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Nat Genet. Author manuscript; available in PMC Mar 27, 2012.
Published in final edited form as:
Published online Aug 2, 2009. doi:  10.1038/ng.421
PMCID: PMC3313685
NIHMSID: NIHMS362193
Genetic variation in the prostate stem cell antigen gene PSCA confers susceptibility to urinary bladder cancer
Xifeng Wu,1 Yuanqing Ye,1 Lambertus A Kiemeney,2,3,4 Patrick Sulem,5 Thorunn Rafnar,5 Giuseppe Matullo,6,7 Daniela Seminara,8 Teruhiko Yoshida,9 Norihisa Saeki,9 Angeline S Andrew,10 Colin P Dinney,11 Bogdan Czerniak,12 Zuo-feng Zhang,13 Anne E Kiltie,14 D Timothy Bishop,15 Paolo Vineis,7,16 Stefano Porru,17 Frank Buntinx,18,19 Eliane Kellen,20,21 Maurice P Zeegers,21,22 Rajiv Kumar,23 Peter Rudnai,24 Eugene Gurzau,25 Kvetoslava Koppova,26 Jose Ignacio Mayordomo,27,29 Manuel Sanchez,28 Berta Saez,30 Annika Lindblom,31 Petra de Verdier,32 Gunnar Steineck,33 Gordon B Mills,34 Alan Schned,35 Simonetta Guarrera,7 Silvia Polidoro,7 Shen-Chih Chang,13 Jie Lin,1 David W Chang,1 Katherine S Hale,34 Tadeusz Majewski,12 H Barton Grossman,11 Steinunn Thorlacius,5 Unnur Thorsteinsdottir,5 Katja K H Aben,4 J Alfred Witjes,3 Kari Stefansson,5 Christopher I Amos,1,36 Margaret R Karagas,8,36 and Jian Gu1,36
1Department of Epidemiology, University of Texas M.D. Anderson Cancer Center, Houston, Texas, USA.
2Department of Epidemiology, Biostatistics & Health Technology Assessment, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands.
3Department of Urology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands.
4Comprehensive Cancer Center East, Nijmegen, The Netherlands.
5deCODE Genetics, Reykjavik, Iceland.
6Department of Genetics, Biology and Biochemistry, University of Torino, Torino, Italy.
7ISI (Institute for Scientific Interchange) Foundation, Villa Gualino, Torino, Italy.
8Epidemiology and Genetics Research Program, Division of Cancer Control and Population Sciences, National Cancer Institute, Bethesda, Maryland, USA.
9Genetics Division, National Cancer Center Research Institute, Tokyo, Japan.
10Department of Community and Family Medicine, Section of Biostatistics and Epidemiology, Dartmouth Medical School, Hanover, New Hampshire, USA.
11Department of Urology, University of Texas M.D. Anderson Cancer Center, Houston, Texas, USA.
12Department of Pathology, University of Texas M.D. Anderson Cancer Center, Houston, Texas, USA.
13Department of Epidemiology, School of Public Health, University of California Los Angeles, Los Angeles, California, USA.
14Section of Experimental Oncology, Institute of Molecular Medicine, St. James’s University Hospital, Leeds, UK.
15Section of Epidemiology & Biostatistics Leeds Institute of Molecular Medicine, St. James’s University Hospital, Leeds, UK.
16Department of Epidemiology and Public Health, Imperial College, London, UK.
17Department of Experimental and Applied Medicine, University of Brescia, Brescia, Italy.
18Department of General Practice, Catholic University of Leuven, Leuven, Belgium.
19Department of General Practice, Maastricht University, Maastricht, The Netherlands.
20Leuven University Centre for Cancer Prevention, Leuven, Belgium.
21Department of Public Health and Epidemiology, University of Birmingham, Birmingham, UK.
22Department of Complex Genetics, Cluster of Genetics and Cell Biology, Nutrition and Toxicology Research Institute, Maastricht University, Maastricht, The Netherlands.
23Division of Molecular Genetic Epidemiology, German Cancer Research Center, Heidelberg, Germany.
24National Institute of Environmental Health, Budapest, Hungary.
25Environmental Health Center, Cluj, Romania.
26State Health Institute, Banska Bystrica, Slovakia.
27Division of Medical Oncology, University Hospital, Zaragoza, Spain.
28Division of Urology, University Hospital, Zaragoza, Spain.
29Nanotechnology Institute of Aragon, Health Science Institute, Zaragoza, Spain.
30Health Science Institute, Zaragoza, Spain.
31Department of Molecular Medicine and Surgery, Karolinska Institutet, Stockholm, Sweden.
32Department of Oncology and Pathology, Karolinska Institutet, Stockholm, Sweden.
33Department of Oncology, Sahlgrenska University Hospital, Goteborg, Sweden.
34Department of Systems Biology, University of Texas M.D. Anderson Cancer Center, Houston, Texas, USA.
35Department of Pathology, Dartmouth Medical School, Hanover, New Hampshire, USA.
Correspondence should be addressed to X.W. (xwu/at/mdanderson.org)
36These authors contributed equally to this work.
We conducted a genome-wide association study on 969 bladder cancer cases and 957 controls from Texas. For fast-track validation, we evaluated 60 SNPs in three additional US populations and validated the top SNP in nine European populations. A missense variant (rs2294008) in the PSCA gene showed consistent association with bladder cancer in US and European populations. Combining all subjects (6,667 cases, 39,590 controls), the overall P-value was 2.14 × 10−10 and the allelic odds ratio was 1.15 (95% confidence interval 1.10–1.20). rs2294008 alters the start codon and is predicted to cause truncation of nine amino acids from the N-terminal signal sequence of the primary PSCA translation product. In vitro reporter gene assay showed that the variant allele significantly reduced promoter activity. Resequencing of the PSCA genomic region showed that rs2294008 is the only common missense SNP in PSCA. Our data identify rs2294008 as a new bladder cancer susceptibility locus.
Bladder cancer is the fourth most common cancer in men in the United States, with an estimated 68,810 new cases and 14,410 deaths from this disease in 2008 (ref. 1). The main environmental risk factors for bladder cancer are cigarette smoking and occupational exposure. There is also compelling evidence for a genetic component to the etiology of bladder cancer. In a large twin study, it was estimated that inherited genetic susceptibility contributes to 31% of bladder cancer risk2. Case reports have described familial clustering of bladder cancer3. Epidemiological studies showed that the risk of the disease increased by 50%–100% in first-degree relatives of individuals with bladder cancer47. However, genetic loci that account for most familial risk of bladder cancer remain elusive. A recent segregation analysis suggested a ‘no major gene’ model8. Candidate gene association studies have shown that NAT2 slow acetylator and GSTM1 null genotypes are associated with increased bladder cancer risks9. A recent genome-wide association study (GWAS) identified two bladder cancer susceptibility loci10. To identify additional bladder cancer susceptibility loci, we conducted a GWAS using the Illumina HumanHap610 Beadchip.
We performed the primary screen using an ongoing case-control study in Texas. We restricted study participants to self-reported Caucasians to minimize confounding by ethnicity. After applying strict quality control criteria (see Online Methods for details) to remove problematic samples and SNPs, we analyzed 556,429 SNPs in 969 cases and 957 controls. A quantile–quantile plot of observed versus expected χ2 test statistics showed no evidence for inflation of chi-squared tests (inflation factor λ = 1.002; Supplementary Fig. 1). None of the SNPs reached genome-wide significance at this stage (Supplementary Fig. 2). After we removed highly linked SNPs, three SNPs had a P-value < 10−5 and 50 SNPs showed a P-value < 10−4 (Supplementary Table 1). We adjusted the results for population substructure using Cochran-Mantel-Haenszel tests, eigenvectors and permutation tests, and the P-values were consistent (Supplementary Table 1).
We used three more US sets to perform a fast-track replication of the top 50 SNPs (P < 10−4) and the top 10 additional SNPs in 8q24 (P < 5 × 10−3), a region associated with genetic susceptibility to several cancers1014. One SNP (rs2294008) was consistent across the US discovery and the replication sets (P = 7.34 × 10−4 and 3.53 × 10−5, respectively; Table 1). The P-value was 1.07 × 10−7 in the combined US populations (Supplementary Table 2). We next used nine European populations to replicate this SNP. The overall P-value for the combined European populations was 9.83 × 10−5. Combining all US and European subjects (6,667 cases, 39,590 controls), the P-value was 2.14 × 10−10 (Table 1). The allelic odds ratio (OR) was 1.15 (95% confidence interval (CI), 1.10–1.20). There was no significant heterogeneity among the ORs of all the populations (P for heterogeneity = 0.423). We also performed a multivariable logistic regression analysis, adjusting for age, gender and smoking status (5,038 cases and 9,363 controls, excluding three European populations with missing individual age or smoking data for controls; Table 2). The OR for individuals carrying one copy of the variant allele (T) was 1.30 (95% CI, 1.18–1.42) and for those carrying two copies was 1.40 (95% CI, 1.25–1.56). We tested the equality of the ORs for heterozygous and homozygous carriers of the variant allele and observed a nonsignificant P-value of 0.14, suggesting a dominant model. The OR for individuals carrying at least one variant allele was 1.33 (95% CI, 1.22–1.45). We also performed stratified analyses for age, gender and smoking status and observed similar associations across different strata (Table 2). The association was also similar in superficial and invasive bladder cancer (for US populations only; data not shown). The risk allele (T) frequency is 46%, and individuals carrying the homozygous risk genotype (TT) account for about 22% of the control population.
Table 1
Table 1
Summary results of rs2294008 in discovery and replication populations
Table 2
Table 2
Multivariate logistic regression analysis of rs2294008
rs2294008 is a missense SNP located in exon 1 of the PSCA gene. Linkage disequilibrium (LD) analysis of all HapMap SNPs in the vicinity of rs2294008 showed that it maps to an 11-kb LD block on chromosome 8q24 (Fig. 1). We imputed genotypes within 1 Mb of rs2294008 for SNPs in the HapMap database but not on the HumanHap610 chip. rs2294008 was among the top SNPs showing the strongest association (Fig. 1a). To identify unknown variants, we resequenced the genomic region of PSCA in 106 individuals of European ancestry. We found 27 SNPs, 23 of which had a minor allele frequency (MAF) > 5% (Supplementary Table 3). Several of these SNPs are listed in the dbSNP database, but this study is the first to validate them (SNPs no. 4, no. 5, no. 9 and no. 16). We also validated an insertion/deletion polymorphism (no. 7) and identified two new low-frequency SNPs (no. 1 and no. 2). All of the high frequency SNPs are in strong LD (D′ > 0.9) with rs2294008. We genotyped seven of these SNPs in our discovery set and observed nearly identical ORs compared to rs2294008 (Supplementary Table 4).
Figure 1
Figure 1
The 8q24 locus encompassing rs2294008. (a) Results of SNP association from the genome-wide screen. Observed results from genotyped SNPs are in red and imputed results are in black. All known genes in this region are also shown. (b) LD structure of this (more ...)
A recent study15 found that the T allele of rs2294008 resulted in a significant reduction in transcriptional activity of the PSCA promoter in gastric cell lines. We obtained four constructs representing the top four 5′ upstream haplotypes (including promoter and exon 1 region) in our populations (Fig. 2). Except for the wild-type haplotype, the other three haplotypes all contained the variant T allele at rs2294008. We found that in three different bladder cancer cell lines (UC1, UC3 and UC13) the T allele-containing haplotypes showed significantly lower promoter activity (P < 0.001 for all comparisons between wild-type and the other three constructs; Fig. 2b). Furthermore, substitution of a single nucleotide in the wild-type haplotype (rs2294008 C to T) significantly reduced promoter activity, whereas a single substitution of rs2294008 T to C in UP-H1 increased promoter activity (Fig. 2c). These results are in complete concordance with data in gastric cancer15, providing compelling evidence that rs2294008 is a functional variant in vitro. We then used real-time PCR to detect PSCA mRNA expression in nine bladder cancer cell lines. We found that UC9 (TT genotype) had the highest expression, whereas three CT-genotype cell lines (UC1, UC5 and UC7) showed intermediate expression, and three CC genotype cell lines (UC3, UC13 and UC17) and two TT cell lines (UC12 and UC18) showed very low expression (data not shown). This lack of genotype–expression correlation in cancer cell lines is likely due to somatic changes because PSCA is upregulated in most bladder tumors16. By contrast, expression of PSCA in most normal tissues is very low, except for prostate, esophagus and stomach17,18. We did not have normal bladder tissues and thus could not measure PSCA expression in them. We used 135 lymphoblastoid cell lines with different genotypes of rs2294008 (TT, CT and CC) and attempted to determine PSCA mRNA expression, but PSCA mRNA was not detectable (data not shown). Finding sufficient numbers of normal tissue samples, particularly of tissues abundantly expressing PSCA (prostate, esophagus and stomach), is warranted in order to compare endogenous PSCA expression for the different rs2294008 genotypes and determine whether the T allele reduces PSCA gene expression in vivo.
Figure 2
Figure 2
In vitro reporter assay of the four most frequent haplotypes of the PSCA 5′ upstream region (nucleotides −3236 to +28). (a) The four SNPs that comprise these haplotypes are rs2976387, rs6471587, rs13262164 and rs2294008. The frequencies (more ...)
Previous GWASs have identified several independent susceptibility alleles at 8q24 for prostate, breast and colorectal cancers1114. None of these SNPs showed significant associations with bladder cancer risk in our discovery set (Table 3). The power calculation showed that we had adequate power (>80%) to detect an OR of 1.33 for an additive model and 1.37 for a dominant model when the MAF was greater than 0.10 in our discovery set. These 8q24 SNPs have also been examined in the nine European studies previously10, and the results were consistent with ours. The 8q24 SNP (rs9642880) identified from the previous bladder cancer GWAS10 was validated in our US populations (OR = 1.14; 95% CI, 1.06–1.22; P = 2.1 × 10−4; Table 3). When we performed a meta-analysis of all the US and European populations, the OR was 1.19 (95% CI, 1.14–1.24; P = 1.35 × 10−14; Supplementary Fig. 3a). We also validated a second SNP (rs710521) at 3q28 (OR = 1.16, 95% CI, 1.10–1.22; P = 1.09 × 10−8; Supplementary Fig. 3b).
Table 3
Table 3
Previously reported cancer susceptibility alleles on 8q24 and bladder cancer riska
PSCA was initially identified as a prostate-specific cell-surface marker17. PSCA is overexpressed in prostate cancer, and the level of expression increases with tumor grade and stage17,19. It may be involved in cell proliferation, and migration as monoclonal antibodies to PSCA could inhibit tumor growth and metastasis formation in animal models20,21. PSCA is expressed at low levels in the transitional epithelium of normal bladder but is overexpressed in the majority of bladder cancers16. Immunocytochemical analysis of PSCA in voided urine was shown to be a complementary marker for cytological diagnosis of bladder cancer22. The expression level of PSCA was an independent predictor of recurrence in superficial bladder cancer23. These observations provide biological plausibility for the association between PSCA and bladder cancer risk.
rs2294008 is a missense variation that alters the start codon of PSCA. The first report on PSCA protein17 was based on the cDNA sequence carrying the T allele (the longer, 123-amino-acid protein). In the cDNA sequence carrying the C allele, the translation is predicted to start from the next ATG codon, resulting in a nine-amino-acid truncation. In vitro translation showed that cDNA sequences containing these two alleles produced proteins of almost the same size, compatible with a difference of only nine amino acids15. PSCA is a member of the Thy-1/Ly-6 family of glycosylphosphatidylinositol (GPI)-anchored cell surface proteins. All GPI-anchored proteins undergo complex cellular processing before becoming mature protein24. The 123-amino-acid primary PSCA translation product, like other GPI-anchored proteins, has two signal sequences: an N-terminal signal sequence (20 amino acids) for endoplasmic reticulum targeting and a C-terminal sequence that directs the GPI-anchoring15. Both these signal sequences are removed in the endoplasmic reticulum, and the GPI-anchored form is then carried through a secretory pathway to the cell surface. Therefore, the mature proteins are the same for the two alleles of rs2294008. However, because rs2294008 changes the length of the N-terminal signal peptide, it may change protein folding, intracellular modifications and/or trafficking of PSCA proteins. An in vitro assay using EYFP (enhanced yellow fluorescent protein)-fused PSCA expression vectors transfected into HSC60 cells found no detectable difference in the amount or distribution of the EYFP-fused short and long forms of PSCA protein15. In vivo measurements of PSCA protein have been challenging owing to the low expression of PSCA in most normal tissues and the uncertainty of the sensitivity and specificity of the available PSCA antibody. Furthermore, if the truncated N-terminal signal peptide affects protein folding and/or intracellular modifications, then an antibody could recognize mature PSCA protein processed from the long and short signal sequences differently even though the mature protein sequence is the same. Future efforts are needed to determine the protein expression and physiological function of PSCA and the functional consequence of rs2294008 in vivo.
rs2294008 was recently identified as a susceptibility allele for diffuse-type gastric cancer in Japan15. Notably, the T allele has higher frequency in individuals of European ancestry (MAF = 0.46) and Koreans (MAF = 0.46) than in Chinese (MAF = 0.26) and Africans (MAF = 0.25), based on HapMap data; however, it is a major allele in Japanese (frequency = 0.62). The population genetic history of this SNP and why only Japanese possess a different minor allele remain to be explained. Whether rs2294008 is associated with gastric cancer or any other cancers in individuals of European descent warrants further investigation.
The T allele reduced the transcriptional activity of the PSCA promoter in vitro. It seems paradoxical that the T risk allele reduces transcription, whereas PSCA has been shown to be overexpressed in bladder tumors. Because the physiological function of PSCA and the functional impact of different N-terminal signal lengths on protein function are still unknown the functional consequence of the risk T allele in vivo is unclear, but it would be the cumulative result of transcriptional, translational and post-translational effects. All the previously identified cancer susceptibility variants in 8q24 in individuals of European descent are located near the MYC gene, and the causal variants and biologic mechanisms remain elusive. rs2294008 (position 143758933) is 15 Mb distal from the previous bladder cancer susceptibility locus (rs9642880, position 128787250) on 8q24, and these two SNPs are not in LD (D′ = 0.01, r2 = 0.00), suggesting that rs2294008 is an independent bladder cancer susceptibility locus on 8q24. Future functional studies are warranted to delineate the physiological role of PSCA and the biological mechanisms underlying the association of rs2294008 in PSCA with bladder carcinogenesis.
METHODS
Methods and any associated references are available in the online version of the paper at http://www.nature.com/naturegenetics/.
Supplementary Material
Wu Nat Gen 2009 Supplementary Data
ACKNOWLEDGMENTS
The study was partially supported by NIH grants U01 CA 127615 (X.W.), R01 CA 74880 (X.W.), P50 CA 91846 (X.W., C.P.D.), R01 CA 133996 (C.I.A), P42 ES07373 (M.R.K.) and R01 CA 57494 (M.R.K.), R01 CA 131335 (J.G.) and the Kleberg Center for Molecular Markers at MDACC. We thank the genotyping personnel, study coordinators and interviewers for performing experiments and recruiting participants. We are especially thankful for all the study participants who made the population-based research possible.
Footnotes
Note: Supplementary information is available on the Nature Genetics website.
AUTHOR CONTRIBUTIONS
Texas: X.W. conceived this study and established the M.D. Anderson bladder cancer study, supervised laboratory and statistical analyses and wrote the initial draft of the manuscript. Y.Y. supervised and performed statistical analyses. C.P.D., B.C. and H.B.G. were involved in patient recruitment. J.L. was involved in epidemiologic data collection and database management. D.W.C performed in vitro assays. T.M., G.B.M. and K.S.H. were involved in the validation genotyping. C.I.A. provided guidance in statistical analyses, assisted in the initial development of the research and contributed in manuscript preparation. J.G. was involved in the development of the research and study design, oversaw genotyping and in vitro assays and wrote the initial draft of the manuscript. Other sites: L.A.K. and T.R. organized and supervised the replication efforts in European populations. P.S. performed primary statistical analysis of European populations. G.M., A.E.K., D.T.B., P.V., S. Porru, F.B., E.K., M.P.Z., R.K., P.R., E.G., K.K., J.I.M., M.S., B.S., A.L., P.d.V., G.S., S.G., S. Polidoro, S.T., U.T., K.K.H.A., J.A.W. and K.S. were involved in the subject ascertainment, DNA collection or data collection of European populations. A.S.A., A.S., Z.-f.Z. and S.-C.C. were involved in the subject ascertainment, DNA collection or data collection of US populations. D.S. was involved in the study design and data interpretation. N.S. and T.Y. provided reporter constructs of PSCA promoters and were instrumental in studying and discussing the function of PSCA and rs2294008. M.R.K. assisted in the initial development of the research, established the New Hampshire bladder cancer case control study and contributed to manuscript preparation. All authors contributed to the final paper.
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