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This paper was published with modifications in: Genetics in Medicine 2002; 4(4):258-274.

GJB2 (connexin 26) variants and
nonsyndromic sensorineural hearing loss
Print Version

by Aileen Kenneson, PhD; Kim Van Naarden Braun, MPH; Coleen Boyle, PhD

From the National Center on Birth Defects and Developmental Disabilities, Centers for Disease Control and Prevention, Atlanta, Georgia

Key words: GJB2; connexin 26; hearing loss

Reprint requests to Aileen Kenneson, PhD, National Center on Birth Defects and Developmental Disabilities, 4770 Buford Highway NE, Mailstop F-15, Atlanta, GA 30341-3724

Note: The term hearing loss is used in this article instead of the term hearing impairment, which is considered to be pejorative by people who are deaf or hard of hearing.1 We use the term hearing loss to include all levels of loss (mild to profound) and any age of onset including congenital losses. The word deaf refers to hearing status as determined by an audiogram. The word Deaf refers to the cultural community of people who are deaf and hard of hearing.2

August 15, 2002


 

HuGE Review

gray triangle button Abstract
gray triangle button Gene
gray triangle button Gene Variants
gray triangle button Hearing Loss
gray triangle button Associations
gray triangle button Interactions
gray triangle button Laboratory Tests
gray triangle button Population Testing
gray triangle button Acknowledgements
gray triangle button Appendix
gray triangle button References
gray triangle button Table 1
gray triangle button Table 2, Table 2A
gray triangle button Table 3, Table 3A, Table 3B, Table 3C
gray triangle button Table 4
gray triangle button Internet Sites
gray triangle button Medical Literature Search

Abstract

Despite the enormous heterogeneity of genetic hearing loss, variants in one locus, Gap Junction Beta 2 or GJB2 (connexin 26), account for up to 50% of cases of nonsyndromic sensorineural hearing loss in some populations. This article reviews genetic epidemiology studies of the alleles of GJB2, prevalence rates, genotype-phenotype relations, contribution to the incidence of hearing loss, and other issues related to the clinical validity of genetic testing for GJB2. This review focuses primarily on three alleles: 167ΔT, 35ΔG, and 235ΔC. These alleles are recessive for nonsyndromic prelingual sensorineural hearing loss, and the evidence suggests complete penetrance but variable expressivity.


Gene

The Gap Junction Beta 2 or GJB2 gene (GenBank M86849, OMIM: *121011) resides at the chromosomal location 13q11 and encodes for the protein connexin 26, a beta class gap junction protein expressed in the cochlea and in the epidermis.3 Connexin 26 hexamers form channels between cells that, when open, allow cell-to-cell diffusion of small molecules. This function is necessary for recycling potassium in the cochlea that plays a critical role in sensorineural hearing function.4 The GJB2 gene is small, with the entire coding region of 680 base pairs falling within exon 2.


Gene Variants

Aided by the relatively small size of the GJB2 gene, the flourish of activity on the genetics of hearing loss has resulted in rapid identification of GJB2 variants. Missense, nonsense, frameshift, insertion, and deletion variants have all been reported. To identify published genetic epidemiology studies related to GJB2, we searched the MEDLINE database using the keywords GJB2, connexin 26, and hearing loss to identify relevant studies. References in related studies were also reviewed.

A list of GJB2 variants is presented in Table 1. Some variants are benign polymorphisms, with a high prevalence rate in various populations. For example, the V27I, E114G, and I203T variants were found on 54%, 55%, and 94% of Japanese chromosomes, respectively.5,6

Table 1:  Published GJB2 variants with corresponding change in the connexin 26 protein and the putative allele type.

The 167ΔT, 35ΔG (also known as 30ΔG), 235ΔC, and R143W alleles are the most common hearing loss-associated GJB2 alleles in the Ashkenazi Jewish, Caucasian, Japanese, and Ghanian populations, respectively. The best-characterized population is Caucasian of northern European descent. Table 2, Table 2A presents the heterozygote carrier frequencies of the first three alleles either in the general population (hearing status unknown) or in control groups (without hearing loss). Ascertainment details were generally lacking and are listed in Table 2, Table 2A as described in the publication. Likewise, descriptive information, including sex and age, were generally not provided. Despite these limitations, the studies consistently reported a prevalence of the 35ΔG allele in the range of 1% to 3%. In fact, one population-based study, which genotyped 560 randomly selected neonatal bloodspots in the Midwestern United States, detected a 35ΔG heterozygosity rate of 2.5% in this predominantly Caucasian population.7

Table 2, Table 2A: Heterozygote rates of three GJB2 mutations among control populations.

In addition to Caucasians, published studies have focused on Mediterranean, Japanese, Korean, and Ashkenazi Jewish populations. The 35ΔG allele is common in individuals of Mediterranean descent (1 in 30) and GJB2 testing has begun to be included in prenatal genetic counseling in Greece on a pilot basis.8 Although the numbers were small, and ascertainment and demographic details were generally lacking, studies indicated that 10% of Ashkenazi Jews carry the 167ΔT allele, and 1% of Japanese and Korean individuals carry the 235ΔC allele (Table 2, Table 2A).

The 35ΔG, 167ΔT, and 235ΔC alleles are all recessive alleles associated with nonsyndromic prelingual hearing loss. No homozygotes for any of these three alleles have been reported in control groups. Some of the methods used tested only for specific alleles; therefore, distinguishing between heterozygotes and compound heterozygotes is not possible. This limitation affects the conclusions that can be drawn regarding penetrance in these studies. However, in the studies that did fully characterize the second allele in the control groups, no compound heterozygotes were reported. This finding suggests that for these three common alleles, i.e., 167ΔT, 35ΔG, and 235ΔC, penetrance of hearing loss in homozygotes is complete. However, larger population-based studies are needed to support this model and to characterize the penetrance of the less common alleles.


Hearing Loss

"With over a million essential moving parts, the auditory receptor organ, or cochlea, is the most complex mechanical apparatus in the human body."9 Given this complexity, it is not surprising that sequence variation in any one of hundreds of genes can lead to hearing loss. Hearing loss occurs in approximately 1 to 3 of 1,000 children,10 and is generally attributed to pure genetic factors in approximately 50% of cases.11 In approximately 30% of cases, a specific syndrome can be identified, with more than 400 syndromes claiming hearing loss as a component. The other 70% of cases are nonsyndromic.11,12 Nonsyndromic cases may be familial or sporadic. The nature of familial nonsyndromic prelingual hearing loss is usually described as follows: 75% to 80% are autosomal recessive (designated with the prefix DFNB), 20% to 25% are autosomal dominant (DFNA), and 1% to 1.5% are X-linked (DFN).13 The extraordinary genetic heterogeneity of hearing loss has been demonstrated by linkage studies, which indicated many loci for nonsyndromic hearing loss: 30 autosomal recessive, 29 autosomal dominant, and 8 X-linked.14 Two mitochondrial variants, A1555G and A7445G, also have been implicated in nonsyndromic hearing loss. Several other mitochondrial mutations are associated with syndromic forms of hearing loss.15 One of the autosomal recessive loci, DFNB1, was mapped to chromosome 13q1216 and was attributed recently to the GJB2 gene.3  Although the majority of GJB2 variants are recessive, dominant alleles have been identified that account for the DFNA3 locus mapped to the same region.17,18


Associations

Contribution of GJB2 to hearing loss
Given the extraordinary genetic heterogeneity of nonsyndromic hearing loss, it was surprising to find that sequence variations at the GJB2 locus account for up to 50% of cases of nonsyndromic prelingual sensorineural hearing loss in some populations. A recent model to explain this observation is based on the tradition of intermarriage between individuals with hearing loss in some populations. A gradual increase in the proportion of hearing loss due to a hypothetical autosomal recessive mutation would be a consequence of this assortative mating.19

More than 90 variants of the GJB2 gene have been reported, and many are rare. One variant generally predominates in any given population, such as 167ΔT in the Ashkenazi Jewish population, 35ΔG among Caucasians of northern European descent, 235ΔC in the Japanese population, and R143W in Ghana. Table 3, Table 3A, Table 3B, Table 3C summarizes studies of the prevalence of various GJB2 genotypes among individuals with prelingual hearing loss. The studies presented in this table vary in their ascertainment methods, case definitions, inclusion of presumably acquired cases, and genotyping methods. Because the genotype information that can be gleaned from the studies depends on the methodology, the data must be considered accordingly.

Table 3, Table 3A, Table 3B, Table 3C: Contribution of GJB2 variants to hearing loss: Summary of GJB2 genotypes characterized in cases of hearing loss (familial, non-familial, and both)

Twenty-two of the 30 studies in Table 3, Table 3A, Table 3B, Table 3C used DNA sequence analysis to fully characterize both alleles in each individual. These studies provided the best estimate of the proportion of hearing loss cases associated with GJB2 variants. The percentages of cases of prelingual hearing loss associated with two variants in GJB2 (i.e., homozygotes or compound heterozygotes) for these studies were calculated (Table 3, Table 3A, Table 3B, Table 3C). Sequence variations at the GJB2 locus were detected in approximately 20% of individuals with nonsyndromic prelingual sensorineural hearing loss in populations of Caucasians of northern European descent. GJB2 variants were detected in approximately 5% of individuals with hearing loss in Korea, 14% in Australia, 17% in Tunisia, 20% in Japan, and 43% in Israel. Thus the contribution of GJB2 variants to hearing loss varied between populations. Table 3, Table 3A, Table 3B, Table 3C also demonstrates that the frequencies of the various GJB2 alleles differed between the populations.

Most of these DNA-sequencing studies analyzed exon 2 of GJB2, which contains the entire coding region and 92 of the 94 variants described in Table 1. However, seven of the studies also sequenced exon 1, which contains the 3′ untranslated region and the other two known variants.7,20-25 Only one of these studies detected a sequence variation in exon 1,20 suggesting that exon 2 analysis will detect most of the mutations in GJB2 in individuals with hearing loss. However, studies with larger groups are necessary to determine the frequency of the variants found in exon 1.

Other studies used methods that detect only specific alleles, or reported data only on the major alleles, particularly 35ΔG. Because these data did not fully characterize the second allele, heterozygotes could not be distinguished from compound heterozygotes (Table 3, Table 3A, Table 3B, Table 3C). These studies also tended to include small numbers and generally lacked ascertainment and descriptive details. However, despite these limitations, the study results were consistent with those presented above and indicated that the 35ΔG allele accounts for approximately 10% to 20% of cases of hearing loss in Caucasians of northern European descent, but approximately 30% to 40% of cases in Mediterranean regions.

Population differences in contribution of GJB2 to hearing loss
As indicated above, there are population differences in the distribution of the various GJB2 alleles. Despite that different alleles predominate in different populations, there is a relatively high carrier rate of GJB2 alleles in all described populations. Furthermore, the carrier rate seems to be slightly higher in certain geographical areas, such as the Mediterranean region.26,27 The cause of this high carrier rate is unknown.

A notable gap in the literature is the lack of assessment of the contribution of GJB2 variants to hearing loss over a wide range of populations, as illustrated by the African American population as described below. Characterization of these populations is important to determine (1) the prevalence of GJB2 variants among individuals with nonsyndromic hearing loss, and (2) the prevalence of the different alleles in the control populations. As demonstrated in Table 3, Table 3A, Table 3B, Table 3C and discussed above, both of these measures appear to be population-specific.

The proportion of individuals with nonsyndromic hearing loss in African Americans who are carriers of GJB2 variants has not been determined. However, two studies have looked for specific GJB2 variants among African American control groups. The first group consisted of individuals receiving genetic counseling at the University of Michigan for disorders unrelated to hearing loss. This study tested 173 African Americans for the 35ΔG variant and 171 African Americans for the 167ΔT variant, and found no carriers of either allele.28 The other study looked for 35ΔG variants among 190 African Americans (ascertainment details not reported) and found none.26 These two studies indicated that 35ΔG is significantly less common among the African American population than it is among the Caucasian population (as described above and in Table 2, Table 2A). The rate of nonsyndromic hearing loss is not lower in African Americans than in Caucasians.29 Two possible explanations for these data are (1) the proportion of cases of hearing loss attributed to GJB2 variants is lower in African American than in Caucasian populations, and/or (2) GJB2 alleles other than 35ΔG play a significant role in the African American population.

In support of the latter model, no individuals with the 35ΔG variant were found among 365 students with profound sensorineural hearing loss in Ghana. Likewise, the 167ΔT and 235ΔC variants were not found in this population. Of the 63 individuals in this study who carried GJB2 variants, 51 (81.0%) were homozygous for the R143W allele and 8 (12.7%) were compound heterozygotes for R143W and a second variant allele.25 Assessment of GJB2 variants among non-Caucasian hearing loss and control populations are necessary to address these issues so that the clinical validity can be defined in these populations.

Type of hearing loss
Connexin 26 is expressed in the stria vascularis, spiral ligament, spiral limbus, and between the supporting cells in the cochlea,3 and appears to function in the recycling of potassium that is used by the hair cells to generate an action potential in response to sound waves.4  Consequently, it has generally been presumed that hearing loss associated with mutations in the GJB2 gene will be sensorineural in nature. The nature of GJB2-related hearing loss has not been formally assessed by genetic epidemiologic methods. With one exception, the studies presented in this review either excluded conductive and mixed cases of hearing loss or did not distinguish between the different types of hearing loss. In the study of 99 unrelated children with hearing loss of unknown etiology who were attending an outpatient otolaryngology clinic in Boston, 30 were found to carry one or two GJB2 mutations. Temporal bone abnormalities were identified in four of these individuals (35ΔG/167ΔT, 35ΔG/G12 V, L90P/+, and 35ΔG/+), and conductive or mixed hearing loss was reported for one (E47X/+) and two (both 35ΔG/M34T) cases, respectively.30 These associations may be coincidental, but additional studies are needed to describe the type of hearing loss associated with of GJB2 variants.

Age of onset of hearing loss
GJB2 variants are generally described as causing prelingual hearing loss. However, in most published studies, it is not possible to distinguish between congenital (present at birth) and noncongenital prelingual hearing loss. Only one published study has examined the contribution of GJB2 variants to congenital hearing loss. The prevalence of the 35ΔG genotypes in a Rhode Island newborn population with hearing loss did not differ from other American populations with hearing loss who were ascertained in childhood and who were of similar race/ethnicity (Table 3, Table 3A, Table 3B, Table 3C). More studies of this type, as well as studies including documented noncongenital prelingual hearing loss, are needed to assess the relationship between GJB2 variants and congenital hearing loss. In this regard, the reports of newborns who passed the newborn hearing screen but in whom GJB2-related hearing loss was diagnosed later in infancy are notable.31, 32 Whether these cases represented false-negative results of the newborn hearing screening programs or indicated a late-onset and/or progressive nature of some GJB2-related cases of hearing loss is not clear. Likewise, Orzan et al. reported three Italian children with biallelic GJB2 genotypes who had a sudden onset of hearing loss between 18 and 24 months of age, although it is not clear whether prior hearing status was formally documented or based on parental report.23

Recent research has not focused on rigorous analysis of the possible contribution of GJB2 to postlingual hearing loss. Four published studies have included individuals with postlingual hearing loss. The first consisted of genetic analysis of GJB2 among individuals recruited from consecutive patients at the genetic counseling service for deaf individuals at two hospitals in Paris. Of the participants, 43 of the 88 individuals with prelingual sensorineural hearing loss carried variations in the GJB2 gene, but no changes were found among the 16 individuals with postlingual (before age 20) sensorineural hearing loss.20 Likewise, a study in Israel ascertained individuals with nonsyndromic hearing loss (ascertainment details not reported) and tested them for GJB2 variants. Of the 66 individuals with prelingual hearing loss, 25 were homozygous or compound heterozygous for GJB2 variants, and 4 were heterozygous. No GJB2 variants were found among the 11 cases of postlingual (definition not provided) hearing loss.24 In Japan, 5 of 39 individuals with prelingual hearing loss were homozygous or heterozygous GJB2 variant carriers, but no changes were found among the 39 individuals with postlingual (onset between 3 and 30 years) sensorineural hearing loss (ascertainment details not reported).6

The fourth study, taking place in Austria, found four carriers of GJB2 variants among 16 individuals with postlingual (undefined) hearing loss.22  The genotypes were L90P/I20T (onset in first decade), L90P/35ΔG (onset in first decade), 35ΔG/+ (onset in first decade), and G160S/+ (onset in fourth decade). The L90P allele is of interest in this population because it is seen in 2 of 16 postlingual (undefined) cases, and 3 of 53 prelingual cases of hearing loss. Thus this allele may be a significant contributor to postlingual hearing loss. The failure to detect GJB2 variants in the other three studies may be due to a higher prevalence of the L90P allele in the Austrian population, as this allele was detected only rarely in individuals with hearing loss in France (2 of 135)20, 35 but not at all in Israel (0 of 102)24, 33 or Japan (0 of 94).5, 6, 34

Two dominant alleles have been specifically implicated in noncongenital hearing loss. The C202F allele was observed to cosegregate with postlingual (age of onset at 10 to 20 years) and progressive hearing loss in a 5-generation French family.36 Likewise, the W44C allele cosegregated with progressive hearing loss in an American family of mixed Northern European descent, with age of onset ranging from infancy to age 18 years.37 These alleles were not detected in studies that provided sequence data on controls, including 100 Korean newborns,38 209 Japanese individuals,5, 6, 34 and 204 French individuals.36, 35

These studies suggest that hearing loss associated with the more common GJB2 sequence variations is likely to be prelingual. However, additional population-based studies involving individuals with congenital, noncongenital prelingual, postlingual, and late-onset hearing loss will be needed to fully assess the relationship between GJB2 variants and age of onset, particularly in reference to the less common alleles.

Severity of hearing loss
Hearing loss associated with GJB2 variations generally fall into the moderate to profound range. Three European studies have looked at the severity of hearing loss among children with and without GJB2 sequence changes. In one of these studies, in France (ascertainment described above), GJB2 homozygotes or compound heterozygotes accounted for 31 (55%) of 56 profound ( ≥ 90 dB) cases, 14 (48%) of 29 severe (70-89 dB) cases, 8 (42%) of 19 moderate (40-69 dB) cases, and 1 (14%) of 7 mild (20-39 dB) cases.20 Of the 47 individuals who carried the 35ΔG/35ΔG genotype, the hearing loss was profound in 29 (62%), severe in 10 (21%), moderate in 7 (15%), and mild in 1 (2%).39 The profile for individuals with one 35ΔG and one other allele was two profound (22%), three moderate (33%), three severe (33%), and one mild (11%) Although the latter group is small in size, the results are suggestive of variability in degree of hearing loss between alleles.

In 1999, a United Kingdom (U.K.) group ascertained a group of 284 individuals with nonsyndromic prelingual sensorineural hearing loss from several sources, including otolaryngologists, audiologists, clinical geneticists, and the British Deaf Associations.21 They found biallelic GJB2 carriers among 0 (0%) of 19 mild (20-39 dB) cases, 9 (10%) of 92 moderate (40-69 dB) cases, 11 (17%) of 64 severe (70-94 dB) cases, and 30 (30%) of 100 profound ( ≥ 95 dB) cases. The 35ΔG/35ΔG genotype was present in 6 individuals with moderate, 10 with severe, and 24 with profound hearing loss. Only two 167ΔT/167ΔT individuals were found in this study, and both had moderate hearing loss. Two 35ΔG/167ΔT individuals were found: one with moderate and one with severe hearing loss.

Also in 1999, 94 individuals with nonsyndromic prelingual hearing loss were recruited from Italian audiology and phoniatrics services. Of these individuals with profound hearing loss ( ≥ 95 dB), 63% carried GJB2 variant alleles as did 43% of individuals with severe (70-94 dB) and 33% of those with moderate (40-69 dB) hearing loss. Of the individuals with GJB2 variant genotypes, 27 were homozygous for 35ΔG and 13 were compound heterozygotes. As in the French study discussed above, the 35ΔG homozygotes fell into the moderate or profound range, whereas the compound heterozygotes were dispersed among the three categories (moderate, severe, and profound), suggesting allelic difference in expressivity.23

Seven additional studies presented data regarding the severity of hearing loss among individuals with GJB2 variants. Because the number of alleles and, therefore, the number of possible genotypes was large, the absolute numbers of cases for each genotype in the studies combined were small. Therefore, the data presented here focus on the 35ΔG/35ΔG and 167ΔT/167ΔT genotypes. Two Israeli studies,24,33 two Australian studies,340, 41 one Austrian study,22 and two American studies30, 42 described the level of hearing loss among individuals with GJB2 mutations. In the seven studies combined, information was presented on 50 people with the 35ΔG/35ΔG genotype: 8 moderate (16%), 12 severe (24%), and 30 profound (60%). Likewise, of the 30 people with the 167ΔT/167ΔT genotype, one had mild hearing loss (3.3%), 5 moderate (16.7%), 8 severe (26.7%), and 16 profound (53.3%). These data are consistent with the above reports, and indicate that the hearing loss associated with the 35ΔG/35ΔG and 167ΔT/167ΔT genotypes is generally in the moderate to profound range, with profound hearing loss being the most common manifestation.

Although these data suggest that GJB2 variants tend to be associated with moderate to profound hearing loss, the numbers were small, dB ranges of degrees of hearing loss varied among the studies, and the specific relationship between various GJB2 alleles and severity of hearing loss were not addressed. In addition, the nonpopulation-based approach may have resulted in underascertainment of mild hearing loss. However, the low prevalence of GJB2 biallelic genotypes among the individuals with mild hearing loss in the British21and French20 studies described above suggested that GJB2-associated hearing loss, particularly with the 35ΔG and 167ΔT alleles, tends to be moderate to profound. Likewise, GJB2 biallelic individuals have not been described in the general hearing population. On the other hand, the Australian group described three individuals with mild hearing loss (25-40 dB) with less common GJB2 genotypes: M34T/R184W, 35ΔG/M34T, and 35ΔG/V37I.40 Likewise, the Austrian study reported three individuals with mild hearing loss: L90P/314Δ14, Y155X/+, and G160S/+.22  It is possible that carriers of these alleles do not always have hearing loss, but because these alleles are less common than the 35ΔG and 167ΔT alleles, larger population-based studies are needed to address this issue.

Laterality of hearing loss
The inter-ear difference in severity of hearing loss was described for 54 French children with biallelic GJB2 genotypes. In 48 (89%) of the children, the severity did not differ between the ears. In the other six (11%), the ears differed by one degree of severity (dB ranges described above).20 These children were ascertained through genetic counseling services for deaf individuals at two hospitals in Paris. Therefore, individuals with unilateral hearing loss may have been underascertained. The study included two children with two degrees, and one child with three degrees of difference in severity, none of whom carried GJB2 variants. However, the number of children in these groups was clearly small.

In an analysis of children with nonsyndromic prelingual hearing loss ascertained through Italian audiology services, more than 90% of the 46 children with GJB2 variants demonstrated a symmetrical hearing loss (inter-ear difference of <15 dB at two frequencies or 10 dB at four frequencies). In this study, GJB2 variants were detected in 43 of 75 (66%) individuals with symmetrical hearing loss but in only four of the 19 (21%) of those with asymmetrical hearing loss.23

In the U.K. study,21individuals were ascertained through a variety of sources, including otolaryngologists, clinical geneticists, and Deaf associations. The data were presented as average dB difference in hearing loss between the ears: 6.33 (N = 45, SD = 8.09) for GJB2 homozygotes and compound heterozygotes, 6.66 (N = 26, SD = 8.05) for heterozygotes, and 7.86 (N = 175, SD = 11.19) for individuals without GJB2 variations. There was no significant difference between any of these groups.

In a group of consecutive individuals with sensorineural hearing loss seen at a center for Hearing, Speech, and Voice Disorders in Austria, 24 individuals with GJB2 variant genotypes were identified. Of these, five displayed asymmetry of hearing loss: three by two degrees and two by one degree of severity.22

In all of these studies, the ascertainment of individuals with unilateral hearing loss is unclear. Therefore, although the described GJB2 variants tend to be associated with bilateral hearing loss, population-based data on all individuals with any type of hearing loss are needed to clarify the issue.

Progression of hearing loss
Longitudinal data are lacking on individuals with GJB2-related hearing loss. Follow-up on individuals with GJB2-associated hearing loss over 1 to 20 years indicated no changes in the threshold of hearing loss.3843 However, details about the number of such cases and the timing of repeated testing were not published.

The French group studied children ascertained through genetic counseling services for the deaf in Paris, and described 16 children with biallelic GJB2 genotypes for whom test results were available over a 10-year span. In 11 children, no change ( ≤ 5 dB) in the threshold was noted. Three children (one with severe and two with profound hearing loss) showed slight progression (5-10 dB). Two children (one with moderate to severe, and one with profound hearing loss) had a progression >10 dB.20

Likewise, a retrospective analysis of audiograms (over 2 to 15 years) in Italian children ascertained through audiology services detected a progression of hearing loss in only 1 child of 47 who had a GJB2 variant genotype. Progression was defined as a >15 dB change in two or more frequencies, or a >10 dB change over an average of four frequencies.23

Among 24 Austrian individuals with GJB2-related sensorineural hearing loss, 3 were described as progressive in nature, although the definition was not provided.22 Thus the limited data suggest the GJB2-related hearing loss is primarily nonprogressive in nature.

High-frequency hearing loss
Audiograms of individuals with biallelic GJB2 genotypes tend to be flat or slightly descending, indicating equal loss across all frequencies.20-24, 38, 40, 41, 43-47 Two individuals with the 35ΔG/L90P genotype have been described with high-frequency (2,000-8,000 Hz) hearing loss.
40 This finding suggested that certain alleles, other than the more common and well-studied 35ΔG and 167ΔT, may be associated with high-frequency hearing loss. Most individuals with GJB2 variants have been ascertained through services for individuals with hearing loss, and many of the studies did not assess the high-frequency (2,000-8,000 Hz) range. Thus the contribution of GJB2 variants to high-frequency-only hearing loss has not been well-studied.

M34T allele and hearing loss
In 1997, the M34T variant was reported to cosegregate with three generations of hearing loss in one family in an apparently dominant manner, implicating GJB2 in nonsyndromic hearing loss.3 Several years later, a second variant was characterized in this family, found in trans with the M34T allele in the individuals with hearing loss. This finding suggested that the M34T allele is recessive.48 On the other hand, the M34T allele failed to cosegregate with hearing loss in several families, raising the possibility that M34T is a benign polymorphism.36, 49-51

The prevalence of M34T heterozygote carriers and compound heterozygotes among individuals with hearing loss and control groups is summarized in Table 4. The M34T allele is present in approximately 2% to 3% of the general Caucasian population, but has not been reported in Japan or Korea. Data on other populations are limited.

Table 4: Prevalence of M34T heterozygote carriers and compound heterozygotes in cases and controls in various geographical areas (raw numbers, frequencies, and 95% confidence intervals)

A similar prevalence of the M34T alleles was seen among Caucasian individuals with hearing loss, supporting the model that the M34T allele is a benign polymorphism. However, the M34T allele was sometimes found as a compound heterozygote among individuals with hearing loss,7, 30, 34, 39, 46, 49-52 and more rarely, in the homozygous form in individuals with hearing loss.39, 51, 53 No changes have been reported in the other allele among the M34T carriers in the control groups. Thus, the evidence appears to support the hypothesis that M34T is a recessive allele, although the lack of compound heterozygotes in the control groups may be due to their smaller sample sizes. This example demonstrates the importance of looking at data on genotypes rather than allele frequency. In addition, a recent report of linkage between M34T and an upstream 10-base pair deletion raised the possibility that the association between M34T and hearing loss may be due to linkage disequilibrium.51 Additional studies are needed to clarify the involvement of the M34T allele in hearing loss.

GJB2 variants and Vohwinkel syndrome
The vast majority of GJB2 variants are associated with nonsyndromic hearing loss (see Table 1). Ironically, the original report of a GJB2 allele associated with hearing loss occurred in a family that also displayed palmoplantar keratoderma (PPK). PPK is a form of hyperkeratosis in which the overgrowth is limited to the palms of the hands and the soles of the feet. The M34T allele cosegregated with hearing loss but not PPK in this family.3 Subsequently, it was shown that the PPK in this family was due to a D66H variant in the GJB2 gene, a variant not seen in the 122 unrelated controls.48 The combination of dominant sensorineural hearing loss and hyperkeratosis is also known as Vohwinkel syndrome (OMIM: 124500), and hearing loss with PPK appears to be a mild variant. The D66H allele has been implicated in this syndrome in three additional families.54 Neither group detected the D66H allele among the control groups of 122 and 145 unrelated individuals.

Likewise, the G59A variant cosegregated with Vohwinkel syndrome in a three-generation family. The G59A allele was not detected among 50 hearing controls or among 55 individuals with nonsyndromic hearing loss.55

The R75W variant was described in an Egyptian family with autosomal PPK and congenital deafness. It was also detected in one individual in the control group of 77 Egyptian individuals attending a clinic for reasons unrelated to skin disorders; however, the hearing status of this control individual is unknown. R75W was not found in the 17 Caucasian controls.56

Several other studies included sequence information about control individuals, and none of them detected any carriers of D66H, G59A, or R75W. The studies included 100 Korean newborns,38 209 Japanese individuals,5, 6, 34 and 119 French individuals35 (ascertainment details provided in Table 3, Table 3A, Table 3B, Table 3C). The numbers of controls examined in these studies were small and did not necessarily come from the same population as the cases; therefore, we cannot rule out the possibility that these alleles may present in the general population at a low frequency due to incomplete penetrance.


Interactions

Many study groups reported variations in the degree of hearing loss in individuals with the same genotype (see the Severity of Hearing Loss section above), even within sibships.20, 22, 24, 33, 41, 43 For example, one Israeli family consisted of five siblings with the 167ΔT/167ΔT genotype; three had profound ( ≥ 90 dB), one had severe (70-89 dB), and one had mild (20-39 dB) hearing loss.33 Likewise, in the French report of 16 children with biallelic GJB2 genotypes, the degree of hearing loss differed between the siblings in 50% of the families.20 This finding suggests that other factors, genetic and/or environmental, may be modifying the phenotypic outcome.

Individuals who are carriers of a single variation in GJB2 display evidence of reduced hair cell function57; therefore, it is possible that these individuals are more likely to develop hearing loss in the presence of additional genetic or environmental factors than are noncarriers. This possibility is supported by recent reports of mutations in other genes found at increased incidence among GJB2 carriers with hearing loss: GJB6 (connexin 30)58 and the mitochondrial 12S rRNA59.  Additional studies of this nature are expected to continue to characterize the gene-gene interactions involved in the etiology of GJB2-associated hearing loss.

Many of the studies in this review excluded cases of suspected environmental causes from the genetic analysis. These factors included infections (e.g., meningitis, rubella), low birth weight, ventilator use, ototoxic medications (e.g., aminoglycosides), and hyperbilirubinemia. However, two individuals with hearing loss attributed to rubella infection were later found to be homozygous for the 167ΔT variant.32 Thus, the presence of known environmental factors does not necessarily preclude genetic analysis. Indeed, the proportion of GJB2 cases that have been attributed to other causes has not been elucidated; therefore, the possibility of gene-environment interactions has not been examined.

Likewise, published studies have generally excluded cases of syndromic hearing loss from GJB2 analysis. Thus the possibility that GJB2 variants may be involved in the penetrance and expressivity of hearing loss due to syndromic causes has not been examined.


Laboratory Tests

Many DNA-based methods are available for detecting the various alleles reported for the GJB2 gene. Assays have been developed to rapidly test for specific common variants, including allele-specific polymerase chain reaction (PCR), PCR followed by restriction enzyme digestion, and PCR with allele-specific hybridization. These technologies, once analytically validated in the performing laboratory, are both highly sensitive and specific. However, they will only detect the allele for which they were designed. As some common alleles account for the majority of variants in some populations (e.g., 35ΔG in Greece), these methods offer rapid and economical approaches. They also provide simple and reliable methods for carrier testing in families with known alleles.

Scanning methodologies are often used for allele detection, including denaturing gradient gel electrophoresis, single-strand conformation polymorphism detection, heteroduplex analysis, and denaturing high-performance liquid chromatography. Although scanning technologies have the advantage of screening for many variants at once, they tend to be less reliable than the allele-specific PCR-based techniques in that they are more sensitive to laboratory conditions. They also will miss some alleles, the specific alleles being detected dependent on the method and conditions.

Sequencing of PCR products of the GJB2 gene is a common approach that has the advantage of detecting most alleles, including novel changes. Of the 94 known variants described in Table 1, all but 2 are in exon 2. Both exons 1 and 2 are small and amenable to PCR amplification. Sequencing of exon 1 will pick up these remaining three alleles. As described in the Contribution of GJB2 to Hearing Loss section, only a few published studies have used the method of sequencing both exons 1 and 2. Therefore, information is lacking to accurately determine the relative clinical validity of these two methods.

Laboratories offering clinical testing for GJB2 vary in their methodologies of choice (Kenneson et al., unpublished). Clinical validity thus varies accordingly.


Population Testing

Consistent with the recommendations of the Joint Committee on Infant Hearing,60 a growing number of states are screening newborns for audiologic function so that infants with hearing loss are identified and referred for intervention services very early in life. Some newborn hearing screening programs may in the near future refer individuals for genetic testing for GJB2 variants as part of follow-up services. The role that GJB2 testing will play in conjunction with universal newborn hearing screening programs has not yet been defined. Population-based studies are needed to determine the contribution of GJB2 variants to congenital hearing loss, as well as the association between GJB2 variants and progressive hearing loss.

A continuing challenge for laboratories has been the interpretation of novel sequence variants that may have clinical relevance. In recognition of the need, the American College Medical Genetics (ACMG) has published recommendations for interpreting sequence variants of questionable clinical relevance.61 The report cautions laboratorians to develop any interpretation made based on what is known not only about the sequence variant but also the individual's chance of having the condition, family history, other test results, and the sensitivity and specificity of the test being performed. As GJB2 testing is used more often in the evaluation of children with hearing loss, interpretation of uncommon and novel mutations will be necessary.

Genetic tests are often offered for clinical use before the clinical validity and utility are fully understood.62, 63 Because this is the case for GJB2 testing, research participants need to understand the distinction between genetic research, testing, and screening. The identification of GJB2 variants in infants with hearing loss may prove to have many clinical purposes, including (1) ruling-out risk of syndromic complications, (2) predicting moderate to profound hearing loss requiring aggressive language intervention, (3) indicating sensorineural hearing loss for which cochlear implants may be an intervention option for consideration, and (4) allowing genetic counseling regarding recurrence rates.42, 64, 65 The current literature is not sufficient for a careful review of all of these potential uses of GJB2 testing. Consequently, the child's course of intervention may not be significantly altered by the knowledge of GJB2 genotype at the present time. Although the genetic information may be useful to the family, genetic testing of minors is generally not accepted in the absence of direct intervention benefits for the child.66 However, as more information is collected about GJB2-related hearing loss, and the above-mentioned potential uses are evaluated, GJB2 testing may find a place in medical services that goes beyond reproductive counseling issues.

Genetic testing related to hearing loss is particularly ridden with complex ethical issues. For example, although the ACMG recommends providing genetic services to individuals with hearing loss "to establish the etiology whenever possible,"67 individuals with hearing loss often argue that genetic testing will devalue individuals with hearing loss.68 Furthermore, people with hearing loss often have different attitudes and beliefs about genetic testing for hearing loss which in most cases is reflective of different perspectives. One study surveyed parents with normal hearing who have one or more deaf children and demonstrated an overwhelmingly positive attitude toward genetic testing for hearing loss (96%).69 On the contrary, a survey administered to a group of delegates attending a conference on the "Deaf Nation" reported that 55% thought that genetic testing would do more harm than good and 46% responded that its potential use devalued people with hearing loss.68 The issues raised by the Deaf community provide a unique opportunity by challenging scientists and society to find culturally sensitive methods for genetic research and testing that are acceptable to all cultural groups.


Acknowledgements

This project was supported in part under a cooperative agreement from the Centers for Disease Control and Prevention through the Association of Teachers of Preventive Medicine.


Appendix

Statements


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Internet Sites

Hearing and hearing loss resources
Connexin 26 Homepage 
GeneClinics: Deafness Overview 
Hereditary Hearing Loss Homepage 
National Institute on Deafness and Other Communication Disorders 
Promenade 'round the Cochlea 
The Genetics of Infant Hearing Loss

Genetic Resources
Online Mendelian Inheritance in Man 
GenBank 
Human Gene Mutation Database