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doi:10.1016/j.heares.2005.08.001
Autosomal recessive and sporadic deafness in Morocco: High frequency of the 35delG GJB2 mutation and absence of the 342-kb
GJB6 variant
Bouchaïb Gazzaz a,b , Dominique Weil d , Leïla Raïs c , Omar Akhyat e , Houssine Azeddoug b , Sellama Nadi W a,¤
a Laboratoire de Génétique Médicale, Faculté de Médecine et de Pharmacie, Casablanca, Maroc
b Laboratoire de Biologie Moléculaire, Faculté des Sciences Ain Chock, Casablanca, Maroc
c Département d’ophtalmologie, CHU Ibn Rochd, Casablanca, Maroc
d Unité de génétique des déWcits sensoriels, Institut Pasteur Paris, France
e Laboratoire de Biologie Moléculaire, Faculté des Sciences Ibn Zohr, Agadir, Maroc
Received 27 April 2005; accepted 4 August 2005 Available online 21 October 2005
Abstract
Deafness is a heterogeneous disorder showing di
Verent pattern of inheritance and involving a multitude of di
Verent genes. Muta- tions in the gene, GJB2 Gap junction type 1), encoding the gap junction protein connexin-26 on chromosome 13q11 may be respon- sible for up 50% of autosomal recessive nonsyndromic hearing loss cases (ARNSHL), and for 15–30% of sporadic cases. However, a large proportion (10–42%) of patients with GJB2 has only one GJB2 mutant allele. Recent reports have suggested that a 342-kb dele- tion truncating the GJB6 gene (encoding connexin-30), was associated with ARNSHL through either homozygous deletion of Cx30, or digenic inheritance of a Cx30 deletion and a Cx26 mutation in trans. Because mutations in Connexin-26 (Cx26) play an important role in ARNSHL and that distribution pattern of GJB2 variants di
Vers considerably among ethnic groups, our objective was to
Wnd out the signi
Wcance of Cx26 mutations in Moroccan families who had hereditary and sporadic deafness. One hundred and sixteen families with congenital deafness (including 38 multiplex families, and 78 families with sporadic cases) were included. Results show that the prevalence of the 35delG mutation is 31.58% in the family cases and 20.51% in the sporadic cases. Further screening for other GJB2 variants demonstrated the absence of other mutations; none of these families had mutations in exon 1 of GJB2 or the 342-kb deletion of GJB6. Thus, screening of the 35delG in the GJB2 gene should facilitate routinely used diagnostic for genetic coun- selling in Morocco.
2005 Elsevier B.V. All rights reserved.
Keywords: Deafness; ARNSHL; Sporadic; GJB2 and GJB6; Morocco
1. Introduction
Deafness is a common sensory disorder that a V ects one in 1000 children, and its prevalence increases with the age (Nadol, 1993). In developed countries, about 60% of the cases are attributed to genetic defects (Cohen and Gorlin, 1995). Genetic deafness may be syndromic (associated with additional clinical features) or non-syn- dromic (no other recognizable abnormal associated
Abbreviations: ARNSHL, autosomal recessive non syndromic hearing loss; GJB, gap junction (protein); , subgroup; DFNB, deaf- ness, autosomal recessive; CX, connexin (protein); ACRS-PCR, artiW- cial created restriction site – polymerase chain reaction; DGGE, denaturing gradient gel electrophoresis; GJA, gap junction (protein);
, subgroup; NSHL, non syndromic hearing loss
* Corresponding author. Tel.: +21 222 271630; fax: +21 222 261453.
E-mail address: s.nadiW@caramail.com (S. NadiW).
phenotype). Among hereditary non-syndromic deafness, about 80% of congenital cases are either inherited as an autosomal recessive trait or apparently sporadic (Denoyelle et al., 1999). The identi W cation of genes responsible of hearing impairment has been very fruitful during the last years, and the molecular events of deaf- ness are rapidly being elucidated by genetic analysis of human families and mouse model systems. To date close to 70 ARNSHL loci (DFNB) responsible for non-syn- dromic hearing loss have been mapped and 35 deafness genes have been identi W ed (Van Camp and Smith, 2003).
An important gene that should be considered in congenital or childhood onset ARNSHL is GJB2 (MIM#121011), which encodes the gap junction protein connexin-26 (beta-2, GJB2), originally assigned to chro- mosome 13q12 (Guilford et al., 1994). It account for 50%
of this type of hearing loss (Cohen and Kelley, 1999).
The distribution pattern of GJB2 variants diVers consid- erably among ethnic groups. Some variants occur prefer- entially, but not exclusively, in distinct ethnic groups.
To date, more than 70 mutations in GJB2 gene are known to be associated with deafness (Kenneson et al., 2002; Rabionet et al., 2002; Calvo et al., 2002). One par- ticular variant, the 35delG mutation, accounts for up to 70% of the pathologic alleles in northern and southern European and American Caucasian populations, with a carrier frequency ranging from 1.3% to 2.8% (Gasparini et al., 2000). Mutations in other genes are rare causes of hearing loss, except for a 342-kb deletion in GJB6 (MIM#604418) gene recently identi W ed and found to be very common in patients with NSHL from Spain and Cuba (Del Castillo et al., 2002).
Since data on the occurrence of GJB2 and GJB6 asso- ciated deafness in African populations are scarce (Denoyelle et al., 1997; Brobby et al., 1998; Lench et al., 1998; Ben Arab et al., 2000; Hamelmann et al., 2001;
Gasmelseed et al., 2004), and no reports provided yet a systematic study of the prevalence of Cx26 and Cx30 mutations in Moroccan’s population, we decided to ana- lyse the prevalence of GJB2 and GJB6 variants among individuals with hearing loss in Morocco by analysing families with inherited sensorineural and sporadic deaf- ness.
2. Material and methods 2.1. Patient’s evaluation
The study was conducted on 116 probands a V ected by congenital non syndromic sensorineural hearing impair- ment, ranging from mild to profound forms. These patients were ascertained from the genetic counselling service at the clinical department of Ibn Rochd hospital in Casablanca and from records of Moroccan’s deaf associations in Casablanca and Agadir. Each individual
was the proband of a separate family; therefore there were 116 independent families. Children, their parents and other informative relatives were interviewed.
Informed consent was obtained from all participants.
Both sporadic and familial cases, the latter with appar- ent autosomal recessive mode of inheritance, were included. A detailed history was taken for each subject.
For carrier frequency estimate, 200 unrelated individuals with normal hearing were screened for the presence of the 35delG mutation.
2.2. Clinical evaluation
A V ected subjects were 3–20 years old. For each patient, the complete medical history was explored to record the age of deafness onset and to ensure that hear- ing loss was not the result of environmental causes:
maternofetal infection, perinatal complications, menin- gitis, mumps, prenatal or postnatal drug ototoxicity or acoustic trauma. All families underwent an otoscopic and general examination including a systematic research of signs suggestive of a syndromic form a deafness (espe- cially neurological and diabetes disorders, dysmorphism, integumentary disorders and bronchial, cardiac and thy- roid anomalies). They had also an ophtalmological eval- uation (including fundoscopy) for visions problems, and search for haematuria and proteinuria. Families included in the study were classi W ed as having autosomal recessive inheritance if there were at least two hearing impaired sibs with normal hearing parents. Cases in which a detailed history did not indicate any other child- hood sensorineural hearing loss were referred to as spo- radic.
2.3. Molecular analyses
Blood was obtained by venipuncture from deaf chil- dren and their parents and DNA was extracted using a phenol–chloroform classical method. In the initial screen, all 116 patients were investigated for the presence of a 35delG mutation by a ACRS-PCR as described (Storm et al., 1999). DGGE analysis was performed for the coding exon (exon 2) of the GJB2 gene as previously described (Antoniadi et al., 2000). All results obtained by PCR/restriction fragment analysis and DGGE (samples showing band shifts) were conWrmed by direct genomic sequencing using the dideoxy chain terminator method on an Applied Bio systems DNA sequencer ABI 370.
PCR products of exon 2 (the single coding exon in GJB2 gene) of the Cx26 and X anking acceptor splicing site were ampli W ed using the following primers: GAP1F: 5 ⬘ - CCTATGACAAACTAAGTTGGTTC-3 ⬘ and CX51:
5 ⬘ -CTGAGCACGGGTTGCCTCATCCC-3 ⬘ . Experi- mental conditions for PCR ampli W cation and sequenc- ing were those previously described by Denoyelle et al.
(1997). Probands from multiplex families negative for
GJB2 mutation and all heterozygous probands for GJB2 mutation were analyzed for the presence of the 342-kb deletion in the GJB2 gene using multiplex PCR in these samples.
The junction fragment caused by the deletion was ampli W ed using a primer pair: forward: GJB6-1R 5 ⬘ -TTTAGGGGCATGATTGGGGTGATTT-3 ⬘ and reverse: BKR-1 5 ⬘ -CACCATGCGTAGCCTTAACC- ATTTT-3 ⬘ . The wild-type allele was ampli W ed using the forward primer: STS CX636-S 5 ⬘ -TGCCCACCCCCC- AAGTAGAG-3 ⬘ and reverse: STS-CX636-AS 5 ⬘ -TT- TCGGTTTCATTCATTTTCCCTATT-3⬘ at cycling conditions: After an initial denaturation at 94 °C for 5 min, followed by 35 cycles of 94 °C denaturation for 30 s, 63 °C for 30 s, 72 °C for 45 s, the PCR products were ver- iWed by 2% agarose gel electrophoresis. A positive sam- ple kindly provided by Unidad de Genetica Molecular, Hospital, Ramon Y Cajal, Madrid was included in all PCR reactions and gel runs. DNA samples from hearing control subjects were screened for the presence of the 35delG allele as described above to estimate the carrier frequency. The allele frequency was determined using GENEPOP Version 3.3 (Raymond and Rousset, 1995).
3. Results
As an initial step towards understanding the genetic causes of hearing impairment in Moroccan’s population, 116 individuals, (78 families with sporadic cases, and 38 families with multiplex cases) were enrolled in the study.
The pattern of deafness inheritance in these families was autosomal recessive. For all patients, non-progressive hearing impairment varied from mild to profound, involving all frequencies. Initial screening for the 35delG mutation revealed that in a total of 116 families ana- lysed, the 35delG was found in 24% (28/116) of the fami- lies. Combining the familial and sporadic cases of
congenital deafness brought the allele frequency of this mutation to 21.55%. (50 positives in 232 alleles). In both sporadic and familial cases, homozygosity for the 35delG mutation was observed in 19% of the families, heterozygosity in 5% and 76% of the families lacked this mutation.
The 35delG mutation was found in 31.58% of familial cases of deafness, and in 20.51% of the sporadic cases (Table 1).
In order to evaluate the presence of possible other mutations (not detected by PCR-ACRS), all negative and 6 heterozygous patients for 35delG were analysed using DGGE and direct genomic DNA sequencing (Fig. 1). No other mutations were found after sequenc- ing of the entire coding sequence of GJB2 gene and its Xanking regions. The 35delG mutation was thus respon- sible for 100% of GJB2 alleles mutated in our patients.
Furthermore, these patients were screened for the rare splice site mutation in the non-coding exon 1 of GJB2 gene by direct genomic sequencing. In 83 families (57 sporadic cases and 26 familial), no mutation was found in exon 1. Screening for the 342-kb GJB6 mutation did not reveal a single positive among heterozygote or nega- tive GJB2 for the 35delG mutation. On the other hand, the carrier frequency was 0% in all 200 hearing analysed controls.
Table 1
Distribution of genotypes for 35delG in GJB2 gene in unrelated Moroccan families with non syndromic autosomal recessive (DFNB) and sporadic congenital deafness
Genotypes Multiplex familial Simplex sporadic Total
35delG+/35delG+ 10 (26%) 12 (15.38 %) 22
35delG+/35delG¡ 2 (5%) 4 (5%) 6
35delG¡/35delG¡ 26 (69%) 62 (79%) 88
Total 38 78 116
Fig. 1. Sequence analysis of GJB2 gene. The upper panel (a) shows the normal gene sequence, and the lower panel (b) shows the sequence of a sub- jects with the 35delG/35delG mutation.
4. Discussion
This study is the W rst of its kind, describing the preva- lence GJB2 and GJB6 variants in the Moroccan popula- tion. This analysis shows that the 35delG mutation is the most common GJB2 allele variant with a prevalence approaching 24%. Our data con W rm the importance of GJB2 (essentially 35delG) mutations in hearing impair- ment. Indeed, several studies have shown that the 35delG mutation is present in di V erent ethnic and geo- graphic groups, and accounted for up to 85% of GJB2 mutations (Denoyelle et al., 1999). The relative contribu- tion of the 35delG to non-syndromic recessive and sporadic prelingual deafness is higher in the Mediterra- nean’s population (Table 2), demonstrating genetic het- erogeneity of deafness. However, some studies were based on small numbers of patients and mutation screening methods. In Asiatic populations, the preva- lence of this mutation is nil (0%), as in African Ameri- cans (Morell et al., 1997), Ghanian (Hamelmann et al., 2001) and Kenyan populations (Gasmelseed et al., 2004).
The prevalence is low in Australia (about 4%) (Wilcox et al., 2000) and Sudan (about 2.7%) (Gasmelseed et al., 2004). Moroccan population prevalence is comparable to those of other Mediterranean countries and is largely greater than in African populations as reported for Sudan and Kenya. Such prevalence variations among populations may re X ect an anthropological history of human movements; 35delG mutation is believed to have a Caucasoid origin (Tekin et al., 2000), a conse- quence of a single mutational mechanism appeared in founder chromosome, as concluded from a recent study of hotspot and haplotypic heterogeneity worldwide including many more populations (Rothroch et al., 2003).
Further analysis of families without mutations in the entire coding region of GJB2 gene also showed none in the non coding region (exon 1) of GJB2. The same is true for GJB6 (connexin-30) gene and remarkably for the 342-kb deletion which represents the most common GJB6 variant recently described in patients with non- syndromic hearing loss from Spain and Cuba through either homozygous for the Cx30 deletion or by digenic inheritance of the Cx30 deletion and Cx26 mutation in trans (Del Castillo et al., 2002). We conclude that this deletion is absent in our population and is restricted to some populations; it indicates a founder eVect regarding this deletion. These Wndings suggest the involvement of other genetic or environmental factors playing a major role in the manifestation of the disease in these families.
To date, the molecular genetic basis of those variations remains unknown. A possible explanation could be the involvement of mutations in other connexin genes for example, encoding other gap junction proteins (GJB3, GJA1) that could be functionally related to or interact with GJB2 and/or GJB6 in the promoter region in the inner ear (Liu et al., 2000, 2001), or the existence of a so called modi W er gene (Ruazidin et al., 2000). Conse- quently, these families will need further investigation to look for the presence of mutations in other genes mapped either on chromosome 13 or elsewhere.
Given the high frequency of GJB2 mutations (espe- cially the 35delG) among deaf children with congenital deafness, DNA diagnosis based on screening of this mutation in the GJB2 gene enhances the possibility to detect new-borns with non-syndromic recessive and spo- radic hearing impairment at an early stage of develop- ment. As a consequence, it will improve the current diagnosis and therapeutical option. Thus, the risk for genetic counselling can be quanti W ed more exactly in the majority of cases in Morocco.
Acknowledgements
We are most grateful to the probands for their will- ingness to participate in the study. We thank the director of Moroccan association of deaf in Casablanca for their help. We also thank Dr. Del Castillo from the Unidad Genetica molecular, Hospital, Ramon Y Cajal, Madrid for providing us with a positive control sample for the 342-kb GJB6 deletion, and Sylvie Nouaille from samples sequencing.
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Table 2
Proportion (%) of non syndromic, prelingual, sensorineural deafness due to 35delG mutation in diVerent populations
NI: not identiWed.
n: number of patients tested.
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