Human Molecular Genetics, 2002, Vol. 11, No. 16 1887-1898
© 2002 Oxford University Press
Mutation of the novel gene Tmie results in sensory cell defects in the inner ear of spinner, a mouse model of human hearing loss DFNB6
1Department of Otolaryngology/Kresge Hearing Research Institute and 2Department of Human Genetics, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA and 3The Jackson Laboratory, Bar Harbor, Maine 04609, USA
Received April 22, 2002; Accepted June 7, 2002
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The recessive mutation at the mouse spinner (sr) locus results in hearing loss and vestibular dysfunction due to neuroepithelial defects in the inner ear. Using a positional cloning strategy, we have identified the mutant locus responsible for this pathology. The affected gene (Tmie) lies within a 40 kb deletion in the original sr allele. In a newly identified allele, Tmie contains a nonsense mutation expected to truncate the C-terminal end of its product. The 153 amino acid protein encoded by the gene shows no similarity to other known proteins, and is predicted to contain a signal peptide and at least one transmembrane domain. Tmie transcripts were identified in several tissues, including the cochlea. Loss of function of Tmie results in postnatal alterations of sensory hair cells in the cochlea, including defects in stereocilia, the apical projections of hair cells that are important in mechanotransduction of sound. These morphological defects are associated with a profound failure to develop normal auditory function. Consistent with a conserved role for this gene in the cochlea, the genetic mapping data presented here support human TMIE as the gene affected at DFNB6, a non-syndromic hearing loss locus. The spinner mutant is thus a valuable model for insight into mechanisms of human deafness and development of sensory cell function.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The incidence of hearing loss in humans is substantial, with a frequency of prelingual deafness as high as 0.1–0.2%, and a similar frequency of postlingual deafness before the third decade of life (1,2). In developed nations,
50% of these cases appear to have a genetic basis (1). Inherited deafness in humans is genetically heterogeneous, with effects in any one of more than 100 distinct genes likely to be responsible for non-syndromic hearing loss (1). Despite the difficulties in analysis of genetically heterogeneous conditions, there has been dramatic progress in the localization and identification of a large number of genes associated with hearing loss during the past several years (3–5). Mouse genetic models provide a valuable approach to identify genes which play a role in inherited human hearing loss, and also offer a useful system in which to investigate gene function (6). Identification and analysis of these genes in the mouse has implicated a diverse array of proteins required in the inner ear during early embryonic development and postnatal maturation of the sensory neuroepithelium, and in the adult organ (7). The spinner (sr) mutation arose spontaneously over 40 years ago in a colony of C57BL mice and was first recognized by behavioral dysfunction, including bidirectional circling and head shaking (8). Auditory function in spinner mice was found to be reduced, based upon the lack of a startle reflex to sound at any age. Breeding experiments indicated that these defects were inherited in an autosomal recessive fashion. Low-resolution linkage studies localized sr to distal chromosome 9, with respect to genes encoding two catalytic enzymes, mannose phosphate isomerase 1 (Mpi1) and cytosolic malate dehydrogenase (Mod1) (9). Comparative gene mapping studies have identified a region of conserved synteny between distal chromosome 9 in the mouse and chromosome 3 in humans (10). Analysis of an Indian family with members exhibiting autosomal recessive non-syndromic hearing loss indicated the presence of a deafness locus (DFNB6) on the short arm of chromosome 3 (11). Hearing loss in affected family members was present at birth and was severe to profound across all tested frequencies. Haplotype analysis indicated that DFNB6 was localized in a 14–20 cM region, within 3p21–3p14. The conserved synteny of this region suggested that spinner may be a molecular model for DFNB6.
Light microscopic examination of the inner ears of affected spinner mice indicated that auditory and vestibular dysfunction were likely to be peripheral in origin (8). No obvious defects in gross inner ear morphogenesis or neuroepithelial cell patterning were noted. Sensory cells in the cochlea, however, were decreased slightly in size by postnatal day (P) 15, and were completely degenerated by P40. Later defects were also noted, including degeneration of auditory nerve cells. Vestibular pathology appeared to be restricted to the macula of the sacculus, where progressive loss of sensory hair cells also occurred. These specific defects, along with the apparent lack of affected tissues outside of the inner ear, indicated that spinner belonged to the neuroepithelial class of mouse deafness mutants (12). In the current study, we have used a positional cloning approach to identify mutations in a novel gene as the basis for the spinner phenotype. We have also identified postnatal defects in sensory cell ultrastructure that are associated with profound hearing loss in affected spinner mice.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Previous linkage studies indicated that sr is located on chromosome 9,
20 cM distal of Mod1 (9). In order to localize the mutation with greater resolution, we generated an intersubspecific intercross between (C57BL/6J-srxCAST/Ei) F1 mice. The percentage of affected mice obtained (180/730, 24.7%) was consistent with previous segregation data indicating recessive inheritance of behavioral and auditory defects (8). Analysis of genotypes from the 730 F2 progeny indicated that sr lies within a 0.2 cM interval between D9Mit348 and Ltf (Fig. 1). Gene localization on this map placed the sr locus within a previously identified region of conserved synteny with human chromosome 3 (Fig. 2) (10). Cdc25a was localized proximal of sr, while parathyroid hormone receptor 1 (Pthr1), myosin light chain (Mylc) and teratocarcinoma-derived growth factor 1 (Tdg f1) were non-recombinant with the sr locus. The human orthologs of these four genes were previously localized to 3p21–22 (13–16). Stac was localized 0.6 cM distal of sr; human STAC was previously mapped to 3p22.3 (17). As relative gene order appears to be well conserved across the region in mouse and human (Fig. 2), the likely location of the human ortholog of the spinner gene is therefore 3p21–22, closely linked to MYL3 and PTHR1. Based upon recent assembly of human genomic sequence, this location lies within the DFNB6 candidate region, and is consistent with the gene as a positional candidate for the affected locus, as previously suggested (11).
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0.2 cM in size (95% confidence interval). Distances between markers are indicated (±SE). Gene localization on this map placed the sr locus within a previously identified region of conserved synteny with human chromosome 3 (Using closely linked markers, we identified a large collection of BAC genomic clones across the spinner candidate region on mouse chromosome 9 (Fig. 3). While assessing BAC clone overlap, we found that several markers were deleted from genomic DNA of sr/sr mice (Fig. 3). Through bioinformatics analysis of available genomic sequences, we identified four genes in the immediate region. AK007173 and Tsp50 encode proteins with strong similarity to serine-type proteases. mRn.49018, a gene with high sequence similarity to a predicted rat gene, encodes a protein with a Dbl homology domain. Dbl domains are present in guanine nucleotide exchange factors specific for Rho-family GTPases (18). Mm.87012 is a gene with no significant similarity to other database sequences.
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We confirmed the extent and structure of the deletion by Southern blot and PCR analysis. Probes derived from DNA flanking the deleted region hybridized to BglII restriction fragments of 2.1 kb and 5.2 kb in normal C57BL/6J DNA (probes I and III; Fig. 4B). In sr/sr DNA, both probes instead hybridized to a common-sized junction fragment of 5.8 kb, consistent with deletion of the intervening DNA. A probe derived from the 3'-UTR of Mm.87012 (probe II) hybridized with 1.1 kb fragments of DNA from C57BL/6J and +/sr, but failed to hybridize with DNA from sr/sr, consistent with deletion of this probe region. Primer pairs designed from sequences flanking the centromeric breakpoint (primers 1+2) and the telomeric breakpoint (primers 3+4) of the deletion amplified products of the predicted size from C57BL/6J and +/sr DNA but not from sr/sr DNA (Fig. 4C). Primers flanking the deletion (primers 1 and 4) amplified an sr-specific product only, again consistent with a deletion. The deletion, as assayed using these primer sets, showed absolute co-segregation with the sr locus in the genetic cross (unpublished data). Sequence analysis of the amplification products indicated that the deletion breakpoints occurred within direct repeats of the sequence CTCAG (Fig. 4D). These repeats are separated by
40 kb in the normal C57BL/6J chromosome, based upon genomic sequence comparisons. The centromeric deletion breakpoint is within AK007173 (Fig. 4A); the 3' end of the gene is deleted. Tsp50 and Mm.87012 lie completely within the deletion. The telomeric deletion breakpoint is within mRn.49018; the spinner deletion removes the 5' end of the gene. Despite the complete or partial loss of these four genes, affected spinner mice exhibit no obvious phenotypic defects other than inner ear dysfunction.
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We recently identified a new vestibular mutant that arose spontaneously on the BXA-4 recombinant inbred strain. Like spinner, the new mutant exhibited circling and head-shaking behaviors, and a defective startle response indicative of hearing loss. These defects were inherited in a recessive manner. Analysis of pooled DNA of affected F2 mice from a small CAST/Ei intercross indicated linkage to microsatellite markers on distal chromosome 9. Analysis of 56 individual F2 mice demonstrated the following order: cen—D9Mit111 —5.8±1.6 cM—D9Mit78—1.3±0.8 cM—mutation—0.9±0.6 cM—D9Mit17---tel. This map position suggested potential allelism with the spinner locus. A complementation test was carried out by crossing an affected male homozygote with a female heterozygous for the original sr allele. Seven of 13 progeny exhibited circling behavior and lacked startle responses, consistent with allelism of the two mutations. We therefore named the new mutant sr J.
Given the evidence for allelic mutations, we analyzed in sr J mice the four genes affected by the deletion in the original sr allele. RT–PCR amplification from brain RNA with gene-specific primers indicated that all four genes were expressed in sr J/sr J homozygotes, with no detectable differences in transcript abundance or structure relative to C57BL/6J controls (unpublished data). RT–PCR products spanning the open reading frames of each of the four genes were then sequenced. The sequences in sr J of AK007173, Tsp50 and mRn.49018 were identical to those in C57BL/6J controls. The only variant identified was the single nucleotide substitution C1451T in exon 5 of Mm.87012. This substitution was confirmed in genomic DNA from sr J/sr J homozygotes (Fig. 5). The sequence variant alters an arginine codon in the normal transcript to a stop codon in sr J, truncating the predicted protein. We sequenced the corresponding region in the BXA-4 progenitor strain, eight C57BL-related strains and the A/J strain, and observed the C(1451) nucleotide in each case (Fig. 5, and unpublished data). The nonsense mutation in sr J and the deletion in the original spinner allele indicate that defects in the Mm.87012 gene are responsible for the spinner phenotype.
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Assembly of overlapping cDNA and RT–PCR sequences of Mm.87012 identified a transcript of 2.6 kb. Comparison of cDNA and genomic sequences indicated that the gene is composed of six exons, encompassing
14 kb of genomic DNA (Fig. 6A). A 459 bp open reading frame spans exons three to six and encodes a 153 amino acid protein (Fig. 6B). The amino acid sequence exhibits no significant similarity to any known protein. Two hydrophobic transmembrane (TM) domains were predicted near the N-terminus by pattern recognition algorithms, with one TM domain overlapping a strongly predicted signal peptide at the extreme N-terminus. Following cleavage of the signal peptide and retention of the protein in the membrane via the second TM domain, the mature N-terminus is expected to be extracellular (or lumenal) and the C-terminus cytoplasmic (19). The C-terminal region of the protein is rich in charged residues (36/74), and includes two clusters of lysine residues. The nonsense mutation identified in sr J mice is expected to truncate the protein at amino acid 96 and prevent expression of the majority of the C-terminal charged domain.
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Northern analysis with a probe derived from the 3' UTR of Mm.87012 demonstrated expression of the gene in brain, kidney, liver and lung (Fig. 7A). Kidney expressed two transcripts, 2.8 and 3.2 kb in size. Brain expressed a smaller 2.2 kb transcript, while liver and lung expressed only the larger 3.2 kb transcript. Preliminary evidence suggests that the various transcript sizes derive from the use of alternative promoters and 5' non-coding exons in the different tissues (unpublished data). Expression of the gene was also detected in cochlear RNA by RT–PCR (Fig. 7B). Based upon the predicted transmembrane domains, and its critical requirement in the inner ear, we have renamed this gene Tmie, for transmembrane inner ear.
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Control +/sr mice detected external sound stimuli at P14 as assessed by auditory brainstem response analysis, while sr/sr mice failed to exhibit responses at this age (Fig. 8) or at later time points (unpublished data). Homozygous sr J mice also exhibited a complete lack of evoked brainstem responses (unpublished data). We examined cochlear ultrastructure in sr mutants to determine whether this early functional loss was correlated with morphological defects. General neuroepithelial cell patterning was normal in the cochlea of control +/sr (Fig. 9A) and sr/sr (Fig. 9B) mice at P15. Closer examination, however, revealed irregularities at the apical surfaces of both inner sensory hair cells (IHCs) and outer sensory hair cells (OHCs) in sr/sr (Fig. 9B). Compared to those of +/sr controls (Fig. 9C), IHCs of sr/sr mice exhibited extra rows of maturing stereocilia (Fig. 9D). Deflection of stereocilia plays a fundamental role in the mechanotransduction of sound energy by hair cells, through physical gating of a putative cation channel in the plasma membrane surrounding the actin-rich core of the stereocilia (20). IHCs of sr/sr mice also retained their kinocilia, suggesting a block or delay in maturation (Fig. 9D). IHCs of the controls have lost this transient, microtubule-containing structure, consistent with previous developmental studies (21). On OHCs, individual stereocilia appeared irregular in sr/sr, with portions of each bundle missing or considerably shortened (Fig. 9F). The apical surface areas of sr/sr OHCs (Fig. 9F) were also decreased compared to normal OHCs (Fig. 9E), consistent with original light microscopic studies of spinner (8). The surface areas of supporting cells that surround OHCs in sr/sr were correspondingly increased. We identified indistinguishable pathologies in the cochleae of sr J/sr J mice (unpublished data), indicating that deletion of three additional genes in the original sr allele does not apparently contribute to the inner ear phenotype of the mutant. These defects suggest a critical requirement for Tmie function during postnatal maturation of the neuroepithelium.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The current study provides strong evidence that defects in Tmie are responsible for inner ear dysfunction in the spinner mutant. First, this gene is localized within the small candidate interval defined by high-resolution genetic analysis. Second, mutations were identified in two independent alleles of spinner. The entire Tmie gene is deleted in the original sr allele, while in the sr J allele, the gene contains a single base pair substitution that truncates the expected protein product. Both mutations result in profound loss of auditory function. Our genetic mapping data support the human ortholog of Tmie as an excellent positional candidate for the gene affected at the DFNB6 locus. Indeed, mutations have recently been identified in TMIE in each of five pedigrees segregating hearing loss due to defects at this locus, implicating TMIE as a critical gene in the human inner ear (22). The human mutations include a small deletion and an insertion that alter the normal open reading frame and that are likely to be complete loss-of-function alleles. Finally, Tmie is expressed in the cochlea, consistent with the specific inner ear pathology identified in affected spinner mice.
The postnatal defects present in the cochleae of sr/sr mice suggest a requirement for Tmie during maturation of sensory cells, including the normal development or maintenance of stereocilia bundles. A number of other genes have been implicated in pathways critical for normal stereocilia structure, including those which encode unconventional myosins [Myo7a (23), Myo6 (24), and Myo15 (25)], members of the cadherin superfamily [Cdh23 (26) and Pcdh15 (27–29)], an integrin subunit [Itga8 (30)], and an actin-bundling protein [Espn (31)]. Based upon specific bundle defects in deaf mice carrying mutations in these genes, they appear to regulate pathways in sensory hair cells that are critical for growth, integrity or organization of the bundle. The severe shortening of individual OHC stereocilia in sr/sr mice suggests that the TMIE protein may influence actin filament dynamics in the normal maturing bundle, a process also thought to be affected by MYO15 (32) and ESPN (31). The shortened stereocilia may alternatively reflect a secondary degenerative response to the mutation. Additional studies of earlier developmental time points will help to distinguish between these possibilities. The apparent delay in maturation of IHCs in sr/sr mice appears more subtle than the stereocilia defects found on OHCs. Given that IHC signaling provides the majority of afferent input to the central auditory system (33), the failure of the mutants to develop measurable auditory brainstem responses suggests that IHC function is severely compromised. Although we have not identified any early postnatal defects in auditory nerve morphology (unpublished data), Tmie mutations could also affect brainstem responses through more direct action within auditory neurons. Tmie thus may serve multiple roles during sensory cell development, including critical functions in both stereocilia bundle maturation and sensory transduction in IHCs or in auditory neurons. Alternatively, Tmie may have only a single role, and the different morphological defects in IHCs and OHCs of sr/sr mice may reflect cell type-specific, secondary responses to the mutation. Electrophysiological evaluation of hair cells in spinner mice should provide important information regarding a role for Tmie in transduction.
Tmie joins an expanding set of genes, mutations of which lead to defects in development and homeostasis in the inner ear and cause hearing loss and vestibular dysfunction (34). Based upon sequence similarities and direct biochemical studies, many of these genes belong to recognized families. Others, such as Tmie, exhibit little or no similarity to known genes, and their functions are presently unclear. Recently, mutations that cause human hearing loss have been identified in two other novel genes that encode potential integral membrane proteins. USH3A mutations are responsible for hearing loss and retinal degeneration in a subset of individuals with Usher's syndrome (35). USH3A encodes a small, novel protein with a signal peptide/transmembrane domain structure like that of TMIE (35). Mutations in TMC1 are the basis for non-syndromic hearing loss in individuals with defects at the DFNA36/DFNB7/DFNB11 locus (36). Likewise, defects in Tmc1 are responsible for inner ear dysfunction in the mouse mutants known as deafness and Beethoven (36,37). Like TMIE and USH3A, the predicted integral membrane product of TMC1/Tmc1 has no significant similarities to other database sequences. Its predicted membrane topology (at least six transmembrane domains) and hair cell expression pattern have suggested a potential role for TMC1 as a sensory cell-specific ion channel (36).
While the predicted structure of TMIE is not consistent with a typical channel or receptor, the protein may play some other role in a cellular membrane location. TMIE may reside within an internal membrane compartment and function in pathways such as those involved in protein and/or vesicle trafficking. Alternatively, the mature protein may be localized in the plasma membrane and serve as a site of interaction for other molecules through its highly charged C-terminal domain. The multiple clusters of positively charged residues present in the C-terminus are also found in the cytoplasmic regions of a diverse set of integral membrane proteins that are linked to the cortical actin cytoskeleton through interactions with members of the ERM (ezrin, radixin, moesin) protein family (38). Mutations of these clusters to uncharged residues in the Na–H exchanger NHE1 (39) and in other membrane proteins such as CD44, CD43 and ICAM-2 (40) inhibit ERM protein binding and co-localization at membrane sites associated with actin-rich structures, including microvilli and lamellipodia. In the case of NHE-1, the abundance of these structures appeared to decrease in cells expressing the mutant proteins, consistent with a critical role of the ERM–membrane protein complexes in regulating cytoskeleton–membrane interactions (39). Positively charged residues in the C-terminus of TMIE are likely to be important in protein function, as each of three missense mutations detected in DFNB6 pedigrees replaces a different conserved lysine residue with an uncharged amino acid (22). A role for TMIE in organizing cytoskeleton–membrane interactions in sensory hair cells would be consistent with the stereocilia pathology found in spinner mice, and would predict a localization of the protein at the apical surface of the hair cell. Immunocytochemical studies with antibodies specific for TMIE will help to distinguish the site of action of the protein and its potential functional roles.
Further characterization of the impact of Tmie mutations in the inner ears of spinner mice will provide insight into pathways that are critical for normal maturation of sensory cell function. Analysis of spinner will also help to define the molecular mechanism of hearing loss in humans with defects at the DFNB6 locus, and will provide a system in which to investigate potential therapies that rescue sensory cells and preserve auditory function.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Mice and genetic mapping
C57BL/6J-+/sr heterozygous mice were obtained from the frozen embryo repository at The Jackson Laboratory (Bar Harbor, ME, USA). The original sr mutation arose on C57BL/How (8), and has been maintained on a C57BL/6J background by mating homozygous C57BL/6J-sr/sr mice to C57BL/6J-+/sr heterozygotes. Homozygous sr/sr mice were mated with M. musculus castaneus (strain CAST/Ei) to produce a (C57BL/6J-srxCAST) F1 generation. F1 mice were intercrossed to produce an F2 generation. The new sr J allele arose spontaneously in a colony of BXA-4 recombinant inbred mice at The Jackson Laboratory, and was maintained by similar homozygotexheterozygote matings. All mice were maintained in accordance with institutional UCUCA standards.
Genetic markers and genotype analysis
F2 offspring from (C57BL/6J-srxCAST) F1 intercrosses were typed with SSLP (simple sequence length polymorphism) markers from distal chromosome 9 (41). DNA was prepared from tail biopsies of F2 mice as described (42). Affected sr/sr F2 mice were identified at weaning age by their circling and hyperactive behaviors, and lack of a startle reflex in response to loud noise. Animals with ambiguous behavior were subjected to a swimming test; sr/sr animals are completely unable to remain above the surface of the water. Unaffected mice with recombination events in the critical region were retested to confirm their unaffected status. Genotypes at spinner were inferred from the genotype of flanking markers, or determined by test crosses with sr/sr mice. The first 60 F2 mice generated were typed for all markers. An additional 670 F2 mice were first typed with D9Mit212 and D9Mit149; mice that exhibited recombination events within this 4 cM region were then typed with additional markers. Genotype data were analyzed using Map Manager software (43). The most likely gene order was determined by minimizing the total number of crossovers in the region. PCR primers specific for D9Mit markers were obtained from Research Genetics (Huntsville, AL, USA). All other primers were synthesized at the University of Michigan Oligonucleotide Facility. Two D9Mit markers served as surrogate markers for genes based on close physical association: D9Mit37 (Mylc) and D9Mit38 (Ltf). Cdc25a was localized using an SSLP primerset (5'-CACCCTGCCGTTACACTCTTCTG-3', 5'-GCACTTGGCGGCTTTTCTCT-3'; C57BL/6J allele size 185 bp; CAST/Ei allele size 192 bp). PCR for analysis of SSLP markers was carried out under standard conditions; products were resolved on 4% agarose gels and visualized with ethidium bromide fluorescence. For analysis of gene-based markers, single-strand conformational polymorphism (SSCP) analysis was performed with end-labeled primers using standard protocols (44). Reaction products were separated on gels composed of 0.4xMDE (Bio Whittaker, Walkersville, MD, USA)/8% glycerol, using 0.7xTBE as running buffer and electrophoresis under constant power conditions (10 W) for 12–24 h. Gels were vacuum dried at 70°C, and then exposed to X-ray film (X-Omat AR, Kodak). SSCP primer sets: Pthr1 (5'-TGTTGCAGGAAGAATGGGAA ACAGTC-3'; 5'-CCTTGAGCACAACACAGGAAGCCAC-3'); Stac (5'-TTTGGCTGGCAGGATTGTTCTTTCTTTCTC-3'; 5'-CTTCCTTGTCCCCACTCCCGCTTC-3'); Tdgf1 (5'-TTTGTCTTGCTTGAACCTTGTCAGTAAC-3', 5'-TGAGTCGGATGCCCAGAACCA-3'). For localization of the sr J mutation, we performed a genome-wide linkage screen, comparing MIT marker genotypes of F1 DNA with genotypes of pooled DNA from 20 affected F2 mice. Following discovery of linkage to Chromosome 9, 56 individual affected F2 mice (112 meioses) were typed for additional markers on Chromosome 9.
Physical map construction and characterization of the spinner deletion
Hybridization probes were derived from STS markers non-recombinant and flanking the spinner candidate region and pooled to screen the RPCI-23 BAC genomic library (45). DNA from positively hybridizing BACs was purified using standard methods, and insert overlap was evaluated by PCR and/or hybridization with STS and BAC end markers. BAC inserts were sized by pulsed-field gel electrophoresis. The status of markers on the spinner chromosome was determined by PCR and/or hybridization of sr/sr genomic DNA. Marker STS primers were designed from genomic sequence of BAC RP23-79H19 (AC079643): Pthr1 (5'-CACTCCAGCCCT GCCAGCACGATA-3'; 5'-TCCTCCTCCCACCCTGAA CCCACT-3'); 79H19A (5'-CAGGGCGGGACATGCT TACAA-3'; 5'-GGGGATGCTGGGAGTCTGGTC-3'); 79H19B (5'-TGAGGGGCTTTA ACATCAGTAGAGACAT-3'; 5'-CCATAAAGAAGA AACAATAGCAGCAACAAT-3'); 79H19C (5'-ATCCCC TATCTCACCCCCACATC-3'; 5'-CATCTAGCAATCCCGA GAACCAGTC-3'); 79H19D (5'-TTTAG ATGGCCACAGGACAATAGC-3'; 5'-CCCCAGG AAAT CACATCACCA-3'); 79H19E (5'-CAGCC CGCAGT GGAGTGACC-3'; 5'-TGTGATGGGGCAG AATGGAGACC-3'); 195G15-T7 (5'-GTAGT CCGCGCTGGTGCCCTG-3'; 5'-CCTCAAA CTCCTCTCCCCGTCCAG-3'); Tdgf1 (5'-TGGGCAT CCGACTCAGGTTGTAAAG-3'; 5'-CACGGC CACGCTCGGAATAAA-3'); Ltf (5'-GGCAAGGCCAGT TCAGATACAAAGATG-3'; 5'-CACACCCACCGGAAG CAATAAATG-3').
In order to precisely define the extent of the spinner deletion, we used inverse PCR to amplify genomic DNA from sr/sr mice with nested primer sets designed from sequence telomeric to the deletion (primary primer set—5'-GCCCAGCT CCCAATGCACACTCAGA-3'; 5'-CTCTCCCAGC ACGTGCAGAAATAC-3'; secondary primer set: 5'-TGTCC AAAGGCAGGGGTGAGAGC-3'; 5'-TCAGGGG ACCGAGATAAGCCATAGG-3'). Template was prepared by digestion of sr/sr genomic DNA with Tsp509I, followed by ligation at a DNA concentration of 1 mg/ml. Amplification conditions: 97°C, 3 min; 35 cycles of 94°C, 30 s; 65°C, 30 s; 72°C, 60 s; final extension of 5 min at 72°C. The sequence of inverse PCR products was compared to genomic sequence in the region AC079643. Based upon this analysis, primer sets were designed to amplify across the centromeric and telomeric breakpoints in normal genomic DNA (centromeric breakpoint set—primer 1: 5'-CCAGTTACGTCAGATCCTACACC-3'; primer 2: 5'-CACAATCCTGACAGCCAAACCTT-3'; telomeric breakpoint set—primer 3: 5'-CTGCCCCTGA GGAGCTCTATGG-3'; primer 4: 5'-CACAACCGCT GGGCACTCTCAT-3'). For Southern analysis, probes were prepared by PCR amplification of BAC RP23-133F23 with primer sets designed from DNA centromeric to the deletion (probe I: 5'-CCAGTTACGTCAGATCCTACAC-3'; 5'-TAAAACAAAACAAGTGAAGCAAAAGTCTCC-3'; 361 bp product), within the deletion (probe II: 195G15-T7 forward and reverse primers, see above; 446 bp product), and telomeric to the deletion (probe III: 5'-GTGCCC AGCGGTTGTGGTATGTGAC-3'; 5'-GTCTTCCCC AACTCCCCCTCCTCAG-3'; 594 bp product). Genomic DNA from C57BL/6J, C57BL/6J-+/sr, and C57BL/6J-sr/sr mice was digested with BglII, electrophoresed in triplicate (5 mg/lane) on a 0.8% agarose gel, and transferred to Zetaprobe GT using standard techniques. Each of the triplicate filters was hybridized with one of the above probes. Hybridization was performed at 65°C in nylon buffer (0.5 M Na2PO4; 7% Sodium dodecyl sulfate (SDS); 1 mM EDTA) using an 
-[32P]-dATP random labeled PCR product. Final washes were in 0.2x Sodium chloride/sodium citrate (SSC)/0.1% SDS at 65°C. The washed filter was exposed to BioMax film (Kodak) at -80°C.
Gene identification, cDNA sequence assembly, and mutation analysis
The exon content of genomic sequence from BAC RP23-79H19 (AC079643) was evaluated by Genscan analysis (46) and by sequence similarity searches of public databases, including human genome assemblies (47), using the BLAST algorithm (48). Predicted exons were verified by sequence analysis of kidney cDNA clones (IMAGE:1431993 and IMAGE:1432228) and by RT–PCR with gene-specific primer sets. Additional cDNA sequences were identified by 5' and 3' RACE from brain and kidney Marathon cDNA (Clontech, Palo Alto, CA, USA). Total RNA was isolated from adult mouse brain using Trizol reagent (Invitrogen, Carlstad, CA, USA). Poly(A)+ transcripts were purified using PolyATract (Promega, Madison, WI, USA); first-strand cDNA synthesis was performed on 5 mg poly(A)+ RNA using oligo-dT primers with the Superscript II system (Invitrogen). For mutation detection in sr J and control strains, overlapping RT–PCR products were amplified using gene-specific primer sets, and products were gel purified and sequenced as described previously (49). For confirmation of the nonsense mutation identified in sr J, we amplified genomic DNA (100 ng) from sr J/sr J and BXA-4 mice with primers that flank exon 5 of Tmie (5'-CTGTGAGACAAGCTTAAGCAGATGGAGT-3'; 5'-CAATTGGAACAGTGGGAGTGAGGAA-3'), and sequenced the gel-purified products. Domain structure of the conceptual TMIE protein sequence was evaluated using SignalP (50) and TMHMM (19).
Expression analysis
A northern blot containing RNA isolated from adult mouse tissues (Stratagene, La Jolla, CA, USA) was hybridized with a probe derived from the 3' UTR of Tmie (probe II, above). Hybridization was performed as described above for Southern analysis. The filter was stripped by two 15 min incubations in 0.1x SSC/1% SDS at 95°C, and then rehybridized with a human ß-actin probe at 60°C. RNA was prepared from adult mouse cochleae using the SV Total RNA Isolation System (Promega). RT–PCR of cochlear RNA was performed with primers designed to amplify across exons 3–6 of Tmie (5'-GTGTCGCCACGCAGCT GGTA-3'; 5'-GACAACCTGTTCAGT CTTGCCATCCTA-3').
Scanning electron microscopy
Deeply anesthetized mice derived from a C57BL/6J-+/srxC57BL/6J-sr/sr cross were decapitated and cochlea obtained and fixed in 2% glutaraldehyde. Four sr/sr and four +/sr mice at P14 were analyzed. Tissues were prepared for SEM analysis as previously described (32). Samples were analyzed using a Philips XL30 FEG scanning electron microscope.
Electrophysiological analysis
Auditory brainstem analysis was performed on mice from the CAST/Ei intercross, using pure tone stimuli of 1, 8 and 24 kHz, as previously described (29). Two sr/sr mice and two +/sr mice from each of three time points (P14, P25 and P90) were analyzed.
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ACKNOWLEDGEMENTS |
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We thank Margaret Lomax, Hana Odeh, Didi Robins, Sally Camper and Miriam Meisler for critical reading of the manuscript. We are also grateful to Tzy-Wen Gong for generous assistance with preparation of mouse cochlear RNA. This work was supported by grants from the National Organization for Hearing Research (DCK), the American Hearing Research Foundation (DCK), NIH-NIDCD grants DC04410 (DCK), DC02982 (DCK, DFD, and YR) and NIH-NIDCD contract DC62108 (KRJ).
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FOOTNOTES |
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* To whom correspondence should be addressed at: University of Michigan Medical School, Department of Otolaryngology/Kresge Hearing Research Institute, 9301D MSRB III, 1150 West Medical Center Drive, Ann Arbor, MI 48109-0648, USA. Tel: +1 7347639653; Fax: +1 7346472563; Email: dkohrman{at}umich.edu
AF481144, AF481143 and AF481142
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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