Human cochlear expressed sequence tags ... - Semantic Scholar

 1999 Oxford University Press

Human Molecular Genetics, 1999, Vol. 8, No. 3

439–452

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 nonsyndromic 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 α-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

*To whom correspondence should be addressed. Tel: +1 617 732 7980; Fax: +1 617 738 6996; Email: [email protected]

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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′ reads and 3097 3′ 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). 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

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.

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′, 3′ 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 α2 (COL1A2). Eighty six (2.6%) of the cochlear clones represented COL1A2. The next most frequently expressed genes were COL3A1 (38 clones), translation elongation factor 1α (30 clones) and osteonectin/SPARC (16 clones).

441 Human Genetics, 1999, 8, No. NucleicMolecular Acids Research, 1994, Vol. Vol. 22, No. 1 3 Table 2. Known human genes among cochlear ESTs Cell cycle/proliferation/survival

cytochrome c oxidase VIIc

apoptotic protease-activating factor 1

cytosolic selenium-dependent glutathione peroxidase

B cell lymphoma protein 2 (bcl2)

deoxyuridine nucleotidohydrolase

B cell translocating gene 1

diazapam-binding inhibitor

CDC10 homolog

dihydropyrimidinase-related protein 2

cyclin G

dioxin-inducible cytochrome P450

cyclin H

electron transfer flavoprotein β

death-associated protein 5

enoyl-CoA hydratase

defender against death 1 protein (DAD1)

esterase D

early growth response protein

glutamine PRPP amidotransferase

enhancer of rudimentary homolog

glutamine synthetase

growth inhibitor p33ING1

glycinamide ribonucleotide formyltransferase (GART)

Hs-cul-3

GPI transamidase

inhibitor of apoptosis protein 2

isoleucyl-tRNA synthetase

retinoblastoma-binding protein

L-arginine:glycine

wild-type activated p53 fragment 1 (WAF1)

lactate deyhydrogenase A

amidotransferase

Cell surface markers/cell adhesion

lecithin–cholesterol acyltransferase

cadherin-associated protein related (cap-r)

lipocorton III

e-cadherin

lysyl hydroxylase isoform 2

epithelial membrane protein CL-20

malate dehydrogenase

fibronectin receptor β

manganese superoxide dismutase (SOD2)

integrin α6

mitochondrial phosphate carrier

laminin receptor

mitochondrial ubiquinone- binding protein

laminin receptor homolog

NADH:ubiquinone oxoreductase MLRQ

MGC-24

Niemann–Pick C disease gene

MIC2

NRD1 convertase

MUC18

ornithine aminotransferase

osteopontin

OXA1

surface glycoprotein

peroxisomal 70 kDa membrane protein

thrombospondin 2

phospholipase A2

trophinin

phosphoribosylpyrophosphate synthetase-associated protein 39

Cellular metabolism

propionyl-CoA carboxylase α

17-β hydroxysteroid dehydrogenase

prostaglandin D synthase

acetolactate synthase homolog

pyruvate dehydrogenase α

aldehyde dehydrogenase

ribonucleotide reductase subunit M1

aldehyde reductase

seryl-tRNA synthetase

aryl sulfotransferase

spermidine aminopropyltransferase

aspartate aminotransferase

spermidine/spermine N1-acetyltransferase

ATP-dependent mitochondrial RNA helicase

thioredoxin

ATP synthase β-subunit

tyrosinase-related protein

ATP synthase γ-subunit

Channel proteins

ATP synthase subunit 9

connexin 26

ATPase coupling factor 6 subunit

porin

carbonic anhydrase II

sodium potassium ATPase β1

carbonic anhydrase III

sodium potassium ATPase α2

carboxypeptidase E

sodium potassium transporting ATPase β3

CDP-diacylglycerol synthase

vacuolar H+ ATPase

CMP-sialic acid transporter

voltage-dependent anion channel

cytochrome bc-1 complex core protein II

Cytoskeleton

cytochrome c oxidase subunit Vb

actin, β

cytochrome c oxidase subunit VIC

actin, γ

cytochrome c oxidase subunit VII6

actin-related protein ARP3

cytochrome c oxidase VIIa-L

adducin-like protein ADDL

441

442


Table 2. Continued dynein light chain 1

protein kinase related to ERK3

dystroglycan DAG1

protein phosphatase 1 γ

dystrophin

protein phosphatase 2A β

gelsolin

protein phosphatase 2A catalytic subunit

kinectin

protein tyrosine phosphatase (PTP-BAS 2)

laminin B2

protein tyrosine phosphatase (PTP-PEST)

moesin

Ste20-like kinase (MST2)

myosin class Ic

VRK2

myosin regulatory light chain

Neuronal specific

p167

heparin-binding neurite outgrowth-promoting factor

profilin II

myelin basic protein

scaffold protein Pbp1

myelin proteolipid protein

T-plastin

neuronal membrane glycoprotein M6

tubulin α chain

P311

tubulin β chain

peripheral myelin protein 22

vimentin

Protein processing/trafficking

DNA replication/repair

28 kDa heat shock protein

autonomously replicating sequence mRNA


DNA mismatch repair protein MSH2

71 kDa heat shock cognate protein

DNA-dependent protein kinase catalytic subunit


topoisomerase α truncated form

adaptin-related protein 2

topoisomerase I

ADP-ribosylation factor 4

topoisomerase II

a mannosidase II isozyme

UV radiation resistance associated gene

archain

xeroderma pigmentosum group E UV-damaged DNA-binding factor

b-1,2-N-acetyl glucosaminyltransferase II

Extracellular matrix

b-3A adaptin subunit of AP3 complex

collagen type I α1

calnexin

collagen type I α2

carboxypeptidase H

collagen type II α1

chaperonin 10

collagen type III α1

chaperonin protein Tcp20

collagen type IV α1

clathrin coat assembly protein

collagen type IV α2

coatamer protein COPA

collagen type V α1

collagen-binding protein 2

collagen type VI α1

DNAJ homolog

collagen type IX α3

ER-60 protein

collagen type XI α1

m-adaptin-related protein 2

collagenase 3

prolyl 4-hydroxylase α

decorin

reticulocalbin

fibronectin

Sec23B isoform

heparin sulfate proteoglycan core protein

Sec62

laminin B1 chain

secretory granule proteoglycan core protein

lumican

signal recognition particle 14 kDa protein

matrix GLA protein

SRP54

osteopontin

stimulator of TAR RNA binding

reelin

translocon-associated protein β subunit

SPARC

Proteolysis

tissue inhibitor of metalloproteinases 3

26S proteasome regulatory subunit SUG2

Kinase/phosphatase

cathepsin B

cAMP-dependent protein kinase 1

cysteine protease

CDC-like kinase CLK1

ganglioside GM2 activator

cytoplasmic phosphotyrosine phosphatase

MB1

PKU-α

proteasome component C9

protein kinase Cµ

proteasome component HC2

443 Human Genetics, 1999, 8, No. NucleicMolecular Acids Research, 1994, Vol. Vol. 22, No. 1 3 Table 2. Continued proteasome subunit HC2

bone morphogenic protein 3b

proteasome subunit p55

calbindin

proteasome subunit X

calmodulin

sphingolipid proactivator polypeptide

calmodulin-dependent protein phosphatase catalytic subunit

ubiquitin

calpactin 1 light chain

ubiquitin-activating enzyme E1

CASK

ubiquitin C-terminal hydrolase (UHX1)

connective tissue growth factor

RNA processing

EBV-induced G-protein-coupled receptor

A+U-rich element RNA-binding factor

Efs1

ATP-dependent RNA helicase 46

endothelin 1 receptor

G-rich sequence factor 1

endothelin B receptor

hnRNP A1

ERK activator kinase (MEK1)

hnRNP A2/B1

extracellular signal-related kinase ERK3

hnRNP C

EYA1A

hnRNP D

fibroblast growth factor receptor 2

hnRNP E1

FGF1 intracellular binding protein

hnRNP H

frezzled (fre)

hnRNP K

frizzled-related protein

HPBRII-4

glucocorticoid receptor α

poly(A)-binding protein

GTP-binding protein GEM

pre-mRNA splicing factor SF2

guanine nucleotide-binding protein (Gi) α

pre-mRNA splicing factor SR75

guanine nucleotide-binding protein G(S) α

ribonucleoprotein ss-B/La

guanine nucleotide regulatory protein NET1

splicing factor CC1.3

hap mRNA encoding a DNA-binding hormone receptor

splicing factor SRp40-1

HRY

survival of motor neuron-interacting protein 1 (SIP1)

hTGR1

transformer 2 β

insulin-like growth factor 1

U2 snRNA-associated B antigen

insulin-like growth factor-binding protein 5

U4/U6 snRNP hPrp3

interferon γ receptor α

Serum/blood cell proteins

interleukin 1 receptor

apolipoprotein J

interleukin 13 receptor

CD9 antigen

KDR/flk1

coagulation factor XIII a subunit

keratinocyte growth factor

common acute lymphoblastic leukemia antigen (CALLA)

lysophosphatidic acid receptor EDG2

complement component C1s

MacMarcks

complement H factor

macrophage colony-stimulating factor

ferrochelatase

MAL

hemoglobin α

map kinase-activated protein kinase

hemoglobin ε

mitogen-induced nuclear orphan receptor (MINOR)

hemoglobin γ

p62

immunoglobulin light chain

phosphatidyl inositol transfer protein

rhesus polypeptide

PI3-kinase regulatory α subunit

T cell acute lymphoblastic leukemia-associated antigen 1

platelet-derived growth factor receptor

Signaling molecules/growth factors/receptors

pleitrophin

14-3-3 ε

prostacyclin stimulating factor

80 K-L protein

proto-oncogene WNT-5a

activin receptor-like kinase 2

rab11 GTPase

activin receptor type IIB

ragA

adaptor protein p150

Ran-GTP-binding protein 5

adenovirus E3-interacting protein 1

rapamycin-selective 25 kDa immunophilin

adrenomedullin

RasGAP-related protein IQGAP2

amyloid-β protein (APP)

Rho-associated protein kinase

basic fibroblast growth factor

RLIP76

443

444


Table 2. Continued set

proto-oncogene BMI-1

SH3 domain-containing protein SH3P18

putative transcription factor CA150

Src-like adaptor protein

Ran-binding protein 2

stathmin

retinoblastoma susceptibility gene

syntenin

RFXAP

Tis11d

RNA helicase p68

transforming growth factor β1-binding protein

RNA polymerase II elongation factor protein

vasopressin-activated calcium mobilizing receptor-like protein

RNA polymerase II elongation factor-like protein

Yes-associated protein YAP65

telomeric repeat-binding factor

Transcription/nuclear-specific proteins

trans-acting T cell specific transcription factor GATA3

apolipoprotein AI regulatory protein 1

transcriptional regulator homolog RPD3

arginine-rich nuclear protein

v-erbA-related protein ear3

basic transcription factor 2

X box-binding protein 1

basic transcription factor 3a

Y box-binding protein

c-fos cellular oncogene

YY1-associated factor 2 (YAF2)

c-jun proto oncogene

zinc finger protein 7

c-myc transcription factor

zinc finger protein HZF3

CREB-binding protein

zinc finger protein X linked

CRM1

zinc finger protein Zic

DEC1

zinc finger protein ZNF131

DNA-binding protein A


DNA-binding protein B


DNA-binding protein CPBP

Translation/protein synthesis

DNA polymerase ζ (REV3)

acidic ribosomal phospho-protein P2

early growth response protein 1

acidic ribosomal phospho-protein P0

general transcription factor 2I

cap-binding protein

Gu-binding protein

elongation factor 1 α1

hbrm

elongation factor 2

histone acetyl transferase 1

eukaryotic initiation factor 4AII

histone H3

eukaryotic initiation factor 4C

HLH type transcription factor Id3

glutaminyl-tRNA synthetase

HMG box-containing protein 1

glycyl-tRNA synthetase

HMG-1

methionine aminopeptidase

HMG-14

p97

HMG-17

ribosomal protein L11

hypoxia-inducible factor 1α


immediate early response protein NOT


MIP


myocyte enhancer factor 2A


myocyte enhancer factor 2C

ribosomal protein L23a

nascent polypeptide-associated complex


NF-E2-like basic leucine zipper transcriptional activator


nuclear factor I-B2


nuclear factor NF45


nuclear p68 protein (RNA helicase)

ribosomal protein L37a

nuclear pore-associated protein TPR


nucleic acid-binding protein


nucleophosmin


nucleoporin NUP358


nucleosome assembly protein


nucleosome assembly protein 1


pilot

ribosomal protein S12

proline-rich homeobox protein

ribosomal protein S13

445 Human Genetics, 1999, 8, No. NucleicMolecular Acids Research, 1994, Vol. Vol. 22, No. 1 3 Table 2. Continued

ribosomal protein S17 ribosomal protein S18 ribosomal protein S20 ribosomal protein S25 ribosomal protein S27 ribosomal protein S28 ribosomal protein S3A ribosomal protein S4 ribosomal protein S6 translation initiation factor 3 Miscellaneous α crystallin B chain breast cancer suppressor candidate 1 caltractin cyclophilin C CYR61 dentin matrix protein 1 DROER homologous protein ERC-55 ETR101 ferritin heavy chain hkf-1 hSIAH1 Int-6 KDR/flk-1 lipocortin II lysozyme membrane cofactor protein MHC class I HLA-B51 myeloid differentiation primary response protein MyD88 neogenin osteoblast-specific factor 2 phosphatidylethanolamine-binding protein placental anitcoagulant protein protein C inhibitor proto-oncogene homolog pim2 RAB geranylgeranyl tranferase β S-100 protein β subunit selenoprotein P sin3-associated polypeptide p18 SOX9 striated muscle contraction regulatory protein Id2B stromelysin 3 thyroid autoantigen TPRDIII tra1 type 1 procollagen C proteinase enhancer vacuolar ATP synthase subunit C Werner syndrome gene Unknown function 23 kDa highly basic protein 5T4 oncofetal antigen AF-4 AF1q antiquitin B4-2

breast tumor autoantigen C1D CAGH3 CG1 protein cisplatin resistance-associated β protein DCRA DCRR1 DOC2 extracellular protein (S1–5) glycine-rich RNA-binding protein (CIRP) gravin H2K-binding factor 2 hevin histidine-rich calcium binding protein IAI.3B interferon-γ-induced protein (IFI 16) leucine-rich protein leukophysin M phase phosphoprotein 10 melanoma differentiation-associated protein mitogen-responsive phosphoprotein N33 NECDIN-related protein neuroendocrine-specific protein octamer-binding protein 3 (OTF3) OPA-interacting protein OIP2 osteonidogen p54nrb p63 transmembrane protein pancreatic tumor-related protein PEX phorbolin I polyposis locus protein 1 potential laminin-binding protein protein induced by vitamin D prothymosin α putative nucleic acid protein RY-1 putative oral tumor suppressor protein doc1 putative progesterone-binding protein retinal pigment epithelium mRNA RTP Sm-like protein CaSm SMT3A SRcyp protein sui 1 iso 1 tax1-binding protein TFG thymosin β-4 translationally controlled tumor protein TRPM2 TSC-22 protein tumor necrosis factor-inducible mRNA tumor suppressor protein MN1

445

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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. 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 nonhuman 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.

447 Human Genetics, 1999, 8, No. NucleicMolecular Acids Research, 1994, Vol. Vol. 22, No. 1 3

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Table 3. Chromosomal map positions of cochlear ESTs and deafness loci

Continued overleaf

448


Table 3. Continued

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).

449 Human Genetics, 1999, 8, No. NucleicMolecular Acids Research, 1994, Vol. Vol. 22, No. 1 3

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Table 4. Non-human genes with significant homology to human cochlear ESTs Cochlear EST accession no.

Gene namea

GenBank accession no.

Species

% Identityb

N63142

β-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 γ subtype

S55305

rat

89

H88637

acidic calponin

U06755

rat

78

N66965

clathrin heavy chain

J03583

rat

93

N64047

collagen type XII α1

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-β1b

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

γ 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 α subunit

AF008589

pig

81

N63219

non-erythroid β-spectrin

L02897

dog

95

N66989

mannosyl-oligosaccharide α1,2 mannosidase

U04301

rabbit

85

N22012

MALR

X15241

yeast

98

aGene

which the cochlear sequence most closely matches. identity of the cochlear EST sequence to the gene sequence in the aligned region.

bPercentage

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 α1. 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

450


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 (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. 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