Simonetta Guarrera

Sequence variant on 8q24 confers susceptibility to urinary bladder cancer

We conducted a genome-wide SNP association study on 1,803 urinary bladder cancer (UBC) cases and 34,336 controls from Iceland and The Netherlands and follow up studies in seven additional case-control groups (2,165 cases and 3,800 controls). The strongest association was observed with allele T of rs9642880 on chromosome 8q24, 30 kb upstream of MYC (allele-specific odds ratio (OR) = 1.22; P = 9.34 × 10−12). Approximately 20% of individuals of European ancestry are homozygous for rs9642880[T], and their estimated risk of developing UBC is 1.49 times that of noncarriers. No association was observed between UBC and the four 8q24 variants previously associated with prostate, colorectal and breast cancers, nor did rs9642880 associate with any of these three cancers. A weaker signal, but nonetheless of genome-wide significance, was captured by rs710521[A] located near TP63 on chromosome 3q28 (allele-specific OR = 1.19; P = 1. 15 × 10−7).

DNA repair gene polymorphisms, bulky DNA adducts in white blood cells and bladder cancer in a case-control study

Individuals differ widely in their ability to repair DNA damage, and DNA‐repair deficiency may be involved in modulating cancer risk. In a case‐control study of 124 bladder‐cancer patients and 85 hospital controls (urological and non‐urological), 3 DNA polymorphisms localized in 3 genes of different repair pathways (XRCC1‐Arg399Gln, exon 10; XRCC3‐Thr241Met, exon 7; XPD‐Lys751Gln, exon 23) have been analyzed. Results were correlated with DNA damage measured as 32P‐post‐labeling bulky DNA adducts in white blood cells from peripheral blood. Genotyping was performed by PCR‐RFLP analysis, and allele frequencies in cases/controls were as follows: XRCC1‐399Gln = 0.34/0.39, XRCC3‐241Met = 0.48/0.35 and XPD‐751Gln = 0.42/0.42. Odds ratios (ORs) were significantly greater than 1 only for the XRCC3 (exon 7) variant, and they were consistent across the 2 control groups. XPD and XRCC1 appear to have no impact on the risk of bladder cancer. Indeed, the effect of XRCC3 was more evident in non‐smokers [OR = 4.8, 95% confidence interval (CI) 1.1–21.2]. XRCC3 apparently interacted with the N‐acetyltransferase type 2 (NAT‐2) genotype. The effect of XRCC3 was limited to the NAT‐2 slow genotype (OR = 3.4, 95% CI 1.5–7.9), suggesting that XRCC3 might be involved in a common repair pathway of bulky DNA adducts. In addition, the risk of having DNA adduct levels above the median was higher in NAT‐2 slow acetylators, homozygotes for the XRCC3‐241Met variant allele (OR = 14.6, 95% CI 1.5–138). However, any discussion of interactions should be considered preliminary because of the small numbers involved. Our results suggest that bladder‐cancer risk can be genetically modulated by XRCC3, which may repair DNA cross‐link lesions produced by aromatic amines and other environmental chemicals.

Genetic variation in the prostate stem cell antigen gene PSCA confers susceptibility to urinary bladder cancer

We conducted a genome-wide association study on 969 bladder cancer cases and 957 controls from Texas. For fast-track validation, we evaluated 60 SNPs in three additional US populations and validated the top SNP in nine European populations. A missense variant (rs2294008) in the PSCA gene showed consistent association with bladder cancer in US and European populations. Combining all subjects (6,667 cases, 39,590 controls), the overall P-value was 2.14 × 10−10 and the allelic odds ratio was 1.15 (95% confidence interval 1.10–1.20). rs2294008 alters the start codon and is predicted to cause truncation of nine amino acids from the N-terminal signal sequence of the primary PSCA translation product. In vitro reporter gene assay showed that the variant allele significantly reduced promoter activity. Resequencing of the PSCA genomic region showed that rs2294008 is the only common missense SNP in PSCA. Our data identify rs2294008 as a new bladder cancer susceptibility locus.

DNA adduct levels and DNA repair polymorphisms in traffic-exposed workers and a general population sample.

Peripheral blood DNA adducts have been considered an acceptable surrogate for target tissues and possibly predictive of cancer risk. A group of 114 workers exposed to traffic pollution and a random sample of 100 residents were drawn from the EPIC cohort in Florence, a population recently shown to present increased DNA adduct levels (Palli et al., Int J Cancer 2000;87:444–51). DNA bulky adducts and 3 DNA repair gene polymorphisms were analyzed in peripheral leukocytes donated at enrollment, by using 32P‐postlabeling and PCR methods, respectively. Adduct levels were significantly higher for traffic workers among never smokers (p = 0.03) and light current smokers (p = 0.003). In both groups, urban residents tended to show higher levels than those living in suburban areas, and a seasonal trend emerged with adduct levels being highest in summer and lowest in winter. Traffic workers with at least 1 variant allele for XPD‐Lys751Gln polymorphism had significantly higher levels in comparison to workers with 2 common alleles (p = 0.02). A multivariate analysis (after adjustment for age, season, area of residence, smoking, XPD‐Lys751Gln genotype and antioxidant intake) showed a significant 2‐fold association between occupational exposure and higher levels of adducts (odds ratio 2.1; 95% confidence interval 1.1–4.2), in agreement with recent pooled estimates of increased lung cancer risk for similar job titles. Our results suggest that traffic workers and the general population in Florence are exposed to high levels of genotoxic agents related to vehicle emissions. Photochemical pollution in warmer months might be responsible for the seasonal trend of genotoxic damage in this Mediterranean urbanized area.

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.

Polymorphisms/Haplotypes in DNA Repair Genes and Smoking: A Bladder Cancer Case-Control Study

Bladder cancer is associated with tobacco smoking and occupational exposure. The repair of DNA damage has a key role in protecting the genome from the insults of cancer-causing agents. We analyzed 13 polymorphisms in seven DNA repair genes belonging to different repair pathways [X-ray repair cross-complementing group 1 (XRCC1): 26304C>T, 26651A>G, 28152A>G; xeroderma pigmentosum-D (XPD): 23591A>G, 35931A>C; excision repair complementing defective in Chinese hamster, group 1 (ERCC1): 19007C>T; XRCC3: 4541T>C, 17893A>G, 18067C>T; proliferating cell nuclear antigen (PCNA): 6084G>C; ERCC4: 30028C>T, 30147A>G; and XRCC2-31479A>G] in 317 incident bladder cancer patients and 317 controls. After adjustment for age and smoking, the PCNA-6084C variant was significantly associated with an increased risk of bladder cancer [CC + CG versus GG, odds ratio (OR), 1.61; 95% confidence interval (95% CI), 1.00-2.61], as well as the XRCC1-26651G variant (GG+AG versus AA: OR, 1.73; 95% CI, 1.17-2.56). After stratifying by smoking habits, an elevated risk for carriers of the XRCC3-18067T allele was detected both in current (TT versus CC: OR, 2.65; 95% CI, 1.21-5.80; CT versus CC: OR, 1.96; 95% CI, 1.09-3.52) and never smokers (TT versus CC: OR, 4.34; 95% CI, 1.14-16.46; CT versus CC: OR, 2.02; 95% CI, 0.72-5.66), whereas an opposite and slightly weaker effect was associated to the XRCC3-17893G allele in current smokers (GG versus AA: OR, 0.30; 95%CI, 0.11-0.82; AG versus AA: OR, 0.73; 95% CI, 0.42-1.27). XRCC3,XRCC1, ERCC4, and XPD-ERCC1 haplotype frequencies were estimated by the maximum likelihood method. The XRCC3-TAT haplotype was associated with an enhanced risk in the current smokers group (OR, 1.62; 95% CI, 1.15-2.29), whereas a reduction of the risk in the overall sample was observed in the presence of the XRCC3-TAC (OR, 0.69; 95% CI, 0.50-0.97). A significant protective effect of the XPD-ERCC1-ACC haplotype was observed among never smokers (OR, 0.16; 95% CI, 0.03-0.81). Our results suggest that polymorphisms and/or haplotypes in XRCC3, XRCC1, and PCNA genes and spanning XPD-ERCC1 region may modulate bladder cancer risk and that some of these effects may preferentially affect current smokers.

Genomewide association study using a high-density single nucleotide polymorphism array and case-control design identifies a novel essential hypertension susceptibility locus in the promoter region of endothelial NO synthase

Essential hypertension is a multifactorial disorder and is the main risk factor for renal and cardiovascular complications. The research on the genetics of hypertension has been frustrated by the small predictive value of the discovered genetic variants. The HYPERGENES Project investigated associations between genetic variants and essential hypertension pursuing a 2-stage study by recruiting cases and controls from extensively characterized cohorts recruited over many years in different European regions. The discovery phase consisted of 1865 cases and 1750 controls genotyped with 1M Illumina array. Best hits were followed up in a validation panel of 1385 cases and 1246 controls that were genotyped with a custom array of 14 055 markers. We identified a new hypertension susceptibility locus (rs3918226) in the promoter region of the endothelial NO synthase gene (odds ratio: 1.54 [95% CI: 1.37–1.73]; combined P=2.58 · 10−13). A meta-analysis, using other in silico/de novo genotyping data for a total of 21 714 subjects, resulted in an overall odds ratio of 1.34 (95% CI: 1.25–1.44; P=1.032 · 10−14). The quantitative analysis on a population-based sample revealed an effect size of 1.91 (95% CI: 0.16–3.66) for systolic and 1.40 (95% CI: 0.25–2.55) for diastolic blood pressure. We identified in silico a potential binding site for ETS transcription factors directly next to rs3918226, suggesting a potential modulation of endothelial NO synthase expression. Biological evidence links endothelial NO synthase with hypertension, because it is a critical mediator of cardiovascular homeostasis and blood pressure control via vascular tone regulation. This finding supports the hypothesis that there may be a causal genetic variation at this locus.

DNA Repair Polymorphisms Modify Bladder Cancer Risk: A Multi-factor Analytic Strategy

Objectives: A number of common non-synonymous single nucleotide polymorphisms (SNPs) in DNA repair genes have been reported to modify bladder cancer risk. These include: APE1-Asn148Gln, XRCC1-Arg399Gln and XRCC1-Arg194Trp in the BER pathway, XPD-Gln751Lys in the NER pathway and XRCC3-Thr241Met in the DSB repair pathway. Methods: To examine the independent and interacting effects of these SNPs in a large study group, we analyzed these genotypes in 1,029 cases and 1,281 controls enrolled in two case-control studies of incident bladder cancer, one conducted in New Hampshire, USA and the other in Turin, Italy. Results: The odds ratio among current smokers with the variant XRCC3-241 (TT) genotype was 1.7 (95% CI 1.0–2.7) compared to wild-type. We evaluated gene-environment and gene-gene interactions using four analytic approaches: logistic regression, Multifactor Dimensionality Reduction (MDR), hierarchical interaction graphs, classification and regression trees (CART), and logic regression analyses. All five methods supported a gene-gene interaction between XRCC1-399/XRCC3-241 (p = 0.001) (adjusted OR for XRCC1-399 GG, XRCC3-241 TT vs. wild-type 2.0 (95% CI 1.4–3.0)). Three methods predicted an interaction between XRCC1-399/XPD-751 (p = 0.008) (adjusted OR for XRCC1-399 GA or AA, XRCC3-241 AA vs. wild-type 1.4 (95% CI 1.1–2.0)). Conclusions: These results support the hypothesis that common polymorphisms in DNA repair genes modify bladder cancer risk and highlight the value of using multiple complementary analytic approaches to identify multi-factor interactions.

Polymorphic DNA repair and metabolic genes: a multigenic study on gastric cancer

Risk factors for gastric cancer (GC) include inter-individual variability in the inflammatory response to Helicobacter pylori infection, in the ability of detoxifying DNA reactive species and repairing DNA damage generated by oxidative stress and dietary carcinogens. To evaluate the association between polymorphic DNA repair genes and GC risk, a case-control study including 314 histologically confirmed GC patients and 548 healthy controls was conducted in a GC high-risk area in Tuscany, Italy. Polymorphic variants of base excision repair (APE1-D148E, XRCC1-R194W, XRCC1-R399Q and OGG1-S326C), nucleotide excision repair (XPC-PAT, XPA-23G>A, ERCC1-19007T>C and XPD-L751Q), recombination (XRCC3-T241M) and alkylation damage reversal (MGMT-L84F) were tested for their potential role in the development of GC by using logistic regression models. The same population was also characterised for GSTT1 and GSTM1 variant alleles to search for possible functional interactions between metabolic and DNA repair genotypes by two-way interactions using multivariate logistic models. No significant association between any single DNA repair genotype and GC risk was detected with a borderline association with the XPC-PAT homozygous genotype [odds ratio (OR) =1.42; 95% confidence interval (CI) 0.94-2.17]. Gene-gene interaction analysis revealed combinations of unfavourable genotypes involving either multiple DNA repair polymorphisms or DNA repair and GST-specific genotypes. The combination of the XPC-PAT and the XPA variant alleles significantly increased GC risk (OR=2.15; 95% CI 1.17-3.93, P=0.0092). A significant interaction was also found between the APE1 wild-type genotype and either the single GSTT1 (OR=4.90; 95% CI 2.38-10.11, P=0.0079) or double GSTM1-GSTT1 null (OR=7.84; 95% CI 3.19-19.22, P=0.0169) genotypes or the XPA-mutant allele (OR=3.56; 95% CI 1.53-8.25, P=0.0012). These findings indicate that a complex interaction between host factors such as oxidative stress, antioxidant capacity and efficiency of multiple DNA repair pathways underlies the inter-individual variability in GC risk.

TP53 and KRAS2 Mutations in Plasma DNA of Healthy Subjects and Subsequent Cancer Occurrence: A Prospective Study

In cancer patients, plasma often contains mutant DNA released by cancer cells. We have assessed the significance of plasma DNA mutations for subsequent cancer development in healthy subjects in a large longitudinal prospective study. The European Prospective Investigation into Cancer and Nutrition study was analyzed with a nested case-control design. Cases were nonsmokers or ex-smokers for >10 years and newly diagnosed with lung, bladder, or upper aerodigestive tract cancers or leukemia accrued after a median follow-up of 6.3 years. Controls were matched 2:1 for follow-up, age, sex, area of recruitment, and smoking status. KRAS2 mutations were detected by mutant-enriched PCR and sequencing (n = 1,098). TP53 mutations were detected by denaturing high-performance liquid chromatography, temporal temperature gradient electrophoresis, and sequencing (n = 550). KRAS2 or TP53 mutations were detected in 13 of 1,098 (1.2%) and 20 of 550 (3.6%) subjects, respectively, 16 of whom developed cancer on average after 18.3 months of follow-up. Among 137 subjects who developed bladder cancer, 5 had KRAS2 mutations [odds ratio (OR), 4.25; 95% confidence interval (95% CI), 1.27-14.15] and 7 had TP53 mutations (OR, 1.81; 95% CI, 0.66-4.97). There was a nonsignificant trend for association between TP53 mutations and bulky adducts in lymphocyte DNA (OR, 2.78; 95% CI, 0.64-12.17). This is the first report of TP53 or KRAS2 mutations in the plasma of healthy subjects in a prospective study, suggesting that KRAS2 mutation is detectable ahead of bladder cancer diagnosis. TP53 mutation may be associated with environmental exposures. These observations have implications for monitoring early steps of carcinogenesis.