Supplemental Materials and Methods
Patients
Hungary
The Hungary case-control series contains 243 bladder cancer cases and 77 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 64, 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
The Wittenberg case-control series (Lutherstadt Wittenberg bladder cancer study) as
described (Golka et al. 2009) was used. In brief, 212 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.
200 controls (86% 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.
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. 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 387 patients with a confirmed bladder
cancer and 167 controls from the Departments of Urology admitted for treatment of benign
urological diseases, enrolled from July 2009 to August 2010. All study groups are still
ongoing. The median age at diagnosis was 71 (range 26-95) years. 78% of the participants
were males. The 167 control individuals (72% males; median age 67, range 22-99) 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 ongoing case-control series consists of 128 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 141 controls from the same Department of Urology,
admitted for treatment of benign urological diseases, enrolled from July 2009 to August 2010.
The median age at diagnosis was 71 (range 35-89) years. 77% of the participants were
males. The 141 control individuals (70% males; median age 67, range 22-99 years) were
cancer free and frequency-matched for age and gender with the cases.
Dortmund bladder cancer study, Klinikum Dortmund, Germany
Thirty-six bladder cancer cases and one control from the Department of Urology, Klinikum
Dortmund, Germany, located in an area of former coal, iron and steel industries, enrolled
from July 2007 to August 2010 were included. The median age at diagnosis was 67 (range
41-84) years. 72% of the participants were males. The control (one male, 70 years) was
cancer free.
Neuss bladder cancer study, Lukasklinik Neuss, Germany
The ongoing case-control series consists of 168 bladder cancer cases and two controls from
the Department of Urology, Lukasklinik Neuss, Germany. The median age at diagnosis was
73 (range 26-93) years. 80% of the participants were males. Data on tumour stage and
grade were obtained through the cancer registry. The two male controls (64 and 73 years)
were cancer free. Data was collected from June 2009 to August 2010.
Düsseldorf bladder cancer study, Heinrich-Heine University, Germany
The ongoing case-control series consists of 39 bladder cancer cases and 15 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. 82% of the participants were males.
The controls (93% males; median age 68, range 27-85 years) were cancer free. Data was
collected from November 2009 to August 2010.
Mainz bladder cancer study, Johannes Gutenberg University, Germany
Sixteen bladder cancer cases and eight controls from the department of Urology at the
Johannes Gutenberg University of Mainz, Germany, were included. The median age at
diagnosis was 62 (range 37-76) years. 75% of the participants were males. Data on tumour
stage and grade were obtained through the cancer registry. The eight control individuals
(75% males; median age 66, range 49-71 years) were cancer free. Data was collected from
January 2010 to August 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)
Eighty-seven 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 68 (range 45-84) years. 84% of
the participants were males.
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 311
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 August 2010 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, 176
patients of the Department of Surgery of the Klinikum Dortmund without any malignancy in
the medical history, 219 patients without malignancies from the St. Elisabeth Hospital in
Iserlohn, Germany, 89 former hard coal miners with pneumoconiosis recognized for an
occupational disease surveyed for the course of their disease, 309 persons participating in
an ongoing study on the impact of enzyme polymorphisms on selected brain functions as
well as 81 staff of the Dortmund institute serving as controls in different studies were
included. In total, 874 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
The Pakistan case-control series contains 98 bladder cancer cases, 100 controls from the
Sindh Institute of Urology and Transplantation, Civil Hospital, Karachi and 119 populationbased controls. All cases and controls are Pakistani, which was confirmed by questionnairebased 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.
Venezuela
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 188 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
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 ND1000 UV/Vis-spectrophotometer (PEQLAB Biotechnologie GMBH, Erlangen, Germany).
Genotyping was performed on an ABI7500 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 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
7300/7500/7500, fast real-time PCR System Allelic Discrimination Getting Started Guide).
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). 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 rs2854744 genotype with UBC were
evaluated applying exact chi-square tests, odds ratios (OR) and 95% confidence intervals
(CI) in case of the global (genotype), recessive, dominant (not shown) and multiplicative
(allelic) model, Cochran-Armitage trend tests, ORs from the logistic regression model and
95% CIs were used for the additive model. 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 chi-square 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 rs2854744, 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 non-exposed persons. The power
analysis (Clayton and Hills 1993) was performed for a chi-square test at  = 0.05, risk allele
frequency of 0.5 and odds ratios of 1.2 to 2.0, in the Caucasian and combined study groups
as well as the study group of Safarinejad et al. (2011) (Table 3) using the PS Power and
Sample Size Program version 3.0.12 (Dupont and Plummer 1998; Dupont and Plummer
1990). The linkage disequilibrium plot of r² for a region around rs2854744 (chromosome 7,
45,900–45,980kb, MAF >0.001, HWE cut-off 0.001) 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 case-control series from Safarinejad et al. (2011). 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 inhomogeneity of the ORs random effect models were applied to
calculated combined P values, ORs and 95% CIs using the inverse variance method. The
meta-analysis, the forest plot and Cochran's Q test were performed using the software R,
version 2.10.1 (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 indicated otherwise. The level of significance was  = 0.05 for all tests and
confidence intervals.
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 Stransferase 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
Clayton D, Hills M (1993) Statistical models in epidemiology. Oxford University Press, New
York
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 84:967978
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
Safarinejad MR, Shafiei N, Safarinejad SH (2011). The association between bladder cancer
and a single nucleotide polymorphism (rs2854744) in the insulin-like growth factor (IGF)binding protein-3 (IGFBP-3) gene. Arch Toxicol [Epub ahead of print]
Saravana Devi S, Vinayagamoorthy N, Agrawal M et al (2008) Distribution of detoxifying
genes polymorphism in Maharastrian population of central India. Chemosphere 70:18351839
SAS/STAT® software, version 9.2. Copyright © 2002-2008, SAS Institute Inc. Cary, NC, USA