Chemical Senses Advance Access originally published online on September 28, 2006
Chemical Senses 2007 32(1):21-30; doi:10.1093/chemse/bjl032
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Mutations in Olfactory Signal Transduction Genes Are Not a Major Cause of Human Congenital General Anosmia
1 Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel 2 Department of Human Molecular Genetics & Pharmacogenetics, MIGAL-Galilee Technology Center, Kiryat Shmona, Israel 3 Department of Otolaryngology–Head and Neck Surgery, Kaplan Medical Center, Rehovot, Israel 4 Outpatient Department and Research Unit, Geha Mental Health Center, Petach Tikva, Israel 5 Department of Otorhinolaryngology & Head and Neck Surgery, Meir Hospital, Kfar-Saba, Israel 6 Present address: Department of Internal Medicine, University of Texas Health Science Center, Houston, TX 77030, USA
Correspondence to be sent to: Prof Doron Lancet, Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel. e-mail: doron.lancet{at}weizmann.ac.il
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Abstract |
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Anosmia affects the western world population, mostly the elderly, reaching to 5% in subjects over the age of 45 years and strongly lowering their quality of life. A smaller minority (about 0.01%) is born without a sense of smell, afflicted with congenital general anosmia (CGA). No causative genes for human CGA have been identified yet, except for some syndromic cases such as Kallman syndrome. In mice, however, deletion of any of the 3 main olfactory transduction components (guanidine triphosphate binding protein, adenylyl cyclase, and the cyclic adenosine monophosphate–gated channel) causes profound reduction of physiological responses to odorants. In an attempt to identify human CGA-related mutations, we performed whole-genome linkage analysis in affected families, but no significant linkage signals were observed, probably due to the small size of families analyzed. We further carried out direct mutation screening in the 3 main olfactory transduction genes in 64 unrelated anosmic individuals. No potentially causative mutations were identified, indicating that transduction gene variations underlie human CGA rarely and that mutations in other genes have to be identified. The screened genes were found to be under purifying selection, suggesting that they play a crucial functional role not only in olfaction but also potentially in additional pathways.
Key words: anosmia, CNGA2, GNAL. ADCY3, linkage analysis, SNP
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Introduction |
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Olfactory deficits have a strong effect on human quality of life, including compromised personal safety and eating disorders (Hummel and Nordin 2005
). Studies in the western world suggest that 
1% of the human population suffers from olfactory disorders (Quint et al. 2001). Other studies show that as much as 5% of all individuals above the age of 45 suffer from anosmia (Bramerson et al. 2004; Landis et al. 2004). Most of the affected people have an acquired condition, which develops during life, due to allergy, viral upper respiratory tract infection, nasal sinus diseases, head trauma, inhalation of noxious chemicals, or medicinal drug intake (Zusho 1982; Apter et al. 1999). A much smaller minority is born without a sense of smell, a rare condition. This may be isolated, appearing as the only deficit observed (Online Mendelian Inheritance in Man [OMIM] %107200), referred to here as congenital general anosmia (CGA). Alternatively, it is syndromic, appearing in conjunction with other deficits or anomalies, as exemplified by the well-studied Kallmann syndrome (OMIM +308700), where general anosmia, secondary to olfactory bulb agenesis, appears together with hypogonadotropic hypogonadism.
The prevalence of CGA has been roughly estimated as 1 in 10 000 (Quint et al. 2001; Temmel et al. 2002). Because there is no positive test for CGA, diagnosis has been based on the complete lack of the sense of smell for an entire lifespan, null performance in olfactory sensitivity tests, and the lack of any alternative medical explanation. Using cranial magnetic resonance imaging (MRI) on CGA patients, morphological abnormalities have been shown to include hypoplastic or aplastic olfactory bulbs and hypoplasia in the olfactory tract in combination with shallow olfactory sulcus (Abolmaali et al. 2002). Biopsies of the olfactory region have found normal respiratory epithelium but total or partial lack of olfactory epithelium (Jafek et al. 1990; Leopold et al. 1992). Interestingly, the olfactory epithelium of 2 anosmic patients with Kallmann syndrome included functionally mature olfactory neurons, suggesting that they are able to differentiate in the absence of fully developed olfactory bulbs (Rawson et al. 1995).
Most of the CGA cases reported so far were sporadic, and only a few were familial. In a previous study that included 22 patients, 8 of them had a familial history of CGA (Leopold et al. 1992). Other sporadic cases have been described mostly in children and adolescents (Vowles et al. 1997; Assouline et al. 1998; Ho and Carrie 2001; Nishida et al. 2004). The published familial cases include a large 4-generation family with 27 affected individuals from the Faroe Islands (Lygonis 1969) and 8 American familial cases described by Leopold (Leopold et al. 1992). More recently, linkage analysis in 2 Iranian families with 9 affected members suggested a 46 cM linkage interval on chromosome 18 (Ghadami et al. 2004). Although the total number of familial CGA cases, reported so far, is small, all the published pedigrees suggest an autosomal dominant mode of inheritance with partial penetrance (Leopold et al. 1992; Ghadami et al. 2004).
To date, no causative gene has been described for isolated human CGA. For some syndromic cases, such as Kallmann and Bardet-Biedl syndromes (OMIM #209900), causative genes have been identified (Keith 1984; Franco et al. 1991; Dode et al. 2003). Interestingly, one mutation in KAL1, the gene responsible for the X-linked form of Kallmann syndrome, was reported to cause either hypogonadism and anosmia or isolated anosmia in 2 brothers (Parenti et al. 1995), making KAL1 a suitable CGA candidate gene. The causative genes for the above-mentioned syndromes encode for developmental (Hardelin 2001) or morphology determining factors (Kulaga et al. 2004) but not for primary transduction pathway components.
In contrast, in a mouse model, inactivation of the 3 major transduction components in the primary sensory neurons revealed behavioral phenotypes consistent with general anosmia. The deleted components included a subunit of the olfactory cyclic nucleotide–gated cation channel (Cnga2) (Brunet et al. 1996), the alpha subunit of a stimulatory olfactory G-protein (Gnal) (Belluscio et al. 1998), and the cyclic adenosine monophosphate–generating enzyme adenylyl cyclase type III (Adcy3) (Wong et al. 2000).
In an attempt to further elucidate the genetic basis of CGA, we have collected 66 CGA families (83 affected subjects) and applied 2 complementary genetic approaches, whole-genome screen for the largest families and direct mutation detection for 3 candidate genes GNAL, CNGA2, and ADCY3 in the entire cohort. We were able to identify neither a linkage interval for this phenotype nor mutations in the 3 transduction genes. We conclude that these 3 genes are not likely to constitute a major genetic basis for CGA.
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Materials and methods |
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Subjects studied
All studies were conducted with the approval of The Institutional Review Board for human experiments in the Meir Hospital, Kfar Saba, Israel, protocol no. T15601. [GenBank] Participants in the present study signed a consent form. Potential anosmic cases were verified by the medical individual's history, physical examination, and olfactory sensitivity screening test. Determination of CGA in deceased persons was according to their relative's recollection. Forty-three anosmic males, 40 anosmic females, and 186 normal family members were recruited, and blood samples or buccal swabs were obtained. Additional control DNA samples were obtained from the National Laboratory for the Genetics of Israeli Populations at Tel-Aviv University (www.tau.ac.il/medicine/NLGIP/nlgip.htm).
Olfactory sensitivity screening
Olfactory sensitivity screening was carried out using a forced-choice 3-way bottle test (Gross-Isseroff et al. 1989, 1992; Fortier et al. 1991). We used 2 odorants (isoamyl acetate and Eugenol, 30 µl/ml) dissolved in mineral oil, avoiding excessively high concentrations of odorants that might lead to trigeminal perception. We used also Eugenol because isoamyl acetate can stimulate the trigeminal nerve at extremely high concentrations (Doty 1975; Doty et al. 1978). The use of both odorants assured that general anosmia would be missed only in individuals with unusually high trigeminal sensitivity for both of the odorants used. We note that only one individual was anosmic to Eugenol but responded correctly to isoamyl acetate, perhaps due to trigeminal stimulation.
For each odorant, 8 trials were conducted. On each trial, subjects were presented with one stimulus and 2 solvent-only blanks. Subjects were asked to sniff and choose (if necessary, guess) the different odor among the 3 samples. Subjects performing at chance level, guessing correctly less than 4 times, were considered anosmics.
Genome scan with markers and linkage analysis
Genomic DNA was extracted from whole blood, whenever available, otherwise buccal swabs were obtained. A genome scan using 400 microsatellite marker loci with an average distance of 10 cM (Linkage Mapping Set v2-MD10, Applied Biosystems, Foster City, CA) was performed, samples run on a DNA sequencer (Model 3100, Applied Biosystems) and allele calling assigned using the software Genescan and Genotyper.
The power to detect a linkage interval was calculated utilizing the slink and msim programs from the LINKAGE package software (http://linkage.rockefeller.edu) (Lathrop and Lalouel 1984; Lathrop et al. 1984), assuming an autosomal dominant inheritance with incomplete penetrance (90%) and a disease allele frequency of 0.001. Slink simulated 100 different genotypes for the given pedigrees under the assumption of linkage. The simulated genotypes were analyzed for linkage in each pedigree.
Two-point linkage analyses were performed using the software package LINKAGE as above, under the assumption of a dominant autosomal model with a partial penetrance of 80% or 90%. Allele frequency for each marker was set as 1/N (where N is the observed number of alleles).
Polymerase chain reaction amplification
Fragments covering the entire coding region and exon/intron boundaries of the 3 candidate genes ADCY3 (NM_004036 [GenBank] .2), CNGA2 (NM_005140 [GenBank] .1), and GNAL isoforms (NM_182978 [GenBank] .1 and NM_002071 [GenBank] .1) were amplified from genomic DNA samples. Two additional exons were predicted in the gene CNGA2 using the partial mRNA AK128186 [GenBank] and the Genescan software (http://genes.mit.edu/GENSCAN.html) and amplified as above. Amplification primers were designed using Oligo software (Molecular Biology Insights, Inc., Cascade, CO) or Primer3 web server (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi). Primer sequences and polymerase chain reaction (PCR) conditions for each amplicon are summarized in Supplementary Table 1. Amplification was performed in a 50-µl reaction volume, using HotStart Taq polymerase (Qiagen, Hilden, Germany), under standard cycling conditions.
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Single nucleotide polymorphism discovery by denaturing high-performance liquid chromatography
For single nucleotide polymorphism (SNP) discovery, 46 fragments, covering the coding regions and exon/intron boundaries of the 3 candidate genes, were analyzed by denaturing high-performance liquid chromatography (DHPLC, using Transgenomic Wave DNA fragment analysis system) (Bercovich and Beaudet 2003; Hasin et al. 2004). This method has been reported to have a sensitivity and specificity exceeding 96% (Xiao and Oefner 2001). In addition, its false-negative rate in several studies was 0 and its false-positive rate was 3% (Colosimo et al. 2002; Ravnik-Glavac et al. 2002; Wu et al. 2003).
The screening was performed in 64 unrelated anosmic individuals (128 chromosomes) and 3 normal control individuals (6 chromosomes) for the genes ADCY3 and GNAL, and in 41 anosmic (41 chromosomes) males and one nonanosmic control (1 chromosome) for CNGA2. Nonsynonymous polymorphisms in the last gene were also screened in 48 normal unrelated individuals, 24 Ashkenazi Jews and 24 Sepharadi Jews. DNA alteration analysis was performed using a WAVE apparatus from Transgenomic Inc. (Omaha, NE). The PCR products were denatured at 95 °C for 5 min and cooled to 65 °C at a temperature gradient of 1 °C/min. The samples were kept at 4 °C until 5 µl were applied to a preheated C18 reversed-phase column based on nonporous poly (styrene-divinyl-benzene) particles (DNA-Sep Cartridge, CAT no. 450181; all DHPLC catalog numbers are from Transgenomic Inc.). DNA was eluted within a linear acetonitrile gradient consisting of buffer A (0.1 M triethylammonium acetate [TEAA], CAT no. SP5890) and buffer B (0.1 M TEAA, 25% acetonitrile, CAT no. 700001). Temperature of heteroduplex detection was deduced from the Transgenomic software (Wavemaker 4.2) and Stanford DHPLC melting program (http://insertion.stanford.edu/meltdoc.html), which analyzes the melting profile of the specific DNA fragment. All the found SNPs were submitted to dbSNP (http://www.ncbi.nlm.nih.gov/SNP/index.html), and their identifiers are NCBI_ss# 52084260-81.
DNA sequencing
For resequencing, PCR fragments were reamplified from the corresponding genomic DNA sample and subjected to direct sequencing using dye terminators. The sequencing reaction was performed at 56 °C, using PCR primers for both the forward and reverse strands. Sequence comparisons and SNP visualization were performed using the Sequencher software (Gene Codes Corporation, Ann Arbor, MI). Each SNP was identified in at least 2 independent PCR amplifications and appeared in at least 2 sequencing reactions.
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Results |
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Population recruitment
The population recruitment was done by advertisements in major newspapers in Israel. A comprehensive examination to exclude cases of acquired general anosmia included an extensive questionnaire about the medical history of each subject, physical examination, and olfactory sensitivity testing. Following the examination, 83 CGA subjects within 66 families were recruited, the largest CGA cohort reported. Based on an estimate of direct and indirect readership of 2 million and of 50% response, this amounts to a CGA prevalence of 1 in 10 000. This is in general agreement with previous estimates of 3% CGA among anosmics (Temmel et al. 2002), which in themselves are estimated as 1% in the general population (Quint et al. 2001). The ethnic segregation of the CGA subjects (40 females and 43 males) and number of affected individuals per family are presented in Figure 1 (see also Supplementary Table 2).
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Linkage analysis
Whole-genome linkage analysis with 400 microsatellite markers was utilized in an attempt to identify genetic markers associated with anosmia. It was performed in families A002 (4 affected individuals) and A003 (5 affected individuals), assuming a dominant mode of inheritance. For these CGA families, such mode is more probable than a recessive one because there are affected individuals in every generation and the assumption of 3 carriers outside the nuclear families in each pedigree is considerably less parsimonious. Partial penetrance is suggested by the following observations (Figure 2): 1) An obligatory carrier was found to be healthy (Family A002, II-2); 2) a hyposmic individual is seen (Family A002, III-2); 3) in generation III of families A002 and A003, only 4 anosmics are seen out of 15 individuals whose parents were anosmics, considerably less than the expected 50%.
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The power to detect a linkage interval in these families was calculated. The probability of reaching a logarithm of the odd (LOD) score of 3 or higher in family A002 was 11% and for family A003 was 33%. Although the power was very low, especially for family A002, the maximum expected LOD scores were 3.7 and 3.1 for families A002 and A003, respectively.
In a first round, genotypes were determined for partial cohorts (Figure 2). Two-point LOD scores were computed employing a dominant autosomal model, with partial penetrance of 80% and 90%. Each family was analyzed separately because CGA is presumed to be genetically heterogeneous, that is, the causative gene in each family could be different. The 9 markers with the highest LOD score values (all ranging between 1.0 and 1.5) were further analyzed in the second step, where the remaining family members, for whom only buccal swabs were available, were genotyped. The newly calculated LOD score values remained similar to the previous ones, (see Supplementary Table 3). Given the possibility that the hyposmia of individual III-2 in family A002 (Figure 2) may have a different cause than the anosmia in the rest of the family, we performed the linkage analysis 3 times, defining individual III-2 as affected, unaffected, and of unknown phenotype. All calculated LOD scores were comparable.
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In view of the following facts, 1) LOD scores did not improve when including in the analysis additional family members in the second step and 2) the highest LOD scores observed were supported by a single marker in each genomic location, we believe these LOD scores are not significant. Combining the LOD scores of the 2 families resulted in maximum LOD scores around 1.7 because the maximum for each family correspond to different genomic locations. In addition, none of these 9 markers were located in the vicinity of potential CGA candidate genes (Table 1). Thus, the results of the genome scan in these 2 families could not define a linkage interval in any genomic location. This result is not surprising in view of the small size of the recruited CGA families and the observed partial penetrance that limited the power of the genetic analysis.
X-linked genetic analyses
Family A001 has 3 affected grandsons descendant from an affected maternal grandfather, hence an X-linked mode of transmission seemed possible, and linkage to chromosome X was tested in this family. Notably, 2 CGA candidate genes, CNGA2 and KAL1, are located on chromosome X. We genotyped 18 microsatellites located along chromosome X in the 3 anosmic siblings and their mother and inferred haplotypes for the mother. Under the parsimonious assumption of no double recombination in 2 siblings, in the region of 50 Mb or less in which the mother is homozygous for 3 contiguous microsatellites, no common haplotype for any region on chromosome X was found in the 3 anosmic siblings (Figure 3). This result excludes the hypothesis that CGA in family A001 might be caused by a gene located on chromosome X.
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Mutation screening by denaturating chromatography in candidate genes
Candidate genes could include genes involved in olfactory development, in olfactory transduction and its termination, in higher levels of olfactory signal processing, and additional genes controlling their expression (Table 1). Some of the genes are considered to be olfactory specific, thus mutations in these components are expected to result in only a sensory deficit.
We performed direct mutation screening in and near exons of the 3 major CGA candidate genes, CNGA2, ADCY3, and GNAL. A total of 46 amplicons covering the coding regions and exon/intron boundaries of the 3 genes were screened by DHPLC (Figure 4). The amplicons of the autosomal genes, ADCY3 and GNAL, were screened in 64 unrelated anosmic individuals and 3 normal control individuals, whereas the X-linked gene CNGA2 was screened in 41 anosmic males and 1 control. An additional control group was added to the screening whenever necessary. Each of the distinct DHPLC elution pattern was verified by resequencing.
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Twenty-two single nucleotide variations were found, of which 12 were in introns or UTRs. The remaining 10 variations were in exons, 6 synonymous and 4 nonsynonymous (Tables 2 and 3). Only 5 of the 22 SNPs were novel polymorphisms, and the rest are included in genomic SNP databases, 3 of them only in Celera Genomics and 14 in dbSNP (Figure 4). All the variations were excluded from being disease-causing mutations as they were found also in the controls. Although for most of the amplicons, the number of controls was small (6 chromosomes), the SNP allele frequencies in anosmic and controls were similar. Our inability to detect causative mutations indicates that for the present CGA cohort, the mutations frequency is less than 2.3% in the ADCY3 and GNAL genes and less than 7.1% in the CNGA2 gene (P value = 0.05).
Interestingly, the number of SNPs found in the 3 major candidate genes is lower than the number observed in a survey performed on 313 genes, where one SNP per 300 bp and one nonsynonymous SNP every 450 bp were detected in coding regions (Stephens et al. 2001; Salisbury et al. 2003; Pungliya et al. 2004) (Table 2).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionSupplementary materialAcknowledgementsReferences |
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We described an effort to uncover the genetic basis of isolated CGA, a sensory deficit that has so far been only scantly analyzed genetically. We describe 66 CGA families that include 83 anosmic individuals, to our knowledge, the largest CGA cohort reported to date. Both the familial distribution of CGA individuals and the estimated prevalence were in agreement with previous studies (Leopold et al. 1992
; Quint et al. 2001; Temmel et al. 2002). We found anosmia cases to be equally represented in all Jewish ethnic groups, and no significant differences were observed between males and females.
Other sensory deficits such as deafness and blindness had been much more extensively studied than CGA. They were typically found to be genetically heterogeneous and showed diverse modes of inheritance, including autosomal recessive, autosomal dominant, and X-linked (Bessant et al. 2001; Petit et al. 2001; Tekin et al. 2001), typically with full penetrance. Similarly, it is not unlikely that CGA would be a genetically heterogeneous condition, with different modes of inheritance for distinct causative genes and characteristically with full penetrance. However, a partial penetrance is observed here in the CGA pedigrees. This reduced penetrance has been previously noted in a 4-generation pedigree from Faroe Islands (Lygonis 1969) and 2 extended Iranian families (Ghadami et al. 2004). These repeated observations hint that CGA might not be an autosomal dominant affection, but an oligogenic one, requiring mutations in two or more loci. The failure to find a linkage interval in the CGA pedigrees could support an oligogenic model of inheritance but also be the result of an underpowered study. Oligogenic inheritance has been reported in other cases of disorders previously thought to be monogenic such as Bardet-Biedl syndrome and Hirschsprung disease (Badano and Katsanis 2002; Gabriel et al. 2002; Katsanis 2004; Van Heyningen and Yeyati 2004).
The 3 main olfactory transduction genes (GNAL, CNGA2, and ADCY3) were screened in the reported CGA cohort because they have been shown to be essential components of olfactory transduction in mice (Brunet et al. 1996; Belluscio et al. 1998; Wong et al. 2000). Mutations in other transduction genes have been related to a number of known inherited diseases, supporting a potential role for components of the olfactory transduction pathway in underlying CGA. Thus, G-protein, adenylyl cyclase, and calcium channel genes have been shown to be involved in developmental abnormalities of bone, hormone resistance and hormone hypersecretion, migraine, and several types of ataxia (Barrett et al. 1989; Abdel-Halim et al. 1998; Pietrobon 2002; Spiegel and Weinstein 2004). Likewise, mutations in each of the genes encoding visual transduction proteins may cause retinal dystrophy (Hims et al. 2003).
Mutations in the 3 major olfactory candidate genes do not seem to be a major cause of human CGA. Although regulatory regions were not screened, it is not likely that the whole anosmic cohort has mutations in regulatory but not in coding regions. Large deletions including one or more exons could disrupt one of the genes under study but would be not detectable by DHPLC.
The ratio of human–mouse divergence at nonsynonymous (amino acid replacement) sites versus synonymous sites (Ka/Ks <<1) (data not shown), together with the low number of SNPs observed in these genes, might hint that the genes are under purifying selection. Purifying selection might indicate that they have a conserved and vital function, olfaction related or otherwise. Examples for such nonolfactory vital functions might be the role of Gnal in the dopamine and adenosine receptor function in mouse basal ganglia (Corvol et al. 2001) and an association between promoter point mutations in Adcy3 and decreased insulin release in a rat model of type 2 diabetes (Abdel-Halim et al. 1998).
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Supplementary material |
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Supplementary Tables 1–3 can be found at http://www.chemse.oxfordjournals.org or at http://bioportal.weizmann.ac.il/HORDE/publications/CGA/.
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Acknowledgements |
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D.L. holds the Ralph and Lois Silver Chair in Human Genomics. This research is supported by the Crown Human Genome Center, the Abraham and Judy Goldwasser Fund, the Israel Ministry of Science and Technology, and the USA National Institute of Deafness and other Communication Disorders.
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Accepted 4 September 2006
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