Analysis of Genes Critical for Growth Regulation Identifies Insulin-like Growth Factor 2 Receptor Variations with Possible Functional Significance as Risk Factors for Osteosarcoma Sharon A. Savage, 1,3 Karen Woodson, 2 Elyse Walk, 2 William Modi, 6 Jason Liao, 1 Chester Douglass, 7 Robert N. Hoover, 4 Stephen J. Chanock 1,5 and The National Osteosarcoma Etiology Study Group 1 Section of Genomic Variation, Pediatric Oncology Branch and 2 Genetics Branch, Center for Cancer Research; 3 Clinical Genetics Branch, 4 Epidemiology and Biostatistics Program, and 5 Core Genotyping Facility, Division of Cancer Epidemiology and Genetics; and 6 Core Genotyping Facility, Science Applications International Corporation-Frederick, Inc., National Cancer Institute, NIH, Bethesda, Maryland; and 7 Harvard School of Dental Medicine, Boston, Massachusetts Abstract Background: Osteosarcoma, the most common malignant primary bone tumor, typically occurs during the adolescent growthspurt.Germ-linegeneticvariationingenescriticalin growth regulation could confer altered risk of osteosarcoma. Methods: Fifty-two common single nucleotide polymor- phisms (SNP) in 13 genes were genotyped in a prospective case-control study of osteosarcoma (104 osteosarcoma cases and 74 orthopedic controls). Genotype data analyzed with contingency tables suggested the strongest association with insulin-like growth factor 2 receptor (IGF2R ) SNPs. Addi- tional SNPs were genotyped to capture IGF2R common haplotypes and resequencing was done across the IGF2R block associated with osteosarcoma risk. Percentage methyl- ation was determined by pyrosequencing of the IGF2R variant allele located in a CpG island. Results: IGF2R Ex16+88G>A (rs998075) and IVS16+15C>T (rs998074) SNPs were associated with increased risk for osteosarcoma compared with orthopedic controls (haplotype oddsratio,2.04;95%confidenceinterval,1.29-3.24).Follow-up genotypingshowedthat IGF2R IVS15+213C>Twasalsoasso- ciatedwithincreasedosteosarcomarisk.Resequenceanalysis identified two additional SNPs linked to the risk-associated SNPs; linkage disequilibrium was strongest in a 1-kb pair regionaroundthem.TheEx16+88G>ASNPislocatedwithina CpGislandandaltersmethylationatthatsite. Conclusion: Thispilotstudyofgerm-linegeneticvariationin growth pathway genes and osteosarcoma identified a haplotype block in IGF2R associated with increased risk of osteosarcoma.ThepresenceofaSNPinthisblockresultsin lossofmethylationataCpGisland,providingcorroborative evidenceofapossiblefunctionalvariant.Ouranalysisofthe IGF2R haplotype structure will be applicable to future studies of IGF2R and disease risk. (Cancer Epidemiol Biomarkers Prev 2007;16(8):1667–74) Introduction Primary malignancies of the bone, with an annual incidence rate of 8.7 per million children <20 years of age, account for f6% of childhood cancer (1). Of these, osteosarcoma is the most common with f400 new cases in the United States per year in individuals <20 years of age (1, 2). The age-specific incidence of osteosarcoma is bimodal, with the first major peak occurring around adolescence and a second smaller peak in adults over age 65 (2, 3). Patients with localized osteosarcoma at presentation have a 60% to 80% long-term survival rate, whereas metastatic disease carries a poorer prognosis (4). Until recently, studies of genetics and bone cancer risk have focused on familial cancer syndromes in which rare, highly penetrant, germ-line mutations have been identified, such as the Li-Fraumeni syndrome (5). Although the sporadic form of osteosarcoma is more common than the familial type, less is known of the genetics of the former (6). Recent advances in the annotation of common genetic variation, together with increased efficiency in scope and cost for high-throughput genotype analyses, have made it possible to investigate the etiologic contribution of the most common type of variant in the genome, the single nucleotide polymorphism (SNP). Only a few preliminary studies of genetic variation in primary bone tumors of childhood have been published to date. In osteosarcoma, the Fok1 polymorphism T>C (rs10735810) of the vitamin D receptor gene, which eliminates a transcription start codon, was associated with increased osteosarcoma risk [odds ratio (OR), 1.78] but no association was observed for SNPs in the estrogen receptor or collagen Ia1 (7). In a separate study, an association was reported between a common variant allele of the TNF promoter and decreased risk for osteosarcoma but not for Ewing’s sarcoma (8). SNPs in the TP53 gene have recently been evaluated in an osteosarcoma case-control study; no strong association with osteosarcoma risk was found (9). Thus, the contribution of germ-line genetic variants to risk of sporadic osteosarcoma is largely unexplored. Suggested osteosarcoma risk factors include height and birth weight, but several small studies have yielded conflicting results (10-13), However, because osteosarcoma most com- monly occurs in long bones during or shortly after the 1667 Cancer Epidemiol Biomarkers Prev 2007;16(8). August 2007 Received 3/8/07; revised 5/24/07; accepted 6/6/07. Grantsupport: Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research, and Division of Cancer Epidemiology and Genetics. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Note: Supplementary data for this article are available at Cancer Epidemiology, Biomarkers & Prevention Online (http://cebp.aacrjournals.org/). The National Osteosarcoma Etiology Study Group is represented by Michael A. Simon (University of Chicago), Marc C. Gebhardt (Massachusetts General Hospital), Mark T. Scarborough (Shands Medical Center, University of Florida), Steven Gitelis (Rush Presbyterian and St. Lukes Medical Center), Jeffrey J. Eckardt (University of California at Los Angeles School of Medicine), James R. Neff (Nebraska Health System), Michael J. Joyce (Cleveland Clinic Foundation), Martin Malawer (Washington Cancer Institute), Michael McGuire (Creighton University), and H. Clarke Anderson (University of Kansas Medical Center). Requests for reprints: Sharon A. Savage, Clinical Genetics Branch, Division of Cancer Epidemiology and Genetics, National Cancer Institute, 6120 Executive Boulevard, EPS/7018, Rockville, MD 20852. Phone: 301-496-5785; Fax: 301-496-1854. E-mail: savagesh@mail.nih.gov Copyright D 2007 American Association for Cancer Research. doi:10.1158/1055-9965.EPI-07-0214 Downloaded from http://aacrjournals.org/cebp/article-pdf/16/8/1667/1751408/1667.pdf by guest on 24 June 2022