MUTATION IN BRIEF
HUMAN MUTATION Mutation in Brief #504 (2002) Online
© 2002 WILEY-LISS, INC.
Received 22 September 2001; revised manuscript accepted 21 February 2002.
GJB2 Mutations in Iranians With Autosomal
Recessive Non-syndromic Sensorineural Hearing Loss
Hossein Najmabadi1, Robert A Cucci2, Solmaz Sahebjam1, Nafiseh Kouchakian1, Mohammad
Farhadi3, Kimia Kahrizi1, Sanaz Arzhangi1, Naiimeh Daneshmandan1, Khalil Javan1, and
Richard JH Smith2
1Genetic Research Center, Welfare Science & Rehabilitation University, Tehran, Iran; 2 Molecular
Otolaryngology Research Laboratories, Department of Otolaryngology Head and Neck Surgery, University of
Iowa; Iowa, IA United States; 3 Department of Otolaryngology, Rasoul Akram Hospital, Iran University of
Medical Sciences, Tehran, Iran.
*Correspondence to: Dr. Hossein Najmabadi, Welfare & Rehabilitation University, Kodakyar St., Beheshti Ave.,
Evin, Tehran, Iran 19834; Tel.: 98212050174; Fax: 98212407814; E-mail: Hnajm@mavara.com
Contract grant sponsors: Iran Deputy of Research and Technology, Ministry of Health & Medical Education;
Contract grant number: P6193; Contract grant sponsor: National Institutes of Health; Contract grant number: R01-
Communicated by Mark H. Paalman
Hereditary hearing loss (HHL) is an extremely common disorder. About 70% of HHL is
non-syndromic, with autosomal recessive forms accounting for ~ 85% of the genetic load.
Although very heterogeneous, the most common cause of HHL in many different world
populations is mutations of GJB2, a gene that encodes the gap junction protein connexin 26
(Cx26). This study investigates the contribution of GJB2 to the autosomal recessive non-
syndromic deafness (ARNSD) load in the Iranian population. One hundred sixty eight
persons from 83 families were studied. GJB2-related deafness was diagnosed in 9 families (4,
35delG homozygotes; 3, 35delG compound heterozygotes; 1, W24X homozygote; 1, non-
35delG compound heterozygote). The carrier frequency of the 35delG allele in this
population was ~1% (1/83). Because the relative frequency of Cx26 mutations is much less
than in the other populations, it is possible that mutations in other genes play a major role in
ARNSD in Iran. © 2002Wiley-Liss, Inc.
KEY WORDS: Autosomal recessive non-syndromic deafness, ARNSD; hereditary hearing loss; HHL; Connexin 26; Cx26;
Genetic hearing impairment is one of the most prevalent inherited sensory disorders, affecting approximately
1/2000 live births (Nance et al., 1997). Autosomal recessive non-syndromic sensorineural deafness (ARNSD) is
the most common form of severe inherited hearing loss (Reardon, 1992). It is an extremely heterogeneous disorder
with at least 30 reported loci (Van Camp et al., 1997; Zbar et al., 1998; Keats and Berlin, 1999, Van Camp and
Smith, 2001; http://www.uia.ac.be/dnalab/hhh).
Serendipitously, mutations in one gene, GJB2 (MIM# 121011), have been shown to be the major cause of
2 Najmabadi et al.
ARNSD in many different populations (DFNB1; MIM# 220290) (Maw et al., 1995; Gasparini et al., 1997; Kelsell
et al., 1997; Zelante et al., 1997; Morell et al., 1998; Park et al., 2000). A single allele variant, the 35delG
mutation, is the most common deafness-causing mutation in people of Northern European heritage and accounts
for about 60% of GJB2-related deafness (Denoyelle et al., 1997; Kelley et al., 1998; Lench et al., 1998; Green et
al., 1999). In this study, we assessed the frequency of GJB2 mutations in Iranian persons with ARNSD and
determined the types of mutations in this heterogeneous population.
PATIENTS AND METHODS
One hundred sixty eight persons from 83 families with presumed hereditary deafness were studied. To be
included in this study, families had to meet the following criteria: 1) hearing loss confirmed by audiologic testing;
2) hearing loss in the absence of other clinical features; 3) a pedigree structure consistent with autosomal recessive
inheritance; 4) both parents with normal hearing; and 5) two or more affected family members. Pure-tone
audiometry was done in a sound chamber. Air and bone conduction were evaluated in frequencies of 250, 500,
1000, 2000, 4000, and 8000 Hz with intensities up to 120 dB. On consenting persons, 10 cc of whole blood was
taken as a DNA source. All procedures were approved by the human research institutional review boards of the
Welfare Science & Rehabilitation University and Iran University of Medical Sciences, Tehran, Iran and the
University of Iowa, Iowa City, Iowa, USA.
Connexin 26 mutation screening
Cx26 mutation screening was completed on DNA extracted from whole blood. The initial screen was an allele-
specific polymerase chain reaction (ASPCR) assay to detect the presence of the 35delG allele variant using
previously described primers (Scott et al., 1998). No further testing was done on those persons identified as
In persons heterozygous for the 35delG allele, the coding sequence of GJB2 (exon 2) was screened for other
allele variants by single strand conformational polymorphism (SSCP) using both MDE (FMC BioProducts,
Rockland, ME) and polyacrylamide gel electrophoresis to compare relative mutation detection rates. All abnormal
band shifts were sequenced; if no abnormal band shifts were identified, the non-coding exon of GJB2 (exon 1) was
sequenced. In those cases in which the initial ASPCR screen failed to demonstrate a 35delG allele, exon 2 was
screened by SSCP, again using both MDE and polyacrylamide gel electrophoresis; samples were sequenced if
shifts were observed. If a single coding sequence mutation was detected, the non-coding exon of GJB2 (exon 1)
was sequenced (Green et al., 1999).
GJB2-related deafness was diagnosed in nine families (11%). In four of these families (44%), deaf persons were
35delG homozygotes; in three of the remaining five families, deaf persons were 35delG compound heterozygotes
(Table 1). Five other GJB2 allele variants were found opposite wild-type alleles. Of these amino acid changes,
three are benign polymorphisms and two have an unknown impact on hearing. We also found one wobble base
(V52V) and one 35delG carrier (Table 2). All of these mutations have been previously described (Estivill and
Table 1. Genotypes in Iranian Patients With GJB2-related Deafness
GJB2 Mutations in Iranian Patients With Hearing Loss 3
Table 2. GJB2 Allele Variants in the Iranian Population
Although HHL is one of the most genetically heterogeneous diseases, mutations in GJB2 are the most common
cause of ARNSD. Mutations in this gene account for about half of all moderate-to-profound deafness in many
world populations (Maw et al., 1995; Gasparini et al., 1997; Kelsell et al., 1997; Zelante et al., 1997; Brobby et al.,
1998; Morell et al., 1998; Fuse et al., 1999; Wilcox et al., 1999; Abe et al., 2000; Kudo et al., 2000; Park et al.,
2000; Rabionet et al., 2000; Sobe et al., 2000; Wilcox et al., 2000). However, most of the populations studied to
date are of Northern European ancestry, suggesting a possible founder effect for GJB2-related deafness and the
35delG mutation in particular. This hypothesis has been confirmed by Van Laer et al. These investigators showed
that the 35delG mutation segregates on a common SNP background and calculated that the mutation arose
approximately 10,000 years ago (Van Laer et al., 2001).
As a corollary to this finding, in populations without a major Northern European heritage, it is reasonable to
expect that the 35delG allele will be less frequent and that the contribution of GJB2 to the ARNSD genetic load
may be different. Consistent with the first point, the 35delG mutation is rare in the Japanese and Chinese
populations, and a different mutation, the 235delC, predominates (Fuse et al., 1999; Abe et al., 2000; Park et al.,
2000; Kudo et al., 2000). Like the 35delG mutation, the 235delC also segregates on a unique genetic background
indicative of a founder effect. Few studies have been reported describing the genetic epidemiology of deafness in
Third World countries.
In this study, we identified GJB2 mutations in 30 of 166 (18%) chromosomes. We also diagnosed 9 families
(11%) with DFNB1, a prevalence much lower than that reported in western populations (Maw et al., 1995;
Gasparini et al., 1997; Kelsell et al., 1997; Zelante et al., 1997; Morell et al., 1998; Park et al., 2000). Although no
studies have been carried out in Arab countries bordering Iran to validate our data, Brown et al. (1996) studied 27
Pakistani families segregating for ARNSD and found only one family linked to GJB2. These data suggest that
there is an ethnic bias in the contribution GJB2 makes to the ARNSD genetic load.
Interestingly, however, of the mutations we did find, the 35delG was most common. It segregated on the
common SNP background reported by Van Laer et al. (2001) indicating some admixture of ancestral Iranian and
European populations. In addition, we found that the carrier frequency of the 35delG allele in the deaf population
was 1.4 %, a figure not significantly different from the 1.7 % carrier frequency reported by Gasparini et al. (2000).
Based on our results, we would expect other genes to make important contributions to the ARNSD genetic load in
Iran. However, since the Iranian population is composed of many different ethnic groups, initially it will be
important to generate ethnic-specific data. Preliminary data suggest that this step is justified based on the
prevalence of the more common allele variants in these ethnic groups.
Factors that have contributed to this degree of diversity include Iran’s place on the route of the Silk Road, a
history of longtime wars with foreign nations, and marked immigration from neighboring countries. Relative
ethnic purity has been maintained by the natural geographic borders within Iran and by an ancient culture that
encourages familial marriages.
That there is an ethnic bias in the contribution specific genes make to the deafness in Iran is consistent with our
finding of heterogeneity in another common disease, beta-thalassemia (Najmabadi et al., 2001). Because studies of
deafness are ethnically biased, results generated in some populations are not applicable to other populations, a
4 Najmabadi et al.
limitation that makes it important to continue investigations like the present study. By generating population-
specific data, improved health care for deafness in countries like Iran will be possible.
We wish to thank our patients and their families for their collaboration to this study. We are also most grateful
to Dr. Y. Shafeghaty, Dr. G.H.R. Baba Mohammadi and Dr. N. Almadani from A.S.M.A. Institute, Tehran, Iran,
for their help in counseling families with hereditary deafness. This work was supported by the Iran Deputy of
Research and Technology Ministry of Health & Medical Education (Grant P 6193) and the National Institutes of
Health (Grant R01-DC02842).
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