Yi-Ping Fu

Mapping of the UGT1A locus identifies an uncommon coding variant that affects mRNA expression and protects from bladder cancer

A recent genome-wide association study of bladder cancer identified the UGT1A gene cluster on chromosome 2q37.1 as a novel susceptibility locus. The UGT1A cluster encodes a family of UDP-glucuronosyltransferases (UGTs), which facilitate cellular detoxification and removal of aromatic amines. Bioactivated forms of aromatic amines found in tobacco smoke and industrial chemicals are the main risk factors for bladder cancer. The association within the UGT1A locus was detected by a single nucleotide polymorphism (SNP) rs11892031. Now, we performed detailed resequencing, imputation and genotyping in this region. We clarified the original genetic association detected by rs11892031 and identified an uncommon SNP rs17863783 that explained and strengthened the association in this region (allele frequency 0.014 in 4035 cases and 0.025 in 5284 controls, OR = 0.55, 95%CI = 0.44-0.69, P = 3.3 × 10(-7)). Rs17863783 is a synonymous coding variant Val209Val within the functional UGT1A6.1 splicing form, strongly expressed in the liver, kidney and bladder. We found the protective T allele of rs17863783 to be associated with increased mRNA expression of UGT1A6.1 in in-vitro exontrap assays and in human liver tissue samples. We suggest that rs17863783 may protect from bladder cancer by increasing the removal of carcinogens from bladder epithelium by the UGT1A6.1 protein. Our study shows an example of genetic and functional role of an uncommon protective genetic variant in a complex human disease, such as bladder cancer.

Genetic Variant as a Selection Marker for Anti–Prostate Stem Cell Antigen Immunotherapy of Bladder Cancer

A monoclonal antibody against prostate stem cell antigen (PSCA) has emerged as a novel cancer therapy currently being tested in clinical trials for prostate and pancreatic cancers, but this treatment is likely to be efficient only in patients with PSCA-expressing tumors. The present study demonstrates that a genetic variant (rs2294008) discovered by bladder cancer genome-wide association studies is a strong predictor of PSCA protein expression in bladder tumors, as measured by two-sided multivariable linear regression (P = 6.46×10−11; n = 278). The association pattern is similar in non-muscle-invasive tumors, stages Ta (P = 3.10×10−5; n = 173) and T1 (P = 2.64×10−5; n = 60), and muscle-invasive tumors, stages T2 (P =.01; n = 23) and T3/4 (P =.03; n = 22). The study suggests that anti-PSCA immunotherapy might be beneficial for bladder cancer patients with high tumor PSCA expression, which is statistically significantly associated with the presence of CT and TT genotypes of a common genetic variant, rs2294008. Future clinical studies will be needed to validate PSCA as a therapeutic target for bladder cancer.

Statistical tests for detecting associations with groups of genetic variants: generalization, evaluation, and implementation.

With recent advances in sequencing, genotyping arrays, and imputation, GWAS now aim to identify associations with rare and uncommon genetic variants. Here, we describe and evaluate a class of statistics, generalized score statistics (GSS), that can test for an association between a group of genetic variants and a phenotype. GSS are a simple weighted sum of single-variant statistics and their cross-products. We show that the majority of statistics currently used to detect associations with rare variants are equivalent to choosing a specific set of weights within this framework. We then evaluate the power of various weighting schemes as a function of variant characteristics, such as MAF, the proportion associated with the phenotype, and the direction of effect. Ultimately, we find that two classical tests are robust and powerful, but details are provided as to when other GSS may perform favorably. The software package CRaVe is available at our website (http://dceg.cancer.gov/bb/tools/crave).

The 19q12 Bladder Cancer GWAS Signal: Association with Cyclin E Function and Aggressive Disease

A genome-wide association study (GWAS) of bladder cancer identified a genetic marker rs8102137 within the 19q12 region as a novel susceptibility variant. This marker is located upstream of the CCNE1 gene, which encodes cyclin E, a cell-cycle protein. We performed genetic fine-mapping analysis of the CCNE1 region using data from two bladder cancer GWAS (5,942 cases and 10,857 controls). We found that the original GWAS marker rs8102137 represents a group of 47 linked SNPs (with r(2) ≥ 0.7) associated with increased bladder cancer risk. From this group, we selected a functional promoter variant rs7257330, which showed strong allele-specific binding of nuclear proteins in several cell lines. In both GWASs, rs7257330 was associated only with aggressive bladder cancer, with a combined per-allele OR = 1.18 [95% confidence interval (CI), 1.09-1.27, P = 4.67 × 10(-5)] versus OR = 1.01 (95% CI, 0.93-1.10, P = 0.79) for nonaggressive disease, with P = 0.0015 for case-only analysis. Cyclin E protein expression analyzed in 265 bladder tumors was increased in aggressive tumors (P = 0.013) and, independently, with each rs7257330-A risk allele (P(trend) = 0.024). Overexpression of recombinant cyclin E in cell lines caused significant acceleration of cell cycle. In conclusion, we defined the 19q12 signal as the first GWAS signal specific for aggressive bladder cancer. Molecular mechanisms of this genetic association may be related to cyclin E overexpression and alteration of cell cycle in carriers of CCNE1 risk variants. In combination with established bladder cancer risk factors and other somatic and germline genetic markers, the CCNE1 variants could be useful for inclusion into bladder cancer risk prediction models.

Abstract 944: Translational implications of the 19q12 bladder cancer GWAS signal for aggressive bladder cancer

Background: A genome-wide association study (GWAS) of bladder cancer identified a genetic marker rs8102137 within the 19q12 region as a novel susceptibility variant. This marker is located upstream of the CCNE1 gene, which encodes cyclin E, a cell cycle protein. Methods: Genetic fine mapping analysis of the CCNE1 region was performed using data from two bladder cancer GWAS, which included a total of 5,942 cases and 10,857 controls. Cyclin E protein expression was evaluated by immunohistochemistry analysis in 265 bladder tumors in relation to CCNE1 genetic variants and tumor characteristics. Functional effects of cyclin E over-expression were evaluated with cell cycle assays. Findings: The original GWAS marker rs8102137 represents a group of 47 linked SNPs (with r2≥0.7) associated with increased bladder cancer risk. From this group we selected a functional promoter variant rs7257330, which showed strong allele-specific binding of nuclear proteins in several cell lines. In both GWAS, rs7257330 was associated only with aggressive bladder cancer (muscle-invasive or high-grade non-muscle-invasive cancer), with a per-allele odds ratio (OR) =1.18 (95%CI=1.09-1.27, p=4.67×10-5). Cyclin E protein expression was increased in aggressive bladder tumors (p=0.013) and, independently, with each rs7257330-A risk allele (ptrend=0.024). Over-expression of recombinant cyclin E in cell lines caused significant acceleration of cell cycle. Conclusions: Molecular mechanisms linking the CCNE1 GWAS signal and aggressive bladder cancer risk are related to cyclin E over-expression and cell cycle regulation. In combination with other genetic and clinical markers, CCNE1 genetic variants may be translationally useful for early prediction of aggressive bladder cancer. Citation Format: Yi-Ping Fu, Indu Kohaar, Lee Moore, Petra Lenz, Jonine D. Figueroa, Wei Tang, Patricia Porter-Gill, Stephen Chanock, Stephen M. Hewitt, Debra T. Silverman, Nathaniel Rothman, NCI-GWAS Bladder Cancer Consortium, Ludmila Prokunina-Olsson. Translational implications of the 19q12 bladder cancer GWAS signal for aggressive bladder cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 944. doi:10.1158/1538-7445.AM2014-944

The 19q12 bladder cancer GWAS signal

A genome-wide association study (GWAS) of bladder cancer identified a genetic marker rs8102137 within the 19q12 region as a novel susceptibility variant. This marker is located upstream of the CCNE1 gene, which encodes cyclin E, a cell-cycle protein. We performed genetic fine-mapping analysis of the CCNE1 region using data from two bladder cancer GWAS (5,942 cases and 10,857 controls). We found that the original GWAS marker rs8102137 represents a group of 47 linked SNPs (with r(2) ≥ 0.7) associated with increased bladder cancer risk. From this group, we selected a functional promoter variant rs7257330, which showed strong allele-specific binding of nuclear proteins in several cell lines. In both GWASs, rs7257330 was associated only with aggressive bladder cancer, with a combined per-allele OR = 1.18 [95% confidence interval (CI), 1.09-1.27, P = 4.67 × 10(-5)] versus OR = 1.01 (95% CI, 0.93-1.10, P = 0.79) for nonaggressive disease, with P = 0.0015 for case-only analysis. Cyclin E protein expression analyzed in 265 bladder tumors was increased in aggressive tumors (P = 0.013) and, independently, with each rs7257330-A risk allele (P(trend) = 0.024). Overexpression of recombinant cyclin E in cell lines caused significant acceleration of cell cycle. In conclusion, we defined the 19q12 signal as the first GWAS signal specific for aggressive bladder cancer. Molecular mechanisms of this genetic association may be related to cyclin E overexpression and alteration of cell cycle in carriers of CCNE1 risk variants. In combination with established bladder cancer risk factors and other somatic and germline genetic markers, the CCNE1 variants could be useful for inclusion into bladder cancer risk prediction models.

Abstract 1658: Post-GWAS genetic and functional studies for bladder cancer: An uncommon protective synonymous coding variant within the UGT1A6 gene explains the GWAS signal and affects cellular detoxification

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Background: A recent genome-wide association study (GWAS) of bladder cancer identified a single nucleotide polymorphism (SNP), rs11892031, within the UGT1A gene cluster on chromosome 2q37.1, as a novel risk factor. UGT1A locus encodes nine UGT proteins that belong to phase II cellular detoxification system. UGTs are functionally important for detoxification of aromatic amines, which are known risk factors for bladder cancer found in industrial chemicals and tobacco smoke. UGTs are composed of shared exons 2-5 and individual alternative first exons that define enzymatic activity and substrate specificity. Materials and results: We sequenced all nine highly similar alternative first exons of UGT genes in more than 2,000 individuals and identified 27 non-synonymous and 19 synonymous known coding variants, but no novel variants. Imputation based on the GWAS dataset, a combined reference panel of HapMap 3 and 1000 Genomes Projects, and a subset of GWAS samples genotyped for all the identified coding variants, generated data for 1,170 SNPs within the whole UGT region. Of these markers, the strongest association was detected for an uncommon (MAF<0.03) protective genetic variant rs17863783 (OR=0.55, 95%CI=0.44-0.69, p=3.3x10-7 in 4,035 cases and 5, 284 controls; D’=0.96, r2=0.23 with rs11892031). No residual association in this region was detected after adjustment for rs17863783. Rs17863783 is a synonymous coding variant Val209Val within the functional UGT1A6.1 splicing form, strongly expressed in the liver, kidney and bladder. We found the protective T allele of rs17863783 to be associated with increased mRNA expression of UGT1A6.1 in in-vitro exontrap assays and in human liver tissue samples. We suggest rs17863783 may protect from bladder cancer by increasing the removal of carcinogens from bladder epithelium by the UGT1A6.1 protein. Conclusions: Our study shows an example of genetic and functional role of an uncommon protective genetic variant in a complex human disease such as bladder cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1658. doi:1538-7445.AM2012-1658

Abstract 4680: Exploring the relationships between genetic variants within the UGT1A locus, cellular detoxification and risk of bladder cancer

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL A recent genome-wide association study (GWAS) for urinary bladder cancer (UBC) has identified multiple novel genetic risk factors (Rothman et. al, Nat Gen, 2010). One of these factors was an intronic SNP rs11892031 (p=1.0×10-7) located within the UGT1A locus on chromosome 2q37. The human cellular detoxification uridine 5’ diphosphate (UDP)-glucuronosyltransferases (UGTs) belong to a superfamily of proteins that conjugate diverse endo and exotoxins, increase their solubility and facilitate their removal via bile and urine. The UGT1A locus includes nine protein-coding genes and four pseudogenes. Each first exon of these genes is regulated by its own promoter and is spliced to four constant exons producing nine UGT1A proteins. Due to the complexity of the region and ∼90-95% similarity between the substrate-binding exon 1 sequences of UGT1A genes, this region is poorly represented in public databases (HapMap, 1000 Genomes, dbSNP). To ensure specificity of detection, we generated long-range amplicons and sequenced all UGT1A exons in HapMap individuals (CEU) and in 44 bladder cancer patients and identified 25 non-synonymous coding variations. All these variations have been genotyped in 1000 bladder cancer cases and 1000 controls from the Spanish Bladder Cancer Study (SBCS). We found one exonic SNP that showed association stronger than the original GWAS variant, and multiple variants that showed some evidence of gene and cigarette smoking interactions. Having ∼20 coding and potentially functional variants within the UGT1A region, we are exploring methods of simultaneous analysis of individual variants and their haplotypes. In conclusion, association of genetic variants within the UGT1A region confirms that altered cellular detoxification of environmental substrates is an important factor for development of bladder cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4680. doi:10.1158/1538-7445.AM2011-4680

Abstract 2783: A novel functional variant is associated with regulation of the prostate stem cell antigen (PSCA) gene and bladder cancer risk in males

Genome-wide association studies (GWAS) identified allele T of SNP rs2294008 within the prostate stem cell antigen (PSCA) gene as a risk factor for diffuse gastric cancer (OR=1.67, p=2.2×10-15) (The Study Group of Millennium Genome Project for Cancer, Nat Genet, 2008) and bladder cancer (OR=1.13, p=4.4×10-11) (Rothman et al, Nat Genet, 2010). In the present study we used data from the Stage 1 bladder cancer GWAS, which was performed on 8,801 individuals of European ancestry (3,529 cases and 5,115 controls), and information from the 1000 Genomes and HapMap 3 projects to impute all other SNPs within +/- 50kb region surrounding PSCA. Association analysis on the combined set of all genotyped and imputed SNPs (n=375) revealed a novel bladder cancer association signal for a variant genotyped in all samples. The association was found only in males (n=7,359, crude OR =1.14, p=1.15×10-4; adjusted for all covariates and rs2294008, OR=1.13, p=1.32×10-3), while no association was observed in females (n=1,442, crude OR=0.98, p=0.81; adjusted for all covariates and rs2294008, OR=0.98, p=0.82). The low linkage disequilibrium between these two SNPs (D’=0.202 and r2=0.019 in 8801 GWAS samples) suggests these variants are independent. A joint analysis showed that these two SNPs have a compound association with bladder cancer dependent on the number of risk genotypes (0, 1 or 2) in males (adjOR=1.18, p=3.93×10-6) but not in females (adjOR=1.01, p=0.95). The risk allele T of rs2294008 is functional as it introduces a novel translation start site that creates a PSCA protein with a leader peptide extended by 11 amino acids. The novel SNP that was found 10 Kb upstream of rs2294008, lies within an alternative untranslated first exon of PSCA that is marked by a H3K4me3 signal and is in a vicinity of an androgen receptor binding site, suggesting a possible role for this variant in regulation of PSCA promoter activity. In conclusion, a novel independent functional variant associated with bladder cancer risk was found within the PSCA gene, which might be important for male-specific regulation of PSCA expression and carcinogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2783. doi:10.1158/1538-7445.AM2011-2783

Abstract LB-350: Tissue and serum miRNA profiling for detection of bladder, breast and prostate cancer

Biomarkers that can differentiate between normal and tumor state, and can be measured in easily accessible body fluids such as blood and urine, can be very important for cancer diagnostics and disease monitoring. MicroRNAs (miRNAs), short non-coding regulatory RNA molecules, are attractive as biomarkers because they are stable in different conditions and are easy to measure with quantitative PCR (qPCR) methods. In this study we aimed to identify a universal panel of miRNAs for cancer screening that can be easily tested in the serum of healthy individuals and patients with different types of cancer. First, we measured expression of ∼800 miRNAs in 40 controls and 60 patients with bladder, breast or prostate cancer using the low density TaqMan expression arrays (Applied Biosystems) and starting from 250 ul of serum. Based on these results we selected a panel of 24 miRNAs that showed best discrimination between normal and cancer samples. These miRNAs were then re-tested as a custom-designed mini-panel in serum samples of 44 healthy controls and cancer patients (31 bladder, 25 breast and 28 prostate) and in relevant normal and tumor tissue samples (42 normal bladder and 43 bladder tumors, 44 normal breast and 42 breast tumors and in 50 normal prostate and 20 prostate tumors). Only miRNAs that expressed in the same direction in serum and tissue samples and showed significant association with cancer in both sample types were used for further analysis. The current panel consists of 16 miRNAs – 14 targets, one positive control and one negative control. Using this panel on serum samples from 44 controls, 31 bladder cancer patients, 25 breast cancer patients and 28 prostate cancer patients we performed ROC analysis and achieved complete discrimination (AUC ∼1.0) between all types of cancers and controls and good discrimination between different types of cancers (minimal AUC 0.89 for breast and bladder samples). Our results prove that miRNA detection from serum might be a promising method of cancer detection. Currently, we are performing validation studies in independent sets of samples. A combination of information on genetic susceptibility factors identified by genome-wide association studies (GWAS), other cancer-specific factors such as PSA for prostate cancer and miRNA expression profiling, might provide additional tools for early disease diagnostics and help to guide treatment options. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-350. doi:10.1158/1538-7445.AM2011-LB-350