Phenotype–Genotype Correlation in 295 Chinese DeafSubjects with Biallelic Causative Mutations in the GJB2 Gene

Fei-Fan Zhao,1 Yu-Bin Ji,2,* Da-Yong Wang,1 Lan Lan,1 Ming-Kun Han,1 Qian Li,1 Yali Zhao,1

Shaoqi Rao,3 Dongyi Han,1 and Qiu-Ju Wang1

Aims: The connexin 26 coding gene (GJB2) is the primary causative gene for nonsyndromic sensorineural hearingimpairment (NSSHI). More than 100 mutations in this gene have been reported to be linked to hearing im-pairment (HI), from mild to profound hearing loss. To precisely estimate the impact of GJB2 mutations in theChinese population, a cross-sectional study was performed to analyze the auditory data of Chinese patients withNSSHI. Results: Two hundred ninety-five unrelated patients with NSSHI with biallelic mutations in GJB2 wererecruited from seven provinces in Northern China from 2004 to 2008. The levels of HI and average pure toneaudiometry were compared across different genotypes by v2 testing. The subjects with the genotypes of com-bined truncating mutations had more cases of severe HI than the subjects with a genotype of several non-truncating mutations. It was also revealed that subjects carrying either c.[79G > A; 341A > G] + [79G > A;341A > G] or c.[109G > A] + [79G > A; 341A > G] had significantly fewer cases of severe HI than the referencegroup of homozygous c.235delC, whereas the subjects carrying c.[235delC] + [176_191del16] had more cases ofsevere HI than the homozygous c.235delC group. Conclusions: This is the first study to clarify the correlationsbetween different GJB2 biallelic genotypes and NSSHI phenotype in the Chinese population. The Chinesesubjects with two truncating mutations in GJB2 were shown to correlate with more severe HI.

Introduction

Hearing loss is a frequent congenital disease with a highoccurrence of 1 in 1000 newborns, and 2 or 3 in 1000 will

develop sensorineural hearing impairment (HI) before the ageof 5 years old (Hilgert et al., 2009). The GJB2 gene, the majormolecular underpin for nonsyndromic sensorineural hearingimpairment (NSSHI), was found to have mutations in morethan 50% of the patients with recessive inheritance in differentpopulations (Gabriel et al., 2001; Li et al., 2007; Putcha et al.,2007; Usami et al., 2008). Now, up to 100 loci in this gene havebeen reported to relate to deafness, in which several hotmutations appeared commonly but distinctly in differentethnic groups. c.[30_35delG] is the primary causative muta-tion with a high carrier rate of 1.89% in European, whereasc.167delT is the leading one in Ashkenazi Jews and Palesti-nians (Kelsell et al., 1997; Shahin et al., 2002). In Indians, threemutations (c.71G > A, c.231G > A, and c.370C > T) are more

frequent (Rickard et al., 2001). Finally, in Koreans, Japanese,and Chinese, c.235delC is the leading mutation (Guo et al.,2008; Lee et al., 2008; Usami et al., 2008).

Some mutations were verified to be pathogenic by func-tional studies in vitro, although the resulting phenotypes weremild HI, which included p.Val37Ile, p.Met34Thr, p.Leu90Pro,p.Arg184Pro, p.Trp77Arg, c.[-23 + 1G > A], and p.Arg127His.Nevertheless, some mutations were thought to be polymor-phisms of high allelic frequency in carriers, such as [p.Va-l27Ile;p.Glu114Gly], but some functional studies found thatcalcium conductance in the cells transfected by plasmidscarrying this mutation was damaged because of dysfunctionof the gap junction protein (Bruzzone et al., 2003; Pilipenkoet al., 2007). Despite these contradictory results, the study ofc.250G > C (p.Val84Leu) (Beltramello et al., 2005) threw a lighton new ways to explore functions of the gap junction protein,which revealed a key function of inositol triphosphate (IP3) indeafness etiology.

1Department of Otorhinolaryngology/Head and Neck Surgery, Chinese People’s Liberation Army Institute of Otolaryngology, ChinesePeople’s Liberation Army General Hospital, Beijing, China.

2Department of Otorhinolaryngology/Head and Neck Surgery, The People’s Liberation Army Second Artillery General Hospital, Beijing,China.

3Department of Medical Statistics and Epidemiology, School of Public Health, Guangdong Medical College, Dongguan, Guangdong,China.

*Joint first author.

GENETIC TESTING AND MOLECULAR BIOMARKERSVolume 15, Number 9, 2011ª Mary Ann Liebert, Inc.Pp. 619–625DOI: 10.1089/gtmb.2010.0192

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For the crucial role of GJB2 in etiology of NSSHI, severalstudies have been performed to reveal the correlation betweengenotypes and the phenotype clinically. It was clarified thatcombination of biallelic truncating mutations (frameshift,nonsense, insertion, and deletion) would lead to more severehearing loss than those of two nontruncating mutations(missense, deletion, or insertion more than 3 times’ nucleo-tides) in combination in the Caucasian and Japanese popula-tions (Martini and Mazzoli, 1999; Oguchi et al., 2005; Yuanet al., 2007). However, this hypothesis has not yet been veri-fied, although it is known that the Chinese population has thedistinct spectrum of GJB2 mutations compared with otherpopulations. Therefore, we enrolled and studied 295 Chinesedeaf subjects carrying GJB2 biallelic mutations to evaluate thecorrelations between genotype and phenotype. These resultswould help precisely estimate the impact of GJB2 mutations inthe Chinese population.

Materials and Methods

Subject recruitment

Subjects with NSSHI were sequentially recruited from ourclinic from 2004 to 2008. Individuals with syndromic, unilateral,acquired, or dominant types of HI were excluded from thisstudy. The clinical information comprised a complete history,physical examinations, and the audiometric examinations. Somealso received radiological examinations to exclude dysplasia.

A total of 307 subjects with NSSHI who were screened withbiallelic GJB2 mutations came from 7 provinces in NorthernChina, of whom 12 subjects were excluded because their au-diometric data were incomplete, such as only data from theauditory brainstem response (ABR) test were available, whichdid not provide complete frequency-specific thresholds. Toavoid potential biases, no familial subjects with identical ge-notypes were enrolled. Finally, detailed audiometric datafrom 295 persons were analyzed in this study. Informationconsent, blood samples, and clinical evaluations were ob-tained from all the participants according to the protocolsapproved by the review boards of the ethics committees of theChinese People’s Liberation Army General Hospital.

Audiological analysis

All subjects in this study were tested by pure tone audi-ometry and acoustic impedance admittance measurements ina sound proof room. Two hundred seventy subjects also re-ceived auditory brainstem response (ABR), and 65 receiveddistortion product otoacoustic emissions (DPOAE). Sixty-foursubjects who did not respond well to ABR also received au-ditory steady-state response (ASSR) tests. According to thepure tone averages (PTA) at frequencies from 0.5 kHz to 4 kHz(PTA0.5–4 kHz), average thresholds in the range of 21–40 dBwere defined as ‘‘mild HI,’’ 41–70 dB as ‘‘moderate HI,’’ 71–95dB as ‘‘severe HI,’’ and > 95 dB as ‘‘profound HI’’(Martini andMazzoli, 1999).

GJB2 mutations analysis

All subjects were subjected to DNA sequencing of two ex-ons in the GJB2 gene. However, two loci were not examined,which included c.[del(GJB6-D13S1830)], because it was sel-dom present in Chinese population (Yuan et al., 2007), andc.[-23 + 1G > A]. The causative mutations listed on the

Connexin-Deafness HomePage and several controversialvariants (e.g., c.109G > A (p.Val37Ile) and c.[79G > A;341A > G]([p.Val27Ile; p.Glu114Gly]) were checked. In addi-tion, some novel mutations with unclear functions werealso screened, including missense mutations and insert mu-tations. Based on their functional implications (i.e., translatedproteins), these loci were classified as nontruncating ortruncating mutations. Missense mutations and deletionsof 3 bp that resulted in a deletion of an amino acid weredefined as nontruncating (NT) type. Similarly, splice sitemutations, insertions, nonsense mutations, duplications, anddeletions of more than 3 bp were defined as truncatingmutations (T).

Statistical analysis

The linear regression of PTA0.5–4 kHz on age was performedto find out whether progression exists in a major genotype.Pearson’s v2 testing using the HI classification of mild, mod-erate, severe, and profound was performed to analyze whe-ther the frequency distribution of HI was randomized indifferent genotype categories (T + T, T + NT, and NT + NT).Then, Fisher’s exact probability testing with 2 · 2 contingencytables of appropriately dichotomized data was performed tofind out the most frequent class of HI in each category. Here,PTA0.5–4 kHz as the grouping variable, the distribution fre-quency of each genotype was compared with the homozy-gous c.235delC group by Fisher’s exact probability testing,which was applied with 2 · 2 contingency tables of appro-priately dichotomized data at the median level (75th percen-tile [P75], 50th percentile [P50], 25th percentile [P25], 5thpercentile [P5]) of the reference group.

Result

Sample information

Totally, 295 subjects (107 women and 188 men) with NSSHIwere found to have two mutations in the GJB2 gene. Agesranged from 5 months to 60 years, with the median age of 14,and the majority was within the age range of 2–21 years. Mostof the subjects were of Han ethnicity, and some were minoritygroups, comprising of four Uighurs (1.3%), two Mongolians(0.7%), one Tibetan (0.3%), and two Hui (0.7%). Since very fewminority were included, we did not separate the analysis byrace. The difference of PTA0.5–4 kHz between two ears wasfrom 0 to 27dB in the sample and mostly less than 15 dB.

Spectrum of mutations

The most frequent loci included six frameshift mutationsc.235delC, c.[299_300delAT], c.[30_35delG], c.[30_35insG],c.[176_191del16], and c.[504_505insAAGG] and three missensemutations c.[79G > A;341A > G] ([p.Val27Ile;p.Glu114Gly]) andc.109G > A(p.Val37Ile). The remaining were several muta-tions with low frequency, including a nonsense mutationc.9G > A(p.Trp3X), three missense mutations c.257C > G(p.Thr86Arg), c.21G > A (p.Gln7Gln), c.427C > T (p.Arg143Trp),and a frameshift mutation c.[559_604dup46].

Spectrum of genotypes

There were totally 19 different genotypes, of which 6 werehomozygous and 13 were compound heterozygous. Eleven

620 ZHAO ET AL.

genotypes were found in more than two subjects (Fig. 1), andthe other 8 genotypes appeared in only one subject (Table 1).In all patients with biallelic mutations, 131 (45.9%) patientshad homozygous c.235delC, and the allele frequency ofc.235delC was 61.9% (365/590), similar to the ratio in theJapanese (Tsukada et al., 2010). However, a study on Chinesepatients showed that frequency of c.235delC carrier in pa-tients and in control group was 22.5% and 1%, respectively(Shi et al., 2004).

Results of DPOAE examination

Sixty-four sporadic subjects were screened with DPOAE.Fifty-four had uncertain conclusions because of their severehearing loss beyond the upper limited threshold for DPOAE.The DPOAE was not recordable in the other ten subjects withPTA0.5–4 kHz less than 60 dB, in whom homozygous c.235delCwere present in six subjects, c.[235delC] + [299_300delAT] inthree, and c.[235delC] + [176_191del16] in one.

Onset age of HI in specific genotypes

The onset age was available in 199 subjects, ranging from 0to 7 years, mainly during the 0–3 years, that is, prelingual. Thenewborns who did not pass the hearing screening were de-fined as onset on 0 year old. There were 11 subjects withpostlingual deafness in this study, and their ages of onsetranged from 4 to 7 years. The genotypes represented inpostlingual deafness included c.[235delC] + [235delC] whichwere present in four subjects, c.[235delC] + [299_300delAT] inthree, c.[109G > A] + [79G > A; 341A > G] in one, c.[79G > A;341A > G] + [79G > A; 341A > G] in two, and c.[30_35delG] +[299_300delAT] in one.

Relationship between age growth and HI

No linear regression was found between the ages and thePTA0.5–4 kHz in each specific genotype. So, no progression ofHI was found in this sample while aging.

Distribution of HI among different genotype categories

Data from 287 subjects were shown in Figure 2. Only ge-notypes of the loci that were present in more than two subjectswere analyzed. Per the nature of the mutations, these geno-types were further grouped into two categories, T + T andNT + NT. As shown in Figure 2, HI values in genotype cate-gories were randomly distributed (v2 = 3.39, p = 0.183). Then,the 2 · 4 contingency (2 meant genotype category, and 4meant HI levels) tables were reduced into 2 · 2 contingencytables to find the distribution frequency of each HI level ineach category, and T + T category was found to have morecases of profound HI than the NT + NT category ( p = 0.074).

FIG. 1. A scatter diagram ofthe PTA0.5–4 kHz of groupswith specific genotypes. ‘‘n’’indicates the number of eachspecific genotype group.‘‘P50’’ indicates the medianPTA0.5-4KhZ of each group.p-values were calculated us-ing v2 test by comparing eachspecific group to c.235delChomozygotes group as thereference. The threshold datawere dichotomized at 108dB(P75 of the reference group),100dB (P50), 86dB (P25), and66dB (P5). PTA, pure toneaverage.

Table 1. The Comparison of PTA Between

the Eight Genotypes Present in One Subject

and the Homozygous c.235delG

Genotype PTA p

NT/NT:c.[427C > T] + [427C > T] 106 1c.[109G > A] + [109G > A] 83 0.25T/NT:c.[235delC] + [109G > A] 113 0.25c.[9G > A] + [257C > G] 98 1c.[235delC] + [79G > A;341A > G] 97.5 1c.[235delC] + [21G > A] 87.5 1T/T:c.[176_191del16] + [176_191del16] 100 1c.[299_300delAT] + [559_604dup46] 103 1

The c.257C > G (p. Thr86Arg) mutant protein did not form gapjunctions. [11] c.9G > A(p.Trp3X) was a nonsense mutation, andc.21G > A(p.Gln7Gln) was a missense mutation. The pathogeniceffect of the latter mutations remained unclear.

Fisher exact probability test was applied, and P values werecalculated by comparing the median PTA0.5–4 kHz of each genotypewith the median PTA0.5–4 kHz (108dB[*P75 of the reference group],100dB[*P50], 86dB[*P25], and 66dB[*P5] of the reference group).

PHENOTYPE–GENOTYPE CORRELATION OF GJB2 MUTATIONS 621

Comparison of HI between other GJB2 genotypesand homozygous c.235delC

First, we delineated the distribution of PTA of 287 subjectsin 11 genotypic groups found in more than two subjects (Fig.1). Then, the P50 PTA for each group was compared with thereference group (homozygous c.235delC). Though NT + NTcategory was not significantly related with the less HI com-paring with the T + T category, there were two genotypes(c.[79G > A; 341A > G] + [79G > A; 341A > G], c.[109G > A] +[79G > A; 341A > G]) representing significantly less HI thanthe homozygous c.235delC. The group with c.[235delC] +[176_191del16] represented more severe HI than the referencegroup.

In the groups with only one subject, Fisher’s exact proba-bility testing was applied to compare the median PTA0.5–4 kHz

with the reference group (Table 1).Though P values did notimply significant difference with the homozygous c.235delCgroup, the conclusion could not be drawn certainly because ofthe small sample size.

Discussion

Correlation between GJB2 genotypes and phenotypes

Since the GJB2 gene plays an important role in the etiologyof NSSHI, it was preferred to the primary screening gene fornewborns. Several multicenter studies looking at the corre-lation between GJB2 mutations and the hearing levels havebeen performed, and some valuable conclusions were drawn.That is, the homozygous c.[30_35delG] genotype was fre-quently associated with profound HI, and the genotypes ofboth inactivating mutations were significantly associatedwith profound and severe HI. Further, the subjects with ge-notypes comprising at least one noninactivating mutation ortwo showed obviously moderate or mild hearing loss (Crynset al., 2004; Snoeckx et al., 2005).

Some results indicated that the level of HI usually dependson the less severe mutation, which meant that patients pre-

senting with significantly milder hearing loss carried at leastone non-inactivating mutation. This point was really sup-ported by some studies, for example, c.[30_35delG] + [109G > A], c.[368C > A] + [368C > A], c.[235delC] + [109G > A],c.[30_35delG] + [-23 + 1G > A], and c.[30_35delG] + [101T > C]were demonstrated to associate with milder clinical symp-toms. In the study sample, some subjects with such genotypeshad the PTA level of 35–70 dB (Cryns et al., 2004; Marlin et al.,2005; Snoeckx et al., 2005).

Though the GJB2 gene is the most frequent causative genein China, the relationship between the phenotypes and thegenotypes has not been reported (Wang et al., 2004). Our studyfirst did this research in a large cohort in Chinese population.As shown in the results, we found three genotypes presentin more than two observations to be associated with signifi-cantly less severe HI than the homozygous c.235delC group;so, our data also supported the popular opinion. Both themild genotypes of ([p.Val37Ile] + [p.Val27Ile; p.Glu114Gly]and homozygous [p.Val27Ile; p.Glu114Gly]) were in theNT + NT category.

In addition, we found that one genotype (c.[235delC] +[176_191del16]) was associated with more significantly se-vere HI than the homozygous c.235delC group. In Cauca-sians, c.[30_35delG] + [del(GJB6-D13S1830)]was found withmore severe HI than the homozygous c.35delG group(Snoeckx et al., 2005).

There was no significantly different HI between some ge-notypes and the homozygous c.235delC group, which werec.[30_35delG] + [299_300delAT], c.[235delC] + [30_35delG], c.[299_300delAT] + [176_191del16], c.[235delC] + [30_35insG],c.[235delC] + [504_505insAAGG], and c.[299_300delAT] + [504_505insAAGG]. It was noted that the P value of homozygousc.[299_300delAT] was 0.064, which showed a tendency todistinguish from the reference group. The result was similarto that in the Caucasian population to some extent, though themap of GJB2 mutations was different between the two dif-ferent populations. If we can deduce that the HI level is re-lated to some specific genotypes, it would be useful to

FIG. 2. Distribution of thethree classes of HI with spe-cific genotype that re-presented in more than twosubjects. The genotypes weredivided into two categories,which were nontruncating +nontruncating (NT + NT) andtruncating + truncating(T + T). The number of pa-tients in each class and thegenotypes are indicated un-der x-axis. The percentile ofcases with different HI levelsin each genotype was showedaccording to y-axis. v2 testindicated that a nonrandomassociation existed betweenthe degree of HI and the ge-notype category. HI, hearingimpairment.

622 ZHAO ET AL.

examine the effect of environmental factors, such as infectionsor medications, and genetic background on hearing loss in-crease in patients with DFNB1 (Wilcox et al., 2000).

A phenomenon emerged that a large scatter of the degree ofhearing loss existed in the three genotypes which had thelargest number of patients tested, as seen in the other study(Cryns et al., 2004). The simplest explanation for the difficultyin correlating a specific genotype to clinical symptoms is thatadditional genetic-, epigenetic-, and/or environmental factorscould modify the phenotype. It was further supported thatsome cases of severe or profound NSSHI are associated with asingle GJB2 mutation (data not shown).

Though some studies showed that no mutations in GJB2were found to relate to postlingual deafness in Japanese, Jews,and the French (Denoyelle et al., 1999; Kudo et al., 2000; Sobeet al., 2000), other studies found that p.Leu90Pro, p.Met34Trp,and p.Trp44Cis may have correlation with postlingual pro-gressive deafness in Poland, Australia, and America, respec-tively (Kenneson et al., 2002; Pollak et al., 2007). It was unclearwhether the progression existed in truncating genotypes. Inour study, we found 11 subjects to be with postlingual deaf-ness. As shown in results, the genotypes scattered in NT + NTand T + T groups, even in four subjects with homozygousc.[235delC] genotype. Our study provided new proof ofpostlingual deafness caused by the GJB2 truncating mutation.It seems contradictory with the putative molecular patho-genic effect of truncating mutation, which could somewhat beexplained as the pathogenic fluctuation or progress, thoughno relationship was discovered between age and HI so far(Cryns et al., 2004). This puzzle requires thorough cellular oranimal model study of GJB2 mutations (Choi et al., 2009).

Contradictory outcomes between functional researchand phenotype

GJB2 codes the Cx26 protein family, which connects the gapjunction to form homotypic or heterotypic channels to main-tain tissue homeostasis and to allow fast intercellular electricalcommunication. An extensive network of gap junctions isfound in two kinds of cells: the cochlear nonsensory epithelialcells and the cochlear connective tissue cells. Cx26 can formhomomeric and homotypic channels. Cx26 and Cx30 havebeen demonstrated to form heteromeric gap junctions in co-expression studies in Hela cells, and it has also been demon-strated that certain Cx26 mutations exert a dominant negativeeffect on Cx30, by affecting Cx30 channel permeability(Marziano et al., 2003).

Plenty of functional studies have been performed to studythe Cx26 protein and the channels the CX26 participated in.Pathogenic changes of different mutations in GJB2 varied,which interfered with the translation or stability of Cx26protein (p.Met1Val, p.Arg184Pro), or disturbed protein traf-ficking and assembling in hemi-channels (p.Leu90Pro,c.[30_35delG], c.[235delC]), or disabled formation of homo-typic gap junction channels (p.Val37Ile, p.Trp77Arg,p.120delGlu, p.Met163Val, and p.Met34Thr). Some mutationswere not thoroughly studied in vitro (c.[559_604dup46],c.[176_191del16], p.Arg32Cis) or were thought to have noeffect on protein function (c.[341A > G], p.Ile203Thr) (HoangDinh et al., 2009).

However, some functional outcomes were discordant withthe phenotype. Though the mutation of c.[-23 + 1G > A] did

not yield any detectable Cx26 protein and was similar to themutation of c.[30_35delG], the subjects with c.[30_35delG] +[-23 + 1G > A] genotype had significantly less severe HI com-paring with c.35delG homozygotes (Cryns et al., 2004).

In this study, we found the mutation p.Val37Ile to be as-sociated with less severe HI compared with c.235delC ho-mozygotes as well in other studies (Lin et al., 2001; Snoeckxet al., 2005). However, some expression functional studieshave demonstrated complete loss of channel activity in cellstransfected with p.Val37Ile (Kudo et al., 2000; Bruzzone et al.,2003), whereas some trials confirmed it was not pathogenic(Hwa et al., 2003; Wattanasirichaigoon et al., 2004). The samecontradiction between functional study and HI level alsoemerged in the study of p.Leu90Pro and p.Met34Tro.

An explanation for this phenomenon was that the p.Va-l37Ile and p.Met34Thr had lower penetrance compared withmutations of undisputed pathogenicity (Pollak et al., 2007).Another explanation was that these studies mainly focused onthe channel properties. The effect of p.Val84Leu on HI wasdiscovered to be another novel way of impairing the perme-ability of IP3, which was essential in mediating the calciumion propagation in cochlear supporting cells (Thonnissenet al., 2002), and suggested that the complicated effect of GJB2mutations in HI should be studied in a model similar to in vivoconditions.

Combined causative mutation of p.Val27Ile(c.79A > G)and p.Glu114Gly(c.341G > A)

In the Chinese population, p.Val27Ile and p.Glu114Glymutation were frequent, and both were thought to be thepolymorphism by majority. However, p.Val27Ile has lowfrequency (less than 2%) in Caucasians (Roux et al., 2004).p.Val27Ile locates in a transmembrane part, and p.Glu114Glysituates in the intracellular loop. The cellular functionalstudies showed that p.Glu114Gly had no effect on protein, butp.Val27Ile caused dysfunction of protein in vitro (Chounget al., 2002; Bruzzone et al., 2003). A recent study reported inthe 30th ARO Annual MidWinter Research meeting discov-ered that dye transfer and calcium conductance was nil in thehomozygous [p.Val27Ile; p.Glu114Gly] injected cells; whereasa compound heterozygote, p.Glu114Gly homozygote, and ap.Glu114Gly heterozygote showed impaired intercellularbiochemical coupling (Pilipenko et al., 2007).

The combination of p.Val27Ile and p.Glu114Gly wasidentified as one causative mutation (Putcha et al., 2007).There were a total of 106 alleles (18.5%) of [p.Val27Ile;p.Glu114Gly] among deaf subjects in our study. We screenedthis mutation in 400 newborns and found that only 5 subjectspresent with it (0.6%). [p.Val27Ile; p.Glu114Gly] appeared farmore frequently in the deaf group than in the normal group.The frequency of this controversial mutation in the deaf groupwas 11.7% in the Altai population and 11.3% in Singapore(Posukh et al., 2005). Our epidemiological data sustained that[p.Val27Ile; p.Glu114Gly] had very close correlation withdeafness.

Mechanism of impairment

The GJB2 mutations primarily affected outer hair cells,because the impairment (detectable by otoacoustic emissions)seems to be constant in all genotypic groups and even in thesubjects carrying only one GJB2 mutated allele. Our DPOAE

PHENOTYPE–GENOTYPE CORRELATION OF GJB2 MUTATIONS 623

data supported this opinion, for we found that normal wavescould not be elicited in patients whose thresholds were lowerthan 60 dB. The impairment was supposed to be caused bylocal intoxication of the organ of Corti by the accumulatingK + ions, which would affect the function or survival of haircells (Lefebvre and Van De Water, 2000). An alternativeopinion was that interruption of K + ions circulation wouldprevent the development of the receptor potential in haircells (Kikuchi et al., 2000). Inner hair cells and nerve im-pairment were found to be variable in the distinct genotypiccombinations analyzed (Engel-Yeger et al., 2003). Anothertwo theories exist that explain the causative mechanismsof CX26 mutations, including a theory of endothelial barrierbreakage and a theory of deficiency in gap-junction protein-facilitated metabolite transportation (Hoang Dinh et al.,2009).

Acknowledgments

We thank the patients and their families for their cooper-ation during this work. This work was supported by grantsfrom the National Natural Science Foundation of China, KeyProject (grant no. 30830104), the National Natural ScienceFoundation of China (grant nos. 30672310, 30771203, and31071166), Beijing Nature Science Technology Major Project(grant no. 7070002), Natural Science Foundation of Guang-dong Province, China (grant no. 8251008901000007), andScience and Technology Planning Project of GuangdongProvince (grant no. 2009A030301004).

Disclosure Statement

No financial conflict of interest relevant to this article wasreported.

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Address correspondence to:Qiu-Ju Wang, M.D., Ph.D.

Department of Otorhinolaryngology/Head and Neck SurgeryChinese People’s Liberation Army Institute of Otolaryngology

Chinese People’s Liberation Army General HospitalBeijing 100085

China

E-mail: wqcr@263.net

PHENOTYPE–GENOTYPE CORRELATION OF GJB2 MUTATIONS 625