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Systematic Review
GJB2-Associated Hearing Loss: Systematic Review of Worldwide
Prevalence, Genotype, and Auditory Phenotype
Dylan K. Chan, MD, PhD; Kay W. Chang, MD
Objectives/Hypothesis: To perform a systematic review of GJB2-associated hearing loss to describe genotype distributions and auditory phenotype.
Data Sources: 230 primary studies identified from Pubmed.
Review Methods: Pubmed was searched systematically to screen broadly for any study reporting on genotype and carrier frequencies for biallelic GJB2-associated hearing loss in defined populations around the world. Genotype and audiometric
data were extracted and subjected to meta-analysis to determine genotype distributions, carrier frequencies, rates of asymmetric or progressive hearing loss, and imaging abnormalities.
Results: A total of 216 articles comprising over 43,000 hearing-loss probands were included. The prevalence of biallelic
GJB2-associated hearing loss was consistent across most of the 63 countries examined, with different mutations being predominant in different countries. Common mutations were found in greater than 3% of the general population worldwide.
Meta-analysis of 48 case-control studies demonstrated a two-fold higher carrier frequency among hearing-impaired individuals compared to normal-hearing controls for truncating alleles, but not V37I. Progression, asymmetry, and imaging abnormalities were present in 14% to 19% of individuals with GJB2-associated hearing loss.
Conclusion: GJB2 mutations are highly prevalent around the world. The multiple predominant mutations present in different populations attest to the importance of this gene for normal cochlear function and suggests an evolutionary heterozygote advantage. The unusually high carrier rate for truncating mutations among hearing-impaired individuals is consistent
with either the presence of complementary mutations or a carrier phenotype. The significant rate of asymmetry and progression highlights the importance of diagnostic workup and close follow-up for this highly variable condition.
Key Words: Hearing loss, connexin 26, genetics.
Laryngoscope, 124:E34–E53, 2014
INTRODUCTION
Hearing loss is the most common congenital sensory
impairment, affecting one in 500 newborns and one in
300 children by the age of 4.1 The most frequent genetic
cause of congenital sensorineural hearing loss (SNHL) is
disruption of GJB2, the gene encoding Connexin 26
(Cx26), the major component of gap junctions in the
cochlea. Ever since the first description of GJB2associated hearing loss in 1997,2 mutations have been
described in many different populations around the
world, with specific genotypes segregating with particular ethnic populations.3 As complete DNA sequencing of
From the Department of Otolaryngology-Head and Neck Surgery
(D.K.C.), University of California, San Francisco; and the Department of
Otolaryngology-Head and Neck Surgery (K.W.C.), Stanford University
School of Medicine, Stanford, California, U.S.A
Send correspondence to Dylan K. Chan, MD, PhD, Pediatric Otolaryngology, 2233 Post Street, 3rd Floor, Box 1225, San Francisco, CA
94115. E-mail: [email protected]
Editor’s Note: This Manuscript was accepted for publication on July 10,
2013.
This manuscript was presented at the Spring Meeting of the Triological Society, Orlando, Florida, U.S.A., April 13, 2013.
The authors have no funding, financial relationships, or conflicts
of interest to disclose.
DOI: 10.1002/lary.24332
Laryngoscope 124: February 2014
E34
the coding region of GJB2 has become routine, variability of auditory phenotype has become increasingly evident. Degree of hearing loss is known to range from
profound deafness at birth to mild, progressive hearing
loss presenting in late childhood, and is highly dependent on genotype.4
The ubiquity of GJB2-associated hearing loss around
the world is notable for a highly prevalent genetic disease. Several mutant alleles have been described as being
particularly prevalent: 35delG,5 found in Europe and the
Middle East; 235delC,6 seen in East Asia; V37I,7 common
in Southeast Asia; and W24X,2 predominant on the
Indian subcontinent. Other mutations such as 167delT8
and R143W9 have been identified in very narrow populations—among the Ashkenazim and Ghanaians, respectively. We are interested in compiling and comparing the
genotype frequencies for GJB2-associated hearing loss in
different populations around the world. The purpose of
this is twofold: 1) with the growing diversity of pediatric
patient populations in urban centers around the world, it
is crucial to understand the variability in GJB2associated hearing-loss phenotype and how it relates to
country of origin; and 2) identifying patterns of GJB2
genotype between different populations may shed light on
the evolutionary history and etiology of this form of
Chan & Chang: Systematic Review of Cx-26-Associated Hearing Loss
congenital hearing loss. Related to this second aim, we
are also interested in examining the carrier frequencies
of different mutant alleles associated with hearing loss.
Finally, we wish to make use of the systematic review of
articles pertaining to GJB2-associated hearing loss to perform meta-analyses of various aspects of the auditory
phenotype in order to better understand the variability
seen in this disorder.
MATERIALS AND METHODS
Systematic Review
A search of Pubmed was undertaken on July 18, 2012,
with the search parameter “(GJB2 OR Connexin 26 OR Cx26)
AND (hearing OR deafness).” These parameters were chosen to
yield a broad, comprehensive review of literature pertaining to
GJB2-associated hearing loss. A total of 1,026 articles were
thus identified. Titles and abstracts were screened, and 245
studies were chosen for full-length review according to the following criteria:
a. Inclusion criteria
i. Primary source
ii. Report of allele and/or genotype frequencies for mutations
in GJB2 in human subjects
b. Exclusion criteria
i. Specific statement in the abstract that complete DNA
sequencing was not performed in all patients for identification of GJB2 mutations. The only exceptions to this criterion were made for articles that specifically state in the
title or abstract that they report carrier rates of 35delG
or V37I in the general population.
ii. Patient population described specifically as having syndromic or autosomal dominant hearing loss, or hearing
loss acquired as a result of known pre-, peri-, or postnatal ototoxic insult due to viral infection, jaundice, meningitis, or other condition.
iii. Only one mutation analyzed or reported
iv. Fewer than ten probands described
v. Sample population reported in previous publication
After full-length review, an additional 15 studies were
excluded for not fulfilling these basic criteria, leaving 230
articles for further consideration for meta-analysis. The investigators (DKC and KWC) independently conducted this review and
agreed upon the final included studies.
GJB2 Genotype Frequency
Using this library of 230 studies as a base, subsets of studies were selected for meta-analysis using the protocols listed
below. An additional 14 studies were ultimately excluded for
not fulfilling the inclusion and/or exclusion criteria for any of
these meta-analyses, leaving 216 articles that were used in various subsets as follows (Fig. 1):
1. Prevalence of biallelic GJB2 hearing loss among all deaf
probands
a. Inclusion criteria
i. Study population consisting of individuals with SNHL
ii. Report of number of patients with biallelic GJB2associated hearing loss
b. Exclusion criteria
i. Original study cohort consisting exclusively of either
DFNB1 or biallelic GJB2-hearing-loss probands
ii. Complete DNA sequencing of all hearing-loss probands not performed
Laryngoscope 124: February 2014
iii. Inability to ascertain the geographic origin for all subjects involved in the study. Specification of ethnic or
racial background was not a requirement.
c. From included studies, the number of hearing-impaired
probands and confirmed biallelic GJB2-associated hearing-loss probands was extracted and combined across
studies on the basis of country of origin. “Country of origin” is defined as the country in which the subject participated in the study, except in specific instances where a
particular ethnic group was being studied, whose ancestral country of origin was reported.
2. Prevalence of the 35delG, 235delC, W24X, and V37I alleles
among all individuals with biallelic GJB2-associated hearing
loss.
a. Inclusion criteria
i. Study population consisting of individuals with SNHL
ii. Specification of GJB2 genotype for all biallelic GJB2hearing-loss probands
b. Exclusion criteria
i. Complete DNA sequencing of all hearing-loss probands
not performed
ii. Inability to ascertain the geographic origin for all studies involved in the study. Specification of ethnic or
racial background was not a requirement.
c. From included studies, the number of 35delG, 235delC,
W24X, and V37I alleles among all homozygotic and compound heterozygotic individuals with biallelic GJB2associated hearing loss was extracted for each allele separately and combined across studies on the basis of country
of origin. For 35delG and V37I, due to their ubiquity,
every included study was used for the analysis. For
235delC and W24X, the analysis was limited to countries
in which either of these alleles was identified as the most
common allele.
3. Carrier frequency of 35delG, 235delC, W24X, and V37I
alleles among the general population
a. Inclusion criterion
i. Description of 35delG, 235delC, W24X, or V37I allele
frequencies among the general population, without bias
to hearing ability. Confirmation of normal hearing in
this population was not required.
b. Exclusion criterion
i. Inability to ascertain the geographic origin for all studies involved in the study. Specification of ethnic or
racial background was not a requirement.
c. From included studies, the number of 35delG, 235delC,
W24X, or V37I alleles among the general control population was extracted for each allele separately and combined
across studies.
4. Case-control comparison of carrier frequency of 35delG,
235delC, W24X, and V37I alleles between hearing-impaired
and general populations
a. Inclusion criterion
i. Rates of simple heterozygosity (carrier rates) reported
for hearing-impaired and control general populations
conducted in the same study.
b. Exclusion criterion
i. None
c. From included studies, the number of 35delG, 235delC,
W24X, or V37I alleles in simple heterozygosity among the
hearing-impaired and general control population was
extracted for each allele. Odds ratios and 95% confidence
intervals were calculated for each study and pooled to
obtain a summary effect using a random effects model
(metan function, Stata 12.1, StataCorp, College Station,
TX).
Chan & Chang: Systematic Review of Cx-26-Associated Hearing Loss
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Fig. 1. Included studies. The primary author (reference number), year of publication, and countries of origin of subjects is noted for each of
the 216 included studies. For each study, the meta-analysis in which that study was included is indicated with an “X.” GJB2: meta-analysis
of prevalence of biallelic GJB2-associated hearing loss among all hearing-loss probands. Allele frequency: meta-analysis of the prevalence
of each specific allele among all biallelic GJB2-associated hearing-loss probands. Carrier frequency: meta-analysis of the prevalence of
carriers of each specific allele among the general population. Case-control: meta-analysis of case-control studies comparing the prevalence of carriers of each specific allele among hearing-impaired cases and general population controls. Progression, symmetry, and
inner-ear anomalies: meta-analysis of the prevalence of each of these phenotypic attributes among all biallelic GJB2-associated hearingloss probands.
GJB2-Associated Hearing-Loss Phenotype
Individual-level meta-analysis of progression, asymmetry,
and inner-ear imaging anomalies in GJB2-associated hearingloss was performed. Studies were included from among the 230
studies identified in the systematic review if they provided
prevalence data for any of these phenotypic findings among
biallelic GJB2-associated hearing-loss probands. Appropriate
data were extracted from studies reporting the prevalence of
each of these properties and combined across studies. Strict definitions for progression, asymmetry, and inner-ear anomalies
were not required, but were recorded when available.
RESULTS
Prevalence of Biallelic GJB2-Associated Hearing
Loss
Of the 230 articles identified from the systematic
review, 175 articles met criteria for meta-analysis of
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GJB2 prevalence on a country-by-country basis (Fig. 1).
An additional four studies were included for larger
continent-wide analysis. These studies encompassed
43,530 hearing-loss probands and 7,518 individuals with
biallelic GJB2-associated hearing loss, for a worldwide
prevalence of 17.3%. The hearing-loss cohort varied from
one study to another, with precise inclusion criteria occasionally available. Four broad categories were identified:
1) all hearing loss, unspecified; 2) congenital hearing
loss; 3) nonsyndromic hearing loss; and 4) autosomal
recessive nonsyndromic hearing loss. The respective
rates of biallelic GJB2-associated hearing loss for these
four groups were 12.3%, 16.9%, 18.1%, and 21.3%, consistent with the expected increase in prevalence as the
patient cohort is narrowed down toward the nonsyndromic autosomal recessive inheritance pattern for
GJB2. Each of these prevalence rates was statistically
significantly different than one another (P < 0.0001,
Chan & Chang: Systematic Review of Cx-26-Associated Hearing Loss
Fig. 1. (Continued)
Chi-squared test), except for the comparison between
congenital and nonsyndromic hearing loss. Because of
the imprecise definitions of patient cohorts in many
studies, we elected to pool all studies regardless of initial
patient cohort for the subsequent meta-analyses.
Sixty-three countries were represented with at least
one study distributed over all six inhabited continents
(Figs. 2, 3). Prevalence was the highest in Europe (27.1%)
and the lowest in sub-Saharan Africa (5.6%), but generally high throughout the world. The most common allele
in each country is described in Figure 2 and Figure 4.
35delG is predominant throughout Europe, North Africa,
and the Middle East, as well as areas populated largely
by immigrants from these regions. The Indian subcontinent features W24X, whereas in East Asia 235delC and
V37I are highly prevalent. Other specific populations
were characterized by other mutations, including 167delC
among the Ashkenazim in Israel and R143W in Ghana.
Prevalence of 35delG- and V37I-Related Genotypes of Bi-Allelic GJB2-Associated Hearing Loss
The allele and genotype frequencies of biallelic
GJB2-associated hearing loss relating to the 35delG and
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V37I alleles were assessed in every included study.
These two alleles were chosen for this analysis because
they represent opposite spectrums of the disease: 35delG
is a truncating mutation predominant in much of the
world and typically gives rise in homozygous form to
profound congenital hearing loss,10 whereas V37I is a
highly prevalent missense allele associated with a very
mild hearing phenotype and high carrier rate in certain
general populations.11
Of the 230 articles identified from the systematic
review, 180 and 169 articles met criteria for metaanalysis of 35delG and V37I allele frequency, respectively, on a country-by-country basis. An additional six
studies were included in larger continent-wide analysis for each allele. The allele frequencies for 35delG
and V37I in each country are shown in Figures 2, 5,
and 6.
Worldwide, among all reported cases of bi-allelic
GJB2-associated hearing loss, 35delG accounts for 57%
of alleles, with allele frequencies in individual countries ranging from 0% in South and East Asian countries to 100% in Europe, North Africa, and the Middle
East. The majority of these alleles (82.4%) are
Chan & Chang: Systematic Review of Cx-26-Associated Hearing Loss
E37
Fig. 1. (Continued)
accounted for in 35delG homozygotes, with the
remainder (17.6%) seen in compound heterozygosity
with another disease-causing GJB2 allele. Overall,
among all hearing-loss probands, the prevalence of
35delG homozygosity is 8.5%, and 35delG heterozygosity (including both simple and compound heterozygotes) is 5.3%.
V37I, on the other hand, accounts for only 5.7% of
all alleles reported worldwide. It is uncommon in most of
Europe, but accounts for a large proportion of the alleles
described in Taiwan, Thailand, Canada, Australia, and
China. A total of 55.2% of alleles are accounted for by
V37I/V37I homozygotes, and 44.8% are involved in compound heterozygosity. The prevalence values of V37I
homozygotes and all heterozygotes among all hearing-loss
probands are 6.9% and 19.6%, respectively.
Prevalence of 235delC and W24X-Related Genotypes of Bi-Allelic GJB2-Associated Hearing Loss
The 235delC and W24X alleles are important in
East Asia and the Indian subcontinent, respectively. We
analyzed the frequencies of these two alleles in each
study where they represent the most common diseaseLaryngoscope 124: February 2014
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causing GJB2 allele (Fig. 2). For 235delC, 31 studies
were thus identified, representing 9,781 hearing-loss
probands, 1,625 (16.6%) of which had biallelic GJB2associated hearing loss. Twenty-five of these studies had
sufficient genotype-specific data to allow meta-analysis.
In seven countries (China, Japan, Korea, Taiwan, Thailand, Mongolia, and Russia), the overall allele frequency
among biallelic GJB2-associated hearing-loss probands
is 38.6%. W24X was identified as a major allele in 12
studies from seven countries (Bangladesh, India, Pakistan, Iran, Czech Republic, Turkey, and Spain). A total
of 224 of 1424 (15.7%) hearing-loss probands had biallelic GJB2-associated hearing loss; among these individuals, the W24X allele accounted for 47.3% of all GJB2
mutations.
Carrier Frequencies of GJB2 Alleles Associated
With Hearing Loss
The frequencies in the general population of GJB2
alleles associated with hearing loss was assessed (Fig.
2). Data from 52,715 normal-hearing controls from 115
studies across 55 countries reveals a worldwide carrier
rate of 1.5% for 35delG. This ranged from 0% in multiple
Chan & Chang: Systematic Review of Cx-26-Associated Hearing Loss
Fig. 1. (Continued)
countries to 5.7% in Belarus. V37I was even more disparate; worldwide, the carrier rate among 20,866 controls across 72 studies from 26 countries is 2.5%,
ranging from 0% in most of these countries to as high as
16.7% in Thailand.
Case-Control Study of Carrier Frequencies
Comparison of carrier rates—specifically, the prevalence of simple heterozygotes of a particular allele—
between hearing-impaired and control populations was
performed. Thirty-five studies were identified that
included carrier rates for 35delG in both hearingimpaired and control populations. The odds ratio (OR)
for being a 35delG carrier given a history of hearing loss
ranged widely, from 0.06912 to 22.3.13 Meta-analysis of
these 35 studies using a random effects model yields a
summary OR of 1.79 (95% CI: 1.29–2.48), indicating that
35delG carriers are relatively overrepresented among
the hearing-loss cohort (Fig. 7).
Two potential confounders for this finding are assortative mating and the presence of complementary genes.
Assortative mating can be controlled for by only including studies where the hearing-loss cohort shows either
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sporadic or autosomal recessive inheritance pattern,
with no history of deafness beyond the proband and his/
her siblings. Limitation of the meta-analysis to 16 studies for which this inheritance pattern could be confirmed
yielded a composite OR of 2.47 (95% CI: 1.55–3.95). This
finding suggests that assortative mating does not play a
role in the increased carrier rate among the hearingimpaired.
The discovery of complementation of GJB2 mutations at the DFNB1 locus by the del(GJB6-D13S1830)
deletion may also confound these results, as patients
reported as simple heterozygotes without assessment of
this locus would not be appropriately allocated. Limitation of the meta-analysis to 12 studies where the
del(GJB6-D13S1830) was assessed yields a composite
OR of 1.42 (95% CI: 0.76–2.65). Further narrowing to
the five studies where both del(GJB6-D13S1830) was
assessed, and hearing-loss probands have no family history of hearing loss gives a summary OR of 2.61 (95%
CI: 0.69–9.80). These results suggest that there either
remain additional complementary genes that have yet to
have been discovered, or that being a carrier for 35delG
indeed is associated with hearing loss, though with very
limited penetrance.
Chan & Chang: Systematic Review of Cx-26-Associated Hearing Loss
E39
Fig. 1. (Continued)
Meta-analysis of four case-control studies for the
W24X allele yielded a similar summary OR of 2.06
(95% CI 0.33–13.07). When nine case-control studies
for the 235delC allele were compared, a larger effect
was seen; the summary OR was 4.55 (95% CI: 1.28–
16.13), even more strongly implicating the presence of
complementary genes or a carrier phenotype. The
majority of these studies did not address del(GJB6D13S1830), as it is very rare in South and East Asian
populations.
Overall, pooling all 48 case-control studies of the
carrier rate of these three truncating mutations among
hearing-impaired and normal control individuals yields
an aggregate OR of 2.27 (95% CI: 1.53–3.36). There are,
therefore, more than twice as many carriers among the
hearing-impaired, as can be explained by the population
allele frequency alone. The overall carrier rate in the
general population across these 48 studies is 1.7%; taking into account the aggregate OR of 2.27, there would
be an additional 2.2% of the hearing-impaired population whose carrier status is not explained by chance
alone. This implies that either there are complementary
genes that act together with GJB2 to account for 2.2% of
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all congenital hearing loss, or there is a carrier phenotype that is penetrant in a small fraction of carriers of
truncating mutations in GJB2.
Interestingly, when 18 case-control studies for carrier frequencies for the nontruncating mutation V37I
were pooled, a summary OR of 0.78 (95% CI: 0.52–1.18)
was observed (Fig. 8), which is significantly different
from that observed for the aggregated truncating alleles.
This suggests three possibilities: 1) hearing loss is not
associated with the V37I carrier state; 2) there are no
additional complementary genes that act in concert with
V37I; or 3) a carrier effect or digenic inheritance occurs,
but the phenotype is either very mild or minimally penetrant so as to be widely unrecognized.
Different Disease-Causing Alleles of GJB2
Account for Similar Rates of Hearing Loss and
Carriers in Disparate Populations
GJB2-associated hearing loss is notable in that
there are multiple independent alleles that are predominant in very disparate populations around the world,
and that each of these alleles represents a major cause
Chan & Chang: Systematic Review of Cx-26-Associated Hearing Loss
Fig. 2. Distribution of common disease-causing variants in GJB2 around the world. For each country, composite values obtained from
meta-analyses of systematically reviewed studies are presented. Most common allele: the most highly prevalent disease-causing
GJB2 variant among all biallelic GJB2 probands. Biallelic GJB2 prevalence: prevalence of biallelic GJB2-associated hearing loss
among all hearing-loss probands. Allele frequency: frequency of each specified allele among all biallelic GJB2-associated hearingloss probands. Data for 35delG and V37I are presented for all countries with available data; for countries where neither 35delG nor
V37I are the most common allele, the allele frequency is presented for the most common allele. Carrier frequency: frequency of simple carriers of each specified allele among the general population. Data for 35delG and V37I are presented for all countries with available data; for countries where neither 35delG nor V37I are the most common allele, the allele frequency is presented for the most
common allele.
of congenital hearing loss in each of these populations.
To quantify and compare this, we grouped countries
according to the most common allele present in that
country and calculated for each group: 1) the prevalence
among all hearing-loss probands of biallelic GJB2 associated hearing-loss; 2) frequency among these individuals
of the most common allele in that population; and 3) carrier frequency among the general population.
Results are shown in Figure 9. 35delG was by far
the most widely distributed allele, accounting for the
majority of alleles in 47 countries. However, multiple
countries, accounting for large geographic areas and
proportions of the world’s population, feature other
alleles, specifically 235delC, W24X, and V37I. Finally,
three other distinct mutations were identified as highly
prevalent in Israel, Ghana, and Mongolia. Focusing on
Laryngoscope 124: February 2014
the three most important truncating mutations—
35delG, 235delC, and W24X—the prevalence of biallelic
GJB2-associated hearing loss (range: 11%–18%), frequency of the allele (50%–52%), and carrier frequency
(0.7%–1.7%) is remarkably similar across all three large
populations. The countries in which these three mutations are endemic together account for 68.4% of the
world’s population; in contrast, countries in which the
rate of GJB2-associated hearing has been reported to
be low or nonexistent only make up 5.1% of the world’s
population. The widespread existence of these mutations as the major cause of congenital hearing loss in
these disparate populations attests to the importance of
this gene in hearing function, and also suggests the
likelihood of a wider evolutionary significance to alterations in GJB2.
Chan & Chang: Systematic Review of Cx-26-Associated Hearing Loss
E41
Fig. 2. (Continued)
GJB2-Associated Hearing-Loss Phenotype
Variability in severity of hearing loss, and its
dependence on genotype, has been well documented previously.4 Progression of hearing loss has varied widely
between studies. Meta-analysis of 28 studies presenting
progression data on 1,140 patients yielded an overall progression rate of 18.7% (range: 0%–56.0%). All but seven
articles provided a precise definition for progression; an
additional four used only patient report of postlingual
onset as a definition for progression. The remaining 17
all had strict audiologic criteria for progression. The rate
of asymmetry was similarly calculated over 19 studies
encompassing 801 biallelic GJB2-associated hearing-loss
probands to be 14.2% (range: 0%–55.6%). A total of 13/19
studies had objective criteria presented to define asymmetric hearing loss. Finally, the incidence of imaging
abnormalities among this population was assessed in 10
studies. A total of 16% (range: 3.1%–76.9%) of the 406
patients tested had some imaging abnormality.
DISCUSSION
Over the past 15 years, tremendous amounts of epidemiologic data have been collected regarding GJB2Laryngoscope 124: February 2014
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associated hearing loss around the world. This study is
an attempt to synthesize these data to serve as both a
reference for clinicians working up and counseling
patients with congenital SNHL, and to provide strong
evidence on a large scale that corroborates findings of 1)
increased carrier rates among the hearing impaired
(which suggests the existence of additional complementary genes or a true carrier phenotype) and 2) widespread prevalence of distinct GJB2 mutations around
the world (which suggests an evolutionary advantage
conferred by mutations in GJB2).
Limitations of the Study
The primary limitation of this study is the heterogeneity of the included studies. Specifically, the starting population of hearing-loss probands was quite varied between
studies, and was not precisely defined for the majority.
This heterogeneity made it impossible to precisely define
meaningful subgroups of hearing-loss probands; therefore,
we elected to group all studies for our analysis. The vast
majority of studies specifically excluded syndromic forms
of hearing loss. Although we included some studies that
did not address inclusion of syndromic hearing loss, we
Chan & Chang: Systematic Review of Cx-26-Associated Hearing Loss
Fig. 3. Prevalence of GJB2-associated hearing loss among all hearing-loss probands. Percentage of individuals with hearing loss having
two disease-causing mutations in GJB2 in shown. Countries with no data are shown in gray. [Color figure can be viewed in the online issue,
which is available at wileyonlinelibrary.com.]
did exclude studies that exclusively looked at syndromic
subjects. Specification of suspected genetic attribution and
the presumed inheritance pattern was very heterogeneous. Commonly excluded acquired forms of congenital
hearing loss were congenital viral infections associated
with hearing loss, neonatal jaundice, and meningitis. A
fraction of the studies limited their cohort to individuals
with an inheritance pattern consistent with autosomal
Fig. 4. The most common GJB2 mutation. The most common GJB2 mutation found in each country is shown. Countries where no instances of biallelic GJB2-associated hearing loss are shown in black. Countries with no data are shown in gray. [Color figure can be viewed in
the online issue, which is available at wileyonlinelibrary.com.]
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Chan & Chang: Systematic Review of Cx-26-Associated Hearing Loss
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Fig. 5. The 35delG allele frequency. The prevalence of the 35delG allele among all cases of biallelic GJB2-associated hearing loss is shown.
[Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
recessive inheritance (either multiple affected siblings
with unaffected parents, or sporadic cases). Other common, specific exclusion criteria among the studies were
the presence of hearing loss that was unilateral, postlingual, or mild-moderate in severity (including a number of
studies specifically on patients undergoing cochlear
implantation). A handful of studies were limited to children, but the majority did not specify age.
Certain specific findings of allele frequencies in different countries are likely highly dependent on the particular population samples, and are thus subject to
sampling bias. For example, the high rate of V37I in
Canada is likely attributable to one study14 with a disproportionately high percentage of East Asian probands,
which is not reflective of the Canadian population as a
whole. Most studies do not specify the ethnic or racial
Fig. 6. The V37I allele frequency. The prevalence of the V37I allele among all cases of biallelic GJB2-associated hearing loss is shown.
[Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
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Chan & Chang: Systematic Review of Cx-26-Associated Hearing Loss
Fig. 7. Carrier-rate meta-analysis for truncating mutations. Odds ratios (ORs) and 95% confidence intervals were calculated for carrier rates of
three truncating mutations—35delG, 235delC, and W24X—in case-control studies examining the carrier rate in hearing-impaired cases and
unaffected general population controls. ORs to the right of the vertical black line indicate higher odds of being a carrier of one of these
mutations in the hearing-impaired population. Meta-analyses using a random-effects model yielded summary ORs and confidence intervals.
*Studies where exclusively sporadic cases of hearing loss were evaluated. # studies in which del(GJB6-D13S1830) was evaluated.
background of their study population, and this analysis
is limited to geography, not ethnicity. This is particularly
important to note in countries in North America, South
America, and Australia, where urban, academic centers
feature high proportions of immigrant populations from
around the world. Conversely, small studies in countries
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with highly compartmentalized ethnic populations, such
as many African and Asian countries, may misrepresent
the GJB2 prevalence in those ethnic groups not represented in the study performed. China, which is itself a
very large and heterogeneous country, was not subjected
to finer analysis based on subregion; in many instances,
Chan & Chang: Systematic Review of Cx-26-Associated Hearing Loss
E45
Fig. 8. Carrier-rate meta-analysis for V37I. Odds ratios (ORs) and 95% confidence intervals were calculated for carrier rates of V37I, a
non-truncating mutation, in case-control studies examining the carrier rate in hearing-impaired cases and unaffected general population
controls. ORs to the right of the vertical black line indicate higher odds of being a carrier of V37I in the hearing-impaired population.
Meta-analysis using a random-effects model yielded a summary OR and 95% confidence interval.
the subject group consisted of individuals from deaf
schools, for whom subregion of origin was not specified.
Both 235delC and V37I were highly prevalent in a number of studies, with a trend toward 235delC being more
common in northern China and V37I prevalent in southern China, consistent with the patterns in neighboring
countries. In general, prevalence data for V37I-related
genotypes may be underreported, especially in older
studies, depending on whether the V37I allele was considered disease-causing. It is not clear in all studies
whether V37I was not reported due to this uncertainty.
Because of the uncertainty about V37I reporting and
subregion of origin, we elected to analyze China as a
whole. Despite these uncertainties with regards to ethnicity, where general patterns are consistent and patient
numbers are high, robust conclusions can be made; this
is certainly true for 35delG in Europe, North Africa, and
the Middle East, 235delC in East Asia, V37I in Southeast Asia, and W24X in the Indian subcontinent.
Finally, other than one study in which progression
was associated with the V37I exclusive genotype,11 phenotypic findings of progression, asymmetry, and concomitant imaging abnormalities were not reported together
with specific genotypes. Rather, these attributes were
calculated for all pooled biallelic GJB2 hearing-loss proLaryngoscope 124: February 2014
E46
bands. Thus, they are subject to the particular genotypic
mix represented in the included studies. This is particularly significant for progression of hearing loss; although
some studies specifically limited the analysis of progression to patients who initially presented with less than
severe hearing loss, the majority did not. Inclusion of
patients in a progression analysis based only on the
presence of multiple audiograms without exclusion of
those with initial severe or profound hearing loss may
lead to underreporting of the rate of progression among
patients for whom progression is more relevant—those
with only mild or moderate hearing loss. Because the
majority of studies where progression was calculated
featured 35delG/35delG as the most common genotype,
and this genotype is typically associated with bilateral
severe-to-profound hearing loss at presentation, the
reported aggregate rate of 18.7% progression is likely an
underestimate for genotypes that present with more
mild hearing loss. Furthermore, owing to the particular
inclusion criteria of this systematic review, which was
not expressly designed to capture all studies addressing
phenotype of GJB2-associated hearing loss, the metaanalyses of progression, symmetry, and imaging abnormalities, while relatively large in sample size, might not
include all relevant studies.
Chan & Chang: Systematic Review of Cx-26-Associated Hearing Loss
Fig. 9. Genotype and allele prevalence rates across mutations. For each of six mutations—35delG, 235delC, W24X, V37I, R143W, and
167delT—data were pooled for each mutation across the countries in which they represented the most common mutation. (A) Prevalence
of biallelic GJB2-associated hearing loss among all hearing-loss probands; (B) carrier frequency among unaffected general population controls; and (C) allele frequency among all biallelic GJB2-associated hearing-loss probands are shown for each of these six mutations. (D)
Percentage of the world’s population accounted for by countries in which each of these six alleles are found in significant abundance
among both the hearing-impaired and the general population. Countries in which GJB2 alleles have been evaluated, and which have very
low or non-existent rates of GJB2 mutations are shown in black. Countries in which GJB2 allele frequencies have not been assessed are
indicated in gray. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Genotype and Ethnicity: An Evolutionary
Advantage Associated With Mutations in GJB2?
Biallelic GJB2-hearing loss is common throughout
the world; it accounts for greater than 10% of all SNHL
probands in all major regions of the world except for
sub-Saharan Africa. Specific alleles correlate highly with
geography. In Europe, the Middle East, and North
Africa, and by extension owing to immigration patterns,
North and South America, and Australia, 35delG is predominant. In East Asia 235delC and V37I are very common, and W24X dominates the Indian subcontinent.
Laryngoscope 124: February 2014
Together with R143W, which is highly prevalent in
Ghana, and 167delT, which is found in high frequency
among the Ashkenazim, these represent a total of six
independently arisen mutations that have come to represent the most prevalent single cause of congenital SNHL
in six disparate populations around the world, which in
aggregate account for almost 70% of the world’s population. The high carrier frequencies of each of these mutations among the general population in which they are
endemic, which range from 0.7% to 16%, comparable to
those of some of the most common genetic disorders,
Chan & Chang: Systematic Review of Cx-26-Associated Hearing Loss
E47
including cystic fibrosis and sickle-cell anemia, further
support the supposition that these mutations are unusually prevalent in so many distinct populations. This finding suggests that there may be an evolutionary
advantage to being a carrier for a mutation in GJB2,
which has led to the widespread persistence of these
multiple mutations after each independently arose in
each of these different populations. Dominant-negative
mutations in GJB2 are associated with hyperkeratosis
in the context of KID syndrome. The expression of Cx26
in the epidermis raises the possibility of a skin-based
protective advantage, perhaps related to susceptibility to
skin barrier breakdown, infection, or neoplasm. Epidermal thickening has been described in both 35delG15and
R143W16 heterozygotes, R143W-transfected epithelial
cells display resistance to cell death,17 and Cx26 signaling has been implicated in Shigella-based bacterial invasion.18 The precise mechanism for this protection merits
further investigation.
Carrier Rate Is Significantly Higher Among
Hearing-Impaired Individuals
Meta-analysis of case-control studies of carrier rates
of pathogenic mutations in GJB2 between hearingimpaired and normal control populations revealed a significantly higher carrier rate among the hearing-impaired
for 35delG, W24X, and 235delC, but not for V37I. Three
possible causes of this disparity could be assortative mating, unidentified complementary mutations, and the presence of a variably penetrant carrier phenotype. Subgroup
analysis suggested that assortative mating does not contribute to this phenomenon. The complementary mutation
hypothesis suggests that there are either unidentified
mutations in regulatory elements that act together with
the known mutation to effectively lead to a biallelic mutation, as has been demonstrated for del(GJB6-D13S1830)
and del(GJB6-D13S1834),19,20 or completely separate
genes that act together with GJB2 in a digenic manner to
lead to hearing loss. Such modifiers and regulatory elements have not yet been identified.21,22
The fact that the disparity in carrier frequencies is
not significantly different between 35delG, W24X, and
235delC suggests that such unidentified mutations
would be widespread, or else multiply independently
arisen in these three populations, which seems unlikely.
Furthermore, the finding that there is a significant difference between the truncating mutations (35delG,
235delC, and W24X) and the nontruncating mutation
(V37I) in terms of the disparity in carrier frequency
makes either a widespread GJB2 regulatory-element
mutation or digenic hypothesis somewhat less attractive,
though one could imagine a complementary mutation
present worldwide that exhibits a digenic inheritance
pattern with severe mutations, but not with a mild
mutation. Assuming, however, that such a complementary mutation would lead to hearing loss in conjunction
with either a truncating or a nontruncating mutation,
the existence of a widespread mutation complementing
35delG, W24X, and 235delC, but not V37I would not fit
with the ethnic distribution of these four alleles: there is
Laryngoscope 124: February 2014
E48
significant population overlap between 235delC and
V37I, which are both found in East Asia, and not
between 235delC, 35delG, and W24X.
More likely is that the three truncating mutations
have a variably penetrant carrier phenotype. That is, in
some individuals, missing one functional copy of the
GJB2 gene leads to hearing loss. This may be due to epigenetic allelic inactivation of the normal copy in some
individuals, or perhaps due to a gene dosage effect. Gap
junctions in the cochlea are formed as heterotypic heteromers between Cx26 and Cx30; altering the gene dosage of Cx26 could alter the stoichiometry of these gap
junctions and affect their function. In contrast to the
truncating mutations, where either gene dosage or inactivation effects could lead to significant effects on gapjunction integrity, the nontruncating mutation V37I,
which even in homozygosity leads only to mild hearing
loss, would not be expected to have a significant effect
on cochlear function and hearing. Overall, however, it is
not possible to definitively distinguish between the
digenic inheritance and carrier phenotype hypotheses in
this study. Further in-depth pedigree analysis and
focused genomic investigation is required to evaluate
this further.
Auditory Phenotype Is Highly Variable
Snoeckx et al.4 demonstrated in a very large cohort
that severity of hearing loss was highly variable and
dependent on genotype. The meta-analyses performed
here on progression, asymmetry, and concomitant imaging abnormalities corroborate the high degree of variability of the audiologic phenotype, and highlight the
importance of complete sequencing of the coding region
of GJB2 in all cases of congenital or early-onset SNHL.
Patients with clearly syndromic or autosomal dominant
hearing loss, as well as those with clearly identified
causes of hearing loss, such as congenital CMV infection,
meningitis, were usually excluded from these studies,
and this meta-analysis cannot address whether GJB2
sequencing is indicated in these cases. Subjects with unilateral hearing loss were also excluded at times; the
finding that 14.2% of subjects reported had some asymmetry, though, suggests that even those with unilateral
hearing loss may be at risk for harboring pathogenic
mutations in GJB2, and should be considered for genetic
analysis.23
The variability in auditory phenotype may reflect
the role of Connexin 26 in cochlear function, and the
pathophysiology of how dysfunction of Connexin 26 leads
to hearing loss. The contribution of Connexin 26-based
epithelial and connective-tissue gap junction networks in
the cochlea to sensory transduction is not completely
described. Important roles in potassium recycling, intercellular calcium signaling, and electrical coupling to support electromotile cochlear amplification have been
proposed.24,25 In most animal models of Connexin 26associated hearing loss, loss of hair cells, which do not
express Connexin 26, is a relatively delayed finding.26–28
There is likely, then, a cascade of pathophysiological
events that must take place for gap-junction dysfunction
Chan & Chang: Systematic Review of Cx-26-Associated Hearing Loss
to lead to hair-cell dysfunction and hearing loss; genetic
and environmental variability in how these mechanisms
propagate would underlie the phenotypic variability.
More investigation into genetic modifiers that underlie
this variability, as well as the pathophysiological mechanisms that are modified, is crucial to understand the
pathway involved and develop treatments that can be
aimed at preventing progression of hearing loss and
even hearing restoration.
CONCLUSION
This systematic review and meta-analysis confirms
that biallelic GJB2-associated hearing loss is nearly universally highly prevalent around the world. Phenotype
is highly variable, and correlates with genotype; genotype, in turn, is highly dependent on geography.
Multiply-arisen, highly-prevalent mutations suggest the
possibility of an evolutionary advantage to being a carrier for a GJB2 mutation. Conversely, analysis of carrier
rates suggests that even carriers of GJB2 mutations are
at higher risk for hearing loss, even without invoking
the presence of additional complementary mutations.
Overall, the wide genetic and phenotypic variability
associated with this most common form of congenital
SNHL invites significant investigation into its genomic
and pathophysiologic mechanisms.
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