Supplementary Materials and Methods

Patients

We used eight case-control series, in total 1,712 bladder cancer cases and 2,020 controls that have been collected by the IfADo and its cooperation partners from 1995 to 2011. The sample collection was approved by the local Ethics Committee and by the IRB (institutional review board). Exclusion criteria were a missing written informed consent, age of less than 20 years and, in case of the German and Hungarian study groups, non-Caucasian descent. Cigarette smoking was defined as non-smokers, former smokers, i.e. smokers that quit smoking at least one year before diagnosis (cases) or examination (controls), and current smokers. Age was defined as “age at diagnosis” for the cases and “age at examination” for the control persons. All bladder cancer cases and controls were genotyped for NAT2 by PCR and RFLP-based methods and/or for the NAT2 tag SNP rs1495741 by TaqMan® assay as part of an ongoing case-control series on genetic and occupational risk factors for urinary bladder cancer. Data on NAT2 7-SNP and tagSNP genotypes and phenotypes was published elsewhere (Selinski et al. 2011). Data on NAT2 7-SNP genotypes was included in Selinski et al. (2013). Data on the NAT2 tagSNP was part of the data set analysed in Schwender et al. (2012).

Hungary

The Hungary case-control series contains 226 bladder cancer cases and 76 controls from the Department of Urology, Semmelweis University, Budapest. The median age at diagnosis was 71 (range 38-95) years. 63% of the participants were males. The controls (74% males, median age 63, range 30-81 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. Caucasian descent was confirmed by questionnaire-based documentation of nationality. 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

The Wittenberg case-control series (Lutherstadt Wittenberg bladder cancer study) as described in Golka et al. (2009) was used. In brief, 216 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. Controls (N=212, 84% males, median age 68, range 29-91 years) 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. Caucasian descent was 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 six 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 stage, grade and treatment were taken from the records. Bladder cancer was diagnosed from July 1979 to January 1999.

West Germany

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. Caucasian descent was confirmed by questionnaire-based documentation of nationality (Lehmann et al. 2010). Data on tumour stage and grade were obtained through the cancer registry. All study groups besides Dortmund-Hörde are still ongoing. 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 six 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 195 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 235 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 71 (range 35-89) years. 75% of the participants were males. The 235 control individuals (77% males) were cancer free and frequency-matched for age with the cases (median age 71, range 22-100).

Dortmund bladder cancer study, Klinikum Dortmund, Germany

Twenty-six bladder cancer cases and five 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 July 2010 were included. The median age at diagnosis was 69 (range 41-84) years. 69% of the participants were males. Data were collected from July 2007 to April 2010. The five controls (three males) were cancer free (median age 71, range 49-83).

Neuss bladder cancer study, Lukasklinik Neuss, Germany

The ongoing case-control series consists of 284 bladder cancer cases and 197 controls from the Department of Urology, Lukasklinik Neuss, Germany. The median age at diagnosis was 73 (range 26-93) years. 78% of the participants were males. Data on tumour stage and grade were obtained through the cancer registry. The 197 male controls (59% males, median age 49, range 21-89 years) were cancer free. Data was collected from June 2009 to November 2011.

Düsseldorf bladder cancer study, Heinrich-Heine University, Germany

The ongoing case-control series consists of 84 bladder cancer cases and 59 controls from the department of Urology at the Heinrich-Heine University of Düsseldorf, Germany. The median age at diagnosis was 70 (range 27-95) years. 80% of the participants were males. The controls (15% males) were cancer free (median age 61, rage 23-85). Data was collected from November 2009 to March 2011.

Mainz bladder cancer study, Johannes Gutenberg University, Germany

Thirty 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 64 (range 35-81) years. 70% of the participants were males. Data on tumour stage and grade were obtained through the cancer registry. The 21 control individuals (62% male) were cancer free (median age 65, range 30-78 years). Data was collected from January 2010 to September 2010.

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)

Ninety-one 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. 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. 85% of the participants were males. Caucasian descent was confirmed by questionnaire-based documentation of nationality.

Dortmund occupational case-series (DO-occupational)

The Occupational case-series (study on patients with suspected occupational bladder cancer) as described by Golka et al. (2009) was used. Details of the ongoing study on 342 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. Bladder cancer was diagnosed from January 1984 to March 2011. 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 workplaces in former decades, 93% of the patients were males. The median age at diagnosis was 60 (range 32-83) 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, urine samples were obtained in addition to the blood samples. Occupational and concurrent non-occupational risk factors for bladder cancer as well as Caucasian descent 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, 191 patients of the Department of Surgery of the Klinikum Dortmund without any malignancy in the medical history, 272 patients without malignancies from the St. Elisabeth Hospital in Iserlohn, Germany, 92 former hard coal miners with pneumoconiosis recognized for an occupational disease surveyed for the course of their disease, 313 persons participating in an ongoing study on the impact of enzyme polymorphisms on selected brain functions as well as 96 staff of the Dortmund institute serving as controls in different studies were included. In total, 964 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. Caucasian descent was confirmed by questionnaire-based documentation of nationality

Pakistan

The Pakistan case-control series contains 106 bladder cancer cases and 61 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. 88% of the participants were males. The controls (85% males, median age 56, range 27-77 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.

Venezuela

The Venezuelan case-control series contains 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. Data on tumour stage and grade were obtained by the cancer registry. Data collected in cases and controls include age, gender, a complete documentation of occupational activities performed at least for six 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.

Subgroups phenotyped for NAT2

A subgroup of 344 Caucasians was phenotyped for NAT2 using the caffeine test consisting of 267 cases from the Dortmund occupational case-control series, 38 cases from the Dortmund hospital based case-control series and 39 healthy controls from the Dortmund control group (IfADo staff). Phenotyping was done routinely for cases from the Dortmund occupational case-control series as part of the medical examination. Cases from the Dortmund hospital based case-control series were phenotyped additionally to NAT2 genotyping. Controls from the IfADo staff served as long-term quality controls to assure comparability of genotyping results over time and methodological or technical alterations.

Analysis of polymorphisms

For differentiating between the homozygous frequent (A/A), homozygous variant (G/G) and heterozygous (A/G) form of the sequence of interest venous blood was taken and frozen at 20°C (Saravana Devi et al. 2008). DNA was isolated out of leucocytes 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). Analysis of A/G substitution (rs1495741) on chromosome 8p23, position 18272881, and differentiating between the homozygous (A/A), homozygous (G/G) and heterozygous (A/G) form of the sequence:

AGCTGAAGGATGATTTTCATAATAAT[A/G]TGGGCATTCACAGTAGCTTCAGGGC was performed on an ABI7500 Sequence Detection System with the use of TaqMan® SNP Genotyping Assay (Applied Biosystems, Darmstadt, Germany). Analysis of data was performed according to the manufacturer’s instructions.

NAT2 genotyping

NAT2 genotyping was performed using PCR- and RFLP based standardized methods (Blaszkewicz et al. 2004, Cascorbi et al. 1995, 1996). A total of seven SNPs, which are adequate to genotype Caucasians for NAT2 (Blaszkewicz et al. 2004), were investigated, namely rs1801279 (G191A), rs1041983 (C282T), rs1801280 (T341C), rs1799929 (C481T), rs1799930 (G590A), rs1208 (A803G) and rs1799931 (G857A). Leucocyte DNA is isolated from a sample of human blood. Amplification of two fragments of DNA with 442 and 559 base pairs (bp) is achieved by means of polymerase chain reaction (PCR). In three aliquots the amplification product from the first PCR is cleaved by three different restriction enzymes (Msp I, Fok I, Dde I), and that of the second PCR with four different restriction enzymes (KpnI, Taq I, Dde I, BamH I; Figure M1, Blaszkewicz et al. 2004). After subsequent gel electrophoresis with the addition of ethidium bromide, the various DNA fragments are detected in UV light. The results are documented by photography, and the alleles are assigned according to an evaluation scheme (Blaszkewicz et al. 2004).