RESEARCH ARTICLE

High Carriers Frequency of an Apparently Ancient Founder p.Tyr322X in the ERCC8 Responsible for 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 (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 (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 (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 -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 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 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. . 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 [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/) 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.

Laugel et al., 2010]. The carrier frequency of CS in this population their pregnancy, both did not have any history of the syndrome in is 6.79% (95% confidence interval: 3.84–9.74). Similar high carrier their families, confirming the high frequency of widely spread frequencies were reported with regard to several other autosomal mutation. Both opted for prenatal diagnosis and in one instance disorders among isolated communities in Israel, including CS we detected an affected fetus. The parents chose to terminate the caused by mutation in the ERCC6 gene in a Druze village [Bach pregnancy. As a result of this pilot study the Christian CS mutation et al., 2007; Basel-Vanagaite et al., 2007; Falik-Zaccai et al., 2008a,b; was included in the Israeli national screening program. 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 REFERENCES to the Druze community in Israel. In contrast, the Christian CS mutation (p.TyrY322X) was detected in carriers in Christian Arabs Bach G, Zeigler M, Zlotogora M. 2007. Prevention of lysosomal storage from all over the Northern part of Israel. Moreover, the same disorders in Israel. Mol Genet Metab 90:353–357. mutation was detected in two CS Australian patients originally Balajee AS, Bohr VA. 2000. Genomic heterogeneity of nucleotide excision from Lebanon [Laugel et al., 2010]. The Israeli Christian Arab repair. Gene 250:15–30. community originated, in part from Lebanon, and finding the same Basel-Vanagaite L, Taub E, Halpern GJ, Drasinover V, Magal N, Davidov B, mutation in patients of Lebanese origin suggests a founder shared Zlotogora J, Shohat M. 2007. Genetic screening for autosomal recessive by the Christian communities in Israel, Lebanon, and perhaps nonsyndromic mental retardation in an isolated population in Israel. Eur J Hum Genet 15:250–253. communities residing in other countries. The frequency of the mutation should be investigated in this community and if demon- Christmann M, Tomicic MT, Roos WP, Kaina B. 2003. Mechanisms of human DNA repair: An update. Toxicology 193:3–34. strated to be true, testing for this mutation should be considered in individuals of Christian Lebanese Arab origin. Colella S, Nardo T, Mallery D, Borrone C, Ricci R, Ruffa G, Lehmann AR, The haplotype analysis demonstrated that the p.Tyr322X CS Stefanini M. 1999. Alterations in the CSB gene in three Italian patients with the severe form of Cockayne syndrome but without clinical photo- mutation is a founder mutation that is ancient, since the shared sensitivity. Hum Mol Genet 8:935–941. haplotype seems to be relatively small. In order to determine the ConteC,D’ApiceMR,BottaA,SangiuoloF,NovelliG.2009.Prenataldiagnosis age of the mutation fine mapping and addition of other markers of Cockayne syndrome type A based on the identification of two novel are needed. mutations in the ERCC8 Gene. Genet Test Mol Biomarkers 13:127–131. Our experience indicates that genetic screening offered to de Boer J, Hoeijmakers JH. 2000. Nucleotide excision repair and human reproducing couples is well accepted among the Israeli Christian syndromes. Carcinogenesis 21:453–460. Arab community, and, indeed, in our pilot study the couples who Falik-Zaccai TC, Laskar M, Kfir N, Nasser W, Slor H, Khayat M. 2008a. were offered a chance to participate (in addition to other tests that Cockayne syndrome type II in a Druze isolate in Northern Israel in are routinely screened in Israel) opted for the test. Already during association with an insertion mutation in ERCC6. Am J Med Genet Part A the phase of the pilot study, we detected two couples at risk for CS in 146A:1423–1429. 3094 AMERICAN JOURNAL OF MEDICAL GENETICS PART A

Falik-Zaccai TC, Kfir N, Frenkel P, Cohen C, Tanus M, Mandel H, Shihab S, Dollfus H. 2010. Mutation update for the CSB/ERCC6 and CSA/ERCC8 Morkos S, Aaref S, Summar ML, Khayat M. 2008b. Population screening in genes involved in Cockayne syndrome. Hum Mutat 31:113–126. aDruzecommunity:Thechallengeand the reward.Genet Med10:903–909. Lehmann AR, Thompson AF, Harcourt SA, Stefanini M, Norris PG. 1993. Graham JM Jr, Anyane-Yeboa K, Raams A, Appeldoorn E, Kleijer WJ, Garritsen Cockayne’s syndrome: Correlation of clinical features with cellular VH, Busch D, Edersheim TG, Jaspers NG. 2001. Cerebro-oculo- facio- sensitivity of RNA synthesis to UV irradiation. J Med Gene 30:679–682. skeletal syndrome with a nucleotide excision-repair defect and a mutated McDaniel LD, Legerski R, Lehmann AR, Friedberg EC, Schultz RA. 1997. XPD gene, with prenatal diagnosis in a triplet pregnancy. Am J Hum Confirmation of homozygosity for a single nucleotide substitution Genet 69:291–300. mutation in a Cockayne syndrome patient using monoallelic mutation Henning KA, Li L, Iyer N, McDaniel LD, Reagan MS, Legerski R, Schultz analysis in somatic cell hybrids. Hum Mutat 10:317–321. RA, Stefanini M, Lehmann AR, Mayne LV, Friedberg EC. 1995. The Nance MA, Berry SA. 1992. Cockayne syndrome: Review of 140 cases. Am J Cockayne syndrome group A gene encodes a WD repeat that Med Genet 42:68–84. interacts with CSB protein and a subunit of RNA polymerase II TFIIH. Cell 82:555–564. Statistical abstracts of Israel. 2009. The central bureau of statistics, Jerusalem, Israel. Laugel V, Dalloz C, Durand M, Sauvanaud F, Kristensen U, Vincent MC, Pasquier L, Odent S, Cormier-Daire V, Gener B, Tobias ES, Tolmie JL, van Hoffen A, Balajee AS, van Zeeland AA, Mullenders LH. 2003. Nucleotide Martin-Coignard D, Drouin-Garraud V, Heron D, Journel H, Raffo E, excision repair and its interplay with transcription. Toxicology 193:79–90. Vigneron J, Lyonnet S, Murday V, Gubser-Mercati D, Funalot B, Brueton Zlotogora J, Carmi R, Lev B, Shalev SA. 2009. A targeted population carrier L, Sanchez Del Pozo J, Munoz~ E, Gennery AR, Salih M, Noruzinia M, screening program for severe and frequent genetic diseases in Israel. Eur J Prescott K, Ramos L, Stark Z, Fieggen K, Chabrol B, Sarda P, Edery P, Hum Genet 17:591–597. Bloch-Zupan A, Fawcett H, Pham D, Egly JM, Lehmann AR, Sarasin A,