Hearing loss associated with an unusual mutation combination in the gap junction beta 2 (GJB2) gene in a Chinese family

Hearing loss associated with an unusual mutation combination in the gap junction beta 2 (GJB2) gene in a Chinese family

International Journal of Pediatric Otorhinolaryngology 78 (2014) 599–603 Contents lists available at ScienceDirect International Journal of Pediatri...

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International Journal of Pediatric Otorhinolaryngology 78 (2014) 599–603

Contents lists available at ScienceDirect

International Journal of Pediatric Otorhinolaryngology journal homepage: www.elsevier.com/locate/ijporl

Hearing loss associated with an unusual mutation combination in the gap junction beta 2 (GJB2) gene in a Chinese family Aiping Huang a,b,1, Yongyi Yuan b,1, Naichao Duan a, Xinxia Jiang a, Baoshan Wang a, Yanping Liu a, Dongyang Kang b, Xin Zhang b, Qingwen Zhu a,*, Pu Dai b,* a b

Department of Otolaryngology, Head & Neck Surgery, The Second Hospital of Hebei Medical University, Shijiazhuang 050018, Hebei, China Department of Otolaryngology, Head & Neck Surgery, Chinese PLA General Hospital, Beijing 100853, China



Article history: Received 30 October 2013 Received in revised form 8 January 2014 Accepted 8 January 2014 Available online 17 January 2014

Objective: To assess the molecular etiology of nonsyndromic sensorineural hearing loss (NSHL) in members of an affected Chinese family. Methods: Common hearing-related genes including gap junction beta 2 (GJB2), SLC26A4, mitochondrial DNA 12S rRNA, GJB3 and GJB6 were examined in a family consisting of a normal hearing father, an NSHLaffected mother, one normal-hearing child and three NSHL-affected children. Specific primers were used in polymerase chain reactions to amplify the coding regions of the above genes from the peripheral blood DNA from each family member, and the genes were analyzed by direct sequencing. The subjects were evaluated for phenotypic characterization using audiometric testing and radiological examination of the inner ear. Results: Pathogenic mutations in the GJB2 gene were identified. The affected mother showed a heterozygous G ! A transition at nucleotide 232, resulting in an alanine to threonine substitution at codon 78 (p.A78T), and the normal hearing father had a c.35insG insertion mutation. The three affected children displayed heterozygosity for the GJB2 mutations, showing a previously unreported combination of c.35insG and c.232G>A. Conclusions: The GJB2 mutations account for a significant proportion of NSHL in affected individuals worldwide. Genetic and audiological data analysis of a Chinese family with NSHL revealed a novel c.35insG/c.232G>A compound heterozygous state. Our results highlight the complexity of the GJB2 genotypes and phenotypes. ß 2014 Elsevier Ireland Ltd. All rights reserved.

Keywords: Hearing loss GJB2 mutation Phenotype

Introduction Hearing loss is one of the most common perception disorders and is a highly heterogeneous sensory disorder. Reported incidences for bilateral hearing loss of at least 40 dB range between 1 and 2 per 1000 newborns in different studies, with about 60% of these cases originating from genetic causes [1]. Postlingual hearing loss is even more common, affecting 10% of the population by 60 years of age and 50% by the age of 80 [2]. Age related-onset hearing loss is a heterogeneous trait with many suspected causes. Genetic factors and environmental factors such as noise exposure might contribute to the trait.

* Corresponding authors at: a, The Second Hospital of Hebei Medical University, Department of Otolaryngology, Head & Neck Surgery, Hebei 050018, China. b, Department of Otolaryngology, Head & Neck Surgery, Chinese PLA General Hospital, Beijing 100853, China Tel.: +86 311 66003788/+86 10 66938119; fax: +86-10-68156974. E-mail addresses: [email protected] (Q. Zhu), [email protected] (P. Dai). 1 Contributed equally to this work. 0165-5876/$ – see front matter ß 2014 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ijporl.2014.01.008

Genetic deafness can be ascribed to various mutations in several genes. To date, over 140 genetic loci associated with NSHL have been mapped (http://hereditaryhearingloss.org). Mutations in the GJB2 gene are the most common cause of hereditary hearing loss in various populations, and a broad spectrum of GJB2 mutations has been identified. Approximately 50% of autosomal recessive NSHL (DFNB1) is caused by mutations in the GJB2 gene [3]. GJB2 is also involved in dominant forms of hearing loss (DFNA3) [4], and in syndromic disorders where deafness can be found associated with skin problems [5]. To date, over 220 mutations, polymorphisms, and unclassified variants have been identified in the GJB2 gene, some of which are frequent and others that are extremely rare [6]. Of these mutations, the c.35delG, c.167delT and c.235delC are common in Caucasian, Ashkenazi Jewish and Oriental populations, respectively (http:// TheConnexin-deafnesshomepage). The c.35delG mutation accounts for up to 70% of all GJB2 pathologic alleles in Northern and Southern Europeans, as well as in American Caucasian populations, with a carrier frequency of 1.3–2.8% [7,8]. Another GJB2 mutation, c.167delT, accounts for 40% of the pathologic alleles

A. Huang et al. / International Journal of Pediatric Otorhinolaryngology 78 (2014) 599–603


in the Jewish deaf population [9], and has a 4% carrier frequency among Ashkenazi Jews [10]. In Chinese populations, the c.235delC mutation, rather than c.35delG, was found to be the most common mutation causing non-syndromic hearing loss [11,12]. Other mutations, such as c.35insG in Italy and Spain [13], c.30delG in Ashkenazi Jews [14], p.V37I(c.109G>A) in eastern Asia [15], p.W24X (c.71G>A) in India or Spain [16], p.R184Q(c.551G>A) in Taiwan [17], p.C202F (c.605G>T) in French [18], p.R143Q(c.428G>A) in Austria/Czech [19] have been reported. The recessive form, DFNB1, usually presents as a stable, severe to profound hearing loss. However, moderate and progressive hearing loss also has been reported by Denoyelle et al. [20]. While there is phenotypic variation across GJB2 genotypes, patients with the c.35delG mutation tend to have the most severe form of hearing impairment [21].Onset is almost always prelingual, but not necessarily congenital [20,22]. In fact, hearing may be normal at birth but progress rapidly during infancy [23,24]. The autosomal dominant form, DFNA3, typically presents during childhood as a progressive, moderate to severe hearing impairment affecting mostly high frequencies. It progresses to involve middle frequencies by middle age [20,22]. As with recessive forms of GJB2, phenotypic variability is present in terms of the severity and evolution of hearing impairment [25].Audiogram shapes were generally flat or sloping, but they were not pathognomonic. Further studies have essentially confirmed these conclusions, but they have also provided insight into some specific issues [1,26]. It is reported that the degree of HI associated with the presence of two truncating mutations was shown to be significantly more severe than that of the HI associated with two non-truncating mutations. The severity of HI associated with truncating/non-truncating genotypes was intermediate between those two groups [27]. Here we examined three generations of a hearing loss-affected family. The male proband, his mother and brother showed lateonset progressive hearing impairment, his younger sister showed prelingual severe hearing loss, his father and an older sister have normal hearing. Common hearing-related genes including GJB2, SLC26A4, mitochondrial DNA 12S rRNA, GJB3 and GJB6 were examined in the family. Finally, a novel c.35insG and c.232G>A compound heterozygous of GJB2 defects were verified to account for the genetic etiology. Patients and methods Clinical features We evaluated a three-generational hearing loss-affected family (Fig. 1) of Han Chinese origin from Xingtai city in Hebei Province. Detailed medical histories of the family members were obtained using a questionnaire regarding the following aspects: subjective

degree of hearing loss, age at onset, evolution of the hearing loss, presence of tinnitus and vertigo, history of head trauma and medication with aminoglycosides, noise exposure, pathologic changes in the ear, goiter and other relevant clinical manifestations, family history and the pregnancy/labor process. All the procedures were approved by the Ethics Review Committee of the Second Hospital of Hebei Medical University and were carried out after a written informed consent had been obtained from each individual or from parents of the children under 18 years of age. Careful medical examinations revealed no clinical features other than hearing impairment. All of the affected members were examined by temporal bone computed tomography (CT) scan for diagnosis of EVA or inner ear malformation based on a diameter of >1.5 mm at the midpoint between the common crus and the external aperture. The procedure were performed at the Second Hospital of Hebei Medical University. Hearing was evaluated through otological examination and audiological evaluations including pure-tone audiometry (Madsen Conera, at frequencies from 250 to 8000 Hz), immittance (GSI Tympstar), auditory brainstem response (ABR) and distortion product otoacoustic emissions (DPOAE). Pure tone thresholds at 0.5, 1, 2, and 4 kHz of the better ear were averaged. The degree of SNHL was classified as mild (21–40 dB), moderate (41–70 dB), severe (71–95 dB) and profound (>95 dB) [28]. Mutational analysis Genomic DNA was extracted from peripheral blood leukocytes using a commercially available DNA extraction kit (Qiagen Inc., Valencia, CA) according to the manufacturer’s instructions. Ultraviolet spectrophotometry was used to measure the DNA concentration and purity. DNA was stored at 70 8C at high concentrations for later use, or at 20 8C in 20 ng aliquots for immediate use. Mutation screening was conducted using polymerase chain reaction (PCR) amplification and the exons were directly sequenced. The GJB2 gene primers used were: exon2-forward: 50 -TTGGTGTTTGCTCAGGAAGA-30 and exon2-reverse: 50 -GGCAT CTGGAGTTTCACCTG-30 to amplify the coding region of the gene. PCR conditions were as follows: 4 min of denaturation at 94 8C; incubation at 94 8C for 30 s, 30 s at 58 8C, 30 s at 72 8C for 30 cycles, then for 7 min at 72 8C for final extension, and storage at 4 8C. The primers and PCR conditions for the exon 1 of GJB2, SLC26A4, mtDNA 12S rRNA, GJB3 and GJB6 were described in detail in our previous published paper [29]. The resultant sequence data were compared with the evaluation samples for alignment with the National Center for Biotechnology Information reference (NCBI) sequence, GJB2 (no. NM_004004), SLC26A4 (no.NM_000441), mtDNA (no.NC_ 012920), GJB3 (no. NM_024009) and GJB6 (no. NM_006783). Results Clinical evaluations

Fig. 1. Pedigree of the family.

The clinical history and audiological findings of hearing loss in the family members revealed a form of post-lingual bilateral SNHL (excluding II-5). The reported onset of hearing problems in most cases (excluding II-5) was in the second decade of life with subsequent gradual progression from mild to severe. The hearing loss in these cases affected high frequencies initially and then developed to the middle and low frequencies. Except for individual II-5(26 years old) who was affected with prelingual deafness and no define nosogenesis. The earliest clinical evidence of hearing loss in the family was obtained from individual II-3(29 years old) at the age of 23 years (Fig. 2). His hearing loss has been aggravated in the preceding 6 years (Fig. 2). A similar form of hearing loss was

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Fig. 2. Tonal audiometric curves of II-3 at the ages of 23 (a) and 29 (b) years.

present in his mother (I-2, 57 years old) and a brother (II-7, 24 years old). Affected individuals had sloping, flat or residual audiograms (Table 1). The hearing levels in the I-1(59 years old) and II-1(31 years old) individuals are currently normal. DPOAE testing in all the affected individuals showed cochlear dysfunction. The temporal bone CT and physical examinations of the affected members were normal. The family reported no history of infections, ototoxic drug use, trauma and vascular pathologies, diabetes mellitus and so on. Genetic analyses We analyzed the GJB2, SLC26A4 and mtDNA 12S rRNA including c.1494C>T and c.1555A>G. Finally, pathogenic mutations in the GJB2 gene were identified. The propositus, II-3, was found to carry the compound heterozygosity of c.35insG (Fig. 3) and c.232G>A (Fig. 4) in the GJB2 gene. II-5 and II-7 carried the same gene mutations as II-3. Individual I-2 showed heterozygosity for c.232G>A. Individual I-1 and II-1 carried c.35insG in GJB2 gene (Table 1). The GJB2 mutations in generation III are as follows, III-1 (2 years old, c.35insG), III-2 (5 years old, c.232G>A), III-4 (5 years old, c.35insG), III-6 (1 years old, c.35insG). No pathogenic mutations in SLC26A4 and mtDNA 12S rRNA were detected in the family. Then we analyzed GJB3 and GJB6 in I-2, but found no mutation. Discussion NSHL is the most common type of neurosensory deafness, in which the majority of patients have highly diversified genetic defects. GJB2, encoding gap junction beta 2 protein (connexin 26, Cx26). The Cx26 protein has four transmembrane segments

(TM1–4), two extracellular loops (E1 and E2), a cytoplasmic loop, an N-terminal helix (NTH) and a C-terminal segment. Cx26 aggregates in groups of six around a central 2–3 nm pore to form a doughnut-shaped structure called a connexon. The connexons from contiguous cells that are covalently bonded to form intercellular channels. Aggregations of connexons, called plaques, are constituents of the gap junction. The gap junctions are specialized membrane regions containing hundreds of intercellular communication channels that allow the exchange of molecules such as ions, metabolites, nucleotides and small peptides [30]. To date, more than 25 different GJB2 deafness-causing mutations have been identified in Chinese patients with nonsyndromic deafness, including a missense mutation and small deletions or insertions across all Cx26 protein domains [11]. The frame-shift mutation of c.35insG has been reported several times in previous epidemiological investigations. Several studies have shown that the c.35insG mutation is a recessive allele that causes mild-to-profound hearing impairment in affected individuals, with most patients showing severe or profound hearing loss [31–33]. However, the c.232G>A mutation which located upstream of the commonly mutated loci c.233–235delC, was reported rarely. The c.232G>A loci was mentioned in 2003 by Li et al. [34] in a Chinese family. He found a compound heterozygous deletions (c.235delC/ c.232G>A) in two affected individuals in one pedigree. The affected individual’s father carried the heterozygous GJB2 mutation c.232G>A with normal hearing, the mother carried the GJB2 mutation c.235delC with hearing impairment. But the clinical characteristic about the affected individuals and their parent were not described in detail. This mutation causes the substitution of alanine to threonine at position 78 of the Cx26 protein (p.A78T), which occurs in the second transmembrane domain, M2.

Table 1 Phenotypes and genotypes of the family members.

1 2 3 4 5 6

Family member


(I-1) (I-2) (II-1) (II-3) (II-5) (II-7)


Age (years)

59 57 31 29 26 24



Allele 1

Allele 2

Nucleotide change


Nucleotide change


c.35insG N c.35insG c.35insG c.35insG c.35insG

Frame-shift N Frame-shift Frame-shift Frame-shift Frame-shift

N c.232G>A N c.232G>A c.232G>A c.232G>A

N Missense N Missense Missense Missense

M: male, F: female, N: normal, PTA: pure-tone audiometry.

Age of Onset (years)

PTA (left) (dB)

PTA (right) (dB)

Temporal bone CT

– 20 – 23 Birth 21

N 28.75 N 53.75 Profound 62.5

N 38.75 N 71.25 Profound 70

– N – N N N


A. Huang et al. / International Journal of Pediatric Otorhinolaryngology 78 (2014) 599–603

Fig. 3. The GJB2 gene mutation, c.35insG.

Fig. 4. Nucleotide sequence of the GJB2 heterozygous variant, c.232G>A.

In this report, we describe a family with compound heterozygosity of GJB2 mutations, c.35insG and c.232G>A. The mother carried the heterozygous GJB2 mutation c.232G>A, while the father carried the heterozygous mutation c.35insG of GJB2. The three affected children were compound heterozygous c.35insG/ c.232G>A. The fact that both the normal-hearing father (I-1) and the proband’s normal-hearing older sister (II-1) had the c.35insG insertion mutation indicates that they are carriers. This also demonstrates that the mutation of c.35insG displays a recessive pattern, consistent with previous reports. The mother (I-2) and her two sons (II-3, II-7) showed later-onset hearing loss, involving the high frequencies at earlier ages that later developed to all frequencies, with the degree of impairment increasing gradually. However, II-5 had the GJB2 c.35insG/c.232G>A gene mutations with profound hearing loss from prelingual, indicating the phenotype complexity associated with the GJB2 gene mutations. In this family, the grandparents were described as having normal hearing, but they were not alive to be screened. We are currently following up the hearing level of the third-generation and hope to clarify the inheritance pattern. If GJB2 c.232G>A is a dominant mutation, the inheritance of the family is dominant. The proband and his affected siblings inherited the mutation from their mother. Though the mother seems to be not so affected as their children even she is 57 years old, the fact that the hearing degree of impairment increasing gradually is similar with that of her sons. In this family, c.35insG has pathogenic role in the compound heterozygotes. First, one of the c.35insG/c.232G>A compound heterozygotes (II-5) shows prelingual profound hearing loss, suggesting a clear role for the c.35insG mutation in the pathogenesis in the family. Second, a dominant mutation may exert its effect on the other allele, but if the other allele is fully inactivated, as it is likely the case of c.35insG, there is no dominant effect possible. Just like the Cx26 channels in those children are only made of Cx26-p.Ala78Thr, not mixtures of Cx26-wt and Cx26-pAla78Thr found in the mother. That may explain why the carrier mother has a slightly milder phenotype than her two compound heterozygous sons. The

phenotype caused by the dominant mutation varies from prelingual severe hearing loss to late-onset progressive post lingual hearing loss, perhaps due to variable expressivity. If GJB2 c.232G>A is a recessive mutation, the compound heterozygous mutations c.232G>A/c.35insG are the genetic cause of the proband and the affected siblings. However, the molecular etiology of their mother with hearing impairment carried a heterozygous GJB2 c.232G>A needs clarification. The mother’s hearing impairment may be due to a different gene defect or other causes. We sequenced the GJB2 exon 1, its flanking donor splice site and the GJB2 basal promoter regions, but found no mutations. Different connexin genes can interact to cause hearing loss in digenic heterozygotes in humans. del Castillo et al. [35,36] reported that a 342 kb, now verified to be 309 kb, deletion involving the connexin-30 gene (GJB6) were found in trans with mutations in the GJB2 gene (connexin-26) in subjects with DFNB1 non-syndromic hearing impairment. Our previous study revealed that GJB2 and GJB3 can interact to cause hearing loss in digenic heterozygotes in humans [37]. We sequenced the coding areas of GJB3 and GJB6 to investigate a possible digenic inheritance mode, but found no mutation. The temporal bone CT scan of the affected members showed normal. And the family reported no history of ototoxic drug use. Also no pathogenic mutations in SLC26A4 and mtDNA 12S rRNA were detected. Both the clinical and related genetic testing support that the molecular etiology in this family are not due to defects in SLC26A4 or mtDNA 12S rRNA. An interesting result of our study is the finding that the hearing deterioration in the affected individuals was more profound in the right ear than in the left. Further studies are required to provide an explanation for this observation. Identifying the etiology of hearing loss can be complex, and the detection of Cx26 mutations does not always implicate the gene as the cause of deafness; some deaf patients have a mutation in only one allele, while others have mutations that are not known definitively to be pathologic. Genetic counselors must be aware of diagnostic pitfalls and be careful when providing information to the families. Most GJB2 mutations are inherited as an autosomal recessive pattern, and if these mutations are found alone (heterozygous), they are less important for the genetic counselor. However, when a mutation is demonstrated to show an autosomal dominant pattern, the risk of inheritance will increase. Therefore, genetic counselors and otologists should attempt to prioritize the evaluation and prevention of this disorder in patients. Acknowledgments This work was supported by grant from the Minister of Science and Technology (2012BAI09B02) and Key Projects in the National Science & Technology Pillar Program during the Twelfth Five-year Plan Period (2012BAI12B00/2012BAI12B01), Chinese National

A. Huang et al. / International Journal of Pediatric Otorhinolaryngology 78 (2014) 599–603

Nature Science Foundation Research Grant (30728030, 31071099, 81230020), and State 863 High Technology R&D Key Project of China (SQ2010AA0221634001) to Dr. Pu Dai, Natural Science Foundation of Hebei Province of China (C2010000571), and Medical applicable Technical Track Project of Hebei Province of China (GL 2011-35) to Dr.Qingwen Zhu, Chinese National Nature Science Foundation Research Grant (81371098), Beijing Natural Science Foundation (7132177), Beijing Nova programme (2009B34) to Dr. Yongyi Yuan. We wish to thank the patients and their families for their cooperation during this work and thank all the medical staff from Chinese PLA Institute of Otolaryngology for their great help during this study.







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