Download Study of 35delG in Congenital Sensorineural non-syndromic Hearing Loss in Egypt

Journal of Applied Sciences Research, 4(6): 621-626, 2008
© 2008, INSInet Publication
Study of 35delG in Congenital Sensorineural non-syndromic Hearing Loss in Egypt
1
1
Nagwa A. Meguid, 2Motaza H. Omran, 1Ahmed A. Dardir, 1Ehab R. Abdel-Raouf,
3
Iman A. Ghorab, 1Hatem R. Abdel-Raouf and 2Wael T. Elgarf
Dept. of Research on Children with Special Needs, National Research Center, Cairo, Egypt.
2
Dept. of Biomedical Technology, National Research Center, Cairo, Egypt.
3
Hearing and Speech Institute, Cairo, Egypt.
Abstract: Hearing loss is the most common form of sensory impairment, with approximately one
infant/1000 born with congenital deafness. A pre-lingual bilateral sensorineural hearing impairment poses
a substantial problem as it negatively impacts on the subject’s ability to conduct a normal social life.
Mutations in the connexin 26 gene (GJB2) cause a significant proportion of prelingual non-syndromic
autosomal recessive deafness in all populations studied so far. One specific mutation, 35delG, has
accounted for the majority of the mutations detected in the GJB2 gene in Caucasian populations. The aim
of the present study is to investigate 35del G mutation in GJB2 gene among Egyptians with recessive
non-syndromic congenital sensorineural hearing impairment. Of fifty nine Egyptian patients (25 families)
with prelingual non-syndromic hearing loss enrolled in this study, 66.1% of cases had severe to profound
hearing loss. Using PCR amplifying the specific coding region of GJB2 gene and direct DNA sequencing
to analyze mutations, only 6 cases (10.17%) had deletion of G at position 35. All cases were homozygous
for the mutation. Compared to other populations, 35delG mutation comprises a low frequency among
Egyptian families with SNHL. This confirms genetic heterogeneity and ethnic variation of nonsyndromic
SNHL. Finally, identification of our patient’s specific mutation provides important information for genetic
counseling, including prognosis and recurrence risk.
Key words: 35delG, Connexin 26, Sensorineural hearing loss, Egypt
locus, the GJB2 gene (OMIM 121011) and the GJB6
(OMIM 604418). Both of them encode connexins,
proteins commissioned to connect electrophysically
neighboring cells through gap-junction connections[4] .
Connexin 26 (Cx26) and 30 (Cx30) are expressed in
the inner ear and are thought to be important for the
passage of small metabolit es, ion trafficking,
homeostasis and the maintenance of the endocochlear
potential, by providing a cell–cell pathway for the entry
of potassium to the stria vascularis[5] .
The GJB2 gene, which encodes Cx26, has a simple
genomic structure comprising two exons, the first of
which is non-coding. Mutations in this gene are
responsible for approximately 60% of prelingual, nonsyndromic, recessive hearing loss in the Caucasian
population, with a carrier frequency varying from 2 to
5% depending on the ethnic groups[6] . More than 100
different deafness-causing GJB2 mutations have been
described. In the Caucasian population a single founder
mutation, 35delG, accounts for the majority of GJB2
mediated hearing loss[7] . Carrier frequency of mutation
35delG in the GJB2 gene among the Mediterranean
European population was found to be 1 in 35 [8].
INTRODUCTION
Hearing loss affects about 4% of people under 45
years of age and comprises a broad spectrum of
clinical presentations (congenital or late onset,
conductive or sensorineural and syndromic or nonsyndromic)[1] . The incidence of pre-lingual hearing loss
is about 1 in 1,000 newborns in Western Europe. At
least half of these cases are genetically determined. The
non-syndromic forms of deafness account for 70% of
these cases, of which about 85% are autosomal
recessively inherited [ 2]. In the past decade, remarkable
progress has been made in the identification of the
molecular basis of hearing loss. Approximately 120
different gene loci associated with non syndromic
hearing impairment have been identified. Presently 67
gene loci associated with autosomal recessive mode of
inheritance have been identified. Of these, 20 genes
have been characterized for autosomal recessive
(DFNB)[3] . Moreover, more than 50% of families with
autosomic recessive non-syndromic hearing loss present
an alteration in the DFNB1 locus (13q11-q12; OMIM
220290). Two related genes have been cloned in this
Corresponding Author:
Nagwa A. Meguid. Prof. of Hu ma n G e netics & Head of Dept. of Research on Children with
Special Needs, National Research Center, Cairo, Egypt.
E-Mail: [email protected]
621
J. Appl. Sci. Res., 4(6): 621-626, 2008
This mutation is a deletion of a single guanine residue
(G) in a stretch of 6 Gs at nucleotide position 30–35
of the coding region of the GJB2 gene. This single
deletion shifts the reading frame, resulting in a
premature chain termination product comprising only
12 amino acids. According to audiometric examinations
there is a correlation between specific GJB2 mutations
and the phenotype in non-syndromic deafness. GJB2
mutations, which completely inactivate the connexin 26
generally cause severe to profound hearing loss[9] .
The aim of this study is to investigate 35del G
mutation in human GJB2 (connexin 26) gene among
Egyptians with recessive non-syndromic congenital
sensorineural hearing impairment.
the affected children. Inclusion criteria for this study
were: severe to profound sensorineural hearing loss
with no associated acquired etiology or genetic
syndrome. Thus, patients with a history of acquired
etiology, syndromic deafness, otoacoustic trauma or any
neonatal disease causing acquired deafness, were
excluded.
Laboratory Investigations: All patients were suspected
to analysis of IgM and IgG for CMV, rubella and
toxoplasma to rule out deafness due to infectious
agents. These tests were carried out by Enzyme-Linked
Immunosorbent (DiA. PRO-Italy):
Molecular Analysis:
DNA Extraction: DNA extraction was carried out by
using:
MATERIALS AND METHODS
Patients: Fifty nine Egyptian sensorineural deaf
patients from twenty five families were enrolled in this
study. W e recruited patients from the genetic
counseling service for deaf people at the Clinic of
Children with Special Needs, National Research Center,
Dokki, Cairo. Age of the patients varied between 2 and
20 years.
C
C
C
C
C
C
Mini
Kit,
Determination of DNA Concentration: DNA samples
were and diluted the optical density of each diluted
s a m p l e w a s r e c o r d e d a t 2 6 0 n m u sin g
spectrophotometer:
All cases were subjected to:
C
By using QIAAmp DNA Blood
QIAGEN; Germany).
By using DNA salting-out method.
Detailed history was taken for each patient that
included: Obstetric history, perinatal history, Job of
parents, address, consanguinity, occupation, onset,
course and duration of hearing loss, history of
repeated ear discharge, starting time of use of
hearing aids if present, history of chronic diseases
like diabetes, and thyroid malfunction, history of
drug intake, noise exposure with specially the kind
and duration of noise, history of trauma, fever or
ear operations.
Three generations pedigree construction was
analyzed for each case.
Careful clinical examination had been done to
exclude syndromic deafness, and other associated
anomalies.
Ear, nose and throat (ENT) examination.
Full basic audiological evaluation: pure-tone
auditometry (PTA) ; speech audiometry ( if
possible ) was done for all cases to determine the
degree of hearing for both ears. Auditory brain
stem response (ABR) was done to confirm hearing
threshold and Otoacoustic emission (OAEs) was
done in some cases to exclude central auditory
system affection
DNA Amplification by Using Polymerase Chain
Reaction (PCR): Connexin 26 coding region
amplification was achieved by using the following
primers:
C
Forward primer: 5´ TCT TTT CCA GAG CAA
ACC GC 3´
C Reverse primer: 5´GAT GAG GCA ACC CGT
GCT CA 3´
PCR reaction was performed in a final volume of
50 ml, containing 200 ng genomic DNA, 10mM
Tris-Hcl
(P H 9.0 at 25ºC), 50mM KCl and 0.1%
®
Triton X-100, 1.5 mM MgCl2, 200 mM dNTPs, 10
mM of each primer and 2.5 units of Taq polymerase
(Promega- USA). Then the thermal cycler was
programmed according to the following steps to
undergo the amplification reaction for Cx26 gene
coding region. First; samples were denatured at 94 °C
for 5 minutes. Subsequently; 40 cycles of denaturation
were achieved at 94 °C for 15 seconds, annealing was
carried out at 55 °C for 30 seconds and extension
was done 72 °C for 1 minute, followed by 5 minutes
of post extension.
Detection and Visualization of Amplified PCR
Products: PCR products were loaded to an agarose gel
(2%); the desired band of the coding region of Cx26
was visualized using Ethidium Bromide florescence
The hearing disorder was bilateral and the onset
was congenital (pre-lingual) in all cases. Written
informed consents were obtained from all parents of
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J. Appl. Sci. Res., 4(6): 621-626, 2008
under ultraviolet light. Band size was determined by
loading DNA marker Qx 174 D N A / Hae III
(FINNZYMES, Finland) to one lane with PCR
products. The desired band appears at 680 bp (Fig 1).
Audiological evaluation of the six cases with
mutation revealed that severe to profound hearing loss
(> 70 dB) was detected in 3 patients (50%) from 2
families, moderate to severe hearing loss in one patient
(16.7% ) from one family, and moderate hearing loss in
2 patients (33.3%) from 2 families (Table 2).
About one child in 1000 is born with hearing loss
which is detected at infancy or early childhood
and about 50% of cases is attributed to genetic defects.
The inheritance pattern of monogenic prelingual
nonsyndromic hearing loss is autosomal recessive in
approximately 77% of patients, autosomal dominant in
~22%, X-linked in ~1% and mitochondrial in <1% [10] .
Advances in genomics have been catalytic in clarifying
the genetic heterogeneity of non-syndromic hearing
impairment. Allele variants of the gene that encodes
the protein connexin 26 cause half of moderate-toprofound non-syndromic autosomal recessive deafness
in many world populations[8]. During the last 10 years,
there has been a virtual explosion in the number of
genes that have been localized or even cloned.
Elucidating the functional biology of these genes may
facilitate our efforts in hearing habilitation.
It has been demonstrated that connexin 26 (GJB2)
gene is a major gene for congenital sensorineural
deafness. A single mutation (named 35delG) was found
in most recessive families and sporadic cases of
congenital deafness, among Caucasians. A carrier
frequency for 35delG of 1 in 35 in southern Europe
and 1 in 79 in central and northern Europe had been
detected. The 35delG carrier frequency of 1 in 51 in
the overall European population clearly indicates that
DNA Purification: DNA samples were purified from
agarose gels:
C
Using kit Invisorb® Spin DNA Extraction Kit
(Invitek; Germany).
Sequencing: Direct DNA sequencing was carried out
in both directions using the forward and reverse
primers on the Automated Sequencer “ABI Prism
310
Genetic
Analyzer”. The cycle–sequencing
reaction
was
performed in volume of 20 µl
containing: 8 µl of the terminator ready reaction, 3.2
pmole of either the forward primer or the reverse
primer and 30 ng of purified PCR product. The thermal
cycle protocol on the “P erkin Elmer, Gene Amp PCR
System 9700” was 95 0C for 4 minutes followed by 25
cycles of (96 0 C for 10 seconds, 50 0C for 5 seconds and
600 C for 4 minutes). Centri-Sep Columns (Princetion
Separations, Adelphia) were used for effective and
reliable removal of excess Dye Deoxy T M terminators
from completed DNA sequencing reactions. Data were
compared and aligned with different sequences of
d if f e r e n t s t r a ins using t h e N C B I in t e r f a c e
(http://ncbi.nlm.nih.gov/ blast).
RESULTS AND DISCUSSION
Of fifty nine Egyptian patients with prelingual nonsyndromic hearing loss enrolled in this study, 39 cases
(66.1%) had severe to profound hearing loss and 10
cases (16.9%) had severe hearing loss (Table 1).
Positive parental consanguinity was present in all
studied families (25 families).
The 35delG mutation was detected in four (16%)
of the twenty five Egyptian families with prelingual
non-syndromic hearing loss. Among these four families,
only 6 cases (10.17%) had deletion of G at position 35
resulting in a frameshift at the 12 t h amino acid, leading
to a premature termination of the protein. To evaluate
the accuracy of our mutation detection, all samples
were directly sequenced using the primers in both
directions, forward and reverse primers. The results
were aligned and compared to different strains in the
gene bank. All cases were homozygous for the
mutation as shown by DNA sequencing (fig 2).
Therefore, the total 35delG mutation frequency of
GJB2 was 10.17% (6/59) in the Egyptian patients with
congenital non-syndromic sensorineural hearing loss.
Hearing assessment
sensorineural HL
Degree of hearing loss
Mild to moderate
Moderate HL
Moderate to sever HL
Severe HL
Severe to profound HL
Profound HL
Total
Table 1:
Table 2: Audiological
Patients
First Family:
Male
Female
Second Family:
Male
Female
Third Family:
Female
Fourth Family:
Male
623
of
patients
with no n-s ynd ro mic
Affected Cases
1
3
5
10
39
1
59
Percent
1.7%
5.1%
8.5%
16.9%
66.1%
1.7%
100%
assessment of positive 35delG patients
Age
Degree of hearing loss
15 ys
4 ys
Bilateral moderate to severe
Bilateral moderate
16 ys
2 ys
Bilateral severe to profound
Bilateral severe to profound
12 ys
Bilateral moderate
2 ys
Bilateral severe to profound
J. Appl. Sci. Res., 4(6): 621-626, 2008
Wilcox et al.[15] and Engel-Yeger et al. [ 1 6 ] stated that
GJB2 mutations cause variable degrees of audiometric
hearing loss, ranging from moderate to profound among
mutations of 35delG. Among the recessive mutations,
the single base deletion mutation, 35delG is responsible
for 80% of DFNB1 related hearing loss in European
and American populations. Zelante et al.[17] first
reported this common connexin 26 mutation in
Mediterranean European population. The carrier
frequency of the 35delG mutant allele varies in
different populations, may be as prevalent as 4% in
particular ethnic groups[6] and 2.8% in the American
population [18] .
The total 35delG mutation frequency of GJB2
gene in the present study was 10.17% (6/59) in the
Egyptian patients with congenital non-syndromic
sensorineural hearing loss. Homozygosity was found
in 100% of cases with 35delG mutation. Another study
in the literature reported that carrier frequency of the
35 del G mutation in healthy Egyptian subjects was
1/54 add ref 19. However, we reviewed the literature
and found that this particular mutation is common in
many other populations and not so common in others
(Table 3). Early diagnosis, evaluation and treatment
of
childhood deafness are essential for a child’s
normal growth. The link between connexin26 and
hereditary deafness will facilitate our understanding of
the biology of normal and abnormal hearing, the
molecular basis of deafness, the genetic counseling of
familial deafness and may provide new strategies for
prevention, diagnosis and management of this common
genetic disorder.
Frequency of 35delG mutations in different p o p ulations of
the world
Country
Frequency
Reference
Egypt
10.17%
The present study
Egypt
10.8%
Snoeckx et al., 2005[1 9]
Palestine
14.0%
Shahin et al., 2002[2 0]
Tunisia
42.0%
Ben Arab et al., 2000[2 1]
Morocco
31.58%
Gazzaz et al., 2005[2 2]
Iran
44.0%
Najmabadi et al., 2002[2 3]
Turkey
76%
Kalay et al., 2005[2 4]
Italy
39.4%
Gualandi et al., 2002[2 5]
Greece
42.2%
Pampanos et al., 2002[2 6]
Greece
44.4%
Riga et al., 2005[2 7]
Germany
70.0%
Kupka et al., 2002[2 8]
France
75.1%
Marlin et al., 2001[2 9]
Spain
82.0%
Rabionet et al., 2000[3 0]
Czech Republic
82.8%
Seeman et al., 2004[3 1]
USA
26.0%
Prasad et al., 2000[3 2]
Switzerland
25%
Gurtler et al., 2003[3 3]
Australia
50.0%
Dahl et al., 2001[3 4]
Austria
72.1%
Janecke et al., 2002[3 5]
Brazil
84.2%
Oliveira et al., 2002[3 6]
Venezuela
7.1%
Angeli et al., 2000[3 7]
India
2.2%
Maheshwari et al., 2003[3 8]
China
1.2%
Liu et al., 2002[3 9]
Japan
0%
Ohtsuka et al., 2003[4 0]
Taiwan
0%
Wang et al., 2002[4 1]
Korea
0%
Park et al., 2000[4 2]
Table 3:
this genetic alteration is a major mutation for
autosomal recessive deafness in Caucasians[6] . Either a
single origin for 35delG somewhere in Europe or in the
Middle East, has been suggested. The meta-analysis
done by Lucotte and Diéterlen [11] showed that the
relatively more elevated value for the 35delG
frequencies in southern Europe concerns the
Mediterranean region, with Greece as the focus. Since
mutation frequency and distribution is variable
throughout different populations, a systematic study of
different loci must be established for each population.
The purpose of this study was to investigate the
frequency and the features of 35del G mutation in
GJB2 gene among Egyptian patients with congenital
non-syndromic sensorineural hearing loss.
In the present study, six cases out of 59 were found to
have 35del G mutation in GJB2 gene. Audiological
evaluation of cases with 35delG mutation in the present
study revealed that 50% had severe to profound hearing
loss, 16.7% had moderate to severe hearing loss and
33.3% had moderate hearing loss. Similar to our
results, previous reports have shown that GJB2
mutations have a consistent picture of hearing loss:
prelingual, bilaterally symmetrical, and usually being
severe or profound with a wide variability in the extent
of hearing loss[12,13]. Although the genotype does not
predict the hearing of the examined child, as even
intrafamilial variations of the severity of hearing loss
are common, 30—57% of children with GJB2
mutations
are expected to have a profound and
another 26—30% severe hearing loss[14] . However,
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