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Mutation scanning using high-resolution melting curve analysis (HR-melt) is an effective and sensitive method to detect sequence variations. However, the presence of a common SNP within a mutation scanning amplicon may considerably complicate the interpretation of results and increase the number of samples flagged for sequencing by interfering with the clustering of samples according to melting profiles. A protocol describing simultaneous high-resolution gene scanning and genotyping has been reported. Here, we show that it can improve the sensitivity and the efficiency of large-scale case-control mutation screening. Two exons of ATM, both containing an SNP interfering with standard mutation scanning, were selected for screening of 1,356 subjects from an international breast cancer genetics study. Asymmetric PCR was performed in the presence of an SNP-specific unlabeled probe. Stratification of the samples according to their probe-target melting was aided by customized HR-melt software. This approach improved identification of rare known and unknown variants, while dramatically reducing the sequencing effort. It even allowed genotyping of tandem SNPs using a single probe. Hence, HR-melt is a rapid, efficient, and cost-effective tool that can be used for high-throughput mutation screening for research, as well as for molecular diagnostic and clinical purposes.

Interleukin-4 (IL-4) and interleukin-4 receptor (IL-4R) modulate inflammation and are associated with the colorectal adenoma-carcinoma progression and the metastatic capacity. IL-4 also causes a dose-dependent reduction of proliferation in colorectal cancer cells. The aim of the study was to evaluate whether genetic variants within IL4 and IL4R could affect the individual risk to develop colorectal cancer. We genotyped all the polymorphisms coding for an aminoacidic change in IL4R and we used a haplotype-tagging SNP approach for IL4 . We carried out a case-control association study by genotyping, with the 5′ nuclease assay, two common SNPs within IL4 (−588C>T, Ex1-168G>A) and five SNPs within IL4R (I75V, C431R, S436L, S503P, Q576R) in 377 cases of colorectal cancer and 326 controls from Spain. No statistically significant association between the SNPs investigated and colorectal cancer risk was found, as main effects. When the sub-analyses were carried out, the homozygotes for IL4 −588C>T or for Ex1-168G>A showed an increased risk for colon cancer only, with the odds ratios of 4 (95% CI 0.97–16.6; P -interaction=0.016 and 4.66 (95% CI 1.16–18.77; P -interaction=0.023), respectively. Moreover, women showed a significant increased risk associated to the IL4 rare alleles and this was clearly greater than that in men (for Ex1-168G>A: OR=1.96; 95% CI=1.11–3.47; P -interaction=0.006). However, when sub-groups are analysed, the findings should be taken with caution for the weakening of the statistical power.

Glutathione S-transferases (GST) are an important part of cell defense against numerous genotoxic compounds and ROS. In order to test the possibility of association between the GSTT1 and M1 null allele variant, and the risk of TCO (thyroid carcinoma with cell oxyphilia), a case-control study was carried out. The rationale for our study was that according to the important roles of GST enzymes in cells and association of GST null genotypes with many types of tumors, inactivating polymorphisms may be genetic susceptibility factors in the etiology of oxyphilic thyroid tumors characterized by mitochondrial dysfunction, increased ROS production and resistance to chemio- and radio-therapy. We found the frequency of GSTT1 null genotype of 19.2% in cases and 15.7% in controls, with an adjusted odds ratio (OR) of 1.4 (95% confidence interval (CI), 0.70-2.81), and a frequency of GSTM1 null genotype of 59% in cases with oxyphilic tumors and of 55.6% in controls (OR 1.24; 95% CI, 0.62-2.48), indicating that the GSTT1 and M1 null genotypes do not increase the risk of development of oxyphilic tumors.

Objectives: We have undertaken a comprehensive study of common polymorphisms in genes of DNA repair, exploring both the risk of developing colorectal cancer and the prognosis of patients. Methods: Subjects from a case-control study (377 cases and 329 controls) designed to assess gene-environment interactions were genotyped by use of an oligonucleotide microarray and the arrayed primer extension technique. Twenty-eight single nucleotide polymorphisms in 15 DNA repair genes were included. The candidate genes belong to different DNA repair pathways: base excision repair (OGG1, LIG3, APEX, POLB, XRCC1, PCNA, and MUTYH), nucleotide excision repair (ERCC1, ERCC2, ERCC4, and ERCC5), double-strand breaks repair (XRCC2, XRCC3, and XRCC9), and reversion repair (MGMT) genes. Results: Polymorphism OGG1 S326C was associated with an increased risk of colorectal cancer [odds ratio (OR), 2.3; 95% confidence interval (95% CI), 1.1-5.0], the risk being higher in younger individuals. A haplotype of ERCC1 was associated with increased risk (OR, 2.3; 95% CI, 1.0-5.3). POLB P242R was also associated with decreased risk (OR, 0.23; 95% CI, 0.05-0.99), although the number of variant allele carriers was low. In the univariate analysis, adjusted for age, sex, and Dukes' stage, three polymorphisms were significantly associated with better prognosis: XRCC1 R399Q [hazard ratio (HR), 0.38; 95% CI, 0.17-0.85], XRCC3 T141M (HR, 0.66; 95% CI, 0.45-0.97), and MGMT L84F (HR, 0.14; 95% CI, 0.02-0.99). ERCC1 19007T>C was associated with worse prognosis (HR, 1.51; 95% CI, 1.01-2.27). In a multivariate analysis, only XRCC1 R399Q and ERCC1 19007T>C remained significant. These associations were stronger among patients receiving adjuvant chemotherapy. Conclusions: Although the overall effect of DNA repair genes in colorectal cancer etiology seems limited, their influence in the response to chemotherapy and prognosis may be more relevant. This knowledge may help to clarify the utility of specific adjuvant treatments according to the individual genetic background.

It has been demonstrated that polymorphisms within inflammation-related genes are associated with risk of gastric carcinoma in Helicobacter pylori-infected individuals. Recently, several studies have reported conflicting results regarding the association between the interleukin (IL)8-251*T/*A polymorphism and risk of gastric carcinoma. In this study, we performed a case-control analysis, including 693 controls, 187 chronic gastritis cases and 333 gastric carcinoma cases, to determine the association between the IL8-251 polymorphism and risk of chronic gastritis and gastric carcinoma in the northern Portugal population. We found no significant association between the IL8-251 polymorphism and increased risk of chronic gastritis or gastric carcinoma, in agreement with that reported in other populations of white origin. The retrospective analysis of published data shows that the association between the IL8-251 polymorphism and risk of gastric carcinoma tends to be reproducible in populations of Asian origin. The estimated effect of the polymorphism under analysis was not significantly different in subgroups of gastric carcinoma cases defined by histologic type and anatomic site of the tumours, and by sex and age of the participants. In conclusion our results indicate that although the IL8-251 polymorphism might be a relevant host susceptibility factor for gastric carcinoma development, this association is likely to be ethnic-specific.

A key fatty acid synthesis enzyme, acetyl-CoA carboxylase α (ACC-α), has been shown to be highly expressed in human breast cancer and other tumor types and also to specifically interact with the protein coded by one of two major breast cancer susceptibility genes BRCA1. We used a comprehensive haplotype analysis to examine the contribution of the ACC-α common genetic variation (allele frequency >5%) to breast cancer in a case-control study (1,588 cases/2,600 controls) nested within the European Prospective Investigation into Cancer and Nutrition. We identified 21 haplotype-tagging polymorphisms efficiently capturing common variation within 325 kb of ACC-α and surrounding sequences using genotype data from the HapMap project and our resequencing data. We found an effect on overall risk of breast cancer in homozygous carriers of one common haplotype [odds ratio (OR), 1.74; 95% confidence interval (95% CI), 1.03-2.94]. When the data were subdivided by menopausal status, we found statistical evidence of heterogeneity for two other common haplotypes (P value for heterogeneity = 0.016 and 0.045). In premenopausal women, the carriers of these haplotypes, compared with noncarriers, had an altered risk of breast cancer (OR, 0.70; 95% CI, 0.53-0.92 and OR, 1.35; 95% CI, 1.04-1.76). These findings were not significant after adjustment for multiple testing and therefore should be considered as preliminary and evaluated in larger independent studies. However, they suggest a possible role of the ACC-α common sequence variants in susceptibility to breast cancer and encourage studies of other genes involved in fatty acid synthesis. (Cancer Epidemiol Biomarkers Prev 2007;16(3):409–15)

The growth hormone receptor (GHR) is potentially involved in prostate cancer through its role in stimulating insulin-like growth factor I production and its cellular effects on prostate epithelium. We have used a haplotype-based tagging approach within CAncer Prostate Sweden, a large retrospective case-control study of 2,863 cases and 1,737 controls to investigate if genetic variation in the GHR gene influences prostate cancer risk. One haplotype in the 3′ region of the GHR gene was found associated with prostate cancer risk in elderly men (>65 years old at the time of diagnosis), with heterozygote haplotype carriers having an odds ratio of 1.65 (95% confidence interval, 1.21-2.16; P = 0.0009, Pcorrected = 0.03). GHR function has been implicated in the determination of body mass index. Interestingly, the same haplotype associated with risk in the 3′ end of the GHR gene was also associated with a decrease in body mass index in controls (P = 0.003, Pcorrected = 0.05), possibly indicating some functionality with this haplotype. These results suggest that whereas genetic variation in the GHR gene does not seem to play a major role in prostate cancer etiology, one haplotype in the 3′ region may be potentially relevant to cases with later onset of prostate cancer. (Cancer Epidemiol Biomarkers Prev 2007;16(1):169–73)

Background Chronic inflammation is a risk factor for colorectal cancer and polymorphisms in the inflammatory genes could modulate the levels of inflammation. We have investigated ten single nucleotide polymorphisms (SNPs) in the following inflammation-related genes: TLR4 (Asp299Gly), CD14 (-260 T>C), MCP1 (-2518 A>G), IL12A (+7506 A>T, +8707 A>G, +9177 T>A, +9508 G>A), NOS2A (+524T>C), TNF (-857C>T), and PTGS1 (V444I) in 377 colorectal (CRC) cancer cases and 326 controls from Barcelona (Spain). Results There was no statistically significant association between the SNPs investigated and colorectal cancer risk. Conclusion The lack of association may show that the inflammatory genes selected for this study are not involved in the carcinogenic process of colorectum. Alternatively, the negative results may derive from no particular biological effect of the analysed polymorphisms in relation to CRC. Otherwise, the eventual biological effect is so little to go undetected, unless analysing a much larger sample size.

Helicobacter pylori (HP) infection affects over 50% of the world's population. The prevalence is over 90% in populations at high risk for gastric cancer, but clinical outcomes of the infection are highly variable and thus host genetic factors have been suggested to play a role in its outcomes in addition to bacterial factors. In this study, we examined the effects of common functional genetic polymorphisms of several proinflammatory cytokines known to be overexpressed in HP-infected gastric mucosa on the risk of various stages of gastric premalignant lesions. The odds ratios (ORs) and 95% confidence intervals (CI) for atrophic gastritis, intestinal metaplasia and dysplasia were estimated by multinominal logistic regression analysis among 2,033 Venezuelan subjects. There was a significant effect of IL8 -251A allele on the prevalence of dysplasia (p = 0.021). The OR associated with the A-allele was 1.34 (95% CI: 0.82–2.18) for heterozygotes and 2.00 (95% CI: 1.13–3.56) for homozygotes, compared with the TT genotype. Furthermore, there was a statistically significant interaction between the number of A-alleles and HP cag A genotype (p = 0.009), suggesting that the A-allele increased the risk of dysplasia only when cag A was present. The OR for the AA compared with TT genotype was 3.22 (95% CI: 1.60–6.52) in this group. There were no associations with other proinflammatory cytokines studied, i.e., IL1β, IL6, monocyte chemoattractant protein 1 (MCP1) and TNFα, or with other stages of premalignant lesions. The present study provides important evidence suggesting host–bacterial interactions in the development of gastric precancerous lesions. © 2006 Wiley-Liss, Inc.

As Helicobacter pylori (HP) is a Gram-negative bacterium, we investigated the associations between several functional polymorphisms in genes involved in lipopolysaccharide (LPS) signaling and the prevalence of various stages of gastric premalignant lesions in a Venezuelan population. The two NOD2 polymorphisms, del3020insC and Gly908Arg, were too infrequent to study their associations with gastric lesions. The risk of intestinal metaplasia (IM) was significantly increased among subjects with the CD14 T-260 allele compared to those without this allele. A similar, but nonsignificant increase in risk for dysplasia was observed among homozygotes of this allele. There was no association between TLR4 Asp299Gly polymorphism and any type of lesions, except for a slight nonsignificant increase in risk of IM associated with the AA genotype among subjects with a higher histological HP score. These results suggest that genetic polymorphisms in HP LPS signaling may be implicated in the development of intermediate stages of gastric premalignant lesions.

While DNA of good quality and sufficient amount can be obtained easily from whole blood, buccal swabs, surgical specimens, or cell lines, these DNA-rich sources are not always available. This is particularly the case in studies for which biological specimens were collected when genotyping assays were not widely available. In those studies, serum or plasma is often the only source of DNA. Newly developed whole genome amplification (WGA) methods, based on ϕ29 polymerase, may play a significant role in recovering DNA in such instances. We tested a total of 528 plasma samples kept in storage at -40°C for approximately 10 years for 8 single nucleotide polymorphisms (SNPs) using the 5′ exonuclease (TaqMan®) assay. These specimens yielded undetectable levels of DNA following extraction with an affinity column but produced an average 52.7 µg (standard deviation of 31.2 µg) of DNA when column-extracted DNA was used as a template for WGA. This increased the genotyping success rate from 54% to 93%. There were only 3 disagreements out of 364 paired genotyping results for pre- and post-WGA DNAs, indicating an error rate of 0.82%. These results are encouraging for expanding the use of poor DNA resources in genotyping studies.

Sporadic colorectal cancer is considered a multifactorial disease in which multiple exposures interact with the individual genetic background resulting in risk modulation. Recent experimental data suggest a role of dopamine and dopamine receptors in the control of proliferation of the cells of colon and gastrointestinal tract. To investigate whether polymorphisms within dopamine receptors genes could have a role in modulating the risk of sporadic colorectal cancer, we did a case-control association study and genotyped 370 cases and 327 controls for seven single-nucleotide polymorphisms (SNP) of DRD2 (−141Cdel, 957T>C, TaqIB, TaqIA, 1412A>G, S311C, and 3208G>T) by a microarray-based technique. Three SNPs within DRD2 were associated with colorectal cancer, with a maximum odds ratio of 2.28 (95% confidence interval, 1.38-3.76) for carriers of the functional SNP −141Cdel. The haplotype which includes −141Cdel, together with the variants 957C and 1412G, shows an odds ratio of 2.86 (95% confidence interval, 1.58-5.18), as compared with the most frequent haplotype. The SNPs within DRD2 associated with colorectal cancer are known to be related to reduced levels of D2 dopamine receptor. Thus, our data point to a possible role of dopamine receptor DRD2 in modulating the risk of colorectal cancer. Future studies on dopamine receptor–mediated signal transduction may provide new insight into the mechanisms of colorectal cancer and suggest new therapeutic strategies.