Vol. 11, 1449–1453, November 2002 Cancer Epidemiology, Biomarkers & Prevention 1449 Multiple Single Nucleotide Polymorphisms on Human Chromosome 19q13.2–3 Associate with Risk of Basal Cell Carcinoma1 Jiaoyang Yin, Eszter Rockenbauer, previously reported that polymorphisms in the gene XPD seemed Mohammad Hedayati, Nicklas Raun Jacobsen, associated with the occurrence of BCC (1). This finding has later Ulla Vogel, Lawrence Grossman, Lars Bolund, and been corroborated by the association of polymorphisms in XPD Bjørn A. Nexø2 with BCC in a different population (2) and by the association of Institute of Human Genetics, University of Aarhus, DK-8000 Aarhus C, markers in the same gene with three other cancers, malignant Denmark [J. Y., E. R., L. B., B. A. N.]; Department of Medical Genetics, melanoma (3), glioma (4), and lung cancer (5). Shenyang Medical College, Shenyang 110031, Liaoning, People’s Republic of In this paper, we present evidence that alleles of several other China [J. Y.]; Department of Biochemistry, Johns Hopkins School of Public polymorphisms in the chromosomal region 19q13.2–3, encom- Health, Baltimore, Maryland 21205 [M. H., L. G.]; and Institute of Occupational Health, Lersø Parkalle 105, DK-2100 Copenhagen O, Denmark passing the genes RAI and XPD, are associated with occurrence of [N. R. J., U. V.] BCC. We are therefore convinced that a chromosomal variation influencing the risk of getting BCC and possibly other cancers must be located in this region, and we see the present paper as a Abstract first step toward identifying this variation. In this paper, we present evidence that alleles of several Of the genes that we have investigated, XPD, ERCC1, and polymorphisms in the chromosomal region 19q13.2–3, LIG1 relate to DNA repair and are probably directly involved encompassing the genes RAI and XPD, are associated in preventing cancer. FOSB, which is a homologue of an with occurrence of basal cell carcinoma in Caucasian oncogene, and GLTSCR1, which may be a tumor suppressor Americans. The association of one of these, RAI-intron1, gene, might well also be involved in carcinogenesis or its is sufficiently strong to make mass significance unlikely prevention. For RAI, which seems to be involved in control of -␹2). We interpret our combined data to transcription, one might construct a relation to cancer preven ,0.004 ؍ P) indicate that a specific haplotype partly defined by the tion, but no experimental data to that effect are available. The alleles of three single nucleotide polymorphisms, RAI remaining genes are unlikely to play a direct role in cancer. intron1G, RAI exon6T, and XPD exon 6C, is associated They were chosen simply because they were located in the with a protective gene variant in a region spanning from chromosomal region of interest. XPD to ERCC1. Materials and Methods Introduction Study Groups. The groups of Caucasian Americans with and SNPs3 are the plankton of genetics. Individually they are tiny, without BCC have been described previously (2, 6). DNAs for 1-base differences in a 3-gigabase human genome, but by their the analyses were purified from frozen lymphocytes obtained multitude they constitute a rich source for genetic research. In from blood. the wake of the Human Genome Project, myriads of SNPs have Determination of Polymorphisms by LightCycler. Geno- surfaced, and it is now estimated that a SNP occurs approxi- types of polymorphisms in CKM exon 8 (position 20076, mately every 1000 bases. So no matter which part of the rs#4884) and RAI intron 1 (position 875, rs#1970764), ERCC1 genome you are interested in, there are likely to be multiple exon 4 (position 19007; Ref. 7), FOSB exon 4 (position 34621, SNPs located in the vicinity. rs#1049698), SLC1A5 exon 8 (position 60620, rs#1060043), SNPs often effectively dichotomize human populations, LIG1 exon 6 (position 111, rs#20580), and GLTSCR1 exon 1 making them useful for tracking genes of importance to disease (position 20775, rs#1035938) were detected using LightCycler in outbred populations by linkage disequilibrium. Specifically, (Roche Molecular Biochemicals, Mannheim, Germany; Ref. 8). typing of SNPs may facilitate the locating of regions of previ- The positions refer to the following accession numbers in ously unknown importance for cancer risk. Thus, we have GenBank: RAI, L47234; CKM, AC005781; ERCC1, M63796; FOSB, M89651; SLC1A5, AC008622; LIG1, L27710; and GLTSCR1; AC010519. The rs numbers refer to National Center for Biotechnology Information’s database over SNPs, dbSNP. Received 9/21/01; revised 6/25/02; accepted 7/3/02. The costs of publication of this article were defrayed in part by the payment of PCR was performed by rapid cycling in a reaction volume of 20 page charges. This article must therefore be hereby marked advertisement in ␮l with 0.5 ␮M each primer, 0.045 ␮M anchor and sensor probe, accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 3.5 mM MgCl 7 ng of genomic DNA, and 2 ␮l of LightCycler 1 2, Supported by The Karen Elise Jensen Foundation, the Danish Cancer Society DNA Master Hybridization probe buffer (Roche Molecular (Grant 9810028), the Danish Medical Research Council (Grant 9600259), the Danish SUE program (Grant 9800647-67), and the Novo Nordisk Foundation. Biochemicals, catalogue number 2158 825). This buffer con- 2 To whom requests for reprints should be addressed, at Institute of Human tains Taq DNA polymerase, dNTP mix, and 10 mM MgCl2. Genetics, Bartholin Building, University of Aarhus, DK-8000 Aarhus C, Den- Table 1 shows the design of primers and fluorogenic probes for mark. Phone: 45-8942-1686; E-mail: [email protected]. LightCycler. Some of the primers were copied from Ref. 8. 3 The abbreviations used are: SNP, single nucleotide polymorphism; BCC, basal cell carcinoma; dNTP, deoxynucleotide triphosphate; SAP, shrimp alkaline Hobolth DNA (Hillerød, Denmark) and TIB-Molbiol (Berlin, phosphatase. Germany) synthesized the primers and probes, respectively. In Downloaded from cebp.aacrjournals.org on October 1, 2021. © 2002 American Association for Cancer Research. 1450 Chromosome 19q13.2–3 and BCC Table 1 Design of primers and fluorogenic probes for LightCycler Table 2 Design of primers and SNaPshot primers for sequenator RAI intron 1 XRCC1 exon 7 Forward primer: 5Ј-TGGCTAACACGGTGAAACC Forward primer: 5Ј-GTCCCATAGATAGGAGTGAAAG Reverse primer: 5Ј-GGAATCCAAAGATTCTATGATGG Reverse primer: 5Ј-CCCTAGGACACAGGAGCACA Anchor probe: 5Ј-GGGAGGCGGAGCTTGCAGTGA-fluorescein SNaPshot primer: 5Ј-TGCATAGCTAGGTCCTGC Sensor probe: 5Ј-LCRed 640-CTGAGATCGCACCACTGCAC-p XRCC1 exon 17 CKM exon 8 Forward primer: 5Ј-GCCAAGCAGAAGAGACAAA Forward primer: 5Ј-TTGAAACTGGAACTCTGAGAAGG Reverse primer: 5Ј-GAGTGGCTGGGGAGTAGGA Reverse primer: 5Ј-TGGTGGATGGTGTGAAGCA SNaPshot primer: 5Ј-AACTGACRAAACTAGCTCTATGGGGTGGTGC- Anchor probe: 5Ј-LCRed 640-CCTTTCTCCAACTTCTTCTCCATTTCC- CGCA ACC-p RAI exon 6 Sensor probe: 5Ј-GGGGATCATGTCGTCAATGGACT-fluorescein Forward primer: 5Ј-CCTACCACCATCATCACATCC ERCC1 exon 4 Reverse primer: 5Ј-GCCTTGCCAAAAATCATAACC Forward primer: 5Ј-AGGACCACAGGACACGCAGA-3Ј SNaPshot primer: 5Ј-CCTCTCCCCAATTAAGTGCCTTCACACAGC Reverse primer: 5Ј-CATAGAACAGTCCAGAACAC-3Ј XPD intron 4 Anchor probe: 5Ј-LCRed640-TGGCGACGTAATTCCCGACTATG- Forward primer: 5Ј-CGCAAAAACTTGTGTATTCACC TGCTG p-3Ј Reverse primer: 5Ј-CCCATTTTTATCATCAGCAACC Sensor probe: 5Ј-CGCAACGTGCCCTGGGAAT-fluorescein SNaPshot primer: 5Ј-CTGGCTCTGAAACTTACTAGCCC FOSB exon 4 Forward primer: 5Ј-AGGCTCAACAAGGAAAAATGC Reverse primer: 5Ј-GCTAGACAGTCAAGGAGGGACG Ј Anchor probe: 5 -LCRed 640-AAAGGGTGGGTGTGGGAGACATTGG-p Table 3 Design of primers and probes for Taqman Sensor probe: 5Ј-AAACCAACCTAGGCACCCCAAA-fluorescein SLC1A5 exon 8 XRCC1 exon 10 Forward primer: 5Ј-CAGTGTCCAAAGAGCACC Forward primer: 5Ј-GCT-GGA-CTG-TCA-CCG-CAT-G Reverse primer: 5Ј-CTACCCCTTTAGCGACC Reverse primer: 5Ј-GGA-GCA-GGG-TTG-GCG-TG a a Anchor probe: 5Ј-LCRed 640-TCCTGCCCCCAGAGCGTCACC-p Probe (A): 5ЈFAM -TGC-CCT-CCC-AGA-GGT-AAG-GCC-T-TAMRA a a Sensor probe: 5Ј-GTACGGTCCACATAATTTTGGAGGA-fluorescein Probe (G): 5ЈVIC -CCC-TCC-CGG-AGG-TAA-GGC-CTC-TAMRA LIG1 exon 6 a Tradenames of Applied Biosystems the bold underlined letter represents the Forward primer: 5Ј-ATGCCCTGTAGGTTCAATGG polymorphic position. Reverse primer: 5Ј-TGGAGGTCTTTAGGGGCTTG Anchor probe: 5Ј-GGCTGGTCCCCGTCTTCTCCTTCC-fluorescein Sensor probe: 5Ј-LCRed 640-TCTCTGTTGCCACTTCAGCCTC-p GLTSCR1 exon 1 ␮ Ј Biolabs, Beverly, MA), and 9 l of PCR reaction in a total Forward primer: 5 -CGACGAACTTCTCTGAAGCGAA ␮ Reverse primer: 5Ј-AGCGACACGGGCATCTGG volume of 14 l of water. The reactions were incubated at 37°C Anchor probe: 5Ј-ATGAGCGTCCACCTCCTGAACC-fluorescein for 60 min and 72°C for 15 min. The SNaPshot reactions Sensor probe: 5Ј-LCRed 640-AGGCAGCAGCATCGTCATCCCC-p contained 1 ␮l of SNaPshot Ready Reaction Mix (Applied Biosystems), 0.5 ␮l of each of the SNaPshot primers (XRCC- e7-ss1, 4 pmol/␮l; XPD-i5-cp1, 0.5 pmol/␮l; RAI-e7-cp1, 1 pmol/␮l; XRCC-e17-ss1, 2 pmol/␮l), 2 ␮l of the purified PCR some cases, the reaction mixture also contained 5% DMSO. product, and 1.5 ␮l of buffer [200 mM Tris-HCl and 5 mM The temperature cycling consisted of denaturation at 95°C for MgCl2 (pH 9.0)]. The reactions were cycled 25 times: 96°C for 2 s, followed by 46 cycles consisting of2sat95°C, 10 s at 10 s, 50°Cfor5s,60°C for 30 s. The primers and dNTPs were 57°C, and 30 s at 72°C. The last annealing period at 72°C was removed in a reaction containing 1 unit of SAP, 0.8 ␮lof10ϫ extended to 120 s. The melting profile was determined by a SAP buffer, and 5 ␮l of SNaPshot reaction in a total volume of temperature ramp from 50°Cto95°C with a rate of 0.1 de- 8 ␮l of water. Two ␮l of purified product were added to 10 ␮l gree/s. For RAI intron 1, we ran the melting profile three times of concentrated deionized formamide (Amresco), incubated for and used the last curve. 5 min at 95°C, and analyzed on the sequenator. The two Determination of Polymorphisms by Sequenator. The poly- markers in XRCC1, in exon 7 and exon 17, could not be reliably morphisms in XRCC1 exon 7 (position 26651; Ref.