Meinolf Blaszkewicz

A sequence variant at 4p16.3 confers susceptibility to urinary bladder cancer

Previously, we reported germline DNA variants associated with risk of urinary bladder cancer (UBC) in Dutch and Icelandic subjects. Here we expanded the Icelandic sample set and tested the top 20 markers from the combined analysis in several European case-control sample sets, with a total of 4,739 cases and 45,549 controls. The T allele of rs798766 on 4p16.3 was found to associate with UBC (odds ratio = 1.24, P = 9.9 × 10−12). rs798766 is located in an intron of TACC3, 70 kb from FGFR3, which often harbors activating somatic mutations in low-grade, noninvasive UBC. Notably, rs798766[T] shows stronger association with low-grade and low-stage UBC than with more aggressive forms of the disease and is associated with higher risk of recurrence in low-grade stage Ta tumors. The frequency of rs798766[T] is higher in Ta tumors that carry an activating mutation in FGFR3 than in Ta tumors with wild-type FGFR3. Our results show a link between germline variants, somatic mutations of FGFR3 and risk of UBC.

The enhanced bladder cancer susceptibility of NAT2 slow acetylators towards aromatic amines: a review considering ethnic differences

Human bladder cancer may be caused by exposure to aromatic amines. The polymorphic enzyme N-acetyltransferase 2 (NAT2) is involved in the metabolism of these compounds. Two classical studies on chemical workers in Europe, exposed in the past to aromatic amines like benzidine, unambiguously showed that the slow acetylator status is a genetic risk factor for arylamine-induced bladder cancer. In the former benzidine industry in Huddington, Great Britain, 22 of 23 exposed cases with bladder cancer, but only 57% of 95 local controls without bladder cancer were of the slow acetylator phenotype. In Leverkusen, Germany, 82% of 92 benzidine-exposed chemical workers with bladder cancer were of the slow acetylator phenotype, whereas only 48% of 331 chemical workers who had worked at that plant were of the slow acetylator phenotype. This is in line with several smaller studies, which also show an over-representation of the slow acetylator status in formerly arylamine-exposed subjects with bladder cancer. Some of these studies included also subjects that were exposed to aromatic amines by having applied dyes, paints and varnishes. These European findings are in contrast to a large study on Chinese workers occupationally exposed to aromatic amines. In this study, only five of 38 bladder cancer cases occupationally exposed to arylamines were of the slow acetylator genotype. This is much lower than the ratio of slow acetylators to the general population in China. This points to different mechanisms of susceptibility for bladder cancer upon exposure to aromatic amines between European (Caucasian) and Chinese populations.

Simultaneous analysis of ochratoxin A and its major metabolite ochratoxin alpha in plasma and urine for an advanced biomonitoring of the mycotoxin.

Ochratoxin A (OTA) is a frequent mycotoxin contaminant found worldwide in foods and feedstuffs. Biomonitoring has been used to assess internal OTA exposure resulting from dietary intake and from other sources. Mycotoxin levels in blood and/or urine provide good estimates of past and recent exposure since OTA binds to serum proteins and is also partly excreted via the kidney. But, measuring OTA alone does not reflect its biotransformation. In light of scarce data on its metabolites in humans, it was the aim of this study to develop a method that allows analysis of OTA and its detoxication product ochratoxin alpha (OTα) in urine and in blood plasma. The method involves enzymatic hydrolysis of conjugates, liquid-liquid extraction, and analysis of sample extracts by liquid chromatography with fluorescence detection. Application of the validated method in a pilot study with 13 volunteers revealed the presence of OTA and OTα in all samples (limit of quantification: 0.05 ng/mL in urine, and 0.1 ng/mL in plasma). In line with negative findings of others, an OTA glucuronide was not detected, neither in urine nor in plasma. By contrast, conjugates of OTα (glucuronide and/or sulfate) are major products in these samples. This was confirmed by mass spectrometry detection. As OTα represents a large fraction of ingested mycotoxin, we propose to include analyses of this metabolite in future biomonitoring studies, also in light of the observed variations for urine OTα-levels that suggest different interindividual abilities for OTA-detoxification in humans.

Genetic variants in urinary bladder cancer: collective power of the “wimp SNPs”

In recent years, genome-wide association studies (GWAS) have identified more than 300 validated associations between genetic variants and risk of approximately 70 common diseases. A small number of rare variants with a frequency of usually less than 1% are associated with a strongly enhanced risk, such as genetic variants of TP53, RB1, BRCA1, and BRCA2. Only a very small number of SNPs (with a frequency of more that 1% of the rare allele) have effects of a factor of two or higher. Examples include APOE4 in Alzheimer’s disease, LOXL1 in exfoliative glaucoma, and CFH in age-related macular degeneration. However, the majority of all identified SNPs have odds ratios between 1.1 and 1.5. In the case of urinary bladder cancer, all known SNPs that have been validated in sufficiently large populations are associated with odds ratios smaller than 1.5. These SNPs are located next to the following genes: MYC, TP63, PSCA, the TERT-CLPTM1L locus, FGFR3, TACC3, NAT2, CBX6, APOBEC3A, CCNE1, and UGT1A. It is likely that these moderate risk or “wimp SNPs” interact, and because of their high number, collectively have a strong influence on whether an individual will develop cancer or not. It should be considered that variants identified so far explain only approximately 5–10% of the overall inherited risk. Possibly, the remaining variance is due to an even higher number of SNPs with odds ratios smaller than 1.1. Recent studies have provided the following information: (1) The functions of genes identified as relevant for bladder cancer focus on detoxification of carcinogens, control of the cell cycle and apoptosis, as well as maintenance of DNA integrity. (2) Many novel SNPs are far away from the protein coding regions, suggesting that these SNPs are located on distant-acting transcriptional enhancers. (3) The low odds ratio of each individual bladder cancer-associated SNP is too low to justify reasonable preventive measures. However, if the recently identified SNPs interact, they may collectively result in a substantial risk that is of preventive relevance. In addition to the “novel SNPs” identified by the recent GWAS, at least 163 further variants have been reported in relation to bladder cancer, although they have not been consistently validated in independent case–control series. Moreover, given that only 60 of these 163 “old SNPs” are covered by the SNP chips used in the recent GWAS, there are in principle 103 published variants still awaiting validation or disproval. In future, besides identifying novel disease-associated rare variants by deep sequencing, it will also be important to understand how the already identified variants interact.

Susceptibility to urinary bladder cancer: relevance of rs9642880[T], GSTM1 0/0 and occupational exposure.

Recently, a genome-wide single nucleotide polymorphism association study has identified a sequence variant 30 kb upstream of the c-Myc gene (allele T of rs9642880) that confers susceptibility to bladder cancer. However, the role of exposure to bladder carcinogens has not been considered. This prompted us to analyse the relevance of this polymorphism in 515 bladder cancer cases and 893 controls where the quality and quantity of occupational exposure to bladder carcinogens has been documented. When we analysed a hospital-based case-control series not selected for occupational exposure, rs9642880[T] was influential, in contrast to GSTM1 0/0. However, in a case-control series of patients that have been occupationally exposed to aromatic amines and polycyclic aromatic hydrocarbons, rs9642880[T] was not influential but GSTM1 0/0 was significantly associated with bladder cancer risk. Therefore, the degree to which rs9642880[T] and GSTM1 0/0 confer susceptibility to urinary bladder cancer seems to depend on the extent of exposure to urinary bladder carcinogens.

The impact of solvent mixtures on neurobehavioral performance: conclusions from epidemiological data.

The review of epidemiological studies investigating the neurobehavioral effects of occupational exposure to solvent mixtures sought to contribute to the following issues: (1) Identification of affected cognitive and motor functions. (2) Identification of sensitive neuropsychological tests. (3) Analysis of exposure-effect relationships. The approach was based on the meta-analytical method of effect size estimates. Fifty-three groups from occupational studies were included in the meta-analysis. Forty-eight neuropsychological performance variables could be analyzed as they were included in at least three studies. Seventeen articles provided detailed information on the constituents of mixtures, thereby enabling the computation of an exposure index that allowed the comparison of different mixtures. Significant negative effect sizes were obtained for 12 test variables measuring attention, memory, motor performance and constructional abilities. The greatest proportion of lower performance scores in the exposed groups was shown by different tests of attention: significant effect sizes between d=-0.16 and -0.46 were calculated. Tests of cognitive processing speed, response alternation and inhibition seemed to be sensitive tools for the detection of poorer performance. Exposure-effect relationships were mainly characterized by inconsistent patterns. Crude and inappropriately calculated exposure measures were blamed for this outcome. A healthy worker effect was suggested more consistently: studies examining groups with longer exposure duration obtained smaller effect sizes. Indications of confounding were observed; however, they did not seem sufficient to question consistent effect size patterns. Paying greater attention to the measurement of exposure and including measures of confounding is advisable for future studies and would enhance the explanatory power of cross-sectional studies and meta-analyses.

Genotyping NAT2 with only two SNPs (rs1041983 and rs1801280) outperforms the tagging SNP rs1495741 and is equivalent to the conventional 7-SNP NAT2 genotype.

Genotyping N-acetyltransferase 2 (NAT2) is of high relevance for individualized dosing of antituberculosis drugs and bladder cancer epidemiology. In this study we compared a recently published tagging single nucleotide polymorphism (SNP) (rs1495741) to the conventional 7-SNP genotype (G191A, C282T, T341C, C481T, G590A, A803G and G857A haplotype pairs) and systematically analysed if novel SNP combinations outperform the latter. For this purpose, we studied 3177 individuals by PCR and phenotyped 344 individuals by the caffeine test. Although the tagSNP and the 7-SNP genotype showed a high degree of correlation (R=0.933, P<0.0001) the 7-SNP genotype nevertheless outperformed the tagging SNP with respect to specificity (1.0 vs. 0.9444, P=0.0065). Considering all possible SNP combinations in a receiver operating characteristic analysis we identified a 2-SNP genotype (C282T, T341C) that outperformed the tagging SNP and was equivalent to the 7-SNP genotype. The 2-SNP genotype predicted the correct phenotype with a sensitivity of 0.8643 and a specificity of 1.0. In addition, it predicted the 7-SNP genotype with sensitivity and specificity of 0.9993 and 0.9880, respectively. The prediction of the NAT2 genotype by the 2-SNP genotype performed similar in populations of Caucasian, Venezuelan and Pakistani background. A 2-SNP genotype predicts NAT2 phenotypes with similar sensitivity and specificity as the conventional 7-SNP genotype. This procedure represents a facilitation in individualized dosing of NAT2 substrates without losing sensitivity or specificity.

Relation of isoflavones and fiber intake in childhood to the timing of puberty

BACKGROUND It has been suggested that phytoestrogens and dietary fiber can affect puberty timing. OBJECTIVE We examined whether intake of isoflavone and fiber in healthy white children before their pubertal growth spurt [age at take-off (ATO)] was associated with puberty timing. DESIGN Multivariate regression analyses were performed in 227 DONALD (DOrtmund Nutritional and Anthropometric Longitudinally Designed) Study participants with 3-d weighed dietary records and information on potential confounders at baseline (1 and 2 y before ATO). In a subsample (n = 111), urinary isoflavones were determined in 24-h urine samples by gas chromatography-mass spectrometry analysis. Puberty timing was examined by using ATO and chronologic ages at pubertal stage 2 for breast development (B2) or gonadal development, peak height velocity (PHV), and menarche or voice break. RESULTS Girls whose diet was in the highest dietary isoflavone tertile experienced Tanner stage 2 for breast development ap 0.7 y later and reached PHV ap 0.6 y later than did girls whose diet was in the lowest isoflavone tertile [age (95% CI) at B2: 10.7 y (10.4, 10.9 y) compared with 10.0 y ( 9.7, 10.3 y), respectively; P for trend = 0.04; age at PHV: 11.9 y (11.6, 12.2 y) compared with 11.3 y (11.0, 11.6 y), respectively; P for trend = 0.04; adjusted for body mass index z score and fiber intake]. In boys, dietary isoflavones were not associated with pubertal markers. Urinary isoflavone and dietary fiber intakes were not associated with pubertal markers. CONCLUSIONS Girls, but not boys, with higher prepubertal isoflavone intakes appear to enter puberty at a later age. Fiber intake in this sample of healthy white girls and boys was not relevant for puberty timing.

Melatonin production during childhood and adolescence: a longitudinal study on the excretion of urinary 6-hydroxymelatonin sulfate.

Abstract: Cross‐sectional data on urinary 6‐hydroxymelatonin sulfate (6‐OHMS) excretion in children suggest a constant melatonin secretion during growth. The present longitudinal study concerned, accordingly, the intra‐individual stability of melatonin production during childhood and adolescence. Urine samples collected during a longitudinal investigation of healthy white children and adolescents were analyzed. Forty‐six boys and 38 girls were chosen for the present study. They had passed 3–15 annual examinations between their 3rd and 18th yr of age. Each examination included the collection of urine over 24 hr. The daily urinary output of 6‐OHMS of the overall 621 samples was quantified by enzyme‐linked immunosorbent assay. The analyses clearly revealed for the first time that, despite huge inter‐individual differences, melatonin production remains constant in one and the same individual during childhood and adolescence. Additionally, neither a significant sex difference was observed nor was the 6‐OHMS output affected by season. The dramatic decrease of plasma melatonin levels as described in the literature is mainly related to an increase in body size rather than to decreasing pineal secretion.

Polymorphic Enzymes, Urinary Bladder Cancer Risk, and Structural Change in the Local Industry

In the 1990s, an uncommonly high percentage of glutathione S-transferase M1 (GSTM1) negative bladder cancer cases (70%) was reported in the greater Dortmund area. The question arose as to whether this uncommonly high percentage of GSTM1 negative bladder cancer cases was due to environmental and/or occupational exposure decades ago. Thus, 15 years later, another study on bladder cancer was performed in the same area after the coal, iron, and steel industries had finally closed in the 1990s. In total 196 bladder cancer patients from the St.-Josefs-Hospital Dortmund-Hörde and 235 controls with benign urological diseases were assessed by questionnaire and genotyped for GSTM1, glutathione S-transferase T1 (GSTT1), and the N-acetyltransferase 2 (NAT2) tag SNP rs1495741. The frequency of the GSTM1 negative genotype was 52% in bladder cancer cases and thus lower compared to a previous study performed from 1992 to 1995 in the same area (70%). NAT2 genotypes were distributed equally among cases and controls (63% slow acetylators). Fewer GSTT1 negative genotypes were present in cases (17%) than in controls (20%).