|Year : 2011 | Volume
| Issue : 4 | Page : 145-148
Connexin 26 mutations in congenital SNHL in Indian population
SS Nayyar1, S Mukherjee1, N Moorchung2, E James1, MD Venkatesh1, PS Sukthankar1, K Sabarigirish1, RB Batra1
1 Department of ENT, Armed Forces Medical College, Pune, India
2 Department of Pathology, Armed Forces Medical College, Pune, India
|Date of Web Publication||29-Mar-2012|
Department of ENT, Armed Forces Medical College, Sholapur Road, Pune-411 040, Maharashtra
Source of Support: None, Conflict of Interest: None
Introduction: Hearing impairment is a sensory disability that affects millions of people all over the world. Fifty percent of these cases are hereditary. Two genes have been localized to DFNB locus (GJB2 & GJB6) on chromosome 13 which have been commonly implicated in autosomal recessive causes of deafness among which the Connexin 26 mutation is the most common. Materials and Methods: Twenty-seven unrelated Indian patients between the ages of 1 and 23 years with nonsyndromic congenital sensorineural hearing loss for GJB2 mutations, specifically for W24X. Analysis was done by the polymerase chain reaction (PCR) Restriction fragment length polymorphism RFLP and sequencing methods. Results: Seven out of these 27 subjects were found to have the W24X mutation, implying a frequency of 26% (7/27). Conclusion: Our results are in concordance with what has been reported in world literature. We also showed a 100% concordance between the PCR RFLP and sequencing methods.
Keywords: Connexin 26, Nonsyndromic hearing loss, Congenital hearing loss, DFNB1
|How to cite this article:|
Nayyar S S, Mukherjee S, Moorchung N, James E, Venkatesh M D, Sukthankar P S, Sabarigirish K, Batra R B. Connexin 26 mutations in congenital SNHL in Indian population. Indian J Otol 2011;17:145-8
|How to cite this URL:|
Nayyar S S, Mukherjee S, Moorchung N, James E, Venkatesh M D, Sukthankar P S, Sabarigirish K, Batra R B. Connexin 26 mutations in congenital SNHL in Indian population. Indian J Otol [serial online] 2011 [cited 2019 Mar 22];17:145-8. Available from: http://www.indianjotol.org/text.asp?2011/17/4/145/94491
| Introduction|| |
Hearing is one of the most important and vital functions of the body. Hearing impairment is a sensory disability that affects millions of people all over the world. Impairment of hearing right from birth results in defective or nondevelopment of speech. Speech is an important element required for social communication.
Deafness occurs due to several reasons. Overall, about 1 in 1000 children are born with a significant degree of sensorineural hearing loss (SNHL).  Fifty percent of these cases are hereditary.  Most cases of hereditary hearing loss are nonsyndromic hearing loss (NSHL), with autosomal recessive forms accounting for 85% of these cases. , Fifty percent of persons with autosomal recessive NSHL have mutations in DFNB1 locus on chromosome 13q11. Two genes localized to this chromosomal region have been implicated in deafness, including connexin 26 (CX26, gene symbol GJB2)  and connexin 30 (CX30, GJB6). ,,
Congenital causes carry social importance, as the child is born deaf and mute. The physical handicap of congenital deaf mutism remains unseen for a long period. With increase in health infrastructure, deafness of infective and/or environmental origin is decreasing while that due to hereditary causes is increasing.
In India, consanguineous marriages are still very common, which leads to further increased chances of expression of autosomal recessive causes, leading to congenital deafness.
Connexins are gap junction proteins that oligomerize as hexamers to form transmembrane channels called connexons. Connexons from the cell membranes interdigitate to form direct intercellular communications pathways, the gap junction channels. Connexins have a highly conserved form of transmembrane domains separating two extracellular loops from a middle cytoplasmic loop and the N- and C-terminal cytoplasmic ends. In the inner ear, CX26 is expressed in the supporting cells, stria vascularis, basement membrane, limbus, and spiral prominence of the cochlea.  The sensory hair cells of the cochlea allow potassium ions to pass through during the mechanosensory transduction process of normal hearing. These potassium ions are recycled across the supporting cells and fibrocytes at the base of hair cells through the gap junctions of the stria vascularis and back to the K+ rich endolymph. It is believed that mutations in the GJB2 gene would lead to complete or partial loss of function of the CX26 protein, interfering with recycling of potassium ions and thus hampering the normal process of hearing. 
Many studies from various parts of the world have documented the incidence of GJB2 mutations in the deaf population. Updates covering all the CX26 mutations are provided by the Connexin-Deafness Homepage (http:// www.crg.es/deafness/). Several of these studies included subjects from the Indian subcontinent. ,,, One Indian study from the southern and western parts of India documented 17.7% of congenital deaf probands having biallelic GJB2 mutations, and of which 95% were due to W24X gene mutations. 
It is unfortunate that there is inadequate knowledge about hereditary causes leading to congenital deafness in India. With more and more advances in gene therapy, it becomes even more imperative to pinpoint the genetic causes leading to congenital deafness so that in the future newer gene therapy approaches can be developed to actually treat such cases.
In our study, an attempt has been made to evaluate the association of the most common autosomal recessive cause, CX26 gene mutation due to W24X, in congenital deafness with NSHL in heterogenous Indian population from all over the country.
| Materials and Methods|| |
We screened 27 unrelated Indian patients between the ages of 1 and 23 years with nonsyndromic congenital SNHL for GJB2 mutations, specifically for W24X. Patients participating in the Cochlear Implantation Program at the Department of Otolaryngology in Armed Forces Medical College (AFMC), Pune (India), were included for this study. However, in view of the cosmopolitan nature of patients reporting to AFMC OPDs, these patients did not represent any particular ethnic group but included patients from all across India. Most of the patients showed severe-to-profound congenital hearing loss on pure-tone audiography and evoked auditory brainstem response evaluation. Patients who had conductive hearing loss; syndromic hearing loss; had taken ototoxic drugs; history of otorrhoea, head trauma, meningitis, NICU admissions, kernicterus, any other perinatal pathology, maternal complications during pregnancy, or history of maternal consumption of ototoxic drugs during pregnancy; or any other known causes of hearing loss were not included in this study.
Five milliliters of blood samples were collected from each of the 27 patients, in bottles containing EDTA, after obtaining written informed consent. Frozen blood samples were thawed at room temperature. High molecular weight DNA was extracted by using the salting out method.  Polymerase chain reaction (PCR) was performed using the primer sequences noted in [Table 1]. PCR was performed with 150 ng genomic DNA, 25 pmol primers, 10 mmol/L Tris-Cl, 50 mmol/L KCl, 1.5 mmol/L MgCl 2 , 800 mmol/L dNTPs, and 2.5 U Taq DNA polymerase (Bangalore Genei) in a total reaction volume of 50 mL for 40 cycles (each cycle of 94°C for 30 seconds, 60°C for 30 seconds, and 72°C for 60 seconds). PCR products were purified using Qiaquick columns (Qiagen) and sequenced using the ABI 3730xl sequencer.
In addition to sequencing, we also attempted to look for the mutation using a PCR RFLP. The confirmation of a successful PCR was done by running the products on a 2% agarose gel. If the products were present, they were digested with 10 U of Alu1 at 37°C for 3 h. Fragments were analyzed by electrophoresis on 3% agarose gel and stained with ethidium bromide. AluI digestion of DNA derived from unaffected subjects produced a single fragment of 286 bp, W24X homozygotes produced two fragments of 182 bp and 104 bp, and W24X heterozygotes produced three fragments of 286 bp, 182 bp, and 104 bp.
| Results|| |
Independently ascertained probands (n=27) from patients from different parts of India were collected from the ages of 1-23 years with a mean age of 7.6 years. There were a total of 13 females and 14 males in this sample group [Figure 1].
|Figure 1: Bar graph showing the sex ratio in cases of sensorineural hearing loss|
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Seven out of these 27 subjects were found to have W24X mutation [Figure 2], implying a frequency of 26% (7/27) [Figure 3]. We also compared the results of sequencing with the PCR RFLP analysis. We found 100% concordance between the results of the PCR RFLP and the PCR-Sequencing analysis.
|Figure 2: Sequence of the GJB2 mutation showing a normal sequence on the top panel and a mutated sequence in the lower panel|
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|Figure 3: Seven out of these 27 subjects were found to have W24X mutation, implying a frequency of 26% (7/27)|
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| Discussion|| |
This study associates the CX26 W24X gene mutations in a heterogenous group of deaf patients from various parts of India.
Gap junctions are believed to play a role in the recycling of potassium ions back to the endolymph of the cochlear duct after stimulation of the sensory hair cells. The loss of CX26 would be expected to disrupt this potassium ion flow, thereby leading to hearing loss. , The mutation of the CX26 gene is a major contributor to autosomal recessive deafness as well as a small percentage of autosomal dominant deafness.  The 35delG mutation accounts for the most common type of nonsyndromic recessive deafness among White populations.  Several types of CX26 mutations cause recessive deafness according to ethnic background, as in the 167delT in Ashkenazi Jews,  the R143W in Africans,  and the 235delC in the Japanese population.  In India, the most common cause of CX26 gene mutation is W24X leading to 95% of mutations of GJB2 and with a frequency of 18.1% among congenitally deaf population.  Our research has shown similar statistics with an overall frequency of 26% of W24X gene mutations in a proband of 27 congenital nonsyndromic SNHL.
In southern Europe and the United States, between 35% and 50% of congenital cases of deafness have biallelic GJB2 mutations,  whereas only 16% to 18% of congenital deaf probands in India have biallelic GJB2 mutations.
Establishing this association is very important, especially in the light of new research showing much better rehabilitative outcome of cochlear implantation in patients with GJB2-related deafness. 
The study has also established the role of PCR RFLP as a cheap and reproducible method for the analysis of the CX26 W24X gene mutations. In the future, it is hoped that this method will replace the standard sequencing methods for the diagnosis of the mutations.
This paper has the following points to offer:
- The prevalence of Connexin 26 mutations in India is comparable to what is seen in other populations.
- PCR RFLP is an accurate and cheaper method to look for Connexin 26 gene mutations.
| References|| |
|1.||Morton NE. Genetic epidemiology of hearing impairment. Ann N Y Acad Sci 1991;630:16-31. |
|2.||Marazita ML, Ploughman LM, Rawlings B, Remington E, Arnos KS, Nance WE. Genetic epidemiological studies of early-onset deafness in the US school-age population. Am J Med Genet 1993;46:486-91. |
|3.||Kelsell DP, Dunlop J, Stevens HP, Lench NJ, Liang JN, Parry G, et al. Connexin 26 mutations in hereditary non-syndromic sensorineural deafness. Nature 1997;387:80-3. |
|4.||Lerer I, Sagi M, Ben-Neriah Z, Wang T, Levi H, Abeliovich D. A deletion mutation in GJB6 cooperating with a GJB2 mutation in trans in non-syndromic deafness: A novel founder mutation in Ashkenazi Jews. Hum Mutat 2001;18:460. |
|5.||del Castillo I, Villamar M, Moreno-Pelayo MA, del Castillo FJ, Alvarez A, Tellería D, et al. A deletion involving the connexin 30 gene in nonsyndromic hearing impairment. N Engl J Med 2002;346:243-9. |
|6.||Zelante L, Gasparini P, Estivill X, Melchionda S, D'Agruma L, Govea N, et al. Connexin 26 mutations associated with the most common form of nonsyndromic neurosensory autosomal recessive deafness (DFNB1) in Mediterraneans. Hum Mol Genet 1997;6:1605-9. |
|7.||Lautermann J, Frank HG, Jahnke K, Traub O, Winterhager E. Developmental expression patterns of connexin 26 and 30 in the rat cochlea. Dev Genet 1999;25:306-11. |
|8.||Cohen-Salmon M, Ott T, Michel V, Hardelin JP, Perfettini I, Eybalin M, et al. Targeted ablation of connexin 26 in the inner ear epithelial gap junction network causes hearing impairment and cell death. Curr Biol 2002;12:1106-11. |
|9.||Scott DA, Kraft ML, Carmi R, Ramesh A, Elbedour K, Yairi Y, et al. Identification of mutations in the connexin 26 gene that cause autosomal recessive nonsyndromic hearing loss. Hum Mutat 1998;11:387-94. |
|10.||Lin D, Goldstein JA, Mhatre AN, Lustig LR, Pfister M, Lalwani AK. Assessment of denaturing high-performance liquid chromatography (DHPLC) in screening for mutations in connexin 26 (GJB2). Hum Mutat 2001;18:42-51. |
|11.||Rickard S, Kelsell DP, Sirimana T, Rajput K, MacArdle B, Bitner-Glindzicz M. Recurrent mutations in the deafness gene GJB2 (connexin 26) in British Asian families. J Med Genet 2001;38:530-3. |
|12.||RamShankar M, Girirajan S, Dagan O, Ravi Shankar HM, Jalvi R, Rangasayee R, et al. Contribution of connexin 26 (GJB2) mutations and founder effect to non-syndromic hearing loss in India. J Med Genet 2003;40;e68. |
|13.||Miller S, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 1998;16:1215. |
|14.||Yeager M, Unger VM, Falk MM. Synthesis, assembly and structure of gap junction intercellular channels. Curr Opin Struct Biol 1998:8:517-24. |
|15.||Steel KP. A new era in the genetics of deafness. N Engl J Med 1998;339:1545-7. |
|16.||Estivill X, Fortina P, Surrey S, Rabionet R, Melchionda S, D'Agruma L, et al. Connexin-26 mutations in sporadic and inherited sensorineural deafness. Lancet 1998;351:394-8. |
|17.||Morell RJ, Kim HJ, Hood LJ, Goforth L, Friderici K, Fisher R, et al. Mutations in the connexin 26 gene (GJB2) among Ashkenazi Jews with nonsyndromic recessive deafness. N Engl J Med 1998;339:1500-5. |
|18.||Brobby GW, Muller-Myhsok B, Hostmann RD. Connexin 26 R143W mutation associated with recessive nonsyndromic sensorineural deafness in Africa. N Engl J Med 1998;338:548-9. |
|19.||Fuse Y, Doi K, Hasegawa T, Sugii A, Hibino H, Kubo T. Three novel connexin26 gene mutations in autosomal recessive non-syndromic deafness. Neuroreport 1999;10:1853-7. |
|20.||Lustig LR, Lin D, Venick H, Larky J, Yeagle J, Chinnici J, et al. GJB2 gene mutations in cochlear implant recipients-prevalence and impact on outcome. Arch Otolaryngol Head Neck Surg 2004;130:541-6. |
[Figure 1], [Figure 2], [Figure 3]