Update of the spectrum of GJB2 gene mutations in 152 Moroccan families with autosomal recessive nonsyndromic hearing loss

Update of the spectrum of GJB2 gene mutations in 152 Moroccan families with autosomal recessive nonsyndromic hearing loss

Amina Bakhchane ^a

¹ , Amale Bous fi ha ^a

¹ , Hicham Charoute ^a , Sara Salime ^a ,

Mustapha Detsouli ^a , Khalid Snoussi ^a , Sellama Nadi fi ^b , Mostafa Kabine ^d , Hassan Rouba ^a , Hind Dehbi ^b , Rachida Roky ^c , Majida Charif ^a , Abdelhamid Barakat ^a

aInstitut Pasteur, laboratoire de genetique moleculaire humaine, 20360 Casablanca, Morocco

bGenetics and Molecular Pathology Laboratory, Medical School of Casablanca, Casablanca, Morocco

cUniversite Hassan II Ain Chock, Laboratoire de Physiologie et genetique moleculaire, Km 8 Route d’El Jadida, B.P 5366 Maarif, Casablanca, 20100, Morocco

dLaboratoire de Biochimie et Biologie Moleculaire, Universite Hassan II, Faculte des Sciences Aïn Chock, Casablanca, Morocco

a r t i c l e i n f o

Article history:

Received 15 December 2015 Received in revised form 26 April 2016

Accepted 4 May 2016 Available online 8 May 2016

Keywords:

GJB2 Mutation Hearing loss Morocco

a b s t r a c t

Deafness is one of the most common genetic diseases in humans and is subject to important genetic heterogeneity. The most common cause of non syndromic hearing loss (NSHL) is mutations in theGJB2 gene. This study aims to update and evaluate the spectrum ofGJB2 allele variants in 152 Moroccan multiplex families with non syndromic hearing loss. Seven different mutations were detected: c.35delG, p.V37I, p.E47X, p.G200R, p.Del120E, p.R75Q, the last three mutations were described for thefirst time in Moroccan deaf patients, in addition to a novel nonsense mutation, the c.385G>T which is not referenced in any database. Sixty six families (43.42%) have mutations in the coding region ofGJB2, while the ho- mozygous c.35delG mutation still to date the most represented 51/152 (33.55%). The analysis of the geographical distribution of mutations located inGJB2gene showed more allelic heterogeneity in the north and center compared to the south of Morocco. Our results showed that theGJB2gene is a major contributor to non syndromic hearing loss in Morocco. Thus, this report of theGJB2mutations spectrum all over Morocco has an important implication for establishing a suitable molecular diagnosis.

©2016 Elsevier Masson SAS. All rights reserved.

1. Introduction

Hearing loss is the most common sensory disorder occurring in approximately 0.2% of newborns (Hilgert et al., 2009a). It has a severe impact on speech and language development in children and cause social and vocational problems for adults (Lasak et al., 2014). The early detection of hearing impairment in

uences the educational outcome of affected infants and rapid intervention maximizes the development of speech, thus facilitating the acqui- sition of normal cognitive, social and motor skills (Hilgert et al., 2009b).

Deafness is very heterogeneous and can occur from external auditory canal, sound conduction mechanism, cochlea, cochlear nerve or central auditory pathways (Lasak et al., 2014). Over half of all cases of congenital hearing loss have a genetic cause (Brown and

Rehm, 2012). It is believed that more than one hundred genes could be involved in non syndromic hearing impairment including 90 various genes previously reported (http://hereditaryhearingloss.

org). Nevertheless a single locus, DFNB1, accounts for about 50%

of cases (Smith et al., 1993). The most concerned gene in this type of deafness is

26 expressed in the inner ear and is thought to be important in maintaining endocochlear potential (Hilgert et al., 2009b). The degree of deafness varies from moderate-to-profound (Najmabadi and Kahrizi, 2014). Mutations in

gene are extremely preva- lent in the world, attesting to its importance for normal cochlear function (Chan and Chang, 2014). The most frequent

mutation in Morocco is c.35delG, it was detected in 29 out of 81 families (35.80%) (Abidi et al., 2007). In this study, we presented the fre- quency of

gene mutations in 152 families with nonsyndromic hearing loss (NSHL) and have also extended the mutational spec- trum of

gene in Morocco. Genetic analysis will be helpful to evaluate the impact of the

mutations as the principal cause of congenital NSHL in our population, which facilitates the diagnosis

*Corresponding author.

E-mail address:hamid.barakat@pasteur.ma(A. Barakat).

1 Equal level contributors.

Contents lists available atScienceDirect

European Journal of Medical Genetics

j o u r n a l h o me p a g e :h t t p : / / w w w . e l s e v i e r . c o m/ l o ca t e / e j m g

http://dx.doi.org/10.1016/j.ejmg.2016.05.002

1769-7212/©2016 Elsevier Masson SAS. All rights reserved.

of congenital hearing loss and allows early care of patients.

2. Patients and methods 2.1. Patients

A large cohort of 152 Moroccan families, with at least two sib- lings with hearing loss from all over the country took part of this study. Written informed consent was obtained from all patients, and the genetic study was approved by the committee on research ethics of Pasteur Institute of Morocco. Audiological evaluation disclosed moderate to severe hearing loss. The degree of hearing loss was assessing by the World Health Organization

s standard:

“

slight/mild (26

40 dB), moderate (41

70 dB), severe (71

90 dB), and profound impairment (

90 dB)

. Further clinical examination of subjects disquali

ed any symptom or malformation that could be suggestive of a syndromic form of hearing loss.

2.2. Genetic analysis

DNA was extracted from whole peripheral blood by using standard phenol/chloroform protocols. The

coding exon was ampli

ed by PCR using the forward primer 5

AGAG TGGTGTTTGCTCAGGA3

and the reverse primer 5

GACTGAGCCTT GACAGCTGA3

amplifying a 900 bp region. Exon 1 (362 pb) was ampli

ed with forward primer 5

GCCGCCCC CTCCGTAACTTTC3

and reverse primer 5

CGTGTGTTGGTCCAGCCCCCC3

. PCR were carried out in a 15 m ^l

nal reaction volume containing 25 ng of genomic DNA, 7 pmol for each primer, 200 m M of dNTP, 1.5 mM of MgCl

, 1 PCR buffer and 0.75U m l of Taq DNA polymerase. PCR conditions were denaturation of genomic DNA at 95

C for 5 min and at 94

C for 30 s, annealing at 64

C for 35 s (Exon 1, 60

C for 35 s) and 72

C for 40 s for 35 cycles followed by a 7 min

nal extension at 72

C. All PCR products were directly sequenced using the ABI Big-Dye Terminator v 1.1 sequencing standard Kit and run on an ABI 3130 Genetic Analyzer. Sequencing data was analyzed by SeqScape v2.5 Software. Impacts of novel variants on protein structure were predicted by SIFT (Sorting Intolerant From Tolerant), PolyPhen-2 (Polymorphism Phenotyping) and Proven prediction software packages.

3. Results

Among a large cohort of 152 NSHL families, 66 (43.42%) had mutations in the coding region of

observed in 53 families (34.87%), compound heterozygosity in 4 families (2.63%), and heterozygosity with mono-allelic mutation in 9 families (5.92%). Six different mutations were detected: c.35delG, p.V37I, p.E47X, p.G200R, p. Del120E, p.R75Q, the last three muta- tions were described for the

rst time in Moroccan deaf patients. In addition, we identi

ed a novel rare variant: the c.385G

T (p.E129X), and was submitted to ClinVar database (accession number SCV000267101) (Table 1). All these mutations have an autosomal recessive mode of inheritance, except the p.R75Q mu- tation which is transmitted in an autosomal dominant manner. The genotype of all

mutations in Moroccan patients, are summa- rized in Table 2. Testing for the IVS1

1G

A splice site mutation in the 86 families with no

mutation in the coding region, and in the heterozygous

mutants showed that this mutation is not involved in the occurrence of Hearing loss in Morocco. The 152 families recruited in this study were originated from different re- gions of Morocco. The analysis of the geographical distribution of

gene mutations shows homogeneous distribution for the c.35delG mutation through Moroccan regions. All four newly described mutations (p.G200R, p. Del120E, R75Q and c.385G

T) in

Morocco were found in the North and the center, demonstrating a gradual decrease in the frequency of

mutations from the north-center to the south of Morocco (Fig. 1).

4. Discussion

Genetic testing to con

rm a conclusive diagnosis of

gene, must be the

rst step of molecular analysis of deafness. In the connexin homepage (http://davinci.crg.es/deafness/), over 200 different mutations have been described in the

gene, hence the necessity to sequence the entire coding region of this gene.

In this study, we reported the spectrum

gene mutations and evaluated their prevalence in the Moroccan population, 152 families with autosomal recessive hearing loss were included. Our results con

rmed that autosomal recessive deafness in the Moroccan population is mainly due to mutations in

gene with a frequency of 44.33% (65/152 families). The contribution of the

gene on NSHL in Morocco is relatively similar to that reported in the neighboring country of Tunisia (39%) (Riahi et al., 2013), but too high compared to Algeria (18.97%) (Ammar-Khodja et al., 2007).

This may be due of the higher number of recruited families in our study (152 families) as well as in the Tunisia study (131 families) compared to Algeria (50 families). The c.35delG is the most frequent pathogenic mutation causing hearing loss in our cohort with a frequency of 33.55% (52/152 families), in accordance with other EuroMediterranean countries like Lebanon (31.25%) (Mustapha et al., 2001), Italy (30.18%) (Murgia et al., 1999), Greece (28, 9%) (Iliades et al., 2002) and Tunisia (35%) (Riahi et al., 2013). In this work, the proportion of the c.35delG mutation of all

mutant alleles accounts for about 87%, similarly to the most other population, 88% in Italy (Rabionet et al., 2000), 76.9% in Austria (Janecke et al., 2002), and 64% in Portugal (Nogueira et al., 2011).

Some studies have raised the possibility of a founder effect of an ancestral mutation expanded throughout the Mediterranean shore.

In the Moroccan population, the estimated age of the c.35delG

Allele frequencies ofGJB2mutation in Moroccan ARNSHL families.

Mutation State Number of families

Alleles frequencies %

Proportion of allele frequencies in families withGJB2mutation % c.35delG Homozygous 51 107/304 (35.20%) 107/123 86.99%

c.35delG Heterozygous 5

p.V37I Homozygous 1 10/304 (3.29%) 10/123 8.13%

p.V37I Heterozygous 8

p.E47X Heterozygous 1 1/304 (0.33%) 1/123 0.81%

c.385G>T Heterozygous 1 1/304 (0.33%) 1/123 0.81%

p.G200R Homozygous 1 2/304 (0.66%) 2/123 1.63%

p.Del120E Heterozygous 1 1/304 (0.33%) 1/123 0.81%

p.R75Q Heterozygous 1 1/304 (0.33%) 1/123 0.81%

Table 2

Genotypes ofGJB2gene occurring in 152 Moroccan ARNSHL families.

Genotype Frequency (%)

35delG/35delG 33.55

35delG/p.E47X 0.658

35delG/c.385G>T 0.658

35delG/p.R75Q 0.658

35delG/Wt 1.316

p.V37I/p.V37I 0.658

p.V37I/Wt 4.605

p.DeL120E/p.V37I 0.658

p.G200R/p.G200R 0.658

Wt/Wt 56.58

mutation was 2700 years ago (135 generations) (Abidi et al., 2008a).

Conversely, the c.35delG mutation is absent in Japanese patients (Fuse et al., 1999; Abe et al., 2000). However, the two nucleotide deletions c.235delC and c.167delT are the most common

mutations in Asian populations (Dai et al., 2007; Han et al., 2008) and Ashkenazi Jews (Morell et al., 1998), respectively. Among the 5 patients found heterozygotes for c.35delG in this study, 3 in- dividuals are carriers of a second mutation in

gene, while for the 2 other patients, deafness may be not related to c.35delG mu- tation, suggesting that they might be simple carriers since in the control Moroccan population, the carrier frequency of c.35delG was estimated to be 2.65% (Abidi et al., 2007).

The missense variant p.V37I was recently more studied for its high prevalence in East Asians (Li et al., 2012; Kim et al., 2013). It was initially described as a polymorphism because of its high prevalence in normal hearing individuals (Kelley et al., 1998). But over the time, The p.V37I was reported in homozygous and com- pound heterozygous patients and lead to mild or moderate hearing loss (Kim et al., 2013), revealing the pathogenic effect of this mu- tation. In this work, the p.V37I was the second most prevalent variant in

gene with an allele frequency of 3.29%, this mutation was found at homozygous and compound heterozygous state in two families respectively with moderate hearing loss, while 7 pa- tients were observed at the heterozygous state. In the normal hearing Moroccan population, this variant was found at the het- erozygous state with a frequency of 1.43% (2/140) (Abidi et al., 2008b).

As regards the less common mutation, the p.E47X was found at compound heterozygous state with c.35delG in one patient with a frequency of 0.658% (1/152). This mutation is deleterious and responsible for hearing loss in patients, it was also found in China

and Turkey with an allele frequency of 1% and 1.40% respectively (Snoeckx et al., 2005; Dai et al., 2009).

Besides, three pathogenic mutations have been identi

ed for the

rst time in Morocco: p.G200R, p.Del120E, p.R75Q. The missense p.G200R was described in two previous studies: in Iran (Hashemzadeh et al., 2007) and recently in Pakistan (Sha

que et al., 2014), this pathogenic mutation is located in the fourth trans- membrane domain (TM4) of Cx26, affecting a residue involved in the formation of intramolecular disulphide bonds. The c.358- 360delGAG, known as p.delE120, is a deletion of codon 120, encoding glutamic acid. This pathogenic mutation has a negative effect on formation of gap junctions leading to a complete loss of channel activity. Deafness, due to homozygous mutation have been previously reported all over the world (Hashemzadeh et al., 2007;

Tekin et al., 2005; Oliveira et al., 2002). In this work, the p.delE120 was found at compound heterozygous state with the p.V37I with a frequency of 0.658%. Compound heterozygosity for p.delE120 mutation especially with the c.35delG, was previously described (Gualandi et al., 2002; Marlin et al., 2005).

A few

mutations have been reported to cause dominant hearing loss hearing loss or with skin diseases, one of them is the p.R75Q. This missense mutation was described for the

rst time in a Turkish family (Uyguner et al., 2002) with HL and PPK, and recently in a Chinese family (Jiang et al., 2014). The p.R75Q was reported also to cause dominant HL without cutaneous manifestations in a south Indian family (Pavithra et al., 2015). In this work, the p.R75Q mu- tation was related to HL and PPK, and found at compound hetero- zygous state with the c.35delG with a frequency of 0.658%.

One novel variant was identi

ed in this work: the c.385G

T,

p.E129X. This nonsense mutation is located within the intracellular

domain 2 (IC2), it converts a glutamic acid residue (GAA) at codon

129 to a stop codon (TAA) of the connexin26 gene, leading to the deletion of the last 97 amino acids of the protein. This mutation is not referenced in any database. The p.E129X was found at com- pound heterozygous state with the c.35delG with a frequency of 0.658%.

5. Conclusions

To sum up,

mutations consider being involved in 43.42% of the Moroccan patients with ARNSHL. The c.35delG is the most frequent pathogenic mutation causing hearing loss in Morocco, This report of the

mutation spectrum across all Morocco has an important implications for the establishing a suitable molecular diagnosis.

Conflict of interest

The authors declare that they have no con

ict of interest.

Acknowledgments

We thank all the families for their participation in this study, and clinicians for their help with this research. This work was funded by the Pasteur Institute of Morocco.

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