-5877.

22. Denoyelle, F., Weil, D., Maw, M.A., Wilcox, S.A., Lench, N.J., Allen-Powell, D.R., Osborn, A.H. et al). (1997) Prelingual deafness: high prevalence of a 30delG mutation in the connexin 26 gene. Hum. Mol. Genet., 6, 2173-2177. MEDLINE Abstract

23. Carrasquillo, M.M., Zlotogora, J., Barges, S. and Chakravarti, A. (1997) Two different connexin 26 mutations in an inbred kindred segregating nonsyndromic recessive deafness: implications for genetic studies in isolated populations. Hum. Mol. Genet., 6, 2163-2172. MEDLINE Abstract

24. Zelante, L., Gasparini, P., Estivill, X., Melchionda, S., D'Agruma, L., Govea, N., Mila, M. et al). (1997) Connexin 26 mutations associated with the most common form of nonsyndromic neurosensory autosomal recessive deafness (DFNB1) in Mediterraneans. Hum. Mol. Genet., 6, 1605-1609. MEDLINE Abstract

25. Lench, N., Houseman, M., Newton, V., Van Camp, G. and Mueller, R. (1998) Connexin 26 mutations in sporadic nonsyndromic sensorineural deafness. Lancet, 351, 415. MEDLINE Abstract

26. Riedner, E.D., Levin, L.S. and Holliday, M.J. (1980) Hearing patterns in dominant osteogenesis imperfecta. Arch. Otolaryngol., 106, 737-740. MEDLINE Abstract

27. Knowlton, R.G., Weaver, E.J., Struyk, A.F., Knobloch, W.H., King, R.A., Norris, K., Shamban, A., Uitto, J., Jimenez, S.A. and Prockop, D.J. (1989) Genetic linkage analysis of hereditary arthro-ophthalmopathy (Stickler syndrome) and the type II procollagen gene. Am. J. Hum. Genet., 45, 681-688. MEDLINE Abstract

28. Richards, A.J., Yates, J.R.W., Williams, R., Payne, S.J., Pope, F.M., Scott, J.D. and Snead, M.P. (1996) A family with Stickler syndrome type 2 has a mutation in the COL11A1 gene resulting in the substitution of glycine 97 by valine in alpha-1(XI) collagen. Hum. Mol. Genet., 5, 1339-1343. MEDLINE Abstract

29. Abdelhak, S., Kalatzis, V., Heilig, R., Compain, S., Samson, D., Vincent, C., Weil, D., Cruaud, C., Sahly, I., Leibovici, M., Bitner-Glindzicz, M., Francis, M., Lacombe, D., Vigneron, J., Charachon, R., Boven, K., Bedbeder, P., Van Regemorter, N., Weissenbach, J. and Petit, C. (1997) A human homologue of the Drosophila eyes absent gene underlies branchio-oto-renal (BOR) syndrome and identifies a novel gene family. Nature Genet., 15, 157-164. MEDLINE Abstract

30. Chen, A., Wayne, S., Bell, A., Ramesh, A., Srisailapathy, C.R., Scott, D.A., Sheffield, V.C., Van Hauwe, P., Zbar, R.I., Ashley, J., Lovett, M., Van Camp, G. and Smith, R.J.H. (1997) New gene for autosomal recessive nonsyndromic hearing loss maps to either chromosome 3q or 19p. Am. J. Med. Genet., 71, 467-471. MEDLINE Abstract

31. Monuki, E.S., Kuhn, R., Weinmaster, G., Trapp, B.D. and Lemke, G. (1990) Expression and activity of the POU transcription factor SCIP. Science, 249, 1300-1303. MEDLINE Abstract

32. de Kok, Y.J.M., van der Maarel, S.M., Bitner-Glindzicz, M., Huber, I., Monaco, A.P., Malcolm, S., Pembrey, M.E., Ropers, H.H. and Cremers, F.P.M. (1995) Association between X-linked mixed deafness and mutation in the POU domain gene POU3F4. Science, 267, 685-688. MEDLINE Abstract

33. Xiang, M., Gan, L., Li, D., Chen, Z.Y., Zhou, L., O'Malley, B.W. Jr, Klein, W. and Nathans, J. (1997) Essential role of POU-domain factor Brn-3c in auditory and vestibular hair cell development. Proc. Natl Acad. Sci. USA, 94, 9445-9450. MEDLINE Abstract

34. Robertson, N.G., Skvorak, A.B., Yin, Y., Weremowicz, S., Johnson, K.R., Kovatch, K.A., Battey, J.F., Bieber, F.R. and Morton, C.C. (1997) Mapping and characterization of a novel cochlear gene in human and in mouse: a positional candidate gene for a deafness disorder, DFNA9. Genomics, 46, 345-354. MEDLINE Abstract

35. Mouse Genome Database (MGD) (1997) http://www.informatics.jax.org/ . The Jackson Laboratory, Bar Harbor, ME.

36. Lennon, G.G., Auffray, C., Polymeropoulos, M. and Soares, M.B. (1996) The I.M.A.G.E. Consortium: an integrated molecular analysis of genomes and their expression. Genomics, 33, 151-152. MEDLINE Abstract

37. Hillier, L., Lennon, G., Becker, M., Bonaldo, M.F., Chiapelli, B., Chissoe, S., Dietrich, N. et al). (1996) Generation and analysis of 280,000 human expresssed sequence tags. Genome Res., 6, 807-828. MEDLINE Abstract

38. Skvorak, A.B., Robertson, N.R., Yin, Y., Weremowicz, S., Lynch, E.D., Her, H., Beisel, K.W., Bieber, F.R., Beier, D.R. and Morton, C.C. (1997) Identification of an ancient conserved gene expressed in the human inner ear: chromosomal mapping of human and mouse antiquitin and analysis of expression. Genomics, 46, 191-199. MEDLINE Abstract

39. Cox, D.R., Burmeister, M., Price, E.R., Kim, S. and Myers, R.M. (1990) Radiation hybrid mapping: a somatic cell genetic method for construction of high resolution maps of mammalian chromosomes. Science, 250, 245-250. MEDLINE Abstract

*To whom correspondence should be addressed. Tel: +1 617 732 7980; Fax: +1 617 738 6996; Email: ccmorton@bics.bwh.harvard.edu

This page is run by Oxford University Press, Great Clarendon Street, Oxford OX2 6DP, as part of the OUP Journals
Comments and feedback: www-admin{at}oup.co.uk
Last modification: 6 Feb 1999
Copyright©Oxford University Press, 1999.
Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Hum Mol GenetHome page
N. G. Robertson, C. W.R.J. Cremers, P. L.M. Huygen, T. Ikezono, B. Krastins, H. Kremer, S. F. Kuo, M. C. Liberman, S. N. Merchant, C. E. Miller, et al.
Cochlin immunostaining of inner ear pathologic deposits and proteomic analysis in DFNA9 deafness and vestibular dysfunction
Hum. Mol. Genet., April 1, 2006; 15(7): 1071 - 1085.
[Abstract] [Full Text] [PDF]

Home page
 Physiol. GenomicsHome page
K. W. Choy, C. C. Wang, A. Ogura, T. K. Lau, M. S. Rogers, K. Ikeo, T. Gojobori, D. S. C. Lam, and C. P. Pang
Genomic annotation of 15,809 ESTs identified from pooled early gestation human eyes
Physiol Genomics, March 13, 2006; 25(1): 9 - 15.
[Abstract] [Full Text] [PDF]

Home page
 Hum Mol GenetHome page
L. M. Peters, D. W. Anderson, A. J. Griffith, K. M. Grundfast, T. B. San Agustin, A. C. Madeo, T. B. Friedman, and R. J. Morell
Mutation of a transcription factor, TFCP2L3, causes progressive autosomal dominant hearing loss, DFNA28
Hum. Mol. Genet., November 1, 2002; 11(23): 2877 - 2885.
[Abstract] [Full Text] [PDF]

Home page
 J. Physiol.Home page
S. Heller
Application of physiological genomics to the study of hearing disorders
J. Physiol., August 15, 2002; 543(1): 3 - 12.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
X. Li, R. Li, X. Lin, and M.-X. Guan
Isolation and Characterization of the Putative Nuclear Modifier Gene MTO1 Involved in the Pathogenesis of Deafness-associated Mitochondrial 12 S rRNA A1555G Mutation
J. Biol. Chem., July 19, 2002; 277(30): 27256 - 27264.
[Abstract] [Full Text] [PDF]

Home page
 Hum Mol GenetHome page
C. C. Morton
Genetics, genomics and gene discovery in the auditory system
Hum. Mol. Genet., May 15, 2002; 11(10): 1229 - 1240.
[Abstract] [Full Text] [PDF]

Home page
 IOVSHome page
M. Buraczynska, A. J. Mears, S. Zareparsi, R. Farjo, E. Filippova, Y. Yuan, S. P. MacNee, B. Hughes, and A. Swaroop
Gene Expression Profile of Native Human Retinal Pigment Epithelium
Invest. Ophthalmol. Vis. Sci., March 1, 2002; 43(3): 603 - 607.
[Abstract] [Full Text] [PDF]

Home page
 J. Clin. Endocrinol. Metab.Home page
L. Fugazzola, D. Mannavola, N. Cerutti, M. Maghnie, F. Pagella, P. Bianchi, G. Weber, L. Persani, and P. Beck-Peccoz
Molecular Analysis of the Pendred's Syndrome Gene and Magnetic Resonance Imaging Studies of the Inner Ear Are Essential for the Diagnosis of True Pendred's Syndrome
J. Clin. Endocrinol. Metab., July 1, 2000; 85(7): 2469 - 2475.
[Abstract] [Full Text]

Home page
 NEJMHome page
P. J. Willems
Genetic Causes of Hearing Loss
N. Engl. J. Med., April 13, 2000; 342(15): 1101 - 1109.
[Full Text] [PDF]

Home page
 Hum Mol GenetHome page
J.-M. Claverie
Computational methods for theidentification of differential and coordinated gene expression
Hum. Mol. Genet., September 1, 1999; 8(10): 1821 - 1832.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (49)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Skvorak, A. B.
Right arrow Articles by Morton, C. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Skvorak, A. B.
Right arrow Articles by Morton, C. C.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?