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RESEARCH ARTICLE
High Carriers Frequency of an Apparently Ancient
Founder Mutation p.Tyr322X in the ERCC8 Gene
Responsible for Cockayne Syndrome Among Christian
Arabs in Northern Israel
Morad Khayat,1 Hagar Hardouf,1 Joel Zlotogora,2 and Stavit Allon Shalev1,3*
1
Genetics Institute, Ha’Emek Medical Center, Afula, Israel
Department of Community Genetics, Public Health Services, Ministry of Health, Hebrew University Jerusalem, Jerusalem, Israel
2
3
Rappaport Faculty of Medicine, Technion - Israel Institute of Technology, Haifa, Israel
Received 27 July 2010; Accepted 16 September 2010
Most autosomal recessive diseases are rare in the general
population, but in genetically isolated communities specific
condition might be frequent, mainly due to founder effect.
Recognition of common inherited disorders in defined populations may be effective in improving public health care. Cockayne
syndrome (CS) is a rare autosomal recessive disorder common in
Christian Arabs due to a p.Tyr322X mutation. Genetic screening
of the p.Tyr322X mutation of the ERCC8 gene in this population
documented a carrier frequency of 6.79% (95% confidence
interval: 3.84–9.74%). The haplotype analysis data, as well as
the high carriers frequency of CS, suggested that the Israeli
Arab Christian CS mutation (p.Tyr322X) is an ancient founder
mutation that may have originated in the Christian Lebanese
community. As a result of this pilot study the Christian CS
mutation was included in the genetic screening program offered
to the Israeli Arab Christian community. Ó 2010 Wiley-Liss, Inc.
Key words: Cockayne syndrome; DNA repair mechanism;
genetic screening; founder mutation
INTRODUCTION
Cockayne syndrome (CS, OMIM# 133540, 216400) is a rare autosomal recessive disorder, comprising severe growth retardation,
progressive neurological dysfunction, and accelerated aging. The
condition is clinically variable and genetically heterogeneous
with a wide range of severity of phenotypes that present mainly
with progressive retinal degeneration and congenital cataracts,
sensorineural hearing loss, cachectic growth retardation, cutaneous
photosensitivity, skeletal anomalies, and mental retardation
[Nance and Berry, 1992]. The primary cause of CS is a defect in
one of the DNA repair systems [de Boer and Hoeijmakers, 2000].
The nucleotide excision repair (NER) mechanism involves the
action of about 30 proteins and is responsible for removing a variety
of DNA lesions, such as helix distorting adducts caused by exposure
to endogenous genotoxic agents. These materials include oxygen
Ó 2010 Wiley-Liss, Inc.
How to Cite this Article:
Khayat M, Hardouf H, Zlotogora J, Shalev SA.
2010. High carriers frequency of an
apparently ancient founder mutation
p.Tyr322X in the ERCC8 gene responsible for
Cockayne syndrome among Christian Arabs
in Northern Israel.
Am J Med Genet Part A 152A:3091–3094.
free radicals or exogenous agents such as ultraviolet (UV)
light [Balajee and Bohr, 2000; de Boer and Hoeijmakers, 2000;
Christmann et al., 2003].
Deficiencies of NER in humans lead to the rare human disorders
xeroderma pigmentosum (XP), trichthiodystrophy (TTD), and
CS [Nance and Berry, 1992; de Boer and Hoeijmakers, 2000;
Christmann et al., 2003].
Most CS patients belong to two complementation groups
namely CSA and CSB [Colella et al., 1999; Graham et al., 2001].
CSA and CSB genes (also published as ERCC8 and ERCC6 genes,
respectively) have been cloned and characterized biochemically.
Their exact function is incompletely understood, but they probably
have a crucial role in the first step of transcription-coupled repair
(TCR), namely the recognition of a lesion [van Hoffen et al., 2003].
CSA belongs to ‘‘WD repeat’’ family of proteins with regulatory
roles but without enzymatic activity [Henning et al., 1995].
In a recent review, 84 CS kindreds were reported, about 62%
(52 kindreds) had a mutation in the ERCC6 gene, while 38% (32
kindreds) had a mutation in the ERCC8 gene [Laugel et al., 2010].
*Correspondence to:
Stavit Allon Shalev, M.D., Genetics Institute, Ha’Emek Medical Center,
Afula 18101, Israel. E-mail: [email protected]
Published online 24 November 2010 in Wiley Online Library
(wileyonlinelibrary.com).
DOI 10.1002/ajmg.a.33746
3091
3092
AMERICAN JOURNAL OF MEDICAL GENETICS PART A
Among the 88 reported mutations that cause CS, 61 mutations
were identified in the ERCC6 gene and the other 27 mutations
were identified in the ERCC8 gene.
Herein, we describe the high frequency of a previously described
mutation p.Tyr322X identified in the ERCC8 gene of Christian
Arab CS patient from Northern Israel [McDaniel et al., 1997].
Linkage analysis was performed using microsatellite markers
located on cytoband 5q11 in order to construct haplotypes for
CS patients and carriers.
55 C for 30 sec and at 72 C for 10 sec; and at 72 C for 5 min.
The mutation revokes a restriction site for the DdeI restriction
enzyme. Amplified fragments of DNA containing the mutation
were digested with DdeI (New England Biolabs, Beverly, MA)
restriction enzyme and electrophoresed on 1% SeaKem LE Agarose/
2% Metaphor Agarose gels (lonza, Rockland, ME).
Normal alleles consisted of 133 and 78 bp fragments, heterozygous carriers showed 211, 133, and 78 bp bands, and homozygous
affected individuals showed 211 bp fragments.
MATERIALS AND METHODS
The Christian Arabs in Israel
Haplotype Analysis
Approximately 117,000 Christian Arabs live mainly in urban
areas in northern Israel, including Nazareth, Shfar’am, and Haifa
[Statistical Abstracts, 2009]. Although many denominations are
represented, most are Greek Orthodox and Roman Catholic.
The Patients
We studied five polymorphic markers spanning about 5.3 cM
around the ERCC8 locus [Conte et al., 2009] of genomic DNA
from two CS patients and 19 carriers. Two markers were proximal
to ERCC8 (D5S1715 and D5S2080), and three were distal to ERCC8
(D5S624; D5S1990; and D5S427). The sense set of the PCR primers
was labeled fluorescently. We analyzed the PCR products of these
reactions using the GeneScan system of the ABI PRISM 3010xl.
In the last decades CS was diagnosed in eight different families
of Christian Arab origin. The families reside mainly in Nazareth
and nearby smaller towns, with no known family links between
most of them. After the characterization of the mutation p.Tyr322X
(c.966 C>A) in the ERCC8 gene in some affected individuals,
we examined some of the other patients and found the homozygous
state of the same mutation.
RESULTS
Carrier Frequency
Blood Sample Collection and Genomic DNA
Isolation
Detection of Couples at Risk
Our pilot study was a prerequisite of the Ministry of Health
before considering the test as part of the national screening
program. This study was approved by the Ha’Emek Medical Center
Human Studies Ethics Committee in accordance with the Helsinki
Declaration.
The test was offered to all couples of Christian Arab origin
asking for genetic screening as a clinical service, free of charge, after
obtaining an informed consent. The result of the screening was
given to the individuals tested.
Blood was collected from 280 healthy individuals belonging to
the Arab Christian population from Northern Israel and genomic
DNA was extracted using the FlexiGene DNA kit (Qiagen, Hilden,
Germany) according to the manufacturer’s instructions.
Carrier Detection Assay
A molecular assay consisting of DNA amplification followed
by restriction enzyme analysis was developed to allow accurate
and rapid detection of carriers of the p.Tyr322X mutation in the
ERCC8 gene.
Carrier detection was performed by amplification of a 211 bp
fragment containing the mutation from genomic DNA. The
sense primer 50 -AATCCTACAGGTGAACTATG-30 , and antisense
primer 50 -TACCTGGAAATTTGACTGAA-30 were used under
the following PCR conditions: denaturation at 95 C for 5 min;
35 subsequent amplification cycles performed at 95 C for 15 sec, at
Among the 280 unrelated individuals we identified 19 carriers.
Therefore, the carrier frequency of the p.TyrY322X mutation in
the Christian Arab population in northern Israel is 6.79% (95%
confidence interval: 3.84–9.74%).
During the screening study, two couples were identified at risk
for having a child homozygous for the p.Tyr322X mutation.
Following genetic counseling, both couples decided to perform
prenatal diagnosis. The first one demonstrated an affected fetus and
the parents elected to terminate the pregnancy. The second prenatal
diagnosis results indicated a normal heterozygous (þ/) fetus.
Haplotype Analysis
The two affected individuals were found to be homozygous for all
five markers that were tested as well as the p.TyrY322X mutation
(Table I). However, the patients shared only the 256 bp allele of the
marker D5S2080 that is located 1.2 cM away from the gene. Among
the 19 carriers, the same 256 bp allele was present in 18 cases, while it
was present in only in 24 of 50 non-carrier Arab Christians
(P ¼ 0.018).
DISCUSSION
The CS patients present manifestations including growth failure, a
cachexia, bird-like face, mental retardation, microcephaly, retinal
degeneration, deafness, photosensitivity, and dental anomalies
[our clinical data; Lehmann et al., 1993; McDaniel et al., 1997;
Laugel et al., 2010].
All CS individuals were homozygous for the p.Tyr322X mutation
in the ERCC8 gene (data not shown) including the CS patient
described previously [Lehmann et al., 1993; McDaniel et al., 1997;
KHAYAT ET AL.
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TABLE I. Haplotype Analysis Using Five Informative Markers Spanning About 5.3 cM Around the ERCC8 Locus
Patient 1
Marker
D5S1715
D5S2080
ERCC8
D5S624
D5S1990
D5S427
Allele 1
257
256
p.Tyr322X
155
243
295
Mother
Allele 2
257
256
p.Tyr322X
155
243
295
Allele 1
257
256
p.Tyr322X
155
243
295
Father
Allele 2
264
263
þ
157
233
293
Allele 1
257
256
p.Tyr322X
155
243
295
Patient 2
Marker
D5S1715
D5S2080
ERCC8
D5S624
D5S1990
D5S427
Allele 1
253
256
p.Tyr322X
157
224
279
Allele 2
264
263
þ
157
233
293
Mother
Allele 2
253
256
p.Tyr322X
157
224
279
Allele 1
253
256
p.Tyr322X
157
224
279
Allele 2
257
256
þ
146
224
293
Bold fonts indicate the disease allele.
Laugel et al., 2010]. The carrier frequency of CS in this population
is 6.79% (95% confidence interval: 3.84–9.74). Similar high carrier
frequencies were reported with regard to several other autosomal
disorders among isolated communities in Israel, including CS
caused by mutation in the ERCC6 gene in a Druze village [Bach
et al., 2007; Basel-Vanagaite et al., 2007; Falik-Zaccai et al., 2008a,b;
Zlotogora et al., 2009].
The Druze CS mutation was detected only among the residents of
one specific isolated village, but not in other individuals belonging
to the Druze community in Israel. In contrast, the Christian CS
mutation (p.TyrY322X) was detected in carriers in Christian Arabs
from all over the Northern part of Israel. Moreover, the same
mutation was detected in two CS Australian patients originally
from Lebanon [Laugel et al., 2010]. The Israeli Christian Arab
community originated, in part from Lebanon, and finding the same
mutation in patients of Lebanese origin suggests a founder shared
by the Christian communities in Israel, Lebanon, and perhaps
communities residing in other countries. The frequency of the
mutation should be investigated in this community and if demonstrated to be true, testing for this mutation should be considered in
individuals of Christian Lebanese Arab origin.
The haplotype analysis demonstrated that the p.Tyr322X CS
mutation is a founder mutation that is ancient, since the shared
haplotype seems to be relatively small. In order to determine the
age of the mutation fine mapping and addition of other markers
are needed.
Our experience indicates that genetic screening offered to
reproducing couples is well accepted among the Israeli Christian
Arab community, and, indeed, in our pilot study the couples who
were offered a chance to participate (in addition to other tests that
are routinely screened in Israel) opted for the test. Already during
the phase of the pilot study, we detected two couples at risk for CS in
their pregnancy, both did not have any history of the syndrome in
their families, confirming the high frequency of widely spread
mutation. Both opted for prenatal diagnosis and in one instance
we detected an affected fetus. The parents chose to terminate the
pregnancy. As a result of this pilot study the Christian CS mutation
was included in the Israeli national screening program.
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