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* 1Genetics Institute, Ha’Emek Medical Center, Afula, Israel 2Department of Community Genetics, Public Health Services, Ministry of Health, Hebrew University Jerusalem, Jerusalem, Israel 3Rappaport 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 How to Cite this Article: condition might be frequent, mainly due to founder effect. Khayat M, Hardouf H, Zlotogora J, Shalev SA. Recognition of common inherited disorders in defined popula- 2010. High carriers frequency of an tions may be effective in improving public health care. Cockayne apparently ancient founder mutation syndrome (CS) is a rare autosomal recessive disorder common in p.Tyr322X in the ERCC8 gene responsible for Christian Arabs due to a p.Tyr322X mutation. Genetic screening Cockayne syndrome among Christian Arabs of the p.Tyr322X mutation of the ERCC8 gene in this population in Northern Israel. documented a carrier frequency of 6.79% (95% confidence Am J Med Genet Part A 152A:3091–3094. 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 free radicals or exogenous agents such as ultraviolet (UV) community. As a result of this pilot study the Christian CS light [Balajee and Bohr, 2000; de Boer and Hoeijmakers, 2000; mutation was included in the genetic screening program offered Christmann et al., 2003]. to the Israeli Arab Christian community. Ó 2010 Wiley-Liss, Inc. Deficiencies of NER in humans lead to the rare human disorders xeroderma pigmentosum (XP), trichthiodystrophy (TTD), and Key words: Cockayne syndrome; DNA repair mechanism; CS [Nance and Berry, 1992; de Boer and Hoeijmakers, 2000; genetic screening; founder mutation 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, INTRODUCTION respectively) have been cloned and characterized biochemically. Cockayne syndrome (CS, OMIM# 133540, 216400) is a rare auto- Their exact function is incompletely understood, but they probably somal recessive disorder, comprising severe growth retardation, have a crucial role in the first step of transcription-coupled repair progressive neurological dysfunction, and accelerated aging. The (TCR), namely the recognition of a lesion [van Hoffen et al., 2003]. condition is clinically variable and genetically heterogeneous CSA belongs to ‘‘WD repeat’’ family of proteins with regulatory with a wide range of severity of phenotypes that present mainly roles but without enzymatic activity [Henning et al., 1995]. with progressive retinal degeneration and congenital cataracts, In a recent review, 84 CS kindreds were reported, about 62% sensorineural hearing loss, cachectic growth retardation, cutaneous (52 kindreds) had a mutation in the ERCC6 gene, while 38% (32 photosensitivity, skeletal anomalies, and mental retardation kindreds) had a mutation in the ERCC8 gene [Laugel et al., 2010]. [Nance and Berry, 1992]. The primary cause of CS is a defect in *Correspondence to: one of the DNA repair systems [de Boer and Hoeijmakers, 2000]. Stavit Allon Shalev, M.D., Genetics Institute, Ha’Emek Medical Center, The nucleotide excision repair (NER) mechanism involves the Afula 18101, Israel. E-mail: [email protected] action of about 30 proteins and is responsible for removing a variety Published online 24 November 2010 in Wiley Online Library of DNA lesions, such as helix distorting adducts caused by exposure (wileyonlinelibrary.com). to endogenous genotoxic agents. These materials include oxygen DOI 10.1002/ajmg.a.33746 Ó 2010 Wiley-Liss, Inc. 3091 3092 AMERICAN JOURNAL OF MEDICAL GENETICS PART A Among the 88 reported mutations that cause CS, 61 mutations 55C for 30 sec and at 72C for 10 sec; and at 72C for 5 min. were identified in the ERCC6 gene and the other 27 mutations The mutation revokes a restriction site for the DdeI restriction were identified in the ERCC8 gene. enzyme. Amplified fragments of DNA containing the mutation Herein, we describe the high frequency of a previously described were digested with DdeI (New England Biolabs, Beverly, MA) mutation p.Tyr322X identified in the ERCC8 gene of Christian restriction enzyme and electrophoresed on 1% SeaKem LE Agarose/ Arab CS patient from Northern Israel [McDaniel et al., 1997]. 2% Metaphor Agarose gels (lonza, Rockland, ME). Linkage analysis was performed using microsatellite markers Normal alleles consisted of 133 and 78 bp fragments, heterozy- located on cytoband 5q11 in order to construct haplotypes for gous carriers showed 211, 133, and 78 bp bands, and homozygous CS patients and carriers. affected individuals showed 211 bp fragments. MATERIALS AND METHODS Haplotype Analysis The Christian Arabs in Israel We studied five polymorphic markers spanning about 5.3 cM Approximately 117,000 Christian Arabs live mainly in urban around the ERCC8 locus [Conte et al., 2009] of genomic DNA from two CS patients and 19 carriers. Two markers were proximal areas in northern Israel, including Nazareth, Shfar’am, and Haifa to ERCC8 (D5S1715 and D5S2080), and three were distal to ERCC8 [Statistical Abstracts, 2009]. Although many denominations are (D5S624; D5S1990; and D5S427). The sense set of the PCR primers represented, most are Greek Orthodox and Roman Catholic. was labeled fluorescently. We analyzed the PCR products of these reactions using the GeneScan system of the ABI PRISM 3010xl. The Patients In the last decades CS was diagnosed in eight different families RESULTS of Christian Arab origin. The families reside mainly in Nazareth and nearby smaller towns, with no known family links between Carrier Frequency most of them. After the characterization of the mutation p.Tyr322X Among the 280 unrelated individuals we identified 19 carriers. (c.966 C>A) in the ERCC8 gene in some affected individuals, Therefore, the carrier frequency of the p.TyrY322X mutation in we examined some of the other patients and found the homozygous the Christian Arab population in northern Israel is 6.79% (95% state of the same mutation. confidence interval: 3.84–9.74%). Blood Sample Collection and Genomic DNA Detection of Couples at Risk Isolation During the screening study, two couples were identified at risk Our pilot study was a prerequisite of the Ministry of Health for having a child homozygous for the p.Tyr322X mutation. before considering the test as part of the national screening Following genetic counseling, both couples decided to perform program. This study was approved by the Ha’Emek Medical Center prenatal diagnosis. The first one demonstrated an affected fetus and Human Studies Ethics Committee in accordance with the Helsinki the parents elected to terminate the pregnancy. The second prenatal Declaration. diagnosis results indicated a normal heterozygous (þ/À) fetus. The test was offered to all couples of Christian Arab origin asking for genetic screening as a clinical service, free of charge, after Haplotype Analysis obtaining an informed consent. The result of the screening was The two affected individuals were found to be homozygous for all given to the individuals tested. five markers that were tested as well as the p.TyrY322X mutation Blood was collected from 280 healthy individuals belonging to (Table I). However, the patients shared only the 256 bp allele of the the Arab Christian population from Northern Israel and genomic marker D5S2080 that is located 1.2 cM away from the gene. Among DNA was extracted using the FlexiGene DNA kit (Qiagen, Hilden, the 19 carriers, the same 256 bp allele was present in 18 cases, while it Germany) according to the manufacturer’s instructions. was present in only in 24 of 50 non-carrier Arab Christians (P ¼ 0.018). Carrier Detection Assay A molecular assay consisting of DNA amplification followed DISCUSSION by restriction enzyme analysis was developed to allow accurate and rapid detection of carriers of the p.Tyr322X mutation in the The CS patients present manifestations including growth failure, a ERCC8 gene. cachexia, bird-like face, mental retardation, microcephaly, retinal Carrier detection was performed by amplification of a 211 bp degeneration, deafness, photosensitivity, and dental anomalies fragment containing the mutation from genomic DNA. The [our clinical data; Lehmann et al., 1993; McDaniel et al., 1997; sense primer 50-AATCCTACAGGTGAACTATG-30, and antisense Laugel et al., 2010]. primer 50-TACCTGGAAATTTGACTGAA-30 were used under All CS individuals were homozygousfor the p.Tyr322X mutation the following PCR conditions: denaturation at 95C for 5 min; in the ERCC8 gene (data not shown) including the CS patient 35 subsequent amplification cycles performed at 95C for 15 sec, at described previously [Lehmann et al., 1993; McDaniel et al., 1997; KHAYAT ET AL. 3093 TABLE I. Haplotype Analysis Using Five Informative Markers Spanning About 5.3 cM Around the ERCC8 Locus Patient 1 Mother Father Marker Allele 1 Allele 2 Allele 1 Allele 2 Allele 1 Allele 2 D5S1715 257 257 257 264 257 264 D5S2080 256 256 256 263 256 263 ERCC8 p.Tyr322X p.Tyr322X p.Tyr322X þ p.Tyr322X þ D5S624 155 155 155 157 155 157 D5S1990 243 243 243 233 243 233 D5S427 295 295 295 293 295 293 Patient 2 Mother Marker Allele 1 Allele 2 Allele 1 Allele 2 D5S1715 253 253 253 257 D5S2080 256 256 256 256 ERCC8 p.Tyr322X p.Tyr322X p.Tyr322X þ D5S624 157 157 157 146 D5S1990 224 224 224 224 D5S427 279 279 279 293 Bold fonts indicate the disease allele.