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Year : 2017  |  Volume : 14  |  Issue : 3  |  Page : 150-153

Early detection of hearing loss with connexin 26 gene assessment

1 Department of Otorhinolaryngology, IMS and SUM Hospital, Siksha “O” Anusandhan University, Bhubaneswar, Odisha, India
2 Department of Medical Research, IMS and SUM Hospital, Siksha “O” Anusandhan University, Bhubaneswar, Odisha, India
3 Department of Pathology, Apollo Hospital, Bhubaneswar, Odisha, India

Date of Web Publication27-Oct-2017

Correspondence Address:
Santosh Kumar Swain
Department of Otorhinolaryngology, IMS and SUM Hospital, Siksha “O” Anusandhan University, Kalinga Nagar, Bhubaneswar - 751 003, Odisha
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/am.am_13_17

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Hearing loss is the most prevalent type of sensory impairment in human beings. Hearing loss is a global problem. Genetic alteration accounts for 50% of the congenital deafness. The connexin 26 (Cx26) mutations are the most common cause behind the nonsyndromic hearing loss and it is easily identified by polymerase chain reaction. Genes responsible for hearing loss are being mapped and cloned progressively. Mutations in the gene (gap junction beta 2) encoding Cx26 have been associated with congenital hearing loss either alone or as part of a syndrome. Cx26 is a part of a large family of gap junction membrane proteins that help electrical and metabolic coupling between adjacent cells. This review article focuses on genetic analysis of nonsyndromic hearing loss, as genetic alteration in this type of deafness recently begun to be identified, epidemiology, diagnosis criteria, and emphasis in early detection.

Keywords: Congenital hearing loss, connexin 26, genetic diagnosis, genetic mutation, sensorineural hearing loss

How to cite this article:
Swain SK, Sahu MC, Baisakh MR. Early detection of hearing loss with connexin 26 gene assessment. Apollo Med 2017;14:150-3

How to cite this URL:
Swain SK, Sahu MC, Baisakh MR. Early detection of hearing loss with connexin 26 gene assessment. Apollo Med [serial online] 2017 [cited 2018 Mar 24];14:150-3. Available from: http://www.apollomedicine.org/text.asp?2017/14/3/150/217360

  Introduction Top

Hearing loss is the most common birth defect in industrialized countries and most commonly sensorineural type of hearing loss. One out of the 500 newborns has bilateral permanent sensorineural hearing loss with more than 40 decibel hearing loss.[1] In developed countries, genetic alterations are responsible for two-thirds of prelingual deafness. There are no clinical anomalies except hearing loss seen in most of the cases. Inherited nonsyndromic hearing loss is monogenic with more than 100 mapped loci and 46 casually implicated genes.[2] Congenital hearing loss affects approximately 1 in every 1000 live births and is one of the most distressing disorders.[3] Hearing loss affects speech development, language acquisition, and education of child. Hearing loss can be divided into two types - conductive and sensorineural deafness. Conductive deafness is a mechanical defect in conductive system of hearing from pinna to the external auditory canal, tympanic membrane, and middle ear up to stapedial footplate. Sensorineural hearing loss occurs mostly due to the defect in sensory hair cells of the cochlea or sometimes of the auditory nerve or processing center in the brain. Almost 50% of patients with sensorineural hearing loss are congenital and 30% of these congenital sensorineural hearing loss patients have syndromic deafness. More than 400 syndromes are identified while 70% of congenital hearing loss are nonsyndromic.[4] The syndromic or nonsyndromic congenital hearing loss is due to the defect in gene or interaction of genes. Congenital hearing loss can be due to mutation in single gene or a combination of mutations of multiple genes. It can also occur as result of environmental factors such as medical problems, drugs, trauma, and environmental exposure to radiations.[5] Nonsyndromic neurosensory autosomal recessive hearing loss accounts for 50% of pediatric prelingual hearing loss (1/2500).[6] Different genes identify in nonsyndromic sensorineural hearing loss are CDH23, CLDN14, COL11A2, COCH, DFNA5, D1APH 1, DSPP, GJA1, EVA4, GJB2, GJB6, GJB3, MYH9, MY03A, KCNQ4, MY06, MY07A, MY015, OTOA, OTOF, PDS/SLC26A4, POU4F3, STRC, TECTA, TMC1, etc. Mutation of gap junction beta 2 protein (GJB2) is called as connexin 26 (Cx26), which accounts for nearly 50% of recessive nonsyndromic hearing loss. Till now, more than 90 variants of Cx26 genes have been reported.[7] Cx26 dysfunction results in potassium retention and microcirculation disorders, subsequently causing hair cell malfunction and degeneration.[8] In addition to regulating potassium concentrations, studies have shown that channels involving Cx26 may also participate in transporting secondary messenger IP3 which regulates Ca ++ distribution. In cells with channels containing Cx26, Ca ++ move faster than in cells with other types of channels. Mutation of Cx26 genes can cause disorder in Ca ++ movement and spread in tissues.[9] However, Cx26 gene mutations do not affect vestibular functions.

  Basic Understanding of Genetics for Otolaryngologists/clinicians Top

Every cell of human body contains a complete set of gene (genome). There are approximately 30,000 genes inside the chromosomes. There are 23 pairs (46) of chromosomes of which 22 pairs are identical in male and female, called as autosomes whereas 23rd pair, i.e., sex chromosomes such as X or Y is dissimilar in male and female sex. If there are any alterations in genes, it results in mutation and manifests in genetic diseases or genetic deafness. Congenital hearing loss can be either nonsyndromic or syndromic. Nonsyndromic is confined to the inner ear whereas in syndromic, hearing loss is a part of multiple anomalies of the body. Approximately 20% nonsyndromic sensorineural hearing loss is inherited as autosomal dominant, which is caused by DFNA. It has delayed onset of hearing loss. Around 80% nonsyndromic senosrineural hearing loss is inherited as autosomal recessive (DFNB), where hearing loss is congenital, but in some of its form has delayed onset. The remaining types of nonsyndromic hearing loss is either mitochondrial or X-linked (DFN) <1%.[10] Till date, 125 deafness loci including 58 DFNA, 63 DFNB, and 4X linked reported in the literature.[11] Multiple genes are involved for inner ear functions. The physiology and anatomy of the inner ear are unique and unlike to other parts of the body. Genetic mutations that control adhesion of hair cells, neurotransmitter release, intracellular transport, ionic homeostasis, and cytoskeleton of hair cells may cause malfunction of the cochlea.

  Gap Junction Beta 2 Top

GJB2 is a gene located in the chromosome 13q.[11] Its length is about 5.5 kilobases. There are two exons, of which one possess the coding sequence. The mRNA is 2.4 kilobases long which translates into a protein of 226 amino acids. This protein are included in the Cx family, which has more than dozen members.[12] GJB2 encodes the gap junction protein in the cochlea which is responsible for recycling potassium ions that go into the hair cells, helpful for transduction current. Genetic alteration of GJB2, responsible for DFNB1 and DFNA3, is often the cause for inherited hearing loss.[12] There are more than 100 different mutations are identified in GJB2 in patients with nonsyndromic hearing loss. There is a single gene mutation, 35delG, responsible for up to 70% of Northern and Southern Europe as well as American Caucasian populations.

  Connexin 26 Top

Cx26 is the most common cause for the deafness in the world. Cx is a protein [Figure 1] which helps in formation of gap junctions, allow direct transfer of molecules and ions between adjacent cells.[13] Cx26 gene is expressed in different tissues of the body, but at cochlea, this gene product plays an important role for normal hearing by controlling the potassium recycling pathway.[13] Cx are membrane proteins which has four transmembrane domains. Six chains of the protein make a complex (a hexamer) called connexon. Two such hexamer in membrane of neighboring cells make a cell-to-cell channel, called gap junction which helps transporting small molecules and ions between cells.[14] About half of nonsyndromic congenital hearing loss are due to genetic alteration and mutations in the gene GJB2 are the major cause of inherited sensorineural hearing loss.[15] GJB2 gene encodes the gap junction protein Cx26. The clinical outcomes due to mutations in the Cx26 gene is exclusively sensorineural hearing loss (nonsyndromic), whereas with other mutations, the hearing loss is part of a syndrome. Cx26 protein is essential for maintaining high K + concentration in the endolymph of the inner ear. Mutations in the Cx gene located on chromosome 13q11-12 are associated with the autosomal recessive nonsyndromic neurosensory deafness known as “DFNB1.”[16] These studies shows that DFNB1 causes 20% of all childhood deafness and have a carrier rate is around 2.8%. Cx are a class of membrane proteins [Figure 1] that form hexameric connexons that form gap junction channels between adjacent cells.[17] Gap junctions form an intercellular communication which helpful in the exchange of electrolytes, second messengers, and metabolites. Cx may play important roles in development, by coordinating the clonal development of groups of cells. Cx may helpful for complex functional architecture in the developing brain by making cortical circuits that mature by synapse formation.[18] In mammals, there are more than 13 different Cx genes and classified into two classes, i.e., alpha and beta, on the basis of their primary structure. Some forms of Cx are expressed in a majority of tissue, whereas others are restricted to specific cell types. Turnover of Cx seems to be rapid, showing that the levels of most forms are dependent on transcription or mRNA turnover. Cx26 is downregulated in tumor tissue and thus is considered a class II tumor-suppressor gene. During the S and G2 phases of cell cycle, Cx26 is upregulated by synchronized cells. Immunostaining of rat cochlea, with antisera raised against cytoplasmic epitopes of Cx26, showed two groups of cochlear cells use the Cx26 protein.[19] The first group of cells includes inner sulcus cells and spiral limbus interdental cells, organ of Corti supporting cells, outer sulcus cells, and inside the spiral ligament cells near its root process; these are all nonsensory epithelial cells. The 2nd group consists fibrocytes of the spiral ligament and spiral limbus, basal and intermediate cells of the stria vascularis, and mesenchymal tissues covering the scala vestibuli, which are all connective tissue cells. This observation supports the hypothesis that Cx26 helpful in recycling of potassium ions inside the endolymph during the transduction process of the hair cells.[20]
Figure 1: Schematic diagram of a gap junction protein. Six connexins form a connexon. Two connexons of neighboring cells make pores, which allow intercellular transport of molecules

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  Hearing Assessment and Genetic Analysis Top

All newborn including high risk attending the newborn clinic are usually screened for congenital hearing loss [Figure 2]. All are planned for details examination of the ear and head neck area to find an association of syndromic manifestations. History of maternal infections such as TORCH will be documented. Brainstem-evoked response audiometry and otoacoustic emissions (OAE) will be carried out among newborns for confirmation of hearing loss. In OAE, PASS means normal hearing and REFERS means abnormal, i.e., hearing is not within normal limit. Follow-up visits for infants participating in the newborn health screening program will be scheduled until infants up to age of 6 months. Workup of the patient includes the brief history of hearing loss, prenatal, natal, and postnatal history, drug intake, and radiation exposure to pregnant mother, developmental history of newborn, hearing aid trial, any other associated diseases, and family history. Blood samples of the deaf patients will be obtained in a heparinized container. Cultivation will be carried out within 3–6 h of the post aspiration. After an incubation period of 69 h at 37°C, the harvesting will be done, and finally, the metaphases on the slides will be obtained. Thereafter, those slides showing metaphase with good morphology will be selected and will be kept under nonhumid dry wooden boxes with different temperature. After 1 week of harvesting, banding procedure will be done using freshly prepared Giemsa stain and EDTA-trypsin solution. About 30 metaphase plates will be observed in each case and a photograph will be obtained from a good quality metaphase slide with the help of a camera attached to eyepiece of the microscope within 8–15 s. The chromosomal findings will be described with respect to the international standard, and finally, karyotype will be prepared using conventional method by cut and paste technique. The coding exon (exon 2) of Cx26 will be amplified from genomic DNA by both forwarding and reverse primers, which amplify DNA fragments of 286 bp and 519 bp, respectively. Amplification of the noncoding gene and the flanking sequence sites was performed using advantage-GC genomic PCR kit (Clontech) and PCR primers Cx26-3F (50-TCC GTA ACT TTC CCA GTC TCC GAG GGA AGA GG-30) and Cx26-3R (50-CCC AAG GAC GTG TGT TGG TCC AGC CCC-30). The PCR products will be purified using QIAquick columns (Qiagen) and will be sequenced by specific kit method.
Figure 2: Flow chart for identifying congenital hearing loss

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  Epidemiology Top

Hearing loss is a global problem. Mutations in Cx26 are the most common etiology of autosomal recessive deafness in the world. Cx26 gene is thought to have half of cases of hereditary hearing loss. This gene has diverse mutations, but one mutation occurs in the Europe, the 35delG mutation. The high chance of Cx26-associated hearing loss in certain population may be due to tradition of marriages between hearing impaired persons.[21] More than 100 types of mutations have been reported regarding Cx26 with characteristic racial features. Mutations such as c. 235delC or c. 233delC are commonly seen among Asians, c. 35delG or c. 30delG among Caucasians, and c. 167delG among Jews. All are frame shift mutations, causing premature ending of protein synthesis and hence dysfunction of Cx. There are mutations in the Cx26 gene among 49% of the families in France, New Zealand, and Great Britain who presented with severe to profound hearing loss. Familial, sporadic hearing loss due to Cx26 mutations among English–Belgian population was 10%.[22] Consanguineous marriages are still common among Indians, which cause increased chance of expressions autosomal recessive causes, making congenital hearing loss. The deletion of cytosine at location 235 (235delC) mutation is the most common genetic alteration, accounting for around 80% of the pathologic alleles in multiplex cases and 67% in simplex cases.[23] However, the 235delC has not been seen in South Asian populations of India, Pakistan, Bangladesh, and Sri Lanka, where the common GJB2 mutations are W24X and W77X.[24] The prevalence of 235delC mutation among Japanese population has been attributed to a founder effect.[25] High frequency of the 235delC mutation in East Asian populations is due to founder effect and that 235delC among all East Asian populations is derived from a common ancestral founder.[26]

  Diagnosis/assessment Top

Cx is the most studied genetic mutations in hearing research. Nowadays, many medical centers are performing mutation analysis to find involvement of the Cx26 gene in congenital hearing loss. Usually, mutation analysis is done among children with a family history of hearing loss. If mutation in Cx26 is present, genetic counseling is essential for providing etiology and answering likely recurrence in future offspring. If the mutation analysis is positive for Cx26, there is no need for further investigations such as imaging and ophthalmological tests as cause of congenital hearing loss no longer have to be excluded. In case of Cx26 mutation with intact auditory nerve, the patients are suitable for cochlear implant, provided that patient has profound hearing loss. Early diagnosis helps for early treatment, which provides best results with hearing rehabilitations such as cochlear implant or hearing aid.

  Conclusion Top

It is estimated that at least 50% of prelingual hearing loss is caused by genetic alteration. Clinical and family histories are extremely vital for diagnosis of genetic sensorineural hearing loss and also help determine the inheritance patterns. Hearing rehabilitations such as hearing aid or cochlear implantation, in early life can result in good speech and hearing rehabilitation because the central pathways remain relatively intact. In the present scenario, the development of precise and advanced molecular diagnosis for congenital profound sensorineural hearing loss in particular is in the infancy stage. Regeneration of hair cells, gene therapy, and developing certain medication is likely future target in the hearing science. At present, early diagnosis and timely intervention are the most effective strategy for improving hearing among the affected children in the world.

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  References Top

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