The biology of OS pathogenesis is complex and there are limited data on risk factors in the more common sporadic form of OS. Epidemiologic studies of OS suggest that growth and development play a role in etiology [2, 32, 33]. It occurs primarily in adolescents during puberty  when bone growth is rapid and endogenous sex hormones and growth hormones are at their highest, so variation in a gene involved in regulating sex hormones is biologically plausible. Rapidly growing tissue, such as bone during puberty, is known to be highly susceptible to carcinogenesis, possibly due to rapidly proliferating osteogenic cells being more vulnerable to DNA repair errors [4, 34]. In addition, chromosomal aneuploidy is extensive in somatic OS cells, which suggests the presence of chromosomal instability[26, 27]. The increased frequency of OS in genetic predisposition syndromes [13, 15] characterized by mutations in DNA repair pathways suggests that variants in genes involved in DNA repair are also reasonable candidates.
We evaluated the association between OS and 255 candidate genes, including 4836 SNPs, from four functional pathways (growth and hormone metabolism, bone formation, DNA repair, and ribosomal). We used 3 approaches to comprehensively evaluate these biologically plausible pathways: analyses were performed at the individual SNP level, gene level and pathway level with conservative statistical corrections for multiple testing. While no genes or pathways were significantly associated with OS after correction for multiple tests, the SNP based approach identified some potentially important candidates. A total of twelve SNPs in genes from the growth and hormone metabolism, and DNA repair pathways were significantly associated with OS risk after correction for multiple tests. Two genes had multiple significant SNPs associated with risk, FANCM and GH1, after correction for multiple tests.
FANCM contained 4 SNPs significantly associated with a similar 2-fold increased risk of OS using a dominant inheritance model, the most in any gene studied. These SNPs were not correlated in our controls. One SNP is located in exon 14 and the minor or risk allele results in a nonsynonymous change from valine to leucine (Ex14+316, Val878Leu). The minor allele of this SNP is the ancestral allele and is highly conserved among other mammalian species. The three other SNPs in FANCM were intronic. FANCM has DNA-dependent ATPase activity, promotes the dissociation of DNA triplexes, and with other Fanconi anemia-associated proteins, may repair DNA at stalled replication forks [35, 36]. DNA repair must be accurate to preserve genome stability for long-term cellular viability; genetic instability is characteristic of cancer cells, and may be due, at least in part, to mutations or variation in genes that function to ensure DNA integrity .
Two significant SNPs were located downstream of GH1. They appear to be highly correlated in our controls, although one SNP was associated with an increased risk of OS and the other was protective. The variant in IGF1 (rs7956547, IVS2+10605) associated with a decreased risk of OS was another interesting candidate involved in growth. The insulin-like growth factor signaling system is important in the formation and homeostasis of bone, and differential expression of IGF1 has been observed in osteosarcomas [38–40]. IGFI expression is stimulated by growth hormone, and OS incidence peaks during puberty with the release of growth hormone. OS cells have been shown to be IGF1-dependent for growth, and inhibiting growth hormone release in mice decreased IGF1 serum levels and inhibited tumor growth [41–43]. In addition, animal model data from dogs, which develop OS similar to human patients (similar sites, histology and treatment response) and large breeds have a 185-fold increased risk of OS compared with small dog breeds [44, 45], suggest that a SNP in IGF1 is a main determinant of small size in dogs and is virtually absent in giant breeds . The data suggest that GH1 and IGF1 may play a role in the etiology of OS.
We also specifically evaluated the genes found to be associated with OS in other studies. We previously identified a SNP in IGF2R (rs998075; Ex16+88G >A) to be significantly associated with OS risk in study of variation in genes critical in growth regulation . This SNP was also included in our dataset and the significant association replicated in an analysis limited to our BDISO cases and controls (P = 0.01), as expected because it is the same study population. However, the association did not replicate with the addition of our 1363 PLCO controls (P = 0.12), which suggests that the original study may have been limited by the number of controls, or possibly these older PLCO controls have a different ethnic mix within whites related to this polymorphism. Others have found significant associations between OS and polymorphisms in VDR , TGFBR1 , and MDM2 , which were also included in our dataset. We found no significant associations with individual SNPs within VDR or TGFBR1 before or after correction for multiple tests, although our dataset did not include the VDR FokI polymorphism  or TGFBR1*6A variant , or at the gene level. One SNP downstream of the DNA repair gene MDM2, rs1690916, was significantly associated with a decreased risk of OS after correction (OR 0.62, 95% CI 0.45-0.85, P
adj = 0.026), and 3 intronic SNPs were significantly associated with an increased risk of OS before correction (ORs 1.6-1.8, P = 0.008-0.02). The previously identified MDM2 T309G (rs2279744) polymorphism was marginally non-significantly associated with an increased risk of OS (OR 1.31, 95% CI 0.97-1.77, P = 0.07). At the gene level, MDM2 was found to be significantly associated with OS (Gene P = 0.016), but not after correction for multiple tests. As others have shown [8, 23], the current study did not confirm associations between polymorphisms in ESR1/2 (including previously analyzed PvuII and XbaI polymorphisms), COL1A1, or TP53 and OS after correction, or at the gene level.
We also investigated many of the genes with germline mutations in cancer predisposition syndromes associated with an increased frequency of OS , including the DNA repair genes TP53 (mutated in Li-Fraumeni Syndrome), WRN (mutated in Werner syndrome), BLM (mutated in Bloom syndrome), RECQL4 (mutated in Rothmund-Thomson Syndrome), and the ribosomal genes RPS19 and RPS24 (mutated in Diamond-Blackfan anemia) [28, 47, 48]. OS also occurs more commonly in older individuals with Paget's disease. Two genes involved in bone formation, SQSTM1 and TNFRSF11A, have been shown to cause Paget's disease . We found that no common SNPs within these genes that predispose to cancer predisposition syndromes or Paget's disease were significantly associated with OS after correction for multiple tests.
A limitation of the current study was the small number of cases; however, the inclusion of 14 controls per case improved the statistical power to detect SNPs with strong effects. For the additive model and our 96 cases and 1426 controls, we had greater than 80% statistical power to detect an OR of 1.82 for MAFs of 0.1 and an OR of 2.15 for MAFs of 0.05 (with a baseline population risk of 0.0000001 and type 1 error of 0.05). Another potential limitation of our study was the use of controls from PLCO that were older than the BDISO case and control population. However, the PLCO controls had no history of any cancer, including osteosarcoma, they were limited to Caucasians from the continental US, as were the BDISO cases and controls, and we found no evidence of population stratification in between groups.
Strengths of the current study include the detailed genotyping of biologically plausible pathways which give a higher a priori likelihood. We used three statistical approaches to comprehensively evaluate associations with OS (at the SNP level, gene level, and pathway level). In addition, we used a stringent Bonferroni correction and conservatively interpreted our results to reduce the probability of a Type 1 error.