ORIGINAL RESEARCH
published: 18 September 2019
doi: 10.3389/fgene.2019.00841
GJB2 and GJB6 Mutations in Non-
Syndromic Childhood Hearing 
Impairment in Ghana
Samuel M. Adadey 1, Noluthando Manyisa 2, Khuthala Mnika 2, Carmen de Kock 2,  
Victoria Nembaware 2, Osbourne Quaye 1, Geoffrey K. Amedofu 3, Gordon A. Awandare 1 
and Ambroise Wonkam 2*
1 West African Centre for Cell Biology of Infectious Pathogens (WACCBIP), University of Ghana, Accra, Ghana, 2 Division of 
Human Genetics, Faculty of Health Sciences—University of Cape Town, Cape Town, South Africa, 3 Department of Eye, Ear, 
Nose and Throat, School of Medical Sciences, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana
Our study aimed to investigate GJB2 (connexin 26) and GJB6 (connexin 30) mutations 
Edited by: associated with non-syndromic childhood hearing impairment (HI) as well as the 
Zané Lombard, environmental causes of HI in Ghana. Medical reports of 1,104 students attending schools 
University of the Witwatersrand, 
South Africa for the deaf were analyzed. Families segregating HI, as well as isolated cases of HI of 
Reviewed by: putative genetic origin were recruited. DNA was extracted from peripheral blood followed 
Aime Lumaka, by Sanger sequencing of the entire coding region of GJB2. Multiplex PCR and Sanger 
University of Liège, sequencing were used to analyze the prevalence of GJB6-D3S1830 deletion. Ninety-
Belgium 
Colleen Aldous, seven families segregating HI were identified, with 235 affected individuals; and a total 
University of KwaZulu-Natal, of 166 isolated cases of putative genetic causes, were sampled from 11 schools for the 
South Africa deaf in Ghana. The environmental factors, particularly meningitis, remain a major cause of 
*Correspondence: 
Ambroise Wonkam HI impairment in Ghana. The male/female ratio was 1.49. Only 59.6% of the patients had 
ambroise.wonkam@uct.ac.za their first comprehensive HI test between 6 to 11 years of age. Nearly all the participants 
had sensorineural HI (99.5%; n = 639). The majority had pre-lingual HI (68.3%, n = 754), 
Specialty section: of which 92.8% were congenital. Pedigree analysis suggested autosomal recessive 
This article was submitted to 
Genetic Disorders, inheritance in 96.9% of the familial cases. GJB2-R143W mutation, previously reported 
a section of the journal as founder a mutation in Ghana accounted for 25.9% (21/81) in the homozygous state in 
Frontiers in Genetics
familial cases, and in 7.9% (11/140) of non-familial non-syndromic congenital HI cases, 
Received: 28 February 2019
Accepted: 13 August 2019 of putative genetic origin. In a control population without HI, we found a prevalent of 
Published: 18 September 2019 GJB2-R143W carriers of 1.4% (2/145), in the heterozygous state. No GJB6-D3S1830 
Citation: deletion was identified in any of the HI patients. GJB2-R143W mutation accounted 
Adadey SM, Manyisa N, Mnika K, for over a quarter of familial non-syndromic HI in Ghana and should be investigated in 
de Kock C, Nembaware V, Quaye O, 
Amedofu GK, Awandare GA and clinical practice. The large connexin 30 gene deletion (GJB6-D3S1830 deletion) does not 
Wonkam A (2019) GJB2 and account for of congenital non-syndromic HI in Ghana. There is a need to employ next 
GJB6 Mutations in Non-Syndromic 
Childhood Hearing generation sequencing approaches and functional genomics studies to identify the other 
Impairment in Ghana. genes involved in most families and isolated cases of HI in Ghana.
 Front. Genet. 10:841. 
doi: 10.3389/fgene.2019.00841 Keywords: hearing impairment, genetics, GJB2 and GJB6, Ghana, Africa
Frontiers in Genetics | www.frontiersin.org 1 September 2019 | Volume 10 | Article 841
Adadey et al. GJB2 and GJB6 in Hearing Impairment in Ghana
INTRODUCTION geneticist and an ENT specialist, and relevant data extracted, 
including three-generation pedigree and perinatal history. If 
Hearing impairment (HI) is a disabling congenital disease required, a general systemic and otological examination and 
(Neumann et al., 2019), with the highest rate for age- audiological evaluation were performed, including pure tone 
standardized disability of life in the world (Murray et al., 2015; audiometry or auditory brain stem response test. We followed 
Vos et al., 2016). Globally, congenital HI has a prevalence of the recommendation number 02/1 of the Bureau International 
1.3 per 1,000 population (James et al., 2018) and accounts d’Audiophonologie (BIAP), Belgium, to classify the hearing 
for about 1 per 1,000 live births in developed countries, levels (Bureau_International_d’Audiophonologie, 1997; 
with a much higher up to 6 per 1,000 in sub-Saharan Africa Wonkam et al., 2013). After consultation with the medical 
(Olusanya et al., 2014). To improve the cognitive, social, geneticist, individuals with syndromic deafness underwent 
speech, and language development of children living with additional assessment, when possible. As previously reported 
HI, early diagnosis and intervention are recommended (Wonkam et al., 2013), HI was defined as: 1) acquired when 
(Barnard et al., 2015). But in the absence of the widely used associated with a putative environmental factor such a clinical 
new-born screening, the age at diagnosis is usually late in evidence of meningitis; 2) genetic when at least two cases were 
Africa, e.g. 3.3 years in Cameroon (Wonkam et al., 2013). In reported in the same family without obvious environmental 
many populations, nearly half of congenital HI cases have a cause, in case of consanguinity, in case of presence of 
genetic etiology, of which 70% are non-syndromic (Bademci et dysmorphism or developmental problems in addition to HI, 
al., 2016; Sheffield and Smith, 2018). Among non-syndromic or in case of a well-defined syndrome in clinically suspected; 
(NS) HI, nearly 80% of the cases are inherited in autosomal 3) of unknown etiology if either an environmental or a genetic 
recessive (AR) mode (Wu et al., 2018; Zhou et al., 2019). To origin were not clearly established.
date, more than 98 genes have been identified, in ~170 NSHI 
loci mapped (Hereditary Hearing Loss Homepage; http:// Control Participants
hereditaryhearingloss.org/). Nevertheless, in many populations A total of 145 control participants without any personal or 
of European and Asian descent, pathogenic variants in familial history of HI was randomly recruited in Ghana, 
GJB2 (connexin 26 gene) and GJB6 are major contributors from an apparently healthy individual, during a tuberculosis 
to autosomal recessive NSHI (ARNSHI) (Chan and Chang, screening study.
2014), with the GJB6-D13S1830 deletion identified in up to 
9.7%, as the second biggest genetic etiology of NS deafness Molecular Methods
in the European populations (del Castillo et al., 2002; del Peripheral blood was used for genomic DNA extraction, following 
Castillo et al., 2003). the instructions on the manufacturer [QIAamp DNA Blood 
The prevalence of GJB2- or GJB6-related NSHI is very low in Maxi Kit. ® (Qiagen, USA)], in the Laboratory of the Department 
most sub-Saharan African populations (Gasmelseed et al., 2004; of Biochemistry, University of Ghana, Accra, Ghana.
Kabahuma et al., 2011; Bosch et al., 2014; Javidnia et al., 2014; Previously reported, primers for the GJB2 genes were evaluated 
Lasisi et al., 2014). Of interest, previous studies have shown using BLAST® and and other Softwares as recommended (Bosch 
that a common founder mutation accounted for about 16.2% of et al., 2014). The entire coding region of GJB2 genes (exon2) was 
congenital HI was p.R143W in a random sample of Ghanaians amplified, followed by sequencing using an ABI 3130XL Genetic 
affected by hearing loss (Hamelmann et al., 2001). To our Analyzer® (Applied Biosystems, Foster City, CA), in the Division 
knowledge, the contribution of connexin 30 to HI, and the carrier of Human Genetics, University of Cape Town, South Africa.
frequency of the GJB2 mutation in non-affected individuals has Detection of del (GJB6-D13S1830) was performed using the 
not been studied in Ghana (Adadey et al., 2017). In the present method and primers described by del (del Castillo et al., 2002; 
research, we aimed to investigate the putative environmental del Castillo et al., 2003). The entire coding region of GJB6 was 
causes of childhood HI, and revisit the contribution of GJB2, amplified using the method described by (Chen et al., 2012). 
and to investigate GJB6 mutations in carefully selected samples The PCR results were validated by Sanger sequencing of 10% 
of families segregating HI, and in isolated cases of putative of the sample.
genetic origin, as well control populations non-affected by HI, 
in Ghana. Data Analysis
Descriptive statistic and non-parametric test were used for 
comparisons. The level of significance was set at 5%.
METHODS
Patient Participants RESULTS
Hearing impaired patients were recruited from 11 schools 
for the deaf following procedures reported previously in Sex, Age of Onset of Hearing Impairment
Cameroon (Wonkam et al., 2013). Briefly, individuals with A total of 1,104 participants was evaluated (Figure 1). The male/
severe HI diagnosed before 15 years of age were enrolled in this female ratio was 1.49 (660/444). Most deaf participants (59.6%) 
study. For all participants, detailed personal and family history had their first comprehensive HI medical test between the ages 
were obtained, and the medical records reviewed by a medical of 6 to 11 years (Table 1 and Figure S1A). The median age of 
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Adadey et al. GJB2 and GJB6 in Hearing Impairment in Ghana
the students at the first medical diagnosis was 9.0 years, within a TABLE 1 | Age at diagnosis and onset of HI.
range of 2 to 22 years. The majority had pre-lingual HI (68.3%, Age of onset Number of cases, n (%)
n = 754; Figure S1B), of which 92.8% were congenital. Prelingual (before 2 years old) 754 (68.3%)
Perilingual (between 2 and 4 years) 69 (6.3%)
Post-lingual (after 4 years) 281 (25.5%)
Audiometric Characterization of HI Total 1,104
Analysis of the students’ medical data indicated that 642 out of Age at first diagnosis Number of cases, n (%)
the 1,104 students had a comprehensive HI test (otoscopic ear 0–5 157 (14.2%)
examination, pure tone audiometry, and/or tympanometry), 6–11 658 (59.6%)
which characteristics are described in Table S1. Nearly all the 12–17 258 (23.4%)
participants had sensorineural HI (99.5%; n = 639). Only 1 and 2 18–23 31 (2.8)
students had conductive and mixed HI, respectively. Total 1,104
Major Etiologies of Childhood HI in the Familial HI With Possible Patterns of HI 
Study Population Inheritance
The flowchart of the cohort is described in Figure 1, and the We identified 97 families segregating hearing Impairment, in 21.4% 
major cause of HI are displayed in Table 2. A lower frequency of the students. In these families, 50.9% (235/461) of children 
of infectious causes of HI was observed in our present study were living with HI, with an average family size of 6.9. Most of 
compared with other studies from sub-Saharan Africa these familial cases were non-syndromic (92/97). The pedigree 
(Table 2). Convulsion (with undetermined medical cause) analysis of the non-syndromic familial cases suggested autosomal 
was the most common cause of post-lingual HI followed by recessive inheritance in 96.7% (89/92), with only 2 families 
cerebrospinal meningitis (C.S.M.). Other diseases such as exhibiting a pattern compatible with a non-syndromic autosomal 
cerebral/complicated malaria, otitis media, and mumps, were dominant inheritance. One family exhibited a mitochondrial 
also reported as causes of post-lingual HI (Figure S2). Over pattern of inheritance.
60% of the students had congenital HI of unknown origin Waardenburg syndrome, an autosomal dominant condition, 
(Figure S2). was the obvious syndromic and familial condition identified 
FIGURE 1 | Flowchart of the recruitment and Molecular analysis of Hearing Impairment cases in Ghana. GJB2-R143W mutation, previously reported as founder a 
mutation in Ghana accounted for 27.2% (22/81) of familial, and in 7.9% (11/140) of non-familial non-syndromic congenital HI cases.
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Adadey et al. GJB2 and GJB6 in Hearing Impairment in Ghana
TABLE 2 | Comparison of our results to other studies in developing African countries.
Country The Gambia Nigeria Sierra Leone Ghana Cameroon Present study
Year of publication 1985 1982 1998 1988 2013 2018
Reference (McPherson and Holborow, 1985) (Ijaduola, 1982) (Wright, 1991) (Brobby, 1988) (Wonkam et al., 2013)
Number of patients 259 298 354 105 582 1104
Hereditary 8.1% 13.1% – – 14.8% 21.3%
Meningitis 30% 11% 23.9% 8.5% 34.4% 3.9%
Measles 1.9% 13% 4.1% 30% 4.3% 0.9%
Rubella 1.5% 2% – 3.8% 0.5% 0.2%
Mumps – 3% 16.7% 3.5% 2.1% 0.5%
Ototoxicity – 9% 20.8% – 6% –
Prematurity – – – – 0.9% 0.5%
Neonatal jaundice – 5.7% – 1.9% 1.4% 0.3%
Head trauma – – – – 0.3% 1.5%
Other illnesses – – – – – 10.8%
Unspecified illness – – – – – 6.3%
Unknown 54.4% 41.2% 34.8% 40% 32.6% 53.8%
in 5.1% (5/97) of familial cases, with variable expression of population had 2 out of the 145 individuals with mutation GJB2-
heterochromia in affected members (Figure 2). R143W in the heterozygous state.
No GJB6-D3S1830 deletion was identified in the samples 
screened.
Molecular Analysis Result of GJB2 
and GJB6 DISCUSSION
A total of 81 families segregating non-syndromic hearing loss were 
molecularly investigated. Although samples were not collected The present report in the most compressive study of the cause of 
from Adamarobe, the “Deaf village”, 27 out of the 81  HI families childhood HI in Ghana. Moreover, we investigated for the first 
screened for GJB2 and GJB6 were from the Eastern Region of Ghana time, the prevalence of GJB2 mutations in a non-affected group 
(Table S2) where the “Deaf Village” is located (Kusters, 2012). One of individuals from Ghana.
individual from each family was sequenced for GJB2 mutation In this study, we observed HI in more boys than girls, although 
and we found a pathogenic mutation in 27.2% (22/81) with GJB2- gender has not been reported as an associated factor that 
R143W in the majority (21/22) in the homozygous state (Table 3); predisposes children to the development of HI (Foerst et al., 2006; 
GJB2 p.W44* mutation in one case, in the homozygous state. Le Roux et al., 2015). This may be due to the fact that more boys 
In non-familial non-syndromic cases, GJB2-R143W mutation enroll in the schools for the deaf compared to girls, especially in 
was found in 7.9% (11/140) patients (Figure 1). The control resource-limited regions. In often cases, boys with disability have 
FIGURE 2 | Probands with both Waardenburg syndrome, that associate variable degree of hearing impairment, and eyes/skin decoloration. Panels (A) and (C) 
represent patients expressing the typical bilateral striking blue eyes phenotype of Waardenburg syndrome, while (B) and (D) represent asymmetrical heterochromia, 
with patients expressing the phenotype in only one eye.
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Adadey et al. GJB2 and GJB6 in Hearing Impairment in Ghana
TABLE 3 | GJB2 mutations among 365 previously studied and 97 Ghanaians families with profound sensorineural hearing impairment.
Nucleotide Amino acid Number of affected individuals
Previously reported (Hamelmann et al., 2001) Our current report
Familial Isolated/Non-familial cases Controls
cases
35 insG Frameshift 1(0.3%) – – –
236T→C L79P 1(0.3%) – – –
427C→T R143W 59 (16.2%) 21 (25.9%) 11 (7.9%) 2 (1.4%)
533T→C V178A 2 (0.6%) – – –
551G→A R184Q 1(0.3%) – – –
589G→T A197S 1(0.3%) – – –
608TC→AA I203K 1(0.3%) – – –
641T→C L214P 1(0.3%) – – –
131G > A W44* – 1 (1.2%) – –
more priority to formal education compared to girls (Groce, 1997; Mutations in GJB2 were investigated in Ghana 18 years ago and 
Nagata, 2003; Rousso, 2015). Although “female protective model” identified a common founder mutation p.R143W (Hamelmann 
is not common to HI studies, it has been proposed by some et al., 2001). The present study revisited the contribution of GJB2 
researchers to explain the higher prevalence of genetic disorders in mutations and confirm the particularly high proposition of the 
males compared to females (Jacquemont et al., 2014; Werling and founder mutation in more than ¼ of families segregating HI. 
Geschwind, 2015). According to this model, females have a higher This is much higher than what was previously reported (18%) 
rate of possible gene disruption but are mostly not associated with due to the stringent selection of familial cases in the present 
genetic disorders compared to males (Jacquemont et al., 2014). study. Majority of the families with HI and families positive for 
Hearing impairment screening aims at detecting permanent the founder mutation were from the Eastern Region of Ghana. It 
HI at early developmental ages for the appropriate intervention is from this Region that a high prevalence of congenital HI was 
(Sarant et al., 2008; Ching et al., 2017; Ma et al., 2018). There reported and hence the name “Deaf Village” (David et al., 1971; 
is no universal newborn HI screening program in Ghana Kusters, 2012). There was a relatively high proportion of GJB2 
explaining the late diagnosis, as most of the study participants mutations among the isolated case of putative genetics origin. 
had their first comprehensive hearing test at the school age, thus This is an indication of the urgent need to implement this GJB2-p.
6–9 years of age. However, parents/guardians of these children R143W testing in patients with HI clinical practice in Ghana. The 
gave the information on the onset of the condition. The late p.R143W mutation has also been reported in patients with HI in 
diagnostic of HI in Ghanaian children is partly tied to the limited Japan (Zheng et al., 2015; Kasakura-Kimura et al., 2017), South 
number of hearing assessment facilities (Waller et al., 2017). In Korea (Kim et al., 2016), and China (Luo et al., 2017). In addition, 
addition, the majority of the HI students were living in remote we report a variant previously described as Mayan: founder 
rural settlements often with unmemorable roads and hence the GJB2 nonsense mutation (p.W44*) in a Ghanaian family. GJB2 
difficulty of having access to quality health care. p.W44* mutation is the most common GJB2 pathogenic variant 
Post-lingual HI in Africa is often caused by environmental in Guatemala deaf populations and was also reported in Mexico 
factors (Wonkam et al., 2013). Similar to other reports, complicated (Martínez-Saucedo et al., 2015). Ghana is an African exception, 
malaria, cerebrospinal meningitis, and convulsion (with as most studies in Africa have not identify GJB2 as a major cause 
undetermined cause) were identified from our study as major of HI in sub-Saharan African populations (Lebeko et al., 2015; 
environmental factors that contribute to post-lingual HI in Ghana Wonkam, 2015).
(Table 2). There was a high number of congenital cases reported This is the first study to investigate GJB6-D13S1830 mutation 
in our study which may account for the reduced frequency of or coding region variations in Ghana, and we found no mutation, 
infectious causes of HI in our study compared to other studies from which is in line with previous African data (Bosch et al., 2014; 
Africa. Nonetheless, the identified environmental factors can be Wonkam et al., 2015). Equally, GJB6-D13S1830 deletion was 
prevented by good health care systems as well as preventive health not found in populations from China (Jiang et al., 2014), India 
care practices. It is therefore important that governmental policies (Padma et  al., 2009), Turkey (Tekin et al., 2003), and among 
should be implemented to minimize childhood morbidities which African American and Caribbean Hispanics (Samanich et al., 
will eventually reduce the prevalence of post-lingual HI. 2007). Therefore, the present data further support the hypothesis 
Pre-lingual hearing impairment was common in our study that the GJB6-D13S1830 deletion is a founder mutation (del 
population which agrees with other findings (Chibisova et  al., Castillo et al., 2003).
2018). Majority of pre-lingual HI are congenital and are usually The study also indicates more than 2/3 of families with HI 
caused by genetic factors (Wonkam et al., 2013; Behlouli are eligible for next-generation sequencing, due to the highly 
et  al., 2016). Waardenburg syndrome was the most common heterogeneous genetic nature of NSHI and the low proportion 
syndromic HI identified among the congenital cases in line with of families solved with single gene approach applied in this study. 
other African data (Noubiap et al., 2014). Nethetheless, the study did not exclude intronic variants in GJB2, 
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Adadey et al. GJB2 and GJB6 in Hearing Impairment in Ghana
that is a possible limitation. Future research should either use Memorial Institute for Medical Research Institutional Review 
high-throughput sequencing platforms to investigate known Board, the University of Ghana, Accra (NMIMR-IRB-CPN 006/16-
genes (Shearer et al., 2010; Lebeko et al., 2016), or whole exome 17 revd. 2018), and the University of Cape Town’s Human Research 
sequencing that will allow identification of novel genes (Diaz- Ethics Committee, reference 104/2018. Written informed consent 
Horta et al., 2012). Indeed, based on the identification of specific was obtained from all participants if they were 18 years or older, 
inner ear transcripts, it is estimated that more than 1,000 NSHI or from the parents/guardians with verbal assent from children, 
genes are still to be identified (Hertzano and Elkon, 2012). including permission to publish photographs.
To contribute towards the reduction of HI incidence in Ghana, 
policy-makers must consider integrating newborn screening for AUTHOR CONTRIBUTIONS
HI into the health care system such that every child is screened 
for both genetic and acquired HI at birth. Early detection of the Conceived and designed the experiments: GAA, GKA, AW. 
condition may lead to early intervention (Copley and Friderichs, Performed the experiments: SA, OQ, NM, KM. Patients’ 
2010) which will eventually reduce the public health impact of recruitment, samples, and clinical data collection and processing: 
this condition. SA, GKA, Analyzed the data: SA, AW; Contributed reagents/
materials/analysis tools: GAA, VN, CK, AW. Wrote the paper: 
SA, GAA, VN, CK, AW. Revised and approved the manuscript: 
CONCLUSION SA, OQ, GAA, GKA, KM, VN, CK, NM, AW.
The study showed that environmental factors remain a major 
cause of Hearing impairment in Ghana. The study confirms FUNDING
that Connexin 26 (GJB2) mutations are the most common 
cause of familial non-syndromic HI in Ghana, an exception The study was funded by the Wellcome Trust, grant number 
in sub-Saharan Africa where mutations in GJB2 in HI patients 107755Z/15/Z to GAA and AW (co-applicant); NIH, USA, grant 
is generally close to zero. GJB2 p.R143W founder mutation number U01-HG-009716 to AW, and the African Academy of 
accounted more > 25% of familial cases and close to 8% of Science/Wellcome Trust, grant, number H3A/18/001 to AW. The 
isolated cases of putative genetic origin and should be considered funders had no role in study design, data collection and analysis, 
in for implementation in clinical practice, particularly after decision to publish, or preparation of the manuscript.
newborns screening for HI. The frequency of GJB2 p.R143W 
founder mutation in the general population without personal SUPPLEMENTARY MATERIAL
and familial was relatively high: 1.4%. The study did not find 
any GJB6 del(GJB6-D13S1830) deletion. Future studies should The Supplementary Material for this article can be found online at: 
employ whole genome sequencing approaches and functional https://www.frontiersin.org/articles/10.3389/fgene.2019.00841/
genomics studies to identify the other genes involved in most full#supplementary-material
families, and in isolated cases of HI in Ghana.
FIGURE S1 | Onset and time of HI test. (A) Age of deaf students at the first 
medical HI test. (B) Onset of HI. Paired T-test was used to compare the mean 
number of students with pre-lingual (n = 754) and post-lingual (n = 336) HI from 
DATA AVAILABILITY 11 schools for the deaf. There was a significant difference between mean number 
of people with pre- and post-lingual HI with P value of 0.0001 (t = 7.68, df = 10).
All datasets supporting the conclusions of this study are included 
in the manuscript and the Supplementary Files. FIGURE S2 | Major causes of childhood HI in Ghana. (A) Major causes of post-
lingual HI in Ghana. (B) Major causes of Pre-lingual HI in Ghana. Cerebrospinal 
meningitis was represented as C.S.M. The cause of HI labelled accident 
ETHICS STATEMENT comprises of motor accidents and medical accidents such as wrong medication, child birth, and surgery. Diseases such as boil, anemia, Gilbertese, Jaundice, 
measles, mumps, Otitis media, and rubella were captured as other diseases 
The study was performed in accordance with the Declaration while undefined sickness consist of individuals who developed the condition due 
of Helsinki. Ethical approval was obtained from the Noguchi to sickness, but the cause of the sickness was not determined.
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Wonkam, A. (2015). Letter to the editor regarding “GJB2, GJB6 or GJA1 genes Awandare and Wonkam. This is an open-access article distributed under the terms 
should not be investigated in routine in non syndromic deafness in people of of the Creative Commons Attribution License (CC BY). The use, distribution or 
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doi: 10.1016/j.ijporl.2015.01.012 copyright owner(s) are credited and that the original publication in this journal 
Wonkam, A., Bosch, J., Noubiap, J. J. N., Lebeko, K., Makubalo, N., and Dandara, is cited, in accordance with accepted academic practice. No use, distribution or 
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