A Prevalent Mutation with Founder Effect in Xeroderma ... - Core

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ORIGINAL ARTICLE

A Prevalent Mutation with Founder Effect in Xeroderma Pigmentosum Group C from North Africa Nadem Soufir1,11, Cecile Ged2,3,4,11, Agnes Bourillon1, Frederic Austerlitz5, Cećile Chemin2,3,4, Anne Stary6, Jacques Armier6, Daniele Pham6, Khadija Khadir7, Joelle Roume8, Smail Hadj-Rabia9, Bakar Bouadjar10, Alain Taieb2,3,4, Hubert de Verneuil2,3,4, Hakima Benchiki7,11, Bernard Grandchamp1,11 and Alain Sarasin5,11 Xeroderma pigmentosum (XP) is a rare autosomal recessive disorder that is associated with an inherited defect of the nucleotide excision repair pathway (NER). In this study, we investigated the involvement of XP genes in 86 XP patients belonging to 66 unrelated families, most of them consanguineous and originating from Maghreb. Sequencing analysis was performed either directly (44 probands) or after having previously characterized the involved XP gene by complementation assay (22 families). XPC and XPA mutations were respectively present in 56/66 and 8/66 probands. Strikingly, we identified the same homozygous frameshift mutation c.1643_1644delTG (p.Val548AlafsX25) in 87% of XP-C patients. Haplotype analysis showed a common founder effect for this mutation in the Mediterranean region, with an estimated age of 50 generations or 1,250 years. Among 7/8 XP-A patients, we found the previously reported nonsense homozygous XPA mutation (p.Arg228X). Six mutations—to our knowledge previously unreported— (five in XPC, one in XPA) were also identified. In conclusion, XPC appears to be the major disease-causing gene concerning xeroderma pigmentosum in North Africa. As the (p.Val548AlafsX25) XPC mutation is responsible for a huge proportion of XP cases, our data imply an obvious simplification of XP molecular diagnosis, at least in North Africa. JID JOURNAL CLUB ARTICLE: For questions and answers about this article, please go to http://www.nature.com/jid/journalclub Journal of Investigative Dermatology (2010) 130, 1537–1542; doi:10.1038/jid.2009.409; published online 7 January 2010

INTRODUCTION Xeroderma pigmentosum (XP) is a rare, autosomal, recessive syndrome, described as early as 1870, as a sun-hypersensitivity disorder associated with numerous skin abnormalities such as excessive freckling, hyper- and hypopigmentation, poikilodermia, skin atrophy, skin aging, and a very high rate of early and multiple skin cancers (Cleaver, 1968; Takebe et al., 1989; Bootsma et al., 1995). Ocular abnormalities including photophobia, keratitis, ectropion, and tumors are also frequent. About 20–30% of XP patients show major 1

Laboratoire de Biochimie hormonale et geńe´tique, Hoˆpital Bichat, APHP, Universite´ Paris VII, Paris, France; 2INSERM U876, Bordeaux, France; 3 Universite´ V Segalen Bordeaux2, Bordeaux, France; 4CHU Bordeaux, Centre des Maladies Rares de la Peau, Hoˆpital Pellegrin, Bordeaux, France; 5 Laboratoire Ecologie, Systematique et Evolution UMR CNRS, Universite Paris Sud, AgroParisTech 8079, Orsay cedex, France; 6Laboratory of Genetic Stability and Oncogenesis, CNRS FRE 2939, Universite´ Paris-Sud, Institut Gustave Roussy, Villejuif, France; 7Service de Dermatologie, CHU Ibn Rochd, Casablanca, Morocco; 8Service de Geńe´tique me´dicale, Hoˆpital de Poissy, Poissy, France; 9Service de Dermatologie, Hoˆpital Necker, APHP, Universite´ Paris V, Paris, France and 10Service de Dermatologie, CHU de Bab El Oued, Alger, Algeria 11

These authors have equally contributed to this work

Correspondence: Nadem Soufir, Laboratoire de Biochimie hormonale et geńe´tique, Hoˆpital Bichat, APHP, Universite´ Paris VII, 46 Rue Henri Huchard, 75018 Paris, France. E-mail: [email protected] or [email protected] Abbreviations: ALL, acute lymphocytic leukemia; NER, nucleotide excision repair pathway; TCR, transcription-coupled repair; XP, xeroderma pigmentosum Received 5 November 2008; revised 12 November 2009; accepted 13 November 2009; published online 7 January 2010

& 2010 The Society for Investigative Dermatology

neurological disorders characterized by premature neuronal death, progressive mental retardation and microcephaly (Kraemer et al., 1987). Genetic defects in XP involve the repair pathway of bulky DNA damage, such as UV-induced DNA lesions, that are mainly repaired by the nucleotide excision repair pathway (NER) (Stary and Sarasin, 2002). Seven NER XP complementation groups, XP-A to XP-G, have been characterized. Two NER sub-pathways have been described. A fast repair of DNA lesions located on actively transcribed genes, called transcription-coupled repair (TCR) and a slower repair of lesions located on the rest of the genome, called global genome repair (Sarasin and Stary, 2007). In addition, an XP variant group has also been described that involves a DNA translesional synthesis polymerase, Pol eta (Masutani et al., 1999, 2008). These patients have a normal NER level but their cells are hypermutable following UV irradiation. The most prevalent NER defective groups comprising approximately 90% of characterized classical XP patients, are XP-C (Europe and North Africa), XP-A (especially in Japan and North Africa) and XP-D (in Europe and USA). The remaining 10% are composed of groups B, E, F, and G, with less than 40 patients recorded in each category (Stary and Sarasin, 2002). Several XP mutations have been described through different countries with a relatively small number of patients for each analysis (Li et al., 1993; Nishigori et al., 1993; www.jidonline.org 1537

N Soufir et al. Prevalent Mutations in Xeroderma Pigmentosum C

Chavanne et al., 2000; Khan et al., 2006; Cleaver et al., 2007). A recent analysis estimated the incidence of XP for the autochthonic western European populations to be close to 0.9 affected cases per million live births (Kleijer et al., 2008). Yet, incidence is much higher in North Africa and because some of these patients have been treated in France, we aimed at characterizing the spectrum of XP mutations in a large cohort of patients originating mainly from Morocco, Algeria, Tunisia. RESULTS We describe a cohort of 86 patients from 66 unrelated XP families with XP features studied either by direct gene sequencing or following DNA repair analysis and complementation assays. The direct gene-sequencing assay included

44 probands, whereas the DNA repair assay analyzed 22 probands from unrelated XP families. We sequenced the XPC and XPA genes at first, then ERCC2/XPD, XPV/POLH, XPB, XPE, XPF and XPG genes, if no mutation had been detected. All mutations identified in the probands were also present in affected relatives. In this study, the largest ever reported XP cohort, XPC and XPA defects were the most frequent, 56/66 (85%) and 8/66 (12%), respectively (Table 1). In two patients (3%), no mutations were identified in any XP gene. XPA mutations

The same XPA mutation, the nonsense mutation c.682C4T p.Arg228X was observed in 7 of 8 XP-A patients. All patients were homozygous for this mutation and consanguinity was

Table 1. Molecular analysis of XP patients Patient identification

Geographic

Proband

origin

number

Direct sequencing analysis B4-B67-B134-B860 -Bx29-Bx50

Molecular defects

Mutant Gene

alleles

Location

DNA level

Protein level

44 Northern Africa

6

XPA

12

Exon 6

c.682C4T homoz

p.Arg228X

O5-O10-O16-O26-O46-O62-O76-O80- Northern Africa O96-O104-O110-O114-O119-O120O126-O144 -O151-O175-O184-O203O242-O250-B946-B947-B948-B971B973-B950-Bx23-Bx36-Bx40-Bx46Bx52-Bx55

33

XPC

66

Exon 9

c.1643_1644delTG homoz

p.Val548AlafsX25

Spain

1

2

Bx41

Northern Africa

1

XPC

2

Exon 6

c.652delT homoz

p.Phe218SerfsX41

Bx28

Chile/West Indies

1

XPC

1

Exon 9

c.1243C4T

p.Arg415X

1

Exon 13

c.2287delC

p.Leu763CysfsX4

DNA repair assay

22

AS689

Northern Africa

1

XPA

2

IVS4

c.609_2A4G homoz

AS858

Northern Africa

1

XPA

2

Exon 6

c.682C4T homoz

p.Arg228X

AS185-AS188-AS202-AS233-AS373AS629-AS664-AS673-AS694-AS731AS769-AS798-AS802-AS820-AS821

Northern Africa

14

XPC

30

Exon 9

c.1643_1644delTG homoz

p.Val548AlafsX25

p.Arg220X

Spain

1

AS860

Northern Africa

1

XPC

2

Exon 6

c.658C4T homoz

AS268

Greece

1

XPC

2

IVS6

c.779+1_14del12 homoz

AS704

France

1

XPC

1

Exon 9

1 AS314

France

1

XPC

AS751

France

1

XPC

1

Exon 9

1 2

Exon 11

—

—

c.1243C4T

p.Arg415X

c.1399C4T

p.Gln467X

c.1243C4T

p.Arg415X

c.1704T4A

p.Tyr568X

c.2092_2093insGTG p.Val696Val697InsVal homoz

The analysis was performed by sequencing of the coding regions following amplification from genomic DNA. In bold characters: the six mutations identified in this work that are, to our knowledge, previously unreported. In italic characters: the recurrent XPC mutation in exon 9.

1538 Journal of Investigative Dermatology (2010), Volume 130


documented in all corresponding Maghrebi families. In addition, we also characterized a splicing XPA homozygous mutation, c.609_2A4G, which to our knowledge has not been described (Table 1). All but one of XP-A patients displayed neurological symptoms (mental retardation and/or spasticity and/or hyporeflexia and/or ataxia). Interestingly, although XP-A and C patients had both pronounced photosensitivity, XP-A patients had fewer skin cancers (30%) than XP-C patients (75%, P-value o0.05 after adjusting on age). XPC mutations

A single mutation located in the ninth exon of the XPC gene (c.1643_1644delTG, p.Val548AlafsX25) was present at a very high frequency in our XP patients. Mutation frequency was 74% (49 of 66) in all the XP probands tested and 87% (49 of 56) in XP type C patients, mostly from the Maghreb countries. The prevalence of this mutant was 96% (50 of 52) in Maghrebi XP-C patients. Noticeably, because of high consanguinity levels in the Maghreb country, the mutation was present at the homozygous state in all the patients studied. The great majority of the patients with the founder mutation had the same clinical features, i.e., pronounced photosensitivity, numerous skin cancers, and ophthalmological signs. Yet, two of them had neurological symptoms. One patient from a consanguineous family had mental retardation, microcephaly, deafness, spasticity, and ataxia. The only available information for the second patient was the presence of a mental retardation. Six patients, all being less than 5 years old did not have any skin cancer. Three other previously described mutations were observed in four patients (Table 1 and see Supplementary Table S2 online). The nonsense mutation located in exon 9 (c.1243C4T, p.Arg415X) previously reported in one patient (Khan et al., 2006) was found in two patients at the heterozygous state. The homozygous c.658C4T (p.Arg220X) mutation has been already described in a patient (Chavanne et al., 2000). The valine insertion in exon 11 c.2092_2093insGTG (p.Val696_ Val697insVal) reported in two patients (Li et al., 1993; Cleaver et al., 2007) was present in one patient from France at the homozygous state. Five mutations to our knowledge previously unreported are observed in this report including two frameshifts in exons 6 and 13, a splice mutation in intron 6, and 2 nonsense mutations in exon 9 (Table 1 and Supplementary Table S2). The frameshift in exon 6 (c.652del T, p.Phe218SerfsX41) is present at the homozygous state in a consanguineous family from North Africa. The frameshift in exon 13 (c.2287delC, p.Leu763CysfsX4) is observed at the heterozygous state. The second mutant allele is a nonsense in exon 9 (c.1243C4T, p.Arg415X) in a family originating from Chile (mother) and the French West Indies (father’s mother). The splicing mutation c.779 þ 1_14del12, present at the homozygous state in a patient from Greece, abolishes the splicing donor site of exon 6, as shown by the bioinformatics programs ‘‘BDGP (www.fruitfly.org/seq_tools/splice.html)’’. Moreover, this mutation does not appear in public SNP databases. The nonsense mutations (c.1399C4T, p.Gln467X, and c.1704T4A, p.Tyr568X) located in exon 9 are present in two

compound heterozygous unrelated French patients who also carried c.1243C4T (p.Arg415X) as the second mutant allele. Finally, two patients from the direct sequencing study had no mutation in XPC, XPA, XPD/ERCC2, and XPV/POLH genes. Therefore, the remaining XP genes (XPB, XPE, XPF, XPG) were entirely sequenced in these two patients, but no mutation was detected. Haplotype analyses were performed in all patients carrying the XPC c.1643_1644delTG, p.Val548AlafsX25 mutation, and in 49 unrelated healthy controls from North Africa. The common mutation c.1643_1644delTG was systematically associated with a unique rare haplotype including the same allele of the two intragenic XPC microsatellites, along with c.1-27 G, IVS5 þ 44 C, IVS8-22 A, c.1475 G (p.492R), c.1496 C (p.499A), c.2061 G (p.687R), IVS12-37 C, IVS12-6 A, IVS15 þ 28 C, IVS14-40 A, c.2815 C (p.939Q). Further haplotype analysis of 11 microsatellites surrounding XPC (Supplementary Table S1) revealed a frequent common haplotype comprising specific alleles of 22GT3/ 26TG/D3S1554/RH5658/D3S3613 encompassing the XPC locus, present in 1/3 of patients. By using the method developed by Austerlitz et al. (2003), we estimated an age of 50 generations or 1,250 years (confidence interval ¼ (1,090–1,495)), assuming a 25-year generation time, for the common XPC mutation c.1643_1644delTG. This estimate was of 1,000 years (confidence interval ¼ (870–1,200)) assuming a 20-year generation time or 1,500 years (confidence interval ¼ (1,310–1,790)) for a 30-year generation time (Tremblay and Vezina, 2000). DISCUSSION We report here the largest group of XP patients analyzed so far for eight DNA repair genes, XPC, XPA, XPD/ERCC2, XPB, XPE, XPF, XPG, and XPV/POLH. As previously suggested (Cleaver, 1968; Bootsma et al., 1995), we can clearly conclude from this work that XP types C and A are the most frequent XP groups, at least in Southern Europe and North Africa. XPA mutations were present in 12% of XP patients, a prevalent mutation—R228X—being present in 87.5% of XPA patients. The frequency of this mutation had already been described in North Africa, with similar results to our data (Nishigori et al., 1993; Cleaver et al., 1999). XPC mutations were identified in 56 of 66 (85%) probands. The common XPC mutation, c.1643_1644delTG (p.Val548AlafsX25), present at the homozygous state in 87% of XP-C patients, has been previously reported in 24 XP-C patients; 21 homozygous patients originated from North Africa mainly Tunisia (Li et al., 1993; Khan et al., 2006; Mahindra et al., 2008, Ben Rekaya et al., 2009), and three patients from Italy, Egypt, and Africa, respectively (Chavanne et al., 2000; Ridley et al., 2005). Two heterozygous patients (XP132BE and XP30BE) originated from USA and Honduras (Khan et al., 2006). As previously shown, this common mutation results in the absence of XPC protein because it leads to a frameshift mutation (Rezvani et al., 2008). The DNA repair ability induced by this mutation has previously been found only to www.jidonline.org 1539


be 20% of proficient normal cells (Chavanne et al., 2000). At least in our experimental conditions, the level of DNA repair is less than 10% of normal cells and can be totally complemented to the normal level by transduction of the wild-type XPC gene via recombinant retroviruses (data not shown). Two patients carrying this mutation had neurological signs, which were for one patient indistinguishable from those observed in XP-A patients. Neurological impairment was recently described in a consanguineous XP-C family harboring a homozygous mutation in the initiation codon of XPC, whereas neurological symptoms were absent in another family carrying the same mutation (Khan et al., 2009). Possible hypotheses accounting for the neurological abnormalities in these patients could be the presence, in case of consanguinity, of homozygous mutation in another (unidentified) gene, or could result from the interaction of modifier genes with XPC on neurological phenotype. As the mutation was predominant in Maghreb and also characterized in Egyptian, Italian, and Spanish patients, it highly suggested a common founder effect in the Mediterranean region. By using already described mathematical tools based on microsatellites haplotyping, we were able to calculate that the common ancestor mutation occurred 1,250 years ago. Interestingly, this date approximately corresponds to the beginning of the European invasion by Muslims from Arabia (the ‘‘Saracens’’). Four of eight patients who did not originate from North Africa were compound heterozygotes for XPC mutations (Table 1 and Supplementary Table S2). A particular mutation was the nonsense mutation located in exon 9 (c.1243C4T, p.Arg415X), present in three different French families (Table 1). It has been previously described at the heterozygous state in two French siblings (XP314VI and XP315VI) whose second mutant allele was not identified at that time (Khan et al., 2006). The French patient AS704 also carries a nonsense mutation to our knowledge previously unreported on the second allele (c.1399C4T, p.Gln467X). In the third French patient (Bx28) the p.Arg415X mutant had a paternal origin and the father’s mother originates from the French West Indies. The second mutant allele was a frameshift in exon 13 (c.2287delC, p.Leu763CysfsX4), and originated from the mother (Chile). Five XPC mutants to our knowledge previously unreported have been identified in this study (Table 1 and Supplementary Table S1). With the mutations reported in this work, a total of forty-five inactivating XPC mutations have been characterized in XP-C patients (Supplementary Table S2). Most of them are frameshift (deletion and insertion) or nonsense (substitution), leading to a truncated XPC protein. A few splicing and missense mutations are also encountered. However, there is no obvious correlation between genotype and phenotype or survival. In our total of 66 XP probands, we did not find any mutations in the eight-screened XP genes in two families, one of which was consanguineous. Unfortunately, fibroblast cultures were not available to confirm the DNA repair defect. In these families, the genetic defect might be an XP mutation 1540 Journal of Investigative Dermatology (2010), Volume 130

localized outside the coding sequence and/or a complex rearrangement of an XP gene. Xeroderma pigmentosum group C, the most common form of XP in Europe and North Africa, is characterized by high morbidity and early mortality, essentially because of the striking accumulation of skin cancers on the exposed body sites. Early diagnosis in these patients is particularly important, because XP-C patients show a normal neurology and an early full protection against sun-exposure has already shown to allow them to have almost a normal life. The high frequency of the same mutation in patients from North Africa will considerably simplify the molecular diagnosis of XP in patients from Maghrebi descent. We therefore suggest that the XP patients from Maghreb, initially specifically test for the XPA-R228X and XPC-p.Val548AlafsX25 mutations. In the absence of these mutations, unscheduled DNA synthesis on cultured fibroblasts should be performed to confirm the DNA repair defect, complementation analysis using recombinant retroviruses should determine the involved gene followed by sequencing analysis of the corresponding XP gene. MATERIALS AND METHODS We obtained an institutional approval of experiments and written informed consent for each patient included in the study. This study was in conformity to the Helsinki Guidelines.

Families with xeroderma pigmentosum features We studied 86 XP patients belonging to 66 different unrelated XP families clinically diagnosed as possible XP patients. Two types of analysis were performed. A direct sequencing of the XP genes (XPC, XPA, XPD/ERCC2, XPV/POLH and then XPB, XPE, XPF, and XPG) was realized in a group of 57 patients belonging to 44 families (called thereafter ‘‘Direct sequencing analysis’’). A more biological approach, in which the DNA repair level was first quantified by unscheduled DNA synthesis on cultured skin fibroblasts followed by complementation with wild-type DNA repair genes to determine the right gene for sequencing (called thereafter ‘‘DNA repair analysis’’) was performed on 22 distinct families (29 patients in total, 22 probands and seven affected relatives). Informed consent was obtained from all the patients or the parents of minor children. In the direct sequencing analysis, 42 probands originated from the Maghreb region of North Africa, including Morocco (24), Algeria (14) and Tunisia (4); one patient was European (Spain), and one French patient had French West Indies and Chile ancestries. In the DNA repair analysis, 17 families originated from North Africa, three patients from France, one from Spain, and one from Greece. Clinical data were available in 60 of the 66 families and in 73 of the 86 patients. Mean age of XP patients was 16 years (range, 2–45 years). Sex ratio M/F was 0.8. Fourteen patients had no familial history of XP, 31 patients had at least one related affected by XP, and in the 21 remaining families, this information was unknown. Consanguinity was confirmed in 25 of 45 families, but could not be documented in 21 families. XP symptoms began at a mean age of 42 months. Skin photosensitivity was present in most patients, with poikilodermia in 92%, xeroderma in 94%, skin atrophy in 77%, and telangiectasia in 74% of XP patients. Skin tumors occurred in 71% of XP patients,


with BCC in 65%, SCC in 49%, and melanoma in 18% of patients. Atypical fibroxanthomas were observed in three patients, and malignant histiocytofibroma in one patient. Ophthalmological symptoms were present in most patients: photophobia in 81%, conjunctivitis in 79%, and keratitis and ectropion in 29% of XP patients. Ocular tumors occurred in 10% of patients. Neurological symptoms, mainly represented by mental retardation and pyramidal ataxia, were noted in nine patients. In addition, two patients had a cerebral glioma, one patient had a lymphoma and another patient an acute lymphocytic leukemia (ALL).

follows: c.1-27 G4C, c.621 þ 44 C4G, c.891-22 A4G, c.1475 G4A (p.R492H), c.1496 C4T (p.A499V), c.2061 G4A (p.R687R), c.2251-37 C4G, c.2251-6 A4C, c.2604 þ 28 C4G, c.2515-40 A4G, c.2815 C4A (p.Q939K).

Estimate of the age and growth rate of the XPC c.1643_1644delTG (p.Val548AlafsX25) mutation

The response to UV irradiation was analyzed by measuring unscheduled DNA synthesis in primary fibroblast cultures established from biopsies of unaffected skin, as previously described (Sarasin et al., 1992). The characterization of XP genetic defect was carried out by classical complementation assays using recombinant retroviruses as already published (Arnaudeau-Begard et al., 2003).

We used the method developed by Austerlitz et al. (2003) that provides a joint estimate of the age of the mutation—that is, the time elapsed since the appearance of the common ancestor of the mutation carriers in the population and the growth rate of the number of copies since this appearance. For this purpose, 11 microsatellites surrounding the XP locus were identified by using the Human Genome Browser gateway (http://genome.ucsc.edu/cgi-bin/ hgGateway) (Supplementary Table S1). These microsatellites were genotyped in 30 unrelated patients (probands) carrying the XPC c.1643_1644delTG (p.Val548AlafsX25) mutation and in 25 of their relatives. As for XPC intragenic microsatellites, fluorescent PCR products were run on the automatic sequencer and analyzed using the GeneMapper software (Perkin Elmer).

Sequence analyses of XP genes

CONFLICT OF INTEREST

Genomic DNA was extracted from peripheral blood samples or cultured cells (fibroblasts and lymphoblasts) using standard procedures in the different laboratories involved in the study. Complete coding sequences were carried out and repeated at least twice. The XPC gene was amplified by PCR in 15 exonic fragments, encompassing the 16 exons of the gene and the corresponding intron–exon junctions, as described (Khan et al., 2002; Jacobelli et al., 2008) by using Taq Gold DNA polymerase (Perkin Elmer, Courtaboeuf, France). The XPA gene was amplified in six exonic fragments. The 23 exons of the XPD/ERCC2 gene and the 10 coding exons of XPV/POLH were analyzed in patients who carried no mutation of either XPC or XPA gene. Automatic sequencing was performed on both strands using BigDye Terminator Cycle Sequencing Ready Reaction Kit on the ABI PRISM 7700 automated sequencer (Perkin Elmer). Mutations and polymorphisms were identified using SeqScape software (Perkin Elmer). Mutation numbering is based on Genbank cDNA sequences (NM_004628 and NM_000380, for XPC, and XPA, respectively) with a ‘‘c’’ symbol before the number and uses the A of the ATG translation initiation start as nucleotide 1. Protein numbering (‘‘p’’ symbol) starts from the initiating methionine (codon 1). Common polymorphisms of the XPC gene have been previously described (Chavanne et al., 2000; Rivera-Begeman et al., 2007).

The authors state no conflict of interest.

DNA repair investigation

XPC haplotype analysis Two intragenic XPC polymorphic microsatellites were analyzed with the following primers: sat1U-50 F-GCCTAAGATACCTGAGGTACA CTTTCA-30 , sat1L 50 -GAATATACACATCCAAGGACCTCAAGT-30 , sat2U50 -F-GGGTTTGAGACCAGCTTGGGC-30 , and sat2L 50 -CGC CATCATTATGTTGTTGCATTC-30 . Fluorescent PCR products were run on the automatic sequencer and analyzed using the GeneMapper software (Perkin Elmer). In addition, the analysis of 11 bi-allelic polymorphisms, localized both in the coding sequence and the flanking intronic sequences of XPC, was performed in XP-C patients carrying the common mutation and in 49 unrelated healthy controls from North Africa (Algeria and Morocco). The SNPs are localized as

ACKNOWLEDGMENTS We thank Dr E Bourrat, Dr C Blanchet-Bardon, Dr T Leblanc, and Dr F Cordoliani (Hoˆpital Saint-Louis, Paris, France), Dr C Robert and Dr MF Avril (Gustave Roussy Institute, Villejuif, France), Dr J Kaplan (Hoˆpital Necker-Enfants malades, Paris), Dr I Gorin and Dr O Eujobras (Hoˆpital Tarnier, Paris), Dr C Maire, Dr B Catteau, and Dr C Dubois (CHU de Lille, France), Professor N Philip (Hoˆpital de la Timone, Marseille, France), Dr L Faivre (Hoˆpital des enfants, Dijon, France), Dr A Terrier (Hoˆpital neurologique, Lyon, France), Professor P Edery (Hoˆpital Debrousse, Lyon), Dr M Zghal (Tunis, Tunisia), Dr L Pinson (CHU d’Angers, France), Professor P Souteyrand (CHU de Clermont-Ferrand, France), Professor L Lantiery and Dr D Benjoar (CHU H. Mondor, Cre´teil, France), Dr M Jamar (CHU Start Tilman, Liege, Belgium), and Dr F Maazoul (Hoˆpital C. Nicole, Tunis, Tunisia) for patient recruitment and Miss A Riffault for technical assistance. Contract grant sponsor: This work was supported by grants from the French Ministry of Health (Paris), ‘‘l’Agence Nationale de la Recherche (Paris), and ‘‘L’Association des Enfants de la Lune’’ (Tercis, France) to A Sarasin.

SUPPLEMENTARY MATERIAL Supplementary material is linked to the online version of the paper at http:// www.nature.com/jid

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