Selinski Et Al. (2012) UGT1A Genetic Variant Confers Bladder Cancer Risk

Selinski et al. (2012) UGT1A genetic variant confers bladder cancer risk 1

Supplemental Materials and Methods

Patients

Hungary case-control series

The Hungary case-control series contains 245 bladder cancer cases and 78 controls from the Department of Urology, Semmelweis University, Budapest. All cases and controls are Caucasians, which were confirmed by questionnaire-based documentation of nationality. The median age at diagnosis was 71 (range 27-95) years. 60% of the participants were males. The controls (73% males; median age 63, range 30-84 years) were cancer free. Data were collected from 2004 to 2006. Data on tumour stage and grade were obtained through the cancer registry. Controls without malignant disease were frequency-matched for age (time of examination) with the cases. Data collected in cases and controls, include age, gender, a documentation of occupational activities and exposures to known or suspected occupational bladder carcinogens and lifetime smoking habits.

East Germany case-control series

The Wittenberg case-control series (Lutherstadt Wittenberg bladder cancer study LWBCS) as described in (Golka et al. 2009) was used. In brief, 218 patients with a confirmed bladder cancer from the Department of Urology, Paul Gerhardt Foundation, Lutherstadt Wittenberg, Germany, were included. Patients were enrolled from December 1995 to January 1999. The median age at diagnosis was 66 (range 20-91) years. 86% of the participants were males. 212 controls (83% males; median age 68, range 29-91) were from the same department of urology, but were admitted for treatment of benign urological diseases. Exclusion criteria were a malignant disease in the medical history or a missing written informed consent. All cases and controls were Caucasians, which were confirmed by questionnaire-based documentation of nationality. Data were collected from July 2000 to May 2005. Cases and controls were matched for age and gender. Data collected for cases and controls include age, gender, a complete documentation of occupational activities performed at least for 6 months, documentation of work places with known bladder cancer risk over the entire working life, exposures to known or suspected occupational bladder carcinogens, lifetime smoking habits, family history of bladder cancer, numbers of urinary infections treated by drugs during the previous 10 years, place of birth and places of residency for more than 10 years. In the case of bladder cancer cases, data on tumour staging, grading and treatment were taken from the records. Bladder cancer was diagnosed from July 1979 to January 1999. The East Germany case-control series was part of the stage 3 confirmation studies of the GWAS of Rothman et al. (2010). Selinski et al. (2012) UGT1A genetic variant confers bladder cancer risk 2

West Germany case-control series

West Germany-ongoing case-control series (W. Germany-ongoing)

The West Germany ongoing case-control series contains bladder cancer cases and controls from the Department of Urology, St.-Josefs-Hospital Dortmund-Hörde, the Department of Urology, Klinikum Dortmund, the Department of Urology, Lukasklinik Neuss, the Department of Urology at the Heinrich-Heine University of Düsseldorf and from the department of Urology at the Johannes Gutenberg University of Mainz, Germany. All cases and controls are Caucasians, which was confirmed by questionnaire-based documentation of nationality as described (Lehmann et al. 2010). Data on tumour stage and grade were obtained through the cancer registry. The case-control series consists of 646 patients with a confirmed bladder cancer and 524 controls from the Departments of Urology admitted for treatment of benign urological diseases, enrolled from July 2009 to May 2011. All study groups but Dortmund- Hörde are still ongoing. The median age at diagnosis was 71 (range 26-95) years. 77% of the participants were males. The 524 control individuals (70% males; median age 62, range 21-100) were cancer free and frequency-matched for age with the cases. Exclusion criterion was a missing written informed consent. Controls were frequency-matched for age (time of examination) with the cases. Data collected for cases and controls include age, gender, a complete documentation of occupational activities performed at least for 6 months, documentation of work places with known bladder cancer risk over the entire working life, exposures to known or suspected occupational bladder carcinogens, lifetime smoking habits, family history of bladder cancer, numbers of urinary infections treated by drugs during the previous 10 years, place of birth and places of residency for more than 10 years.

Dortmund bladder cancer study, St.-Josefs-Hospital Dortmund-Hörde, Germany

The case-control series consists of 198 patients with a confirmed bladder cancer from the Department of Urology, St.-Josefs-Hospital Dortmund-Hörde, located in an area of former coal, iron and steel industries and 238 controls from the same Department of Urology, admitted for treatment of benign urological diseases, enrolled from July 2009 to December 2010. The median age at diagnosis was 70 (range 35-89) years. 76% of the participants were males. The 238 control individuals (76% males; median age 68, range 22-100 years) were cancer free and frequency-matched for age and gender with the cases.

Dortmund bladder cancer study, Klinikum Dortmund, Germany

Fourty-one bladder cancer cases and six controls from the Department of Urology, Klinikum Dortmund, Germany, located in an area of former coal, iron and steel industries, enrolled from July 2007 to May 2011 were included. The median age at diagnosis was 66 (range 41- 84) years. 73% of the participants were males. The controls (67% males, median age 69, range 49-83) were cancer free. Selinski et al. (2012) UGT1A genetic variant confers bladder cancer risk 3

Neuss bladder cancer study, Lukasklinik Neuss, Germany

The ongoing case-control series consists of 285 bladder cancer cases and 199 controls from the Department of Urology, Lukasklinik Neuss, Germany. The median age at diagnosis was 71 (range 26-93) years. 78% of the participants were males. Data on tumour stage and grade were obtained through the cancer registry. The controls (60% males; median age 48, range 21-89 years) were cancer free. Data was collected from June 2009 to May 2011.

Düsseldorf bladder cancer study, Heinrich-Heine University, Germany

The ongoing case-control series consists of 85 bladder cancer cases and 60 controls from the department of Urology at the Heinrich-Heine University of Düsseldorf, Germany. The median age at diagnosis was 69 (range 27-95) years. 80% of the participants were males. The controls (86% males; median age 59, range 23-85 years) were cancer free. Data was collected from November 2009 to May 2011.

Mainz bladder cancer study, Johannes Gutenberg University, Germany

Thirty-seven bladder cancer cases and 21 controls from the department of Urology at the Johannes Gutenberg University of Mainz, Germany, were included. The median age at diagnosis was 63 (range 35-81) years. 70% of the participants were males. Data on tumour stage and grade were obtained through the cancer registry. The controls (62% males; median age 60, range 30-78 years) were cancer free. Data was collected from January 2010 to May 2011.

West Germany–industrial burdened case-control series (W. Germany-industrial)

The West Germany – industrial burdened case-control series (W. Germany - industrial) consists of two independent case groups and one control cohort.

Dortmund hospital based case-series (DO-hospital)

One hundred-eleven patients with confirmed bladder cancer from the Department of Urology, Klinikum Dortmund, Germany, located in an area of former coal, iron, and steel industries, were included. Exclusion criterion was a missing written informed consent. Data were collected from November 1993 to June 1995. All items of the questionnaire applied in Dortmund were also included in the extended version of the questionnaire presented to the cases and controls in the Lutherstadt Wittenberg group. Bladder cancer was diagnosed from July 1981 to June 1995. The median age at diagnosis was 67 (range 45-84) years. 87% of the participants were males. Selinski et al. (2012) UGT1A genetic variant confers bladder cancer risk 4

Dortmund occupational case-control series (DO-occupational)

The Occupational case-series (study on patients with suspected occupational bladder cancer) as described (Golka et al. 2009) was used. Details of the ongoing study on 372 suspected cases of occupational bladder cancer from Germany, mainly from the Federal State of North Rhine-Westphalia, reported to the authorities and surveyed for recognition of an occupational disease (in Germany named “Berufskrankheit BK 1301”) from February 1996 to May 2011 were reported recently. The individuals were suspected to be exposed to occupational bladder carcinogens, mostly carcinogenic aromatic amines, azo dyes based on carcinogenic aromatic amines or polycyclic aromatic hydrocarbons. According to the situation at work places in former decades, 93% of the patients were males. All patients were Caucasians. The median age at diagnosis was 61 (range 32-84) years. All surveyed bladder cancer patients gave informed consent for genotyping of enzymes relevant for bladder cancer and N-acetyltransferase 2 phenotyping by caffeine metabolites. Therefore, blood and urine samples were also obtained. Occupational and concurrent non-occupational risk factors for bladder cancer were explored by three medical specialists in a personal interview.

Dortmund controls (DO-controls)

The control group consists of persons from the greater Dortmund area, Germany, who did not present a malignancy in the medical history. Dortmund is a city with approximately 600,000 inhabitants located in North Rhine-Westphalia, which is the westernmost and - in terms of population and economic output - the largest Federal State of Germany. Briefly, 181 patients of the Department of Surgery of the Klinikum Dortmund without any malignancy in the medical history, 231 patients without malignancies from the St. Elisabeth Hospital in Iserlohn, Germany, 95 former hard coal miners with pneumoconiosis recognized for an occupational disease surveyed for the course of their disease, 315 persons participating in an ongoing study on the impact of enzyme polymorphisms on selected brain functions as well as 89 staff of the Dortmund institute serving as controls in different studies were included. In total, 911 individuals were combined to a control group representing inhabitants of the greater Dortmund area. The median age at examination was 68 (range 20-94) years and 51 % of the controls were males.

Pakistan case-control series

The Pakistan case-control series contains 101 bladder cancer cases, 102 controls from the Sindh Institute of Urology and Transplantation, Civil Hospital, Karachi and 124 population- based controls. All cases and controls are Pakistani, which was confirmed by questionnaire- based documentation of nationality. The median age at diagnosis was 61 (range 24-82) years. 87% of the participants were males. The controls (80% males; median age 56, range 26-76 years) were cancer free. Data were collected from April 2003 to January 2004. Data on tumour stage and grade were obtained through the cancer registry. Controls without malignant disease were frequency-matched for age (time of examination) with the cases. Data collected in cases and controls include age, gender, a documentation of occupational activities and exposures to known or suspected occupational bladder carcinogens and lifetime smoking habits. Selinski et al. (2012) UGT1A genetic variant confers bladder cancer risk 5

Venezuela case-control series

The Venezuelan case-control series contain 112 bladder cancer cases from Departments of Urology, University Hospital at Central University, Caracas; Domingo Luciani Hospital from the Venezuelan Institute of Social Security, Caracas; Oncologic Hospital “Padre Machado”, Caracas, and Policlínica Metropolitana, Caracas. A total of 190 controls were from the same departments of urology, as well as from the Medical Faculty at Central University, Caracas, and all were free of any type of cancer. All cases and controls are Venezuelan; though some of the patients were residents in Venezuela but were of different origin, mainly Colombian, Ecuadorian, Peruvian, Italian and Portuguese which was confirmed by questionnaire-based documentation of nationality. The median age at diagnosis was 59 (range 29-87) years. 71% of the participants were males. The controls (41% males; median age 31, range 20-91 years) were cancer free. Data were collected from December 2006 to November 2009, the study is still ongoing. Data on tumour stage and grade were obtained by the cancer registry. Controls without malignant disease were frequency-matched for age (time of examination) with the cases. Data collected in cases and controls include age, gender, a complete documentation of occupational activities performed at least for 6 months, documentation of work places with known bladder cancer risk over the entire working life, exposures to known or suspected occupational bladder carcinogens, lifetime smoking habits, family history of bladder cancer, numbers of urinary infections treated by drugs during the previous 10 years, place of birth and places of residency for more than 10 years. In the case of bladder cancer cases, data on tumour staging, grading and treatment were taken from the records. The local ethics committees approved the study plan and design.

Analysis of polymorphisms (rs11892031)

For differentiating between the homozygous frequent (A/A), homozygous variant (C/C) and heterozygous (A/C) form of the sequence of interest approximately 5-8 ml of venous blood was taken into a 9 ml tube (Sarstedt, Nümbrecht, Germany) from the cubital vein with EDTA as the anticoagulant and was frozen at -20°C (Saravana et al. 2008). DNA was isolated using a QIAamp DNA blood maxi kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol (Arand et al. 1996). DNA concentrations were determined using a NanoDrop ND-1000 UV/Vis-spectrophotometer (PEQLAB Biotechnologie GMBH, Erlangen, Germany). Genotyping was performed on an ABI PRISM® 7900HT Sequence Detection System with the use of TaqMan® assays (Applied Biosystems, Darmstadt, Germany). A final reaction volume of 15 μl was used per well of a 96-well plate. The reaction mix for amplification was prepared by mixing 7.5 µl TaqMan® Universal PCR Master Mix (Applied Biosystems, Foster City, CA 94404, U.S.A.) and 0.75 µl Working Stock of SNP Genotyping Assay (Applied Biosystems, Foster City, CA 94404, U.S.A.) per sample. To this reaction mixture 1 µl DNA solution (with a total of 10 ng DNA) and 5.75 µl distilled water were added to achieve a final volume of 15 µl. Amplification was performed using a protocol starting with 2 min at 50 °C and 10 min at 95 °C continuing with 40 cycles, 15 s at 92 °C (denature), 1 min at 60 °C (anneal/extend). An initial hold with 10 min at 95 °C was applied. Analysis of data was performed according to the manufacturer’s instructions (Applied Biosystems ABI PRISM® 7900HT, Sequence Detection System and SDS Enterprise Database User Guide). Selinski et al. (2012) UGT1A genetic variant confers bladder cancer risk 6

Statistical analysis

Cigarette smoking was defined as non-smokers, former smokers, i.e. smokers that quitted smoking at least one year before diagnosis (cases) or examination (controls), and current smokers. Former and current smokers were pooled together as “ever smokers”. Age was defined as “age at diagnosis” for the cases and “age at examination” for the control persons. Genetic models were defined according to (Lewis 2002). All combined results including and excluding the East Germany case-control series from the combined study groups (Caucasians combined, all combined) are given as “East Germany” was already published as part of the stage III of Rothman et al. (2010). Deviations from Hardy-Weinberg equilibrium (HWE) were checked in each study group and separately for cases and controls using exact chi-square tests. Associations of the rs11893021 genotype with UBC were evaluated applying exact chi-square tests, odds ratios (OR) and 95% confidence intervals (CI) considering global genotype differences as well as a, recessive, dominant (not shown) and multiplicative (allelic) mode of inheritance. Cochran-Armitage trend tests, ORs from the logistic regression model and 95% CIs were used for the additive mode of inheritance. For the combined Caucasian study groups and for all study groups combined the method of Mantel-Haenszel was used to adjust for the different study groups (combined Caucasian groups: regions are shown, all combined: ethnicities are shown) checking the homogeneity of the odds ratios (OR) in the multiplicative and the recessive model by Breslow-Day tests. Adjusted ORs and 95% confidence intervals as well as Wald tests adjusted for age, cigarette smoking (non smoker, former, current), gender, and origin in case of combined study groups (combined Caucasian groups: regions are shown, all combined: ethnicities are shown) were calculated using logistic regression. Effects of smoking on UBC as well as associations between rs11893021, smoking habits and urinary bladder carcinogens (in the subgroups East Germany, DO-hospital and DO-occupational) were investigated using exact chi-square tests in a stratified analysis. We stratified for exposure (urinary bladder carcinogens, ever/never smoking) comparing the genotype distribution in cases and controls and we stratified for case-controls status comparing the genotype distribution in exposed and not exposed persons. The power analysis was performed for a chi-square test at  = 0.05, risk allele frequency of 0.5, 0.92 (according to Rothman et al. 2010) and 0.93 (combined IfADo control groups) and odds ratios of 1.13 to 1.4, in the Caucasian and combined study groups (Table 2) using the PS Power and Sample Size Program version 3.0.43 (Dupont and Plummer 1998, 1990). The linkage disequilibrium plot of r² for a region around rs11893021 (chromosome 2, 234,180 - 234,245 kb, MAF >0.001, HWE cut-off 0.001; 234,230 – 234,267 kb, not shown) was obtained using the Haploview V4.2 program (Barrett et al. 2005). The plot is based on HapMap CEU (Utah residents with Northern and Western European ancestry from the CEPH collection) and TSI (Toscans in Italy) data version 3, release R2, locations are from NCBI Genome Build 36. A meta-analysis for the multiplicative model was conducted on the basis the present data set and the discovery GWAS from Rothman et al. (2010). We excluded the East Germany case-control series from the IfADo study groups as it was already part of the stage III study groups of Rothman et al. (2010). Cochran's Q test was applied to test for heterogeneity of the ORs of the multiplicative model in the combined study groups in the meta-analysis. Due to homogeneity of the ORs combined P values, ORs and 95% CIs were calculated using the method of Mantel-Haenszel. The meta-analysis, the forest plot and Cochran's Q test were performed using the software R, version 2.14.1 (R Development Core Team 2008). For all further calculations we used the software package SAS/STAT®, version 9.2 (SAS/STAT® software, version 9.2. Copyright© 2002-2008), if not Selinski et al. (2012) UGT1A genetic variant confers bladder cancer risk 7 indicated otherwise. The level of significance was  = 0.05 for all tests and confidence intervals. Selinski et al. (2012) UGT1A genetic variant confers bladder cancer risk 8

References

Arand M, Mühlbauer R, Hengstler J, Jäger E, Fuchs J, Winkler L, Oesch F (1996) A multiplex polymerase chain reaction protocol for the simultaneous analysis of the glutathione S- transferase GSTM1 and GSTT1 polymorphisms. Anal Biochem 236:184–186

Barrett JC, Fry B, Maller J, Daly MJ (2005) Haploview: analysis and visualization of LD and haplotype maps. Bioinformatics 21:263–265

Dupont WD, Plummer WD (1990) Power and sample size calculations: A review and computer program. Control Clin Trials 11:116–128

Dupont WD, Plummer WD (1998) Power and sample size calculations for studies involving linear regression. Control Clin Trials 19:589–601

Golka K, Hermes M, Selinski S et al (2009) Susceptibility to urinary bladder cancer: relevance of rs9642880[T], GSTM1 0/0 and occupational exposure. Pharmacogenet Genomics 19:903–906

Lehmann ML, Selinski S, Blaszkewicz M et al (2010) Rs710521[A] on chromosome 3q28 close to TP63 is associated with increased urinary bladder cancer risk. Arch Toxicol 84:967– 978

Lewis CM (2002) Genetic association studies: design, analysis and interpretation. Briefings in Bioinformatics 3:146–153

R Development Core Team (2008) R: A language and environment for statistical computing, R Foundation for Statistical Computing, Vienna, Austria

Rothman N, Garcia-Closas M, Chatterjee N et al (2010) A multi-stage genome-wide association study of bladder cancer identifies multiple susceptibility loci. Nat Genet 42:978– 984

Saravana Devi S, Vinayagamoorthy N, Agrawal M et al (2008) Distribution of detoxifying genes polymorphism in Maharastrian population of central India. Chemosphere 70:1835– 1839