Pattern of Connexin 26 (GJB2) Mutations Causing Sensorineural Hearing Impairment in Ghana

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1 HUMAN MUTATION Mutation in Brief #428 (2001) Online MUTATION IN BRIEF Pattern of Connexin 26 (GJB2) Mutations Causing Sensorineural Hearing Impairment in Ghana Christoph Hamelmann 1, Geoffrey K. Amedofu 2, Katrin Albrecht 1, Birgit Muntau 1, Annette Gelhaus 1, George W. Brobby 2, and Rolf D. Horstmann 1, * 1 Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany, and 2 School of Medical Sciences, University of Science and Technology, Kumasi, Ghana *Correspondence to: Rolf Horstmann, Department of Molecular Medicine, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany; Tel: ; Fax: ; horstmann@bni.uni-hamburg.de Contract grant sponsor: Bernhard Nocht Institute for Tropical Medicine Communicated by Henrik Dahl Mutations of the connexin 26 gene (GJB2) were studied in 365 apparently unrelated individuals with profound nonsyndromic, sensorineural hearing impairment from Ghana, West Africa. Among 121 mutated chromosomes found, 110 carried the previously described R143W mutation. A total of 6 novel mutations: L79P, V178A, R184Q, A197S, I203K, and L214P, were identified, whereby I203K was based on a dinucleotide exchange and R184Q appeared to be dominant. The GJB2 variants found in Ghana tend to comprise less nonsense and frameshift mutations and more mutations located in the C-terminal half of the molecule than the variants found in other parts of the world Wiley-Liss, Inc. KEY WORDS: gap junction protein-beta 2; GJB2; connexin 26; Cx26; congenital deafness; neurosensory; Africa INTRODUCTION Connexins are a family of vertebrate proteins which, by undergoing oligomerization into hexamers, form transmembrane channels termed connexons (for a review, see Bruzzone et al., 1996). Connexons dock with counterparts of neighbouring cells to form direct intercellular communication pathways, the gap junction channels. The sequences of the various connexins are highly conserved with four transmembrane domains separating two extracellular loops from a middle cytoplasmic loop and the N- and C-terminal cytoplasmic ends. Initial structurefunction studies indicate that oligomerization and gating control seems to reside in the N-terminal part of the protein, whereas compatibility for heterotypic pairing appears to be governed by the C-terminal extracellular loop. However, these studies at present are too limited to allow reliable conclusions. The identification of mutations in the connexin-26 gene (GJB2: MIM# ) as a cause for profound sensorineural hearing impairment (Kelsell et al., 1997) prompted a series of studies on GJB2 in affected families and subjects from Europe, North America, the Near East, North Africa, and Japan. A considerable number of Received 1 March 2001; revised manuscript accepted 18 April WILEY-LISS, INC.

2 2 Hamelmann et al. GJB2 mutations have been found (Table 1, see also Most of them act in a recessive manner; only W44C, C202F and M34T appeared to be dominant whereby the mode of inheritance of M34T is a matter of discussion (Denoyelle et al., 1998; Morlé et al., 2000; Kelsell et al., 1997; Scott et al., 1998b; Kelley et al., 1998; White et al., 1998). We have previously identified an R143W mutation of GJB2 as the cause for hearing impairment in all of 11 families from a Ghanaian village with an extraordinarily high prevalence of congenital hearing impairment (Brobby et al., 1998). We have now extended our studies to deaf individuals from all over Ghana. Table 1: GJB2 mutations associated with nonsyndromic sensorineural hearing impairment* Mutation Reference Mutation Reference -3170G A Denoyelle et al., 1999 M1V Estivill et al., del14 Murgia et al., del38 Denoyelle et al., delG Zelante et al., insG Estivill et al., 1998 G12V Rabionet et al., del12insA Sobe et al., 2000 S19T Rabionet et al., 2000 W24X Kelsell et al., 1997 M34T Kelsell et al., 1997 V37I Rabionet et al., 2000 W44C Denoyelle et al., 1998 G45E Fuse et al., 1999 E47X Denoyelle et al., delT Zelante et al., 1997 Q57X Wilcox et al., del16 Abe et al., 2000 Y65X Estivill et al., 1998 W77X Kelsell et al., 1997 W77R Carrasquillo et al., delC Fuse et al., delC Abe et al., 2000 V84M Kelley et al., insT Denoyelle et al., 1999 L90P Denoyelle et al., 1999 V95M Kelley et al., delAT Abe et al., del14 Denoyelle et al., del14 Denoyelle et al., del14 Kelley et al., delAA Kelley et al., 1998 S113R Kelley et al., 1998 E114X Rabionet et al., 2000 dele120 Rabionet et al., 2000 Q124X Scott et al., 1998a R127H Estivill et al., 1998 Y136X Fuse et al., 1999 dele138 Denoyelle et al., insA Denoyelle et al., 1999 P173R Rabionet et al., 2000 P175T Denoyelle et al., 1999 R184P Denoyelle et al., 1997 R184W Wilcox et al., 1999 C202F Morlé et al., del GT Kelley et al., 1998 *Included are recessive mutations found homozygous or in compound heterozygosity as well as mutations assumed to be dominant MATERIALS AND METHODS Patients The study design was approved by the Ethics Committee of the School of Medical Sciences of the University of Science and Technology, Kumasi, Ghana. Patients were recruited from P1 to P6 classes of ten schools for the deaf located in nine of the ten provinces (Regions) of the Republic of Ghana, West Africa. Consent to participate in the study was obtained from 379 pupils or, if they were under 16 years of age, from one of their parents or a guardian. They were asked using the school teachers as translators if necessary. Fourteen first- or second-degree relatives were excluded so that a total of 365 individuals were included in the evaluation. Data was obtained about the age, gender, and residency of the pupil and of the hearing ability of its first-degree relatives. Any possible source of information was used including the school teachers, the children s parents, guardians, friends or neighbours. Again, the teachers were used as translators if necessary. Eighty pupils originated

3 Connexin 26 Mutations in Ghana 3 from the Ashanti Region, 28 from Brong Ahafo, 36 from the Central Region, 25 from the Eastern Region, 47 from Greater Accra, 27 from the Northern Region, 14 from Upper East, 12 from Upper West, 43 from the Volta Region, and 53 from the Western Region. Excluded were residents of the village of Adamarobe, who had been studied before (Brobby et al., 1998). Inclusion criteria were (i) lack of evidence for an acquired form of hearing loss such as meningitis, measles, mumps or cerebral malaria, as reported by the pupils parents or guardians, (ii) lack of evidence for a syndromal form of hearing impairment obtained by physical examination, and (iii) audiometric findings compatible with a profound and sensorineural form of hearing impairment, i.e. hearing loss is greater than 90% and the differences between air conduction and bone conduction thresholds were equal to or less than 10dB (Intracoustics, Models AD27 or AS7). A blood sample of 3 ml was taken and mixed with an equal volume of 8 M urea for conservation during transport and storage. The pupils or their representatives were asked for permission to communicate the test results to the teachers and to provide counseling through them. Genetic analysis DNA was extracted from urea-preserved blood using a DNA-adsorption kit (Nucleospin Blood L). Primers had been designed to amplify 683 bp comprising 12 bp of the 5 -region and the entire GJB2 structural gene except for 5 bp at the 3 -end. DNA sequencing was done on an ABI PRISM 377 DNA sequencer. In all individuals found heterozygous for a GJB2 mutation, exon 1 was amplified and sequenced using published primer sequences (Denoyelle et al. 1999); none of them was found to carry a -3170G>A mutation. RESULTS A total of 365 unrelated individuals aged 6 to 20 years with evidence for profound, congenital sensorineural hearing impairment were included in the study. They were recruited from ten schools for the deaf from all over Ghana. PCR amplification and sequencing of the GJB2 coding region revealed that 63 of them (17%) carried missense mutations or insertions in that gene. A total of 51 individuals were homozygous for R143W (Table 2). Another 8 were heterozygous for R143W, and in 4 of them no other GJB2 mutations were found. The remaining 4 individuals with R143W were compound heterozygotes, one each with 35insG, I203K, L79P, and L214P, with I203K being based on a dinucleotide exchange. Whereas 35insG had been described before as a cause for recessive hearing impairment (Estivill et al., 1998), the other 3 mutations have not been found so far. Family studies suggest that all of them also act in a recessive manner (Fig. 1, Families A, B, C). Three more novel missense mutations of GJB2 were found. Two apparently unrelated individuals (Table 2) and a sibling of one of them were homozygous for V178A, suggesting that V178A is another recessive cause for hearing loss. One child each was heterozygous for A197S and R184Q. Whereas no family members could be traced for the pupil with A197S, a family study of R184Q was feasible. It strongly suggested that this mutation is dominant (Fig. 1, Family D). Like all other cases studied, family members affected by R184Q did not show any skin alterations such as keratoderma. DISCUSSION Here we describe 6 novel mutations of the GJB2 gene. All of them were found in individuals with profound sensorineural hearing impairment in Ghana, West Africa. In the light of previous reports on the role of GJB2 mutations it was assumed that the mutations were the cause for hearing loss if this was supported by family data. R143W had been described before as a recessive cause of deafness in Ghana, where it was the sole GJB2 mutation found in a village with an extraordinarily high prevalence of hearing impairment (Brobby et al., 1998). R143W was greatly predominant also in other parts of the country, contributing 90% to all GJB2 mutations seen. In four cases it was found in single heterozygotes; there, it either acts in a digenic mode, or its presence was not related to the cause of hearing impairment. In another four cases, R143W was found in compound heterozygosity, in three of them together with the novel mutations L79P, I203K, and L214P. Family studies revealed that single heterozygotes for L79P and I203K had normal hearing, which indicates that these two mutations are recessive (Fig. 2, Families A and B). No family members could be found who were single heterozygotes for L214P, but it was reported that the mother of the compound-heterozygous siblings had normal hearing, which suggests that L214P also is recessive since she must

4 4 Hamelmann et al. be a carrier of L214P (Fig. 2, Family C). V178A was homozygous in three individuals. Although no further family studies were performed, the exclusive occurrence of V178A in a homozygous form in the study group suggests that it also is recessive. Table 2: GJB2 mutations among 365 apparently unrelated residents of Ghana with profound sensorineural hearing impairment* GJB2 Mutations Number of nucleotide amino acid Chromosomes 35insG (frameshift) 1 236T C L79P 1 427C T R143W T C V178A 4 551G A R184Q 1 589G T A197S 1 608TC AA I203K 2 641T C L214P 1 Total affected 121 Total studied 730 Mutations Affected Individuals R143W/R143W 51 R143W/wt 4 R143W/35insG 1 R143W/L79P 1 R143W/I203K 1 R143W/L214P 1 V178A/V178A 2 R184Q/wt 1 A197S/wt 1 Total affected 63 Total studied 365 *Excluded are residents of Adamarobe, who have been described separately. Substantial evidence indicates that another novel mutation, R184Q, is dominant. A family study revealed that hearing loss co-segregated with heterozygosity for R184Q (Fig. 2, Family D). Moreover, a digenic mode of inheritance appears unlikely because the affected siblings were reported to have two different fathers, which was confirmed by microsatellite typing (data not shown). Thus, R184Q, besides W44C, C202F and possibly M34T, appears to be one of the rare cases of GJB2 deafness mutations acting in a dominant manner (Kelsell et al., 1997; Scott et al., 1998b; Kelley et al., 1998; White et al., 1998; Denoyelle et al., 1998; Morlé et al., 2000). Finally, mutation A197S was seen once in a heterozygous form. Since no family members were available, the role of the mutation remains unclear. It may either be dominant, or act in a digenic mode, or its occurrence coincides with some other cause for hearing loss. Comparing the overall prevalences and patterns of GJB2 mutations, our findings in Ghana differ in several ways from those obtained in other populations: (i) Compared to 30-50% found among families and individuals of European origin (Estivill et al., 1998; Denoyelle et al., 1997; Kelley et al., 1998), the fraction of 16% that GJB2 mutations contribute to the study group from Ghana is relatively small; this may relate to the fact that acquired causes for deafness in early childhood such as meningitis have higher incidences in Africa and therefore contribute a larger portion of cases erroneously considered to be congenital. (ii) Except for a single allele of 35insG, we did not find any nonsense or frameshift mutations whereas these predominate in other parts of the world. (iii) There is a tendency that the Ghanaian mutations cluster in the C-terminal half of the molecule whereas the others do so in

5 Connexin 26 Mutations in Ghana 5 Family A F amily B R143W/W T I2 03K/ WT L7 9P/WT R143W /WT R1 43W/ W T/W T I203K/WT WT/WT I2 03K L7 9P/R1 43W Family C R1 43W/WT R143 W/ WT/WT R143W/W T R143 W/ R143W / R1 43W/WT L21 4P L214P L214P F amily D WT / WT R184Q / W T R1 84Q / WT R1 84Q / WT WT / WT R184 Q / WT W T / W T R184Q / WT W T / W T Figure 1. Families studied to analyse the novel mutations I203K, L79P, L214P, and R184Q. Whereas the phenotypes of members affected with I203K, L79P, and L214P are compatible with a recessive mode of inheritance, R184Q appears to be dominant. the N-terminal half. (iv) The most prevalent mutation among Caucasians, 35delG, causes a gross, N-terminal truncation of the molecule whereas the predominant one in Ghana, R143W, causes a single amino-acid exchange. Taken together, these data might indicate a selection for functional mutants in Ghana. This notion is supported by the finding that I203K, one of the novel mutations found in Ghana, is based on a dinucleotide exchange. More detailed structure-function studies on the connexins and sophisticated clinical phenotypings are needed to identify possible modes of selection. ACKNOWLEDGMENTS The work was supported by the Volkswagen Foundation. We thank Paulina Asante and Grace Occansey for expert technical assistence and Paul Bekyir for skillful driving. The work described in this publication is part of the MD thesis of K. A. REFERENCES Abe S, Usami S, Shinkawa H, Kelley PM, Kimberling WJ (2000) Prevalent connexin 26 gene (GJB2) mutations in Japanese. J Med Genet 37: Brobby GW, Muller-Myhsok B, Horstmann RD (1998) Connexin 26 R143W mutation associated with recessive nonsyndromic sensorineural deafness in Africa. N Engl J Med 338: Bruzzone R, White TW, Paul DL (1996) Connections with connexins: the molecular basis of direct intercellular signaling. Eur J Biochem 238:1-27.

6 6 Hamelmann et al. Carrasquillo MM, Zlotogora J, Barges S, Chakravarti A (1997) Two different connexin 26 mutations in an inbred kindred segregating non-syndromic recessive deafness: implications for genetic studies in isolated populations. Hum Mol Genet 6: Denoyelle F, Weil D, Maw MA, Wilcox SA, Lench NJ, Allen-Powell DR, Osborn AH, Dahl HH, Middleton A, Houseman MJ, Dode C, Marlin S, Boulila-ElGaied A, Grati M, Ayadi H, BenArab S, Bitoun P, Lina-Granade G, Godet J, Mustapha M, Loiselet J, El-Zir E, Aubois A, Joannard A, Levilliers J, Garabedian E-N, Mueller RF, Gardner RJM, Petit C (1997) Prelingual deafness: high prevalence of a 30delG mutation in the connexin 26 gene. Hum Mol Genet 6: Denoyelle F, Lina-Granade G, Plauchu H, Bruzzone R, Chaib H, Levi-Acobas F, Weil D, Petit C (1998) Connexin 26 gene linked to a dominant deafness. Nature 393: Denoyelle F, Marlin S, Weil D, Moatti L, Chauvin P, Garabedian EN, Petit C (1999) Clinical features of the prevalent form of childhood deafness, DFNB1, due to a connexin-26 gene defect: implications for genetic counselling. Lancet 353: Estivill X, Fortina P, Surrey S, Rabionet R, Melchionda S, D'Agruma L, Mansfield E, Rappaport E, Govea N, Mila M, Zelante L, Gasparini P (1998) Connexin-26 mutations in sporadic and inherited sensorineural deafness. Lancet 351: Fuse Y, Doi K, Hasegawa T, Sugii A, Hibino H, Kubo T (1999) Three novel connexin26 gene mutations in autosomal recessive non-syndromic deafness. Neuroreport 10: Green GE, Scott DA, McDonald JM, Woodworth GG, Sheffield VC, Smith RJ (1999) Carrier rates in the midwestern United States for GJB2 mutations causing inherited deafness. JAMA 281: Kelley PM, Harris DJ, Comer BC, Askew JW, Fowler T, Smith SD, Kimberling WJ (1998) Novel mutations in the connexin 26 gene (GJB2) that cause autosomal recessive (DFNB1) hearing loss. Am J Hum Genet 62: Kelsell DP, Dunlop J, Stevens HP, Lench NJ, Liang JN, Parry G, Mueller RF, Leigh IM (1997) Connexin 26 mutations in hereditary non-syndromic sensorineural deafness. Nature 387: Lench N, Houseman M, Newton V, Van Camp G, Mueller R (1998b) Connexin-26 mutations in sporadic non-syndromal sensorineural deafness. Lancet 351:415. Lench NJ, Markham AF, Mueller RF, Kelsell DP, Smith RJ, Willems PJ, Schatteman I, Capon H, Van De Heyning PJ, Van Camp G (1998a) A Moroccan family with autosomal recessive sensorineural hearing loss caused by a mutation in the gap junction protein gene connexin 26 (GJB2). J Med Genet 35: Morell RJ, Kim HJ, Hood LJ, Goforth L, Friderici K, Fisher R, Van Camp G, Berlin CI, Oddoux C, Ostrer H, Keats B, Friedman TB (1998) Mutations in the connexin 26 gene (GJB2) among Ashkenazi Jews with nonsyndromic recessive deafness. N Engl J Med 339: Morle L, Bozon M, Alloisio N, Latour P, Vandenberghe A, Plauchu H, Collet L, Edery P, Godet J, Lina-Granade G (2000) A novel C202F mutation in the connexin26 gene (GJB2) associated with autosomal dominant isolated hearing loss. J Med Genet 37: Murgia A, Orzan E, Polli R, Martella M, Vinanzi C, Leonardi E, Arslan E, Zacchello F (1999) Cx26 deafness: mutation analysis and clinical variability. J Med Genet 36: Rabionet R, Zelante L, Lopez-Bigas N, D Agruma L, Melchionda S, Restagno G, Arbones ML, Gasparini P, Estivill X (2000) Molecular basis of childhood deafness resulting from mutations in the GJB2 (connexin 26) gene. Hum Genet 106:40-44 Scott DA, Kraft ML, Carmi R, Ramesh A, Elbedour K, Yairi Y, Srisailapathy CR, Rosengren SS, Markham AF, Mueller RF, Lench NJ, Van Camp G, Smith RJ, Sheffield VC (1998a) Identification of mutations in the connexin 26 gene that cause autosomal recessive nonsyndromic hearing loss. Hum Mutat 11: Scott DA, Kraft ML, Stone EM, Sheffield VC, Smith RJ (1998b) Connexin mutations and hearing loss. Nature 391:32. Sobe T, Vreugde S, Shahin H, Berlin M, Davis N, Kanaan M, Yaron Y, Orr-Urteger A, Frydman M, Shohat M, Avraham KB (2000) The prevalence and expression of inherited connexin 26 mutations associated with nonsyndromic hearing loss in the Israeli population. Hum Genet 106: White TW, Deans MR, Kelsell DP, Paul DL (1998) Connexin mutations in deafness. Nature 394: Wilcox SA, Osborn AH, Allen-Powell DR, Maw MA, Dahl HH, Gardner RJ (1999) Connexin26 deafness in several interconnected families. J Med Genet 36: Wilcox SA, Saunders K, Osborn AH, Arnold A, Wunderlich J, Kelly T, Collins V, Wilcox LJ, McKinlay Gardner RJ, Kamarinos M, Cone-Wesson B, Williamson R, Dahl HH (2000) High frequency hearing loss correlated with mutations in the GJB2 gene. Hum Genet 106: Zelante L, Gasparini P, Estivill X, Melchionda S, D'Agruma L, Govea N, Mila M, Monica MD, Lutfi J, Shohat M, Mansfield E, Delgrosso K, Rappaport E, Surrey S, Fortina P (1997) Connexin26 mutations associated with the most common form of non-syndromic neurosensory autosomal recessive deafness (DFNB1) in Mediterraneans. Hum Mol Genet 6: