GJB2 Mutations in Patients with Nonsyndromic Hearing Loss from ...

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in the GJB6 gene in 58 unrelated patients with nonsyndromic hearing loss from Croatia. About 44.8% of our patients presented with mutation in the GJB2 gene.
GENETIC TESTING AND MOLECULAR BIOMARKERS Volume 13, Number 5, 2009 ª Mary Ann Liebert, Inc. Pp. 693–699 DOI: 10.1089=gtmb.2009.0073

ORIGINAL ARTICLE

GJB2 Mutations in Patients with Nonsyndromic Hearing Loss from Croatia Ivona Sansovic´,1 Jelena Knezˇevic´,2 Vesna Musani,2 Pavel Seeman,3 Ingeborg Barisˇic´1, and Jasminka Pavelic´ 2

The aim of the study was to determine (1) the frequency and type of mutations in the coding region of the GJB2 gene (sequencing), (2) the frequency of splice site mutation IVS1 þ 1G > A in the GJB2 gene (multiplex ligation– dependent probe amplification analysis), (3) possible copy number changes in the GJB2, GJB3, GJB6, and WFS1 genes (multiplex ligation–dependent probe amplification analysis), and (4) the frequency of del(GJB6-D13S1830) in the GJB6 gene in 58 unrelated patients with nonsyndromic hearing loss from Croatia. About 44.8% of our patients presented with mutation in the GJB2 gene. We identified seven sequence variations. Six of them had previously been reported as disease related (35delG, W24X, V37I, L90P, 313del14, and IVS1 þ 1G > A), and we report here for the first time one novel variant, 24A > C. We detected the greatest frequency of 35delG allele compared to the other alleles (35.3%). Allelic frequencies of other common mutations accounted for 2.6–0.9% of analyzed chromosomes. Neither GJB6 deletion nor copy number changes in the GJB2, GJB3, GJB6, and WFS1 genes were found. The 35delG=35delG genotype was associated with severe to profound hearing loss in 94% of 35delG homozygotes. High mutation rate (44%) indicates that testing of the GJB2 gene will clarify the genetic cause in almost half of the cases of recessive nonsyndromic hearing loss in Croatia.

Introduction

H

earing loss (HL) is the most frequent inherited sensory disorder affecting about 1 in 1000 children. At least 60% of cases have genetic etiology. Nonsyndromic hearing loss (NSHL) is the most common form of genetically determined HL and accounts for 70% of the cases (van Camp et al., 1997). It is inherited in an autosomal recessive pattern. To date, more than 85 loci for NSHL have been mapped, with 39 deafness genes identified (van Camp and Smith, 2007). Despite this heterogeneity, up to 50% of autosomal recessive NSHL worldwide can be attributed to variants in one particular gene, GJB2 (MIM 121011) (Kenneson et al., 2002). The GJB2 gene encodes the gap junction protein connexin 26, which is expressed in the epithelial cell gap junction system and the connective tissue gap junction system in the cochlea (Kikuchi et al., 2000). These two gap junction systems form the route by which potassium ions can be recycled back to the endolymph, which is crucial for the normal function of the hearing system. In addition to connexin 26, connexin 30 (the GJB6 gene) is also expressed in the mammalian cochlea and has very similar patterns of distribution to connexin 26 (Lautermann et al., 1999).

There are over 100 nonsyndromic GJB2 gene variants reported in the literature thus far (Ballana et al., 2005). Some of these are very common and display ethnic specificity— namely, 35delG in Caucasians (Zelante et al., 1997; Estivill et al., 1998; Green et al., 1999); 167delT in Ashkenazi Jews (Morell et al., 1998; Dong et al., 2001); 235delC in Japanese and Chinese (Abe et al., 2000; Liu et al., 2002); V37I in Taiwanese and Thai individuals (Hwa et al., 2003; Wattanasirichaigoon et al., 2004); W24X in India and in Gypsies from different European countries (RamShankar et al., 2003; Minarik et al., 2003; Alvarez et al., 2005); and R143W in Ghana (Hamelmann et al., 2001). The splice site mutation IVS1 þ 1G > A in noncoding exon 1 is frequent among Czech, Turkish, and Hungarian GJB2 heterozygous for an exon 2 mutation (Seeman and Sakmaryova´, 2006; Sirmaci et al., 2006; To´th et al., 2007). The GJB6 gene is unique because of its chromosomal localization within 50 kb of GJB2. A frequent mutation, del(GJB6-D13S1830), in this gene leaves the GJB2 coding region intact but deletes a large region close to GJB2 and truncates GJB6. This deletion is frequently found in compound heterozygosity with a GJB2 mutation, and the associated HL is assumed to be caused either by the deletion of a putative GJB2 regulatory element or by digenic inheritance (del Castillo et al.,

1

Department of Pediatrics, Children’s Hospital Zagreb, University of Zagreb, Medical School, Zagreb, Croatia. Division of Molecular Medicine, Rudjer Bosˇkovic Institute, Zagreb, Croatia. Department of Child Neurology, Charles University Prague, Prague, Czech Republic.

2 3

693

´ ET AL. SANSOVIC

694 2002). The del(GJB6-D13S1830) is most frequent in Spain, France, the United Kingdom, Israel, and Brazil, whereas the frequency is lower in Belgium and Australia (del Castillo et al., 2003). In Central Europe the frequency is very low [in Czech Republic, Seeman et al., 2005 reported only one case of double heterozygosity involving del(GJB6-D13S1830)]. The degree of hearing impairment in persons with biallelic GJB2 mutations varies from mild to profound even within the same family and among persons with the same mutation (Green et al., 1999). It is most commonly nonprogressive and bilateral (Denoyelle et al., 1999). Here, we report on the frequency and the spectrum of GJB2 mutations among 58 unrelated subjects with autosomal recessive NSHL in the Croatian population. We have also tested our deafness population on del(GJB6-D13S1830) in the GJB6 gene. Materials and Methods Patients We have analyzed 58 unrelated patients from Croatia in whom childhood nonsyndromic sensory deafness was diagnosed at Children’s Hospital Zagreb. Clinical geneticists evaluated patients by family history, physical examination, and additional laboratory testing when appropriate. For each patient, a comprehensive questionnaire was filled out to rule out known environmental causes of hearing impairment (infection, ototoxic drugs, prenatal asphyxia, etc.). All patients with signs of an acquired HL, and patients with additional symptoms or malformations suggesting syndromic origin of disorder were excluded from the study. Assessment of severity and pattern of HL was performed by pure-tone audiometry, tympanometry, auditory brainstem response, and evoked otoacoustic emissions. Severity of hearing impairment was classified as follows: 95 dB, profound HL (mean of hearing at 0.5–1–2– 4 kHz). The median age of tested individuals was 3 years (ranging from 1 month to 33 years). Informed consent was obtained from all subjects tested or from their parents. DNA analysis

Screening for del(GJB6-D13S1830) in the GJB6 gene The screening was performed in 32 wild-type homozygotes and two heterozygotes for the mutations in coding region of the GJB2 gene. Testing was done using duplex PCR assay with primers GJB6-1R, BKR-1, and GJB6-1F (del Castillo et al., 2002). PCR conditions were initial denaturation at 958C for 5 min; 35 cycles at 958C for 30 s, 628C for 45 s, and 728C for 1 min; and final elongation at 728C for 7 min. The junction fragment (457 bp) caused by the deletion was amplified using primer pair GJB6-1R=BKR-1. To detect a wild-type allele, we used GJB6-1F primer (located within the deleted region) and GJB6-1R primer to amplify 704 bp PCR fragment. Using these primers together in one duplex PCR test, two different PCR products could be amplified providing distinction between wild-type subjects (704 bp PCR product), homozygotes for the deletion (457 bp PCR product), and heterozygotes (both PCR products). A positive control sample (heterozygote for the deletion) was included in every PCR reaction. The PCR products were verified by electrophoresis in a 3% agarose gel containing 0.5 mg=mL ethidium bromide (Fig. 1). Multiplex ligation–dependent probe amplification analysis Multiplex ligation–dependent probe amplification (MLPA) analysis was done using SALSA MLPA kit P163-B1 GJB (MRC Holland, Amsterdam, The Netherlands), according to the manufacturer’s recommendations. It is designed to detect deletions=duplications of GJB2, GJB3, GJB6, and WFS1 genes. It contains probes for all GJB2 and GJB6 exons as well as three GJB2 probes specific for the 35delG, IVS1 þ 1G > A, and 313del14 mutations (lot 0208 version B1). Two heterozygous and 32 wild-type homozygous patients for the GJB2 exon 2 mutations underwent MLPA analysis to test IVS1 þ 1G > A mutation in GJB2 and possible copy number changes in the GJB2, GJB3, GJB6, and WFS1 genes. We used approximately 100 ng DNA in reaction. 5-carboxyfluoresceinlabeled PCR products were separated by capillary electrophoresis on an ABIPRISM 310 Genetic Analyzer and subsequently analyzed by GeneMapper v4.0 Software. In each run we include referent samples: four healthy controls, three 35delG homozygotes, three 35delG heterozygotes, and three 313del14

Genomic DNA was extracted from peripheral blood leukocytes according to standard salting out procedure. Polymerase chain reaction (PCR)–restriction fragment length polymorphism (RFLP) analysis described previously (Sansovic´ et al., 2005) was used for the detection of two common mutations, 35delG and 167delT. All samples negative for 35delG and 167delT mutation in one or both alleles were additionally screened for other GJB2 mutations by DNA sequencing. Sequencing exon 2 of the GJB2 gene The coding region was amplified by using primer pair 1F=5R (Kupka et al., 2002). The PCR product was purified with ExoSap IT reagent (USB Corporation, Cleveland, OH) and subsequently sequenced with primers 1F, 2F, 3R, 4F, and 5R (Kupka et al., 2002), on an ABIPRISM 310 Genetic Analyzer (Applied Biosystems, Foster City, CA). Detected mutations were confirmed on both DNA strands. Sequences were compared to AF281280 as a reference.

FIG. 1. Electrophoretic separation of products of multiplex polymerase chain reaction (PCR) on 3% agarose gel in screening of the del(GJB6-D13S1830) in the GJB6 gene. Lane 1, molecular size marker IX (Roche, Mannheim, Germany); lane 2, positive control, heterozygote for the del(GJB6D13S1830); lane 3, negative control, wild type (wt=wt genotype) for the del(GJB6-D13S1830); lanes 4 and 5, patients, wild types (wt=wt genotype) for the del(GJB6-D13S1830).

NONSYNDROMIC HEARING LOSS IN CROATIA

695

Table 1. Summary of GJB2 Nucleotide Changes Detected in the Croatian Patients with Nonsyndromic Hearing Loss Nucleotide change 24A > Ca 35delG 71G > A 109G > A 269T > C 313del14 IVS1 þ 1G > A

Protein change

Protein domain

Mutation type

Inheritance

Unknown L10fs W24X V37I L90P K105fs No mRNA

50 UTR IC1 TM1 TM1 TM2 IC2 —

Unknown Deletion=frameshift Nonsense Variant Missense Deletion=frameshift Splice site

Unknown Recessive Recessive Recessive Recessive Recessive Recessive

a

Novel sequence change. NSHL, nonsyndromic hearing loss; 50 UTR, 50 untranslated region; IC, intracellular domain; TM, transmembrane domain.

heterozygotes. Each result was confirmed by two independent experiments. Confirmation and analysis of the IVS1 þ 1G > A mutation in the GJB2 gene The exon 1 and flanking donor splicing site of GJB2 gene was amplified with primers ex1F 50 -acggcgggagacaggtgtt-30 and ex1R 50 -agaaacgcccgctccagaa-30 . PCR products were sequenced using the same primers on an ABIPRISM 310 Genetic Analyzer. MLPA data analysis The P163-B1 GJB probemix contains 41 MLPA probes with amplification products between 124 and 428 nt. The 172 and 265 nt probes only generate a signal on samples containing the GJB2 35delG or IVS1 þ 1G > A mutation. On the contrary, 307 nt probe generates a signal on wild-type sample, negative for 313del14 mutation. Hence, visual comparison of peak profiles was sufficient to easily identify 35delG, IVS1 þ 1G > A, and 313del14 mutations. Comparison of results was performed within one experiment. Confirmation of IVS1 þ 1G > A mutation was done by sequencing GJB2 exon 1 and flanking donor splicing site. Data analysis was performed by exporting the peak areas to a Microsoft Excel file. To detect deletions=duplications of one or more exons, data generated by this probe mix were intranormalized by dividing the peak area of each probe’s amplification product by the total area of only the seven reference probes in this probe mix (block normalization). To detect deviating areas, normalization was achieved by diTable 2. GJB2 Genotypes Identified in the Croatian Patients with Nonsyndromic Hearing Loss Genotype 35delG=35delG 35delG=313del14 35delG=W24X 35delG=L90P 24A > C=L90P V37I=V37I 35delG=IVS1 þ 1G > A L90P=wt wt=wt wt, wild type.

Patients with NSHL (n ¼ 58) 18=58 2=58 1=58 1=58 1=58 1=58 1=58 1=58 32=58

(31.0%) (3.4%) (1.7%) (1.7%) (1.7%) (1.7%) (1.7%) (1.7%) (55.2%)

viding this intranormalized probe ratio in a sample by the average intranormalized probe ratio of all healthy control samples. Peak areas outside the range 0.7–1.3 times the control peak area were considered abnormal, with those below 0.7 representing deletions and those above 1.3 representing duplications. Results About 44.8% (26=58) of our patients presented with mutation in the GJB2 gene with one of them being heterozygous. We identified seven sequence variations. Six of them have previously been reported as disease-related recessive mutations (35delG, W24X, V37I, L90P, 313del14, and IVS1 þ 1G > A), and we report here for the first time one novel variant, 24A > C (Table 1). Of 26 patients positive for GJB2 mutations, 18 were 35delG homozygous, 6 were compound heterozygous, 1 was heterozygous, and 1 was V37I homozygous (Table 2). Among 58 patients (116 alleles), the frequency of 35delG mutation was 35.3%. Allelic frequencies of other common mutations in patients, L90P, 313del14, V37I, W24X, and IVS1 þ 1G > A, accounted for 2.6–0.9% of analyzed chromosomes (Table 3). None of the tested alleles among patients carried 167delT mutation. Of the 18 35delG homozygotes, 8 had profound, 9 had severe, and 1 had moderate HL. Missense mutation L90P was detected in two sporadic cases in a compound hetrozygosity with 24A > C and 35delG mutation and in one heterozygous patient. L90P= 24A > C compound heterozygote had mild hearing impairment. Another one, compound heterozygote, 35delG=L90P, presented with moderate HL on right and profound on left ear. The patient with L90P=wild-type

Table 3. Frequencies of Detected GJB2 Mutation=Polymorphism in the Croatian Patients with Nonsyndromic Hearing Loss GJB2 mutation=polymorphism 35delG L90P V37I 313del14 W24X IVS1 þ 1G > A 24A > C

Patients with NSHL (n ¼ 116 chromosomes) 41=116 3=116 2=116 2=116 1=116 1=116 1=116

(35.3%) (2.6%) (1.7%) (1.7%) (0.9%) (0.9%) (0.9%)

696 genotype had moderate HL. The V37I variant was detected in one homozygous patient with mild to moderate HL. Neither of tested NSHL patients being negative (32) or heterozygous (2) for GJB2 mutations carried the del(GJB6D13S1830) in the GJB6 gene. By applying the MLPA analysis, we detected the mutation IVS1 þ 1G > A in one compound heterozygote patient (35delG=IVS1 þ 1G > A) with progressive moderate to severe HL (Fig. 2). DNA sequencing of the GJB2 exon 1 confirmed the heterozygous mutation. We did not find the deletions= duplications in any other genes included in this MLPA analysis. Also, by MLPA technique we confirmed 35delG and 313del14 mutations identified previously by PCR=RFLP method and sequencing, respectively. Discussion Although HL in people with two GJB2 allele variants ranged from mild to profound, recently studies assessed the certain correlation between genotype=type of mutation and degree of HL in persons with two GJB2 allele variants (Cryns et al., 2004; Snoeckx et al., 2005).

´ ET AL. SANSOVIC Among 50 mutated chromosomes detected in patients with NSHL, 41 carried a 35delG mutation (82%). Similar to other studies, the most common pathogenic genotype, 35delG= 35delG, was associated with severe to profound HL in 94% of 35delG homozygotes (Cryns et al., 2004; Snoeckx et al., 2005). Also, persons with inactivating=inactivating genotype of two truncating or nonsense mutations (35delG=313del14 and 35delG=35delG) were associated with severe to profound HL. Cryns et al. (2004) could not detect significant differences between 35delG homozygotes and 35delG=313del14 and 35delG=W24X genotypes. L90P mutation results in an amino acid change from leucine to proline in TM2 (transmembrane) protein domain. It affects a highly conserved residue of the connexin 26 protein and has been described as a disease associated with a recessive mode of inheritance (Murgia et al., 1999; Rabionet et al., 2000). Several studies have previously reported L90P frequency of 0.7–4.0% in Mediterranean patients (Estivill et al., 1998; Denoyelle et al., 1999; Murgia et al., 1999). Particularly high prevalence of the L90P mutation is found in Austria ( Janecke et al., 2002). It was suggested as a founder effect of this mutation in Central European countries. In this study we

FIG. 2. Capillary electrophoresis pattern from the three samples of approximately 100 ng human DNA analyzed with SALSA MLPA kit P163 GJB (lot 0208): (A) negative control sample, 35delG and IVS1 þ 1G > A negative (patient with normal hearing); (B) positive control sample, 35delG homozygote; (C) compound heterozygote patient, 35delG=IVS1 þ 1G > A. 35delG-specific and IVS1 þ 1G > A–specific probes only generate a signal on samples containing the GJB2 mutations.

NONSYNDROMIC HEARING LOSS IN CROATIA found one compound heterozygote 35delG=L90P with moderate HL on right and profound on left ear. Janecke et al. (2002) reported that the L90P mutation in trans with any other recessive mutation is predominantly associated with mild or moderate hearing impairment. However, according to study reported by Snoeckx et al. (2005), the 35delG=L90P genotype, in the truncating=nontruncating genotype class, was associated with a bimodal distribution of the binaural mean pure-tone averages 0.5–1–2 kHz. Hence, although majority within this genotype had significantly less HL than did the reference group (35delG=35delG), a small group of 35delG= L90P compound heterozygotes had severe to profound HL. Therefore, we can conclude that our result is in accordance with study above. V37I allele is relatively frequent in the Asian population (Liu et al., 2002; Wattanasirichaigoon et al., 2004). In our study sample the V37I variant was detected in one homozygous patient with mild to moderate HL. This mutation was originally described as a polymorphism (Kelley et al., 1998). Two years later two research groups found this mutation in homozygosity in patients, suggesting it is an HL-associated, recessive mutation (Rabionet et al., 2000; Wilcox et al., 2000). This proposal is supported by the fact that the amino acid substitution is localized in a highly conserved protein domain (TM1). According to other studies of this allele (Abe et al., 2000; Cryns et al., 2004; Marlin et al., 2001; Snoeckx et al., 2005), it is very likely that V37I is a pathogenic variant leading to a mild phenotype. The splice site mutation IVS1 þ 1G > A (also known as 3170 G > A) was first reported by Denoyelle et al. (1999). Seeman and Sakmaryova´ (2006) estimated that this splice site mutation represents 4% of pathogenic GJB2 mutations in Czech patients with HL and suggested that similar frequency may also be expected in other Central European or Slavic populations. Testing for this mutation explained deafness in 45% of Czech GJB2 monoallelic patients. Approximately onefourth GJB2-heterozygous Hungarian patients analyzed carried the splice site mutation in GJB2 (To´th et al., 2007). Also, half of Turkish NSHL patients, heterozygous for an exon 2 mutation, were found to have the IVS1 þ 1G > A mutation (Sirmaci et al., 2006). In the multicenter study, Snoeckxy et al. (2005) report that 16 persons segregating the 35delG=IVS1 þ 1G > A genotype had significantly less severe hearing impairment compared to 35delG homozygotes. In this report we found IVS1 þ 1G > A allele in one 35delG heterozygote patient with progressive moderate to severe hearing impairment. It is noteworthy that the first signs of HL were noted at the age of four. We report here for the first time a DNA variant, 24A > C transition in 50 untranslated region of the GJB2 gene. This novel variant has yet unknown consequences on the expression of the GJB2 gene. It was detected in one patient in a compound heterozygosity with the L90P mutation. In this report, none of the tested patients with NSHL was carrying the del(GJB6-D13S1830) in the GJB6 gene. Our result is in accordance with studies on patients with NSHL from Austria, China, Morocco, Turky, Egypt, and Iran, and in cypriotic Greeks, in which this deletion has not been found (Liu et al., 2002; Frei et al., 2004; Neocleous et al., 2006; Abidi et al., 2007; Esmaeili et al., 2007; Evirgen et al., 2008). We conclude that the occurrence of this deletion is restricted to certain populations, indicating a founder effect as it has been ob-

697 served with the common mutations in the GJB2 gene (35delG, 167delT, and 235delC). Despite a small number of analyzed patients, we noticed a similar distribution of the GJB2 allele in our NSHL population and other Caucasian European populations. As we expected, with the frequency of 35.3%, 35delG allele was the most frequent pathogenic allele. Also, high mutation rate (44%) indicates that early testing of the GJB2 gene will clarify the genetic cause in almost half of the cases of recessive NSHL. According to combined data from previous studies (Cryns et al., 2004; Snoeckx et al., 2005) and from this one, it is clear that genotype–phenotype correlations do exist for GJB2-related HL. Persons found to have DFNB1 segregating two truncating= nonsense mutations (35delG=313del14 and 35delG=35delG) were associated with severe to profound HL. MLPA is a single-tube, highly automated method and suitable for high-throughput testing. In this study MLPA has shown to be a fast, reliable, and efficient method for the detection of the GJB2 gene mutation with a mutation-specific probe. In our tested cohort 86% (44=51) mutated alleles could be detected by MLPA P163-B1 GJB probe mix. MLPA might serve as an alternative for the PCR=RFLP method in detecting 35delG, 313del14, and IVS1 þ 1G > A mutations and used prior sequencing. Also, it can be easily used for screening del(GJB6-D13S1830) in the GJB6 gene. The GJB2 mutations generally cause a nonprogressive HL, but progression has been described in a considerable proportion of cases ( Janecke et al., 2002; Pagarkar et al., 2006; Gopalarao et al., 2008; Kokotas et al., 2008). Further, profound HL due to biallelic GJB2 mutations might not always be congenital. In some individuals hearing capacities are better, with normal hearing or mild-moderate HL, in the first months of life. We describe one case of progressive HL with age of onset of HL at four due to biallelic GJB2 mutations, emphasizing that such cases will be missed by neonatal hearing screening programs. This illustrates the need to perform a GJB2 mutation analysis in all cases of childhood idiopathic progressive HL to diagnose postnatal or postlingual onset HL early on. Thus, we recommend rigorous audiologic surveillance and careful genetic counseling for all hearing-impaired subjects with GJB2 mutations. We did not detect any GJB2 gene variation in 55.2% (32=58) of our patients. On the basis of these findings, we can assume that mutations in other noncoding parts of the GJB2 gene, mutations in some other genes related to NSHL, or environmental factors can contribute heterozygote and wild-type homozygote GJB2 genotypes. Acknowledgments We thank the families, patients, and control individuals for their cooperation. This work was supported by Grant 0980982464-2394 from the Ministry of Science, Education, and Sports, Republic of Croatia. Disclosure Statement No competing financial interests exist. References Abe S, Usami S, Shinkawa H, et al. (2000) Prevalent connexin 26 gene (GJB2) mutations in Japanese. J Med Genet 37:41–43.

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Address correspondence to: Jasminka Pavelic, Ph.D. Division of Molecular Medicine Rudjer Boskovic Institute Bijenicka c. 54, POB 180 HR-10000 Zagreb Croatia E-mail: [email protected]