Human Molecular Genetics, 2000, Vol. 9, No. 12 1787-1793
© 2000 Oxford University Press
A biochemical basis for the inherited susceptibility to aminoglycoside ototoxicity
1Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA, 2Division of Human Genetics, Children’s Hospital Medical Center, Cincinnati, OH 45229, USA and 3Ahmanson Department of Pediatrics, Steven Spielberg Pediatric Research Center, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA
Received 11 March 2000; Revised and Accepted 31 May 2000.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The A1555 G mutation in mitochondrial 12S rRNA has been found to be associated with non-syndromic deafness and aminoglycoside-induced deafness. The sensitivity to the aminoglycoside paromomycin has been analyzed in lymphoblastoid cell lines derived from five deaf individuals and five hearing individuals from an Arab-Israeli family carrying the A1555G mutation, and three married-in controls from the same family. Exposure to a high concentration of paromomycin (2 mg/ml), which caused an 8% average increase in doubling time (DT) in the control cell lines, produced higher average DT increases (49 and 47%) in the A1555G mutation-carrying cell lines derived from symptomatic and asymptomatic individuals, respectively. The ratios of translation rates in the presence and absence of paromomycin, which reflected the effect of the drug on mitochondrial protein synthesis, were significantly decreased in the cell lines derived from symptomatic and asymptomatic individuals (by 30 and 28% on average, respectively), compared with the ratios in the control cell lines. These ratios showed, in both groups of mutant cell lines, a significant negative correlation with the ratios of DTs in the presence and absence of the antibiotic. These results have provided the first direct evidence that the mitochondrial 12S rRNA carrying the A1555G mutation is the main target of aminoglycosides. They suggest that these antibiotics exert their detrimental effect through an alteration of mitochondrial protein synthesis, which exacerbates the inherent defect caused by the mutation, reducing the overall translation rate down to and below the minimal level required for normal cellular function (40–50%).
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The use of aminoglycoside antibiotics can cause a rapid irreversible hearing loss in genetically susceptible individuals. These antibiotics are a group of important drugs used in the treatment of infections caused by Gram-negative bacteria. These drugs are known to exert their antibacterial effects at the level of the decoding site of the small ribosomal subunit, causing miscoding or premature termination of protein synthesis (1,2). Aminoglycoside antibiotics are composed of amino sugars linked to a deoxystreptamine ring. The 2deoxystreptamine-containing aminoglycosides of the neomycin group, which bind to the aminoacyl tRNA binding site (A site), include paromomycin, neomycin, ribostamycin and neamine. They have common functional groups on rings I and II and produce a characteristic miscoding pattern (3,4). A considerable amount of evidence indicates that sensitivity to paromomycin, neomycin and related aminoglycosides in bacteria involves their directly binding to a base pair at the penultimate helix of the small ribosomal subunit rRNA (3–8). In particular, in wild-type Escherichia coli sensitive to these aminoglycosides, the nucleotide at position 1491 (G) in 16S rRNA is base-paired with a C at position 1409; mutation or methylation of the 1491 nucleotide disrupts the G-C pairing, producing resistance to aminoglycosides (6–8).
In familial cases of toxic deafness, the aminoglycoside hypersensitivity is often maternally transmitted (9), suggesting the occurrence of a mutation(s) in mitochondrial DNA (mtDNA). Recently, a homoplasmic A
G transition at position 1555 of mtDNA, in a highly conserved region of the 12S rRNA gene (Fig. 1), has been found in a number of pedigrees and sporadic patients with aminoglycoside-induced deafness (10–16). In the absence of exposure to aminoglycosides, the A1555G mutation has also been observed in many families with maternally inherited non-syndromic deafness. The A1555G mutation produces a clinical phenotype, which ranges from severe congenital deafness to normal hearing (10,17–19). In many families, the onset appears to occur in early adult life (18). No abnormalities in any other organ, including the vestibular system, have been observed (20). The nucleotide at position 1555 in the human 12S rRNA (equivalent to position 1491 in the E.coli 16S rRNA) in wild-type cells is A, which, when mutated to a G, as in the Arab-Israeli family, would pair with the C at position 1494 (Fig. 1). This G-C pair is expected to create a new binding site for aminoglycosides, thus facilitating aminoglycoside sensitive (21).
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Previous work had revealed that lymphoblastoid cell lines deriving from either symptomatic or asymptomatic members of an Arab-Israeli family carrying the non-syndromic deafness-causing A1555G mtDNA mutation in the 12S rRNA gene exhibited a specific, significant decrease in growth rate in the presence of the aminoglycosides paromomycin (1 mg/ml) and neomycin (0.5 mg/ml) (22). In the present work, in order to elucidate the biochemical basis of the aminoglycoside-induced ototoxicity, lymphoblastoid cell lines derived from members of the Arab-Israeli family carrying the mutation (including five symptomatic individuals and five asymptomatic individuals) and from three control individuals have been analyzed further. This investigation has confirmed that all mutant cell lines, independently of the hearing state of the cell donor, compared with wild-type cells, exhibited a significant, very similar increase in doubling times (DTs) in the presence of a high concentration (2 mg/ml) of paromomycin. Furthermore, in all mutant cell lines treated with paromomycin, a decrease in mitochondrial protein synthesis rate has been observed, which was more pronounced than that detected in the same cell lines in the absence of the drug. The ratios of translational rate in presence and absence of the aminoglycoside showed, in the cell lines derived from either the symptomatic or the asymptomatic individuals, a significant negative correlation with the corresponding DT ratios. These data strongly support the conclusion that the A1555G mutation is responsible for the sensitivity to paromomycin of both the symptomatic and asymptomatic members of the Arab-Israeli family, and that this antibiotic aggravates the protein synthesis impairment caused by the mutation, so as to bring the overall translation rate down to and below the minimal level required for normal cellular function.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The Arab-Israeli pedigree and derived lymphoblastoid cell lines
The pedigree of the Arab-Israeli family with maternally inherited deafness has been previously described (22,23). Immortalized lymphoblastoid cell lines derived from 10 maternally related members of this family exhibiting the A1555G mutation (22,23) [five asymptomatic individuals (F7H, F12C, F12D, F12E, F12H) and five symptomatic individuals (F6H, F7C, F7D, F12F, F12J)], and from three married-in control individuals (#4, F7A, F7E) were selected for analysis. The maternally related individuals of the Arab-Israeli family were all between 20 and 60 years of age, and the control individuals between 20 and 50 years of age. Among the symptomatic individuals, F6H, F7D and F12F were deaf-mutes with congenital severe deafness, F7C developed a severe hearing loss at age 20 years and F12J exhibited a moderate, early-onset hearing loss. Five asymptomatic individuals (F7H, F12C, F12D, 12E, F12H) had normal hearing. None of the individuals listed above was exposed to aminoglycosides, and there were no other clinical problems in these individuals.
Growth properties of cell lines after treatment with paromomycin
Previous investigations (22) had shown that different lymphoblastoid cell lines deriving from symptomatic or asymptomatic individuals of the Arab-Israeli family carrying the A1555G mutation, were similarly sensitive in their growth to paromomycin (1 mg/ml) or neomycin (0.5 mg/ml), with indications that the mitochondrial inner membrane is not very permeable to these antibiotics. In the present work, to further characterize the role of the mtDNA A1555G mutation in the effect of paromomycin on cell growth, 143B.TK– cells and lymphoblastoid cell lines deriving from five symptomatic and five asymptomatic individuals carrying the mutation and from three controls (22,23) were grown in the special Dulbecco’s modified Eagle’s medium (DMEM) in the presence of 2 mg/ml paromomycin, or in its absence for 4 days. As shown in Figure 2, the population doubling times (DTs) of the different mutant cell lines were significantly higher than those of the controls, in confirmation of the previous finding (22). In particular, in the cell lines derived from symptomatic individuals, the ratios of DTs in the presence and absence of paromomycin were increased by an average (± SE) of 38 ± 3%, relative to the average DT ratio in the control cell lines (P < 0.0002). Similarly, in the cell lines derived from asymptomatic individuals, the DT ratios in presence and absence of the drug were increased by an average of 37 ± 4% (P = 0.0011). It is clear from these data that the sensitivities to the aminoglycoside of the growth rate of the cell lines derived from symptomatic and asymptomatic individuals were almost identical. The DT ratio in presence and absence of paromomycin was also measured in the mtDNA-less cells 
°206 (24), and found to be 1.15, close to the value measured in 143B.TK– cells (DT ratio = 1.10). These findings support the conclusion that the main target of the antibiotic in the mutant cell lines is the mitochondrial 12S rRNA.
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Mitochondrial protein synthesis defect
To examine whether a defect of mitochondrial protein synthesis occurs in the cell lines carrying the A1555G mutation after treatment with paromomycin, cells were grown for 4 days in special DMEM medium in the presence or absence of 2 mg/ml paromomycin, then labeled with [35S]methionine for 30 min in the presence of 100 µg/ml emetine in methionine-free special DMEM with or without paromomycin. Figure 3 shows typical electrophoretic patterns of the organelle-specific translation products of the mutant and control lymphoblastoid cell lines. The patterns of the mitochondrially synthesized polypeptides from the mutation-carrying cell lines grown in the presence or absence of paromomycin appeared to be qualitatively identical, in terms of both electrophoretic mobility and relative labeling of the various polypeptides, to those of the three control lymphoblastoid cell lines and 143B.TK– cells. However, all cell lines carrying the mutation and grown in the presence of paromomycin showed a clear tendency to a decrease in the overall rate of labeling of the mitochondrial translation products relative to that of the cell lines grown in the absence of the drug (Fig. 3a–e).
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Figure 4 shows a quantification of the results of a large number of labeling experiments and electrophoretic runs, which was carried out by densitometric analysis of appropriate exposures of the fluorograms and normalization to the data obtained for the 143B.TK– sample included in each gel. It appears from Figure 4a that the rate of labeling in the absence of paromomycin of the mitochondrial translation products in the mutant cell lines from symptomatic individuals was decreased relative to the mean value measured in the control cell lines by 40–68%, with an average of 56 ± 5% (P = 0.0058). The rate of labeling of the same products in the mutant cell lines from asymptomatic individuals was somewhat less decreased, the reduction ranging between 5 and 68%, with an average of 42 ± 10% (P = 0.0574).
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Most significantly, in the presence of paromomycin the reduction in the total rate of labeling of the mitochondrial translation products in the cell lines derived from symptomatic or asymptomatic individuals, relative to the mean value measured in the control cell lines in the absence of the drug (Fig. 4a), was more pronounced. In particular, as shown in Figure 4b, in the presence of paromomycin the decrease in rate of labeling of the mitochondrially synthesized polypeptides in the cell lines from symptomatic individuals ranged between 62 and 78%, with an average of 71 ± 3% (P = 0.001 for the significance of the difference from the mean rate in control cell lines in the presence of paromomycin), whereas in the cell lines derived from the asymptomatic individuals the rate ranged between 34 and 81%, with an average of 61 ± 8% (P = 0.0092). In contrast, the average rate of labeling of the mitochondrially synthesized polypeptides in the control cell lines in the presence of paromomycin decreased by only ~7% relative to the mean value measured in the control cell lines in the absence of the drug.
The decrease in total rate of mitochondrial protein synthesis observed in the presence of paromomycin in the mutant lymphoblastoid cell lines relative to the control cell lines was expected to result from the combination of the specific action of the aminoglycoside and of the inherent effect of the mutation. In order to determine the specific effect of paromomycin, the ratio, in each cell line, of the rate of labeling of the mitochondrially synthesized polypeptides in the presence of paromomycin to the rate measured in the absence of the antibiotic was calculated. As shown in Figure 4c, in the mutant cell lines from symptomatic individuals, the decrease in rate of labeling of the mitochondrially synthesized polypeptides in the presence of paromomycin, relative to the values in the absence of this antibiotic, was on average 35 ± 4%. The cell lines from the asymptomatic individuals exhibited a similar reduction in the rate of mitochondrial protein synthesis in the presence of paromomycin, relative to the values in the absence of the drug: this reduction was on average 33 ± 2%.
Correlation between growth defect and mitochondrial protein synthesis impairment in paromomycin-treated cells
In all mutant cell lines, the ratios of protein synthesis rate in the presence and absence of paromomycin (Fig. 4c) showed a decrease relative to the average ratio in the control cell lines. The reduction in the cell lines from symptomatic individuals ranged between 21 and 45% (average of 30 ± 4%; P = 0.0018). In this group of cell lines, compared with the control cell lines, the ratios of protein synthesis rates in the presence and absence of paromomycin showed a significant negative correlation with the ratios of DTs in glucose-containing medium (r = 0.80; P < 0.05). Likewise, the reduction in the ratios of protein synthesis rates in the presence and absence of the antibiotic in the cell lines from asymptomatic individuals, relative to the average ratio in the control cell lines, ranged between 22 and 35% (average of 28 ± 2%; P < 0.0001). In this group too, compared with the control cell lines, the ratios of protein synthesis rates in the presence and absence of paromomycin showed a significant negative correlation with the ratios of DTs in glucose-containing medium (r = 0.81; P < 0.05).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The A1555G transition in mitochondrial 12S rRNA, which is expected to create a new G-C base pair, makes the secondary structure of this RNA more closely resemble the corresponding region of the E.coli 16S rRNA. This region, highly conserved in different organisms, from bacteria to mammals (25), is an essential part of the decoding site of the small ribosomal subunit (26). This region is also an important locus of action of the aminoglycosides (3–8). Mutations, which disrupt the 1409–1491 base pair of E.coli 16S rRNA, corresponding to positions 1494 and 1555 of the human mitochondrial 12S rRNA, in fact confer aminoglycoside resistance, and have been shown to have effects on accurate translation (27,28). In human mitochondria, the new G-C pair in 12S rRNA created by the A1555G transition is expected to facilitate the binding of aminoglycosides (12). Recent evidence has indeed shown that the A1555G mutation in mitochondrial 12S rRNA increases the binding of these antibiotics (21). Our previous investigation (22) had revealed that the mutant lymphoblastoid cell lines derived from three asymptomatic individuals and five symptomatic individuals of the Arab-Israeli family, compared with the wild-type cell lines, exhibited a very similar, highly significant increase (30 and 26%, respectively) in DT ratios in the presence and absence of 1 mg/ml paromomycin, and a 16% increase in DT ratios in the presence and absence of 0.5 mg/ml neomycin (22). The results obtained in the present study concerning the effects of paromomycin at 2 mg/ml are in substantial agreement with these observations.
The A1555G mutation of mitochondrial 12S rRNA had been previously found to cause an overall decrease in the rate of mtDNA-encoded protein labeling in the mutant cell lines, after a short [35S]methionine pulse, with an average reduction of 48 or 28% in the cell lines derived from symptomatic or asymptomatic individuals, respectively, compared with the mean for the control cell lines (22). The results obtained in the present work have confirmed these observations. The fact that the presence or absence of the deafness phenotype is correlated with the severity of the biochemical defect suggests that the nuclear genes play an important role in the development of the clinical phenotype (22,29). By interacting with the mutated 12S rRNA or a ribosomal protein binding to the mutation site, the product(s) of a putative nuclear gene(s) could enhance the effect of the mutation, so as to produce the clinical phenotype, or suppress it so as to maintain normal hearing (22). The importance of the nuclear background in explaining the differences in biochemical phenotype between cell lines derived from asymptomatic and symptomatic individuals carrying the A1555G transition has been confirmed recently by mitochondria transfer experiments. In fact in the constant nuclear background of mtDNA-less °206 cells (24), almost identical average reductions in the rate of mitochondrial protein synthesis (37 and 35%), compared with the average value found in the transmitochondrial cell lines derived from three control individuals, have been observed in the cell lines derived from five symptomatic and, respectively, from the three asymptomatic individuals of the Arab-Israeli family (our unpublished data). These results strongly support the conclusion that the A1555G mutation, although it is the primary factor underlying the development of deafness, is not sufficient to produce the clinical phenotype.
In this study, we have examined whether a defect in mitochondrial protein synthesis occurred in lymphoblastoid cell lines derived from individuals carrying the A1555G mutation, compared with those derived from control individuals, when grown in the presence of 2 mg/ml paromomycin. Indeed, a strong decrease in the rate of mitochondrial protein synthesis in the presence of this aminoglycoside relative to the mean rate measured in the control cell lines in the absence of the drug, was found in the cell lines derived from symptomatic and asymptomatic individuals (an average decrease of 71 and 61%, respectively). These decreases were more pronounced than those observed previously (22) and in the present work in the absence of the drug (48–56 and 28–42% in the cell lines derived from symptomatic and asymptomatic individuals, respectively). No difference in the electrophoretic mobility or relative labeling of the mitochondrial translation products after the 30 min [35S]methionine pulse was observed between the mutant and the control cell lines. Moreover, pulse–chase experiments failed to show any evidence of differences in protein stability between the two groups of cell lines (data not shown). These data indicated that the A1555G mutation did not cause misincorporation of amino acids affecting the stability or electrophoretic mobility of the mitochondrially synthesized polypeptides in cells treated with paromomycin.
The contribution of the specific effects of paromomycin to the overall mitochondrial protein synthesis impairment detected in these experiments was determined by comparing the decreases in the overall rate of labeling of the mitochondrial translation products in the mutant cell lines treated with the aminoglycoside with the values obtained in the same cell lines not treated with the drug. A very significant average reduction of 30 or 28% in the ratios of rates of protein synthesis in the presence and absence of paromomycin was observed in the cell lines derived from symptomatic or asymptomatic individuals, respectively, compared with the average ratio in the control cell lines. These data indicated that the defect of mitochondrial protein synthesis specifically associated with the action of paromomycin was independent of the hearing status of the individual from which the cell line was derived. Conceivably this alteration of mitochondrial protein synthesis enhanced the inherent effects of the A1555G mutation, which produced a decrease in translation rate of only 28–42% in the cell lines from asymptomatic individuals, thereby causing a reduction in overall protein synthesis rate down to and below the minimal level required for normal cellular function. This threshold level appears to correspond to 40–50% of the normal rate of protein synthesis, as observed previously (22) and in the present work in the cell lines from symptomatic A1555G mutation-carrying individuals. Since mitochondrial protein synthesis is essential for the assembly of the oxidative phosphorylation apparatus in cell types with high oxidative phosphorylation demands, such as cochlear cells (9), a 50–60% decrease in mitochondrial protein synthesis rate can have disastrous consequences for cell function, so as to produce the clinical phenotype, as observed in the symptomatic individuals (22). Strong evidence for a tight control of respiration at the level of mitochondrial translation has recently been obtained in mouse cell lines (30).
In conclusion, the results reported here provide the first direct biochemical evidence that the mitochondrial 12S rRNA carrying the A1555G mutation is the main target of aminoglycosides, and that these antibiotics exert their effect through an alteration of mitochondrial protein synthesis, which aggravates the defect inherently caused by the mutation. To further elucidate the mechanism of aminoglycoside-induced deafness, it would be of particular interest to analyze the sensitivity to these antibiotics of the cochlear cells derived from symptomatic and asymptomatic individuals carrying the A1555G mutation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Cell cultures
A total of 13 human immortalized lymphoblastoid cell lines derived from 10 members of the Arab-Israeli family (22,23) [five asymptomatic individuals (F7H, F12C, F12D, F12E, F12H), five symptomatic individuals (F6H, F7C, F7D, F12F, F12J) and three married-in control individuals (#4, F7A, F7E)] were grown in specially made DMEM containing 1 mg/ml glucose, 0.11 mg/ml pyruvate and 0.36 mM CaCl2 (hereafter referred to as ‘special DMEM’), supplemented with 10% fetal bovine serum (FBS). The bromodeoxyuridine (BrdU)-resistant 143B.TK– was grown in regular DMEM (containing 4.5 mg of glucose and 0.11 mg/ml pyruvate), supplemented with 100 µg/ml BrdU and 5% FBS. The mtDNA-less
°206 cell line, derived from 143B.TK– (24), was grown under the same conditions as the parental line, except for the addition of 50 µg/ml uridine.
Growth analysis
To test the various cell lines for sensitivity to paromomycin, cells were grown for 4 days in special DMEM, supplemented with 10% FBS, in the presence or absence of 2 mg/ml of the antibiotic.
The population DT of the cell lines in special DMEM, supplemented with 10% dialyzed FBS, was determined from the growth curves or by using the formula (31):
DT = (t–t0)log2/(logN–logN0),
where DT is the doubling time, t and t0 are the times at which the cells were counted, and N and N0 are the cell numbers at times t and t0, respectively.
Analysis of mitochondrial protein synthesis
After incubation for 4 days in special DMEM in the presence or absence of 2 mg/ml paromomycin, samples of cultures of the mutant or control lymphoblastoid cell lines (~5 x 106 cells) and of 143B.TK– (6–8 x 105 cells) were labeled with [35S]methionine-[35S]cysteine (1175 Ci/mmol methionine; in total, 50 µCi/ml of medium) for 30 min, in the presence of 100 µg/ml emetine, in methionine-free special DMEM, supplemented with 10% dialyzed FBS, with or without 2 mg/ml paromomycin. After labeling, the cells were collected by centrifugation, washed twice with buffered saline lacking Mg2+ and Ca2+ (TD), finally resuspended in TD and frozen in aliquots. After protein determination, made by the Bradford method (32), samples containing equal amounts of protein (30 µg) from different lymphoblastoid cell lines, or 15 µg of protein from 143B.TK– cells, were run on SDS–exponential polyacrylamide gradient gels (33). The gels were treated with DMSO/PPO, dried and exposed for fluorography. Quantification of radioactivity was made by scanning all well-resolved peaks in appropriate exposures of the fluorograms with an LKB laser densitometer. For comparison of the data from different gels, the densitometric data obtained for the samples of lymphoblastoid cell lines in each gel were normalized to the data obtained for the 143B.TK– sample in the same gel.
Computer analysis
Variance analysis was carried out by the analysis of variance (ANOVA) test contained in the StatView program for Macintosh (version 5.0; SAS Institue) and entering individual replicate values.
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ACKNOWLEDGEMENTS |
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These investigations were supported by a Start-up Fund from Children’s Hospital Medical Center in Cincinnati to M.-X.G., research grant no. 5R01 DC01402-04 to N.F.-G. from the National Institute on Deafness and Other Communication Disorders, NIH, and NIH grant no. GM11726 to G.A. We are grateful to Dr Anne Chomyn for help in the statistical analysis and for critically reading the manuscript, we thank Benneta Keeley, Arger Drew and Rosario Zedan for technical assistance, and Debbie Williams for clerical support.
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FOOTNOTES |
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+ To whom correspondence should be addressed at: Division of Human Genetics, Children’s Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229, USA. Tel: +1 513 636 3337; Fax: +1 513 636 2261; Email: guar6n@chmcc.org
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REFERENCES |
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