Spectrum of GJB2 mutations in Cypriot nonsyndromic hearing loss subjects

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1 c Indian Academy of Sciences RESEARCH NOTE Spectrum of GJB2 mutations in Cypriot nonsyndromic hearing loss subjects VASSOS NEOCLEOUS 1, CONSTANTINA COSTI 1, CHRISTOS SHAMMAS 1, ELENA SPANOU 2,3, VIOLETTA ANASTASIADOU 3,2, GEORGE A. TANTELES 2,3 and LEONIDAS A. PHYLACTOU 1 1 Department of Molecular Genetics, Function and Therapy, The Cyprus Institute of Neurology and Genetics, 1683 Nicosia, Cyprus 2 Department of Clinical Genetics, The Cyprus Institute of Neurology and Genetics, 1683 Nicosia, Cyprus 3 Department of Clinical Genetics, Makarios III Hospital, 1474 Nicosia, Cyprus [Neocleous V., Costi C., Shammas C., Spanou E., Anastasiadou E., Tanteles G. A.and Phylactou L. A Spectrum of GJB2 mutations in Cypriot nonsyndromic hearing loss subjects. J. Genet. 93, ] Introduction The purpose of this study is to report the spectrum of GJB2 mutations in Cypriots with sensorineural nonsyndromic hearing loss (NSHL) in a cohort of 146 subjects with NSHL compatible with recessive inheritance. Subjects were screened for the c.-23+1g>a splice mutation and the coding exon 2 of the GJB2 gene also including del(gjb6-d13s1830) and del(gjb6-d13s1854). This work confirms that the GJB2 c.35delg is an important pathogenic mutation for hearing loss in the Cypriot population and that the underlying molecular basis of autosomal recessive nonsyndromic deafness in Cyprus is genetically relatively homogeneous. To date more than 130 loci and 40 causative genes for hearing loss have been reported to be associated with syndromic and nonsyndromic hereditary hearing loss ( davinci.crg.es/deafness/). Despite the fact that more than 20 loci have been reported to be associated with autosomal recessive NSHL (DFNB), a single locus, DFNB1, accounts for a high proportion of the cases, with variability depending on the population. Because of the exceptional genetic heterogeneity of NSHL, and large size of most deafness genes, it was thought that no single gene would play a distinctive role in its aetiology. The surprising finding in a series of populations that mutations at the GJB2 (DFNB1) locus account for up to 50% of cases of autosomal recessive NSHL changed this notion and more than 90 mutations have now been described in the literature. The mutation c.35delg is the most frequent mutation accounting for up to 86% of the GJB2 mutant alleles in white populations. On the other hand, c.167delt is the most common For correspondence. laphylac@cing.ac.cy. mutation in the Ashkenazi Jewish population and c.235delc in the Korean and Japanese populations (E. Ballana, M. Ventayol, R. Rabionet, P. Gasparini and X. Estivill. Connexins and deafness. URL: van Camp, R. J. H. Smith. Hereditary hearing loss. URL: Subjects Keywords. sensorineural hearing loss, GJB2; GJB6; nonsyndromic hearing loss. Materials and methods We analysed 146 unrelated subjects of Greek Cypriot origin who were found to have bilateral sensorineural hearing loss (SNHL) and which were compatible with recessive inheritance of NSHL. All patients were clinically evaluated by pure tone audiometry with a diagnostic audiometer and those with SNHL loss were found to have hearing thresholds in the severe to profound range. This study was approved by the Institution Ethics Review Board for human studies and informed consent was obtained from all participants or from the parents of the minors. Mutation analysis - direct sequencing for GJB2 and GJB6 genes Genomic DNA was isolated from peripheral blood leucocytes and DNA fragments containing the entire coding regions of the GJB2 and GJB6 genes were amplified from genomic DNA samples as previously published (Neocleous et al. 2006a). All DNA samples were also screened for the c.-23+1g>a splice mutation. For the identification of c.-23+1g>a splice mutation, an in house protocol was developed that screens the basal promoter and exon 1 of the GJB2 gene. A DNA Journal of Genetics, Vol. 93, No. 2, August

2 Vassos Neocleous et al. fragment of 499 bp containing the basal promoter and exon 1 of the GJB2 gene was amplified using the polymerase chain reaction (PCR) with the following primers: GJB2-ivs1 forward 5 -GGG GCT CAA AGG AAC TAG GA-3,and GJB2-ivs1 reverse 5 -AAG GAC GTG TGT TGG TCC AG 3. The presence or absence of the c.-23+1g>a splice mutation was assessed by restriction digestion with HphI of the GJB2 exon 1 PCR product which is able to recognize restriction sites created by the presence of adenine instead of the normal guanidine. The presence or absence of the c.-23+1g>a splice mutation was also assessed with direct sequencing of the GJB2 gene 499 bp fragment on an ABI 3130XL apparatus (Applied Biosystems, Foster City, USA). Allele specific PCR amplification of 35delG The relatives of subjects who were identified as homozygous for the GJB2 c.35delg mutation by direct sequencing were screened using an allele-specific PCR as previously published (Neocleous et al. 2006b). Multiplex PCR amplification for del(gjb6-d13s1830) and del(gjb6-d13s1854) mutations All DNA samples were screened in an assay specific for confirmation of the Cx kb deletion (also called del(gjb6- D13S1830)). Briefly, the DNA samples were subjected to PCR simultaneously with each of the three sets of primers in a multiplex reaction as previously published (Wu et al. 2003). In a similar fashion, all the DNA samples were also screened in another assay for confirmation of the Cx kb deletion (also called del(gjb6-d13s1854)) (del Castillo et al. 2005). Results Mutation analysis of the GJB2 and GJB6 coding regions, the large 342 kb genomic del(gjb6-d13s1830) and the smaller 265 kb del(gjb6-d13s1854) which affects the GJB6 gene was carried out in all 146 unrelated NSHL subjects. A total of 40/146 subjects with NSHL were identified with mutations in the GJB2 gene only (figures 1 and 2). Thirty subjects were verified with GJB2 mutations in both alleles and with c.35delg as the most dominating one, accounting for 81.7% (49 out of 60 mutated alleles), followed by p.arg184pro (6.7%), p.leu90pro (5.0%), p.glu47stop (1.7%), delglu120 (1.7%), 167delThr (1.7%) and p.val178ala (1.7%) (table 1). Ten subjects with severe SNHL showed only one mutation in the GJB2 gene (table 2). Interestingly, five of these subjects were identified in the heterozygous state with GJB2 missense mutation/variant p.val153ile, while the rest were identified as heterozygous for p.val37ile, c.35delg, p.leu90pro, c.-23+1g>a splice mutation and the novel c.-1g>a 5 UTR (table 2). DNA sequencing analysis of the GJB6 gene coding region and the assays for del(gjb6-d13s1830) and del(gjb6-d13s1854) did not reveal evidence for pathogenic mutations in either the homozygous or heterozygous state. Discussion Genetic testing of the GJB2 and GJB6 genes to ascertain a definitive diagnosis of DFNB1, ought to be a first step of molecular analysis. More than 200 different GJB2 mutations correlated with autosomal recessive NSHL are spread throughout the gene, which necessitates sequence analysis of the entire coding region (exon 2) (The Connexin-Deafness homepage. Available from In the present study, 146 unrelated sensorineural NSHL patients were screened for GJB2 and GJB6 gene mutations. Two pathogenic GJB2 mutations were identified in 30/146 patients and the diagnosis for DFNB1 was therefore established. Homozygosity for c.35delg was found in 21/30 of the genotyped unrelated NSHL patients with the rest 9/30 identified in the compound heterozygote state (table 2a). Ten patients with SNHL were identified with only one pathogenic mutation in the GJB2 gene (table 2b). Normally, heterozygosity for a GJB2 or GJB6 mutation connotes carrier status for NSHL and rarely can cause autosomal dominant hearing loss. Therefore, the possibility that a second mutation remains undetected due to limitations of the methodology used could most likely be the case. Such defects could be found in the regulatory region of the GJB2 and GJB6 genes or even in deep intronic areas. Unexpectedly, five subjects with severe SNHL were heterozygous for the p.val153ile missense alteration. The pathogenicity of p.val153ile is controversial and molecular epidemiological studies revealed a p.val153ile allele frequency in the normal hearing population of 0.49 (Grasso et al. 2007). A report by Marlin et al. (2001) identified the p.val153ile mutation in normal hearing parents in association with c.35delg mutation on the other allele, suggesting a lack of pathogenicity of p.val153ile. On the contrary, the view that p.val153ile should be considered a pathogenic change was supported by two other studies that identified the mutation in deaf patients in the compound heterozygous state with the p.thr8met missense mutation (Wu et al. 2002). Additionally, the pathogenicity of the GJB2 p.val153ile was verified by a functional study where the presence of the mutation prevented the formation of functional channels and produce junctional conductance in a paired Xenopus oocyte assay (Mese et al. 2004). The results of the present study demonstrate that the contribution of the GJB2 gene on sensorineural hearing loss in Cyprus (40/146 of tested subjects with SNHL were identified with one or two mutations in the GJB2 gene, i.e. 27.4%) is similar to that reported in the neighbouring country of Lebanon (33%) (Mustapha et al. 2001). A relatively lower c.35delg allele frequency among those with GJB2 mutations 472 Journal of Genetics, Vol. 93, No. 2, August 2014

3 Inherited hearing loss Figure 1 (contd) was found between the subjects in our study (81.7%) when compared to the subjects from the neighbouring country of Lebanon ( 94%) (Mustapha et al. 2001). Regardless of the fact that the sample size of the present study is relatively small (table 1), the observed proportion of the c.35delg mutation among all presumed NSHL causative GJB2 mutations (81.7%) was similar with respect to most of other European countries, e.g. 95.2% in Greece, 88% in Italy, 76.9% in Austria and 55% in Spain (Rabionet et al. 2000; Janecke et al. 2002; Pampanos et al. 2002). The average carrier rate of c.35delg GJB2 mutation in Europe is about 2% but some important differences exist between various populations (Gasparini et al. 2000). A notable lower carrier frequency exists in northern European countries compared to southern European ones (Lucotte and Dieterlen 2005). In Cyprus the carrier frequency of 2.5% is just above the average c.35delg mutation rate in Europe (Neocleous et al. 2006b). Recent meta analyses on the subject showed that the relatively more elevated incidence value for the c.35delg mutation in Europe concerns the Mediterranean region with Greece as the focus (Lucotte and Dieterlen 2005). In main land Greece a moderately high carrier rate of 3.5% for c.35delg was reported and an extremely high carrier rate of 5.4% in the Greek island of Crete (Yanakakis et al. 2009). A recent study from Belarus for the c.35delg mutation identified a 5.7% carrier rate which is the highest so far to be reported (Danilenko et al. 2012). All these values are surprisingly high and Journal of Genetics, Vol. 93, No. 2, August

4 Vassos Neocleous et al. Figure 1. Electropherograms of all the GJB2 listed mutations in the manuscript. suggest either a founder effect or selective advantage for heterozygotes, or both. A recent report among European individuals heterozygous or homozygous for c.35delg 474 identified a significant higher epidermal thickness in these individuals compared to wild type. Therefore, these data could possibly support the hypothesis that skin phenotype Journal of Genetics, Vol. 93, No. 2, August 2014

5 Inherited hearing loss Table 2. Frequencies of GJB2 mutations in the Cypriot SNHL cohort. (a) GJB2 genotype Number of GJB2 compound heterozygote carriers (n = 30) c.35delg/c.35delg 21 (70%) c.35delg/p.arg184pro 4 (13.3%) c.35delg/p.leu90pro 2 (6.7%) c.35delg/p.glu47stop 1 (3.3%) c.delglu120/p.val178ala 1 (3.3%) c.35delg/c.167delthr 1 (3.3%) Figure 2. The presence or absence of the c.-23+1g>a splice mutation was assessed by restriction digestion with HphI of the GJB2 exon 1 PCR product (499 bp). 1 Normal control; 2 heterozygote for c.-23+1g>a. might counterbalance the evolutionary disadvantage caused by deafness (Guastalla et al. 2009). The GJB6 gene has also been demonstrated to cause recessive NSHL mainly through a large deletion called del(gjb6- D13S1830). This deletion is a common cause of deafness in several European populations and several groups around the world have shown that it is most frequent in Spain, France, the United Kingdom, Israel and Brazil, accounting for % of all the DFNB1 alleles (del Castillo et al. 2005; Marlin et al. 2005). Interestingly, del(gjb6-d13s1830) has not been detected in Turkish, Italian, Austrian and Chinese NSHL subjects (Liu et al. 2002; Del Castillo et al. 2003; Gunther et al. 2003; Uyguner et al. 2003). Similarly, the present study showed that del(gjb6-d13s1830) and GJB6 mutations with an autosomal dominant mode of inheritance were not found in any of the 146 Greek Cypriot subjects in either homozygous or heterozygous state. These results confirm the previous finding by our group that del(gjb6- D13S1830) and autosomal dominant GJB6 mutations are unlikely to be of epidemiological or clinical importance for Greek Cypriots (Neocleous et al. 2006a). The fact that these mutations are also absent from the NSHL subjects of some populations strongly suggest the existence of a possible founder effect in western Europe. In conclusion, the results of the present study have important implications for the diagnosis and counselling of Greek Table 1. GJB2 mutation frequency of affected alleles from 30 unrelated compound heterozygote NSHL patients compatible with recessive inheritance. GJB2 gene mutation Number of alleles Per cent of alleles c.35delg p.r184p p.l90p p.e47stop dele c.167delt p.v178a (b) GJB2 genotype Number of GJB2 heterozygote carriers (n = 10) p.val153ile/x 5 (50%) c.35delg/x 1 (10%) p.val37ile/x 1 (10%) p.leu90pro/x 1 (10%) c.-23+1g>a/x 1 (10%) c.-1g>a 5 UTR/X 1 (10%) Cypriot families with NSHL. Further analysis of the different genes implicated in hearing loss need to be performed in the Greek Cypriot deaf population to determine if there are other common NSHL mutations in this population. In addition our data contributes to knowledge of the geographic distribution of GJB2 and GJB6 mutations in Europe. Acknowledgement This work was supported by the A.G. Leventis Foundation. References Danilenko N., Merkulava E., Siniauskaya M., Olejnik O., Levaya- Smaliak A., Kushniarevich A. et al Spectrum of genetic changes in patients with non-syndromic hearing impairment and extremely high carrier frequency of 35delG GJB2 mutation in Belarus. PLoS One 7, e Del Castillo I., Moreno-Pelayo M. A., Del Castillo F. J., Brownstein Z., Marlin S., Adina Q. et al Prevalence and evolutionary origins of the del(gjb6-d13s1830) mutation in the DFNB1 locus in hearing-impaired subjects: a multicenter study. Am. J. Hum. Genet. 73, del Castillo F. J., Rodriguez-Ballesteros M., Alvarez A., Hutchin T.,LeonardiE,deOliveiraC.A.et al A novel deletion involving the connexin-30 gene, del(gjb6-d13s1854), found in trans with mutations in the GJB2 gene (connexin-26) in subjects with DFNB1 non-syndromic hearing impairment. J. Med. Genet. 42, Gasparini P., Rabionet R., Barbujani G., Melchionda S., Petersen M., Brondum-Nielsen K. et al High carrier frequency of the 35delG deafness mutation in European populations. Genetic Analysis Consortium of GJB2 35delG. Eur. J. Hum. Genet. 8, Grasso D. L., Guerci V. I., Zocconi E., Milanese M., Segat L. and Crovella S MBL2 genetic polymorphisms in Italian children with adenotonsillar hypertrophy. Int. J. Pediatr. Otorhinolaryngol. 71, Journal of Genetics, Vol. 93, No. 2, August

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