Similar to the 10q11 risk allele, other disease risk loci have been shown to affect expression of more than one gene [17,34]. A variant associated with systemic lupus erythematous at chromo- some 8p23, for example, is associated with increased expression of one gene (BLK) and decreased expression of another (C8orf13) in B cell lines [34]. As more genes underlying complex traits are discovered, it may be that certain risk alleles mediate their effects through multiple genes, or alternatively, that two risk variants in tight linkage disequilibrium influence separate genes. The findings at 10q11 highlight the importance of considering multiple genes when analyzing GWAS results. The findings at 10q11 also underscore the importance of evaluating risk loci in a tissue-specific context [35]. It is hypothesized that a fraction of non-protein coding risk alleles will alter disease risk by regulating gene activity, and these variants may exert their effects in a specific genetic and epigenetic context [21,36]. In the present study, the 10q11 risk variant is associated with transcript levels of MSMB and NCOA4 in primary prostate tissue. In contrast, no convincing or consistent association is observed in colon or breast tissue. Similarly, alteration of MSMB and NCOA4 expression significantly affects anchorage-indepen- dence of prostate but not breast epithelial cells. These findings may have implications for future studies attempting to connect risk alleles with target gene(s). Evaluation of GWAS findings will focus, in part, on identifying the genes targeted by risk alleles, as these are the genes likely to drive the trait under study. Our findings suggest that this type of analysis should include evaluation of the tissue at risk for disease, although it is entirely plausible that variants associated with a particular disease may manifest their effects in tissues other than target tissue. Notably, associations between rs10993994 genotype and expression of MSMB and NCOA4 are observed in histologically normal prostate tissue, whereas in tumor tissue an association is detected with only MSMB (albeit at an attenuated level relative to the normal tissue). It is conceivable that increased expression of NCOA4 is associated with tumor initiation, as reflected by its association with risk in solely normal tissue, while decreased expression of MSMB is associated with both tumor initiation and maintenance or progression. More studies, however, will be necessary before a general principle emerges. Cellular context also appears to be an important determinant in the functional analysis of candidate risk genes. This is illustrated by comparing data in the present study to previous work involving NCOA4. The characteristics of two NCOA4 isoforms, alpha and beta, have been studied in functional assays. Upregulation of NCOA4beta increases anchorage-independent growth [11], while overexpression of NCOA4alpha inhibits growth in LNCaP cells [37]. Functional analysis of immortalized prostate epithelial cells presented here demonstrates increased colony growth in the setting of an overexpressed alpha isoform (Figure 2). Distinctions between the cell lines used in these studies may account for these divergent results. LNCaP cells are derived from metastatic prostate lesions. Immortalized prostate epithelial cells, on the other hand, are not tumorigenic and differentiate in the presence of androgen [26], suggesting that these cells are more closely related to normal prostate epithelial cells. The data presented here suggest that tissue type (in this case, prostate versus non-prostate tissue) and cellular states (i.e., normal versus tumor) are likely important factors in the evaluation of complex trait loci. Chromatin context and differential use of gene regulatory elements across tissues and disease states may be the basis for expression effects specific to normal prostate tissue [36]. It can be difficult to accurately model these effects outside of the particular genetic and epigenetic context of specific tissues. Luciferase reporter MSMB and NCOA4 Implicated in Prostate Cancer Risk assays, for example, are often utilized to define a relationship between a polymorphism and a gene. Reporter assays cannot, however, detect situations where a risk variant is associated with opposing transcriptional effects on two loci, as observed with rs10993994. An alternative explanation for the effects on the two transcripts is that two separate variants in linkage disequilibrium are responsible for the different transcriptional effects. In contrast to Mendelian diseases, where resequencing protein- coding regions often reveals the causal gene, common complex trait alleles often occur outside of protein-coding regions. As is the case at 10q11 and other loci [38,39], these alleles may be associated with expression of nearby and/or distal candidate genes. There are also situations in which associations with strong candidate genes, however, cannot be established by measuring steady-state expres- sion at a single point in time [19]. In order to better understand the gene(s) involved in complex trait pathogenesis, experiments will need to integrate and to account for the genetic and epigenetic contexts of the particular tissue type and cellular state. Materials and Methods Ethics statement This study was conducted according to the principles expressed in the Declaration of Helsinki. The study was approved by the Institutional Review Board of Dana-Farber Cancer Institute. All patients provided written informed consent for the collection of samples and subsequent analysis. Cohorts and RNA isolation A total of 180 patients treated with radical prostatectomy (RP) for prostate cancer and 92 patients treated for colon cancer consented to provide tissue. Additionally, histologically normal breast tissue from 56 from women undergoing cosmetic reduction surgery was analyzed in the present study. Fresh frozen radical prostatectomy specimens were available from 121 subjects at the Dana-Farber Cancer Institute (DFCI) and Brigham and Women’s Hospital (Boston, MA) and were reviewed by a pathologist (J.C. or R.F.). Over 95% of the patients in the cohort were diagnosed with Gleason 6 or Gleason 7 disease and median PSA was 5.1 ng/ml. Areas of tumor consisted of .60% tumor cells and areas of benign tissue consisted of .80% non- neoplastic epithelial cells at least 5 mm away from any tumor focus. Biopsy cores of fresh frozen tissue were processed for RNA extraction using a modified Qiagen Allprep DNA/RNA protocol. Archival FFPE blocks were available for 59 men with prostate cancer enrolled in the Physicians’ Health Study (PHS) [22,40]. These men were diagnosed with prostate cancer between 1983 and 2003 and treated by radical prostatectomy. RNA were extracted from paraffin-embedded tumor tissue as described previously [40]. Areas of tumor consisted of .90% tumor cells. Fresh frozen colorectal cancer tissue samples were reviewed by a pathologist (J.C.) and areas of benign tissue were selected where 80% of cells consisted of non-neoplastic epithelium. RNA was extracted using a modified Qiagen Allprep DNA/RNA protocol. Fresh frozen normal breast tissue samples were reviewed to identify tissue blocks containing .40% normal epithelial cells. RNA was extracted using a modified Qiagen RNeasy protocol. Ethnicity was self-reported by most, but not all, subjects. Subjects in the DFCI cohort of unknown ancestry were genotyped for 59 ancestry-informative SNPs in order to ascertain ethnicity. The marker set primarily captured ancestral differences between European and African ancestries (D. Reich, personal communi- cation). Five samples found to be from subjects of African ancestry were excluded from analysis. PLoS Genetics | www.plosgenetics.org 4 November 2010 | Volume 6 | Issue 11 | e1001204