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Carcinogenesis vol.28 no.12 pp.2530–2536, 2007 doi:10.1093/carcin/bgm196 Advance Access publication September 3, 2007 MSR1 variants and the risks of prostate cancer and benign prostatic hyperplasia: a population-based study in China Ann W.Hsing1,, Lori C.Sakoda2, Jinbo Chen3, Anand P.Chokkalingam4, Isabel Sesterhenn5, Yu-Tang Gao6, Jianfeng Xu7 and S.Lilly Zheng7 1 Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA, 2University of Washington, Seattle, WA 98195, USA, 3University of Pennsylvania, Philadelphia, PA 19114, USA, 4University of California, Berkeley, CA 94707 USA, 5Armed Forces Institute of Pathology Washington DC 20306, 6Shanghai Cancer Institute, Shanghai 200032, China and 7Center for Human Genomics, Wake Forest University School of Medicine, Winston-Salem, NC 27157, USA To whom correspondence should be addressed. Tel: þ1 301 496 1691; Fax: þ1 301 402 0916; Email: [email protected] Data from epidemiologic and twin studies suggest an important role of genetic susceptibility in prostate cancer. Variants of the macrophage scavenger receptor 1 (MSR1) gene have been linked to both hereditary and sporadic prostate cancer, although the evidence is inconclusive. Most studies have been conducted on Caucasians. The role of MSR1 in prostate cancer development among Asians, for whom rates of prostate cancer are low but rising rapidly, is unclear. To evaluate further the relationship between MSR1 variants and prostate cancer risk, we sequenced all the 11 MSR1 exons, exon–intron junctions, promoter regions, as well as 5# and 3# untranslated regions (UTRs) in 86 individuals from Shanghai, China. We identified a total of 21 sequence variants, including three novel variants that have not been reported previously. To balance genotyping cost and the capacity to capture sufficient genetic variation, we genotyped four haplotype-tagging variants (P275A, INDEL7, P346P and 3# UTR 70006), which capture 85% of the genetic variation in MSR1 in this population. These four variants, plus two other variants (PRO3 and INDEL1) that have been linked to prostate cancer risk in the previous studies, were typed for all study subjects, which included 130 prostate cancer cases, 130 patients with benign prostatic hyperplasia and 150 controls randomly selected from the population. Three of the six variants were associated with prostate cancer. Men with a P346P (a novel variant) G allele (AG 1 GG) had a significantly reduced risk of total prostate cancer [odds ratio 5 0.47, 95% confidence interval (CI) 0.23–0.96], whereas those with a P275A G allele had a 37% reduced risk of prostate cancer (95% CI 0.39– 1.02), with more pronounced reduction in risk seen for localized cancer cases (odds ratio 5 0.25, 95% CI 0.12–0.52; P 5 0.001). In addition, men with the INDEL7 variant had a 67% reduced risk of localized cancer (95% CI 0.16–0.68). Based on the four tagging variants, we inferred four major haplotypes that accounted for >90% of the haplotype variation in this population. The haplotype frequencies were significantly different between localized prostate cancer cases and controls, with a global P value of 0.004, and the haplotype containing the minor alleles of the P275A and INDEL7 variants was associated with a significantly reduced risk of localized prostate cancer (odds ratio 5 0.28, 95% CI 0.13–0.59), relative to the most common haplotype. These results, although modest and confined mainly to localized prostate cancer, suggest that MSR1 polymorphisms may play a role in prostate cancer etiology in Chinese men. The role of MSR1 warrants further investigation in larger studies and other populations. Abbreviations: BPH, benign prostatic hyperplasia; CI, confidence interval; LD, linkage disequilibrium; MSR1, macrophage scavenger receptor 1; PCR, polymerase chain reaction; SNP, single-nucleotide polymorphism; UTR, untranslated region; WHR, waist-to-hip ratio. Published by Oxford University Press 2007. Introduction Data from epidemiologic, family and twin studies suggest an important role of genetic susceptibility in prostate cancer (1–3). It is estimated that 42% of the risk of prostate cancer may be due to genetic influences (1), including individual and combined effects of rare, highly penetrant genes and more common polymorphisms with mild effects on androgen biosynthesis/metabolism, DNA repair and chronic inflammation pathways (3,4). Emerging epidemiologic evidence suggests that chronic inflammation in the prostate increases the risk of prostate cancer (5–7). For example, a recent meta-analysis of 11 studies on prostatitis and prostate cancer reported a relative risk of 1.6 for all studies combined (8). In addition, chronic inflammation induced by bacterial or viral agents has been implicated as a potential underlying mechanism for the link between sexually transmitted diseases and prostate cancer (9). A population-based case–control study in the USA reported that men who have had three or more episodes of gonorrhea had a 3.3-fold risk of prostate cancer compared with those who had never had gonorrhea (10). In the same study, men who had a history of syphilis infection had a significant 2.6-fold prostate cancer risk. Further supporting the role of chronic inflammation are the reported associations of prostate cancer with polymorphisms of genes involved in the inflammation pathway, including the macrophage scavenger receptor 1 (MSR1) gene (11–22). The MSR1 protein, encoded by the MSR1 gene, is a transmembrane protein that is expressed mainly by macrophages. Through binding to a wide range of ligands, including gram-negative and gram-positive bacteria, oxidized low-density lipoprotein and certain polynucleotides, the MSR1 protein is involved in an array of hormonal and pathological processes, including inflammation, innate and adaptive immunity, oxidative stress and apoptosis (21,22). The initial report of MSR1 showed a strong link between MSR1 mutations and hereditary prostate cancer in men of both European and African descent (22). However, results from subsequent studies have been inconclusive. A recent meta-analysis of eight studies on MSR1 suggests that the R293X and D174Y variants are associated with an increased risk of sporadic prostate cancer, particularly in African Americans (23). Similar studies have not been conducted in Asian populations, who have, in contrast to African Americans, the lowest reported rates in the world, although their incidence has been rising rapidly over the last 10 years (24–27). Reasons for the large racial differences in prostate cancer risk are unclear but may involve a complex interplay of genetic factors, including those involved in inflammation such as the MSR1 gene and lifestyle factors (3). To evaluate further the relationships between MSR1 and prostate cancer, we carried out a comprehensive genetic analysis of MSR1 in a population-based study of prostatic disease in Shanghai, China. We first sequenced MSR1 in a subset of subjects to identify haplotypetagging variants, and then typed all study subjects for the tagging variants in order to estimate the risk of prostate cancer and benign prostatic hyperplasia (BPH) associated with common MSR1 polymorphisms. Materials and methods Study population Details of this population-based case–control study have been reported elsewhere (28–31). Briefly, cases were permanent residents of Shanghai who were newly diagnosed with prostate cancer (ICD-9 185) between 1993 and 1995, identified through a rapid reporting system in 28 collaborating hospitals in urban Shanghai. Cases had no history of any cancer. Four cancer cases were excluded from the study after Chinese and American pathologists jointly confirmed their diagnosis as BPH. The rapid reporting system captured 2530 MSR1 variants and the risks of prostate cancer and benign prostatic hyperplasia .95% of the cases diagnosed in urban Shanghai during the study period. Patients with BPH diagnosed at the same hospitals were randomly selected for inclusion in the study. BPH cases were matched to the index cancer cases by age (within 5 years) and hospital. Pathology materials of both cancer and BPH cases were reviewed by study pathologists in Shanghai and subsequently confirmed by pathologists at the Armed Forces Institute of Pathology in USA. Based on records maintained at the Shanghai Resident Registry, male controls with no history of cancer were selected at random from the 6.5 million permanent Shanghai residents .18 years of age and frequency matched to cancer cases by age (5-year intervals). This study was approved by the Institutional Review Boards at the National Cancer Institute and the Shanghai Cancer Institute and written informed consent was obtained from all study subjects. Interview Using a structured questionnaire, trained personnel conducted in-person interviews to collect information on demographic characteristics, diet and smoking history, consumption of alcohol and other beverages, medical history, family cancer history, physical activity and sexual behavior. Anthropometric measurements were also taken, including waist and hip circumferences, height and weight. For cases, an interview was conducted within 3 weeks of diagnosis. Blood collection and DNA extraction Over 75% of the study subjects provided overnight fasting blood samples. Blood samples were processed and separated within 2 h of collection at a central laboratory at the Shanghai Cancer Institute. The blood fractions were stored at 70°C in Shanghai before shipment to USA on dry ice. DNA was extracted from the buffy coat fractions at the American Type Culture Collection (Manassas, VA). Only subjects with sufficient DNA available were included in the study for genotyping. In total, 130 prostate cancer cases, 130 BPH cases and 155 population controls were included. Overall, 70% of the cases in the original study who provided blood samples had sufficient DNA for the present study. There was no difference in demographic characteristics between those with and without DNA samples. Sequence analysis and genotyping Sequencing. To identify novel variants, we sequenced all 11 MSR1 exons, exon–intron junctions, promoter regions and 5# and 3# untranslated regions (UTRs) (32) in 86 individuals (66 prostate cancer cases and 20 non-cancer cases). As shown in Table I, we identified a total of 21 sequence variants (variants 1–21), including one missense change (variant 11), two synonymous changes (variants 13 and 15) and six variants in the 3# UTR (variants 16–21). Of the 21 variants, three (variants 4, 14 and 15) were novel variants that have not been reported previously. Figure 1 shows the location of and distance between these variants in the MSR1 gene, and Table I shows the position, dbSNP ID and minor allele frequency of these variants in the 86 samples. All polymerase chain reactions (PCRs) were conducted in a volume of 10 ll containing 30 ng of genomic DNA, each primer at 0.2 lM, each deoxynucleoside triphosphate at 0.2 mM, 1.5 mM MgCl2, 20 mM Tris–HCl, 50 mM KCl and 0.5 U of Taq polymerase (Life Technologies, Gaithersburg, MD). PCR cycling conditions were as follows: 94°C for 4 min; 30 cycles of 94°C for 30 s, the specified annealing temperature for 30 s, and 72°C for 30 s and a final extension of 72°C for 6 min. All PCR products were purified using the QuickStep PCR purification kit (Edge BioSystems, Gaithersburg, MD) to remove deoxynucleoside triphosphates and excess primers. All reactions were performed using dyeterminator chemistry (BigDye, ABI, Foster City, CA), with 63.5% ethanol for precipitation. Samples were loaded onto an ABI 3700 DNA Analyzer after adding 8 ll of formamide. Genetic variants were identified using Sequencher software version 4.0.5 (Gene Codes Corporation, Ann Arbor, MI). Haplotype tagging. Of the 21 variants identified, three (variants 3, 13 and 16) had a minor allele of frequency ,5% and one (variant 12, INDEL7) deviated from Hardy–Weinberg equilibrium among control subjects (these four variants were not included in the derivation of haplotype tagging single-nucleotide polymorphisms (SNPs)). Results of pairwise linkage disequilibrium (LD) analysis showed three regions with strong LD (variants 1–2, 6–8 and 14–21). There were a total of 24 estimated haplotypes, with six common haplotypes (frequency .5%) that represented 81% of all haplotypes. Haplotype-tagging analysis showed that four variants (variants 11, 12, 15 and 17) captured 85% of the haplotype diversity in this population. Thus, we typed these four tagging variants (three SNPs and one insertion/deletion) for all study subjects: variant 11 (P275A, rs3747531, a missense change in exon 6), variant 12 (INDEL7, rs3036811, a 3 bp insertion/deletion in intron 7), variant 15 (P346P, a synonymous change) and variant 17 (3# UTR 70006, rs12678016). In addition, we included two variants, variant 3 (PRO3, rs433235, in the promoter region) and variant 8 (INDEL1, rs11274081, a 15 bp insertion/deletion in intron 1), that have been reported in previous studies to be associated with prostate cancer risk. Genotyping. Genotyping of the four SNPs (PRO3, P275A, P346P and 3# UTR 70006) was performed using the MassARRAY system (SEQUENOM), whereas genotyping of the two insertion/deletions (INDEL1 and INDEL7) was performed using the 3700 DNA Analyzer (Applied Biosystems, Foster City, CA). DNA samples were arranged in triplets (cancer case, BPH and control) and identified by specimen ID only. Laboratory personnel had no information on case–control status. In addition to quality control samples included in each batch by the laboratory, blinded quality control samples were included to monitor the reproducibility of the genotyping assays. The genotyping failure rate was low (,2%) and concordance of duplicate samples was .99%. Statistical analysis Differences in selected characteristics between cancer cases, BPH cases and controls were tested using the Fisher’s exact test for categorical variables and the t-test for continuous variables. Single locus associations. Among control subjects, genotype frequencies of each MSR1 variant were examined for departure from Hardy–Weinberg equilibrium, using the Pearson v2 test. Differences in genotype frequencies between BPH cases, prostate cancer cases and controls were assessed with the Fisher’s exact test. Through the use of unconditional logistic regression, ageadjusted odds ratios and 95% confidence intervals (CIs) were derived, comparing BPH patients and cancer patients to control subjects separately, to evaluate the relationship of each MSR1 polymorphism with risks of BPH and prostate cancer. The homozygous genotype of the most common allele was used as the reference category. Tests for linear trend in risk according to the number of copies of the variant allele (0, 1 or 2) were also conducted to assess potential dose–response effects (33). Risk estimates were not calculated for comparisons if the sample size was ,5. When the number of subjects with homozygous genotype of the variant allele was ,5, instead of the test for trend analysis, we tested the disease risk associated with the presence or absence of the minor allele. Haplotype associations. Pairwise D’ (34) and r2 (35) values were calculated to examine the extent of LD between the six MSR1 loci. Using the haplo.stats package (http://mayoresearch.mayo.edu/mayo/research/schaid_lab/software. cfm) in R (version 2.2.0, http://www.r-project.org), haplotype frequencies were estimated for cases and controls applying the expectation-maximization algorithm (36); differences in haplotype frequencies between BPH cases, cancer cases and controls were assessed by a global score test (37) and age-adjusted odds ratios and 95% CIs were generated to examine the association of MSR1 haplotypes with risks of BPH and prostate cancer, with the most common haplotype defined as the referent category. We also investigated potential combined effects of MSR1 polymorphisms with other factors, including abdominal obesity [measured by waist-to-hip ratio (WHR)], body mass index (weight in kilogram divided by height in square meter) and insulin resistance, all of which have been linked to both inflammation and/or prostate cancer (28,30,38–40). The significance of the interaction terms representing the modifying effects of these putative risk factors with MSR1 markers was assessed using the likelihood ratio test. All reported P values are two-sided. Results Table I shows the position and minor allele frequency of the 21 sequence variants in the 86 samples and of the six haplotype-tagging variants in control subjects. The genotype distributions for the six loci among controls were in Hardy–Weinberg equilibrium, with minor allele frequencies ranging from 10.4 to 45.9%. Selected characteristics of cases and controls are shown in Table II. Among cancer cases, most tumors were moderately or poorly differentiated, with about one-third of the cases displaying clinically significant, advanced cancers in remote stages. The mean WHR and insulin resistance ratios differed significantly between cancer cases and controls (P , 0.01). Prostate specific antigen (PSA) levels in cases were high due to infrequent prostate cancer screening in China during the study period. Table III shows prostate cancer risk associated with the six MSR1 loci. As shown, only one marker (P346P) was significantly associated with total prostate cancer, with men having the P346P G allele (AG þ GG genotypes) at a 53% significantly reduced risk of prostate cancer (95% CI 0.23–0.96). Two MSR1 markers, including P275A and INDEL7, were associated with significantly reduced risk of localized prostate cancer. Men with the P275A G allele (CG þ GG) had a 75% reduction in risk (95% CI 0.12–0.52) and those with INDEL7 2531 A.W.Hsing et al. 2532 Table I. Sequence variants in the MSR1 gene identified in 86 samples, Shanghai Variant number Position Region Flanking region Nucleotide change dbSNP ID 1 2 3 4c 5 6 7 8 9 10 11 12 13 14c 15c 16 17 18 19 20 21 15 15 14 14 14 14 14 14 Promoter Promoter Promoter Exon 1 Exon 1 Intron 1 Intron 1 Intron 1 Intron 2 Intron 4 Exon 6 Intron 7 Exon 8 Intron 9 Exon 10 3# UTR 3# UTR 3# UTR 3# UTR 3# UTR 3# UTR CGAGCGGA[C/T]CATGAGGT CGGGTGTG[A/G]TGGCACACG ATTAAGAAAG[A/G]TTTTCAACGC GCAGGAAT[G/C]TGTCATTT AGTGGATA[A/G]ATCAGTGC GATGAATG[C/A]TTTATTGTA ATGCTTTAT[T/A]GTATCAGC TTTATTGTA[INS]TCAGCAATA TTAAATGTA[A/T]GTGGATGT CAAGAGGT[A/G]AGAGTTT AAAGGTCCT[C/G]CTGGACC TGTCCATTC[INS-TTA]TATTATATT GGGGAAAA[G/A]GGGAGTG TTTTTTTTT[A/T]ACTTTACAGC TTTACAGCTCC[A/G]TTTACGA TGCATCATATT[T/G]TCATTCAC TTTAGATTAC[A/G]GGATTAAT ACACCTTCAT[G/A]CTTACTAT ACTTATATGC[T/C]GAATTGAA AAAGGCCAA[G/C]GGTAATAG ATCTCAATGT[G/A]CAATAACTTG C/T A/G A/G G/C A/G C/A T/A 15 bp INDEL A/T A/G C/G 3 bp INDEL G/A A/T A/G T/G A/G G/A T/C G/C G/A rs416748 rs433235 NA rs13306541 NA NA rs11274081 rs414580 rs13306550 rs3747531 rs3036811 rs13306540 NA NA rs12677608 rs12678016 rs12675467 rs13306544 rs13306546 rs918 a 22 34 34 59 59 69 70 70 70 70 70 110 026 742 725 637 468 462 458 196 9534 850 504 639 346 360 888 006 032 116 163 392 Allele frequency among 86 subjects (66 cases and 20 non-cases). Frequency of 155 alleles. c Novel variants identified in this study. b Tag SNP Common name PRO3 INDEL1 X X P275A INDEL7 X P346P X 3# UTR 70006 Allele frequencya (%) C A G C G A A þ A G G A T G G G A C C A 38.6 38.1 8.9 0.6 10.6 7.4 7.5 7.5 36.1 11.4 48.1 55.5 1.2 17.9 8.9 3.0 20.0 20.0 20.0 20.0 20.0 Allele frequencyb (%) G 13.2 þ 12.3 G 37.0 45.9 G 10.4 G 14.4 MSR1 variants and the risks of prostate cancer and benign prostatic hyperplasia Fig. 1. Twenty-one sequence variants in the MSR1 gene in the Shanghai population. Table II. Selected characteristics for cancer cases, BPH, and controls Selected characteristic Control Total, n Age, mean (SD) Education, n (%) None Elementary Middle school High school Graduate and above Marital status, n (%) Married Living together Widowed Divorced Never married Smoking, n (%) Non-smoker Former smoker Current smoker Drinking, n (%) Non-drinker Former drinker Current drinker Differentiation, n (%) Cannot be assessed Well differentiated Moderately differentiated Poorly differentiated Missing data Clinical stage, n (%) Unstaged Localized Regional Remote Total PSA, median Total calories, mean (SD) WHR, mean (SD) Body mass index (kg/m2), mean (SD) Insulin resistance (HOMA-IR), mean (SD) 155 70.8 (8.1) 130 71.9 (7.6) 21 (13.5) 56 (36.1) 38 (24.5) 23 (14.8) 17 (11.0) 9 (6.9) 47 (36.2) 24 (18.5) 25 (19.2) 25 (19.2) P 5 0.13 11 44 24 25 26 139 (89.7) 3 (1.9) 11 (7.1) 1 (0.6) 1 (0.6) 117 (90.0) 0 (0.0) 11 (8.5) 2 (1.5) 0 (0.0) P 5 0.49 128 (98.5) 0 (0.0) 2 (1.5) 0 (0.0) 0 (0.0) P 5 0.05 52 (33.5) 42 (27.1) 61 (39.4) 54 (41.5) 34 (26.2) 42 (32.3) P 5 0.34 66 (50.8) 24 (18.5) 40 (30.8) P 5 0.01 86 (55.5) 10 (6.5) 59 (38.1) 93 (71.5) 15 (11.5) 22 (16.9) P 5 0.0003 84 (64.6) 15 (11.5) 31 (23.8) P 5 0.02 Cancer P 5 0.28 BPH 130 68.6 (6.0) (8.5) (33.8) (18.5) (19.2) (20.0) P 5 0.008 P 5 0.11 24 (18.5) 10 (7.7) 40 (30.8) 53 (40.8) 3 (2.3) 1.5 2330.0 (807.0) 0.89 (0.06) 21.7 (3.1) 1.8 (2.0) 1 (0.8) 47 (36.2) 41 (31.5) 41 (31.5) 84.0 2426.1 (619.5) 0.91 (0.05) 21.5 (2.9) 4.8 (8.1) P P P P 5 5 5 5 0.26 0.005 0.62 0.0001 6.8 2500.3 (670.0) 0.90 (0.05) 22.4 (3.6) 3.7 (6.5) P P P P 5 5 5 5 0.05 0.06 0.08 0.002 P value for t-test. P values of Fisher’s exact test comparing control group to cancer and BPH groups separately. deletion had a 67% reduction in risk (95% 0.16–0.68). Subjects with both the P275 G allele and INDEL7 deletion had 78% reduced risk of localized prostate cancer (95% CI 0.10–0.50) Further adjustment for education, body mass index and WHR did not materially change the results. We found no association between MSR1 polymorphisms and BPH risk. Among control subjects, strong LD was present for four of six markers (PRO3, INDEL1, P275A and INDEL7), with pairwise D’ values ranging from 0.73 to 1.0. Based on these four tagging variants (in the order of P275A, INDEL7, P346P and 3# UTR 70006), we inferred four major haplotypes (Table IV): (i) C-()-A-A, (ii) G-(þ)-A-A, (iii) C-(þ)-A-A and (iv) C-()-G-G, which accounted for .90% of the all possible haplotype variation (16 altogether) (Table IV). The haplotype frequencies were significantly different between cancer cases and controls, with a global P value of 0.01 for advanced cancer and 0.004 for localized cancer. When specific haplotypes were examined, the haplotype G-(þ)-A-A was associated with a 68% reduced risk of localized cancer (95% CI 0.13–0.59; global 2533 A.W.Hsing et al. 2534 Table III. ORs and 95% CIs for prostate cancer and BPH in relation to six MSR1 variants Variant (position) and genotypes Total PRO3 (14 742) AA AG GG AG or GG INDEL1 (14 458)b þ þþ þ or þþ P275A (22 850) CC CG GG P trend CG or GG INDEL7 (34 504)c þ þþ P trend þ or þþ P346P (59 360) AA AG GG AG or GG 3# UTR (70 006) AA AG GG AG or GG Controls Total prostate cancer a a Advanced prostate cancer a 95% CI 108 22 0 22 1.00 0.62 — 0.61 Reference 0.34–1.14 — 0.33–1.10 Reference 0.41–1.59 — 0.52–1.87 105 23 0 23 1.00 0.74 — 0.72 Reference 0.41–1.36 — 0.39–1.31 1.00 1.00 1.31 Reference 0.55–1.82 0.56–3.10 1.00 0.86 1.48 Reference 0.50–1.46 0.69–3.17 1.06 0.62–1.88 50 54 21 0.55 75 0.97 0.59–1.61 1.00 1.13 1.56 Reference 0.57–2.23 0.68–3.59 Reference 0.42–1.37 0.88–3.71 1.23 0.64–2.36 33 54 36 0.13 90 1.00 0.76 1.81 0.16–0.68 17 45 18 0.31 36 0.98 0.56–1.72 1.00 0.65 — 0.61 Reference 0.25–1.70 — 0.23–1.57 74 7 0 7 1.00 0.43 — 0.40 Reference 0.18–1.05 — 0.17–0.97 109 20 1 21 1.00 0.86 — 0.84 Reference 0.45–1.65 — 0.45–1.59 1.00 0.57 — 0.55 Reference 0.22–1.46 — 0.23–1.34 56 23 0 23 1.00 1.50 — 1.26 Reference 0.80–2.81 — 0.68–2.32 90 35 3 38 1.00 1.45 — 1.31 Reference 0.81–2.56 — 0.76–2.26 N 155 130 112 38 1 39 96 26 5 31 1.00 0.81 — 0.94 Reference 0.46–1.43 — 0.54–1.62 34 11 1 12 1.00 0.96 — 1.02 Reference 0.44–2.08 — 0.48–2.17 62 14 4 18 1.00 0.67 — 0.84 Reference 0.34–1.34 — 0.44–1.60 115 35 1 36 96 27 5 32 1.00 0.93 — 1.07 Reference 0.52–1.65 — 0.62–1.85 34 11 1 12 1.00 1.06 — 1.12 Reference 0.48–2.31 — 0.52–2.39 61 15 4 19 1.00 0.80 — 0.99 62 48 15 0.16 63 1.00 0.60 0.78 Reference 0.36–1.00 0.35–1.66 1.00 0.24 0.29 Reference 0.11–0.54 0.08–1.07 0.63 0.39–1.02 32 10 3 0.001 13 0.25 0.12–0.52 29 38 12 0.63 50 1.00 0.69 0.89 Reference 0.39–1.21 0.43–1.85 Reference 0.15–0.70 0.12–1.05 0.74 0.43–1.26 21 16 5 0.01 21 1.00 0.33 0.35 101 38 61 23 0.60 84 0.33 120 27 2 29 116 13 0 13 1.00 0.51 — 0.47 Reference 0.25–1.03 — 0.23–0.96 41 6 0 6 112 31 6 37 95 29 1 30 1.00 1.11 — 0.96 Reference 0.62–1.97 — 0.55–1.67 38 6 1 7 90 37 86 25 95% CI N OR 95% CI 47 OR, odds ratio. a Adjusted for age. P , 0.05; P , 0.01. b ‘þ’ and ‘’ denote with and without the 15 bp sequence ‘QAATGCTTTATTGTA’, respectively. c ‘þ’ and ‘’ denote with and without the 3 bp sequence ‘TTA’, respectively. BPH ORa N 56 72 18 OR Localized prostate cancer N OR 95% CI 82 N 130 MSR1 variants and the risks of prostate cancer and benign prostatic hyperplasia Table IV. ORs and 95% CIs for prostate cancer and BPH in relation to common MSR1 haplotypes Haplotype Controls % Total prostate cancer % Four variantsb Common haplotypes C-()-A-A 44.2 G-(þ)-A-A 32.4 C-(þ)-A-A 7.3 C-()-G-G 6.3 Rare haplotypes 9.8 Six variantsd Common haplotypes A-()-C-()-A-A 34.3 A-()-G-(þ)-A-A 32.0 A-()-C-(þ)-A-A 6.2 G-(þ)-C-()-A-A 9.4 A-()-C-()-G-G 6.2 Rare haplotypes 11.9 a OR Localized prostate cancer a OR Advanced prostate cancer BPH 95% CI % 95% CI % 95% CI % OR OR 95% CI 49.4 1.00 23.2 0.67 12.5 1.33 — — 14.9 0.82 P 5 0.10c Reference 0.43–1.05 0.71–2.50 — 0.50–1.35 63.2 1.00 13.7 0.28 12.4 0.94 4.0 0.37 6.7 0.53 P 5 0.004c Reference 0.13–0.59 0.40–2.20 0.08–1.71 0.20–1.42 41.0 1.00 28.8 1.07 12.7 1.72 — — 17.5 1.17 c P 5 0.01 Reference 0.63–1.80 0.83–3.56 — 0.66–2.06 39.2 1.00 29.8 1.11 14.2 1.99 5.7 1.00 11.1 1.39 P 5 0.26c Reference 0.72–1.71 1.06–3.76 0.43–2.32 0.74–2.62 39.4 1.00 22.7 0.66 11.5 1.41 10.2 1.07 — — 16.2 0.81 P 5 0.15c Reference 0.41–1.05 0.69–2.89 0.57–2.02 — 0.49–1.34 53.3 1.00 13.8 0.27 12.4 1.04 9.8 0.81 — — 10.7 0.39 P 5 0.002c Reference 0.13–0.58 0.42–2.57 0.32–2.09 — 0.18–0.86 31.1 1.00 28.0 1.09 11.1 1.82 10.2 1.31 — — 19.6 1.25 c P 5 0.67 Reference 0.62–1.90 0.78–4.21 0.63–2.72 — 0.69–2.24 29.5 1.00 30.4 1.16 14.5 2.21 8.1 0.90 6.1 1.07 11.4 1.24 P 5 0.29c Reference 0.73–1.83 1.12–4.35 0.44–1.83 0.47–2.43 0.66–2.38 OR, odds ratios. a Adjusted for age. b Haplotypes of four tagging variants (in the order of P275A, INDEL7, P346P and 3# UTR 70006). c _ Global test, OR and 95% CI adjusted for age (categorically, G65, 66–75 and 75þ). d Haplotypes of six variants (in the order of PRO3, INDEL1, P275A, INDEL7, P346P and 3# UTR 70006). P value 5 0.004), relative to the most frequent haplotype (C-()-A-A). In contrast, the haplotype C-(þ)-A-A was associated with a 1.9-fold risk of BPH, although the global test was not statistically significant (P 5 0.26). Since the two non-tagging variants (PRO3 and INDEL1) were not associated with prostate cancer risk individually, including these two in the haplotype analysis did not substantially change the haplotype frequency distribution and risk estimates. There was also no interaction between these two variants with haplotypes inferred by the four tagging variants (data not shown). In addition, there was no interaction among any of the MSR1 variants with body mass index, WHR or insulin resistance in the study. Discussion In this population-based study of Chinese men, we used a haplotypetagging approach based on sequencing data to provide a comprehensive assessment of the association between MSR1 variation and prostate cancer risk. We found that the MSR1 P346P variant was associated with a 53% reduced risk of prostate cancer and the P275A and INDEL7 variants were associated with a reduced risk of localized prostate cancer. Haplotype data further support a link between the P275A and INDEL7 variants and localized prostate cancer. However, we found no evidence of a clear link between MSR1 variation and BPH risk. The P346P variant, associated with total and advanced prostate cancer risk in the study, is a synonymous polymorphism in exon 10. This novel variant has not been reported previously and its function is unclear. Of the 21 variants identified by sequencing in this population, P346P was not strongly linked to any of the other SNPs. It is unclear whether the association of this variant is linked to other unmeasured variants. The P275A (a missense change in exon 6) and INDEL7 (4 bp insertion/deletion of ‘TTA’ in intron 7) variants are more common and have been reported to be associated with prostate cancer in Caucasians, although the evidence is inconclusive. In our study, both variants were associated with a significantly reduced risk of localized prostate cancer risk. The magnitude of the observed associations was similar to the pooled estimated odds ratios of .1000 Caucasian cases and controls in a recent meta-analysis (22), although the prevalence of the minor allele is quite different. For example, the prevalence of the G allele in the P275A marker was 24% in the meta-analysis of mostly Caucasian subjects and 58% in our study. The prevalence of the INDEL 7 ‘þ/ or þ/þ’ was 10.2% in the meta-analysis and 70.9% in our study. In our study, the risk of prostate cancer associated with P275A and INDEL7 was more pronounced but did not reach significant interaction, perhaps due to small numbers. It is noteworthy that the specific haplotype, G-(þ)-A-A, that was associated with a significantly reduced risk contained the minor allele of these two markers, further suggesting a potential role for these two variants. Given the nature of the MSR1 protein, the associations between MSR1 P275A and INDEL7 variants and prostate cancer are biologically plausible. Although the specific functions of these variants are unclear, preliminary functional data suggest that INDEL7 (the 15 bp insertion/ deletion polymorphism in intron 7) affects transcription of the MSR1 gene (data not shown). The missense change of P275A could affect the function of the MSR1 protein because it changes a conserved residue in the first Gly-X-Y repeat of the collagenous domain of the protein (23). Thus, it is possible that these variants may be involved in binding, internalization and processing of a wide range of macromolecules, thereby helping macrophages clean-up cellular debris from bacterial infection or damaged fats or lipids (23). Recent studies also suggest that the degree of macrophage infiltration is associated with prostate cancer prognosis, further strengthening the link between MSR1 and prostate cancer (19). In addition, inflammation and other features such as proliferative regeneration of prostate epithelium in the presence of increased oxidative stress that are associated with MSR1 expression contribute to the development of prostate cancer. A recent meta-analysis examining the results of three rare variants (R293X, S41T and D174Y) and five common variants (PRO3, INDEL1, IVS5-59, P275A and INDEL7) in eight studies revealed that most findings are inconsistent and that a mild risk of prostate cancer is associated with the R293X variant in Caucasian men and with the D174Y variant found only in African American men (23). Both R293X and D174Y are rare variants (,2%) and D174Y is found only in African Americans. In our study of 86 samples, we did not observe any variation in these two markers. Clearly, there is substantial allelic variation in the MSR1 gene by race. For example, although the INVS5-59 marker is considered a common variant in Caucasians, we did not observe this variant in our study. In addition, the frequency for the homozygous GG genotype in the PRO3 marker was 14% in a Caucasian population but only 0.67% in our population (21). In one study, 12.9% of the Caucasians had the P275A CG genotype, whereas 2535 A.W.Hsing et al. 48% of the Chinese men in our study harbored this genotype. Whether racial differences in MSR1 variation partly explain the large racial differences in prostate cancer risk warrants further investigation. Strengths and limitations of the study should be noted. Selection and survival biases should be minimal, since .90% of the eligible cases and controls participated in the study. In addition, demographic and clinical factors did not differ between interviewed subjects who did and did not participate in the blood collection. Although not all study subjects who donated blood had sufficient DNA for genotyping, both clinical and selected characteristics did not differ between subjects with or without sufficient DNA, further suggesting little selection bias. Misclassification of genotype is also probably minimal as evidenced by the .99% concordance in duplicate samples. Our assessment of gene–environment interaction was limited by small numbers, such that we may have been unable to detect modest true interactions. In addition, the population in Shanghai is ethnically homogeneous, so our results may not be generalizable to other populations. However, the effect of population stratification (i.e. different genotype frequencies observed may reflect different genetic background in case subjects and control subjects) is probably to be minimal. In summary, this population-based study, conducted in a low-risk population for prostate cancer, suggests that variants in the MSR1 gene are associated with a lower risk of prostate cancer and provides some support for the role of inflammation in prostate cancer. Although we are unable to generalize our results directly to other populations, similar underlying biological mechanisms may exist for other racial/ ethnic groups. Larger studies in other populations are needed to confirm our results. Funding Intramural Research Program of the National Institute of Health, National Cancer Institute. Acknowledgements We thank J.Cheng of the Shanghai Cancer Institute for specimen collection and processing; collaborating hospitals and urologists for data collection; pathologists for pathology review; L.Lannom, J.Heinrich and M.Bendel of Westat for study management and data preparation; G.Yuan of Information Management Systems and S.Niwa of Westat for data management and analysis and J.Koci of the Scientific Applications International Corporation for management of the biologic samples. Conflict of Interest Statement: None declared. References 1. Lichtenstein,P. et al. (2000) Environmental and heritable factors in the causation of cancer—analyses of cohorts of twins from Sweden, Denmark, and Finland. N. Engl. J. Med., 343, 78–85. 2. Stanford,J.L. et al. (2001) Familial prostate cancer. Epidemiol. Rev., 23, 19–23. 3. Hsing,A.W. et al. (2006) Prostate cancer epidemiology. Front. 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