A candidate gene approach to searching for low-penetrance breast and prostate cancer genes.

Most cases of breast and prostate cancer are not associated with mutations in known high-penetrance genes, indicating the involvement of multiple low-penetrance risk alleles. Studies that have attempted to identify these genes have met with limited success. The National Cancer Institute Breast and Prostate Cancer Cohort Consortium--a pooled analysis of multiple large cohort studies with a total of more than 5,000 cases of breast cancer and 8,000 cases of prostate cancer--was therefore initiated. The goal of this consortium is to characterize variations in approximately 50 genes that mediate two pathways that are associated with these cancers--the steroid-hormone metabolism pathway and the insulin-like growth factor signalling pathway--and to associate these variations with cancer risk.

A major susceptibility locus to murine lung carcinogenesis maps on chromosome 6.

Lung tumours represent a major cause of death in humans, and although smoking represents the main pathogenetic factor, inheritance also plays a part. However, the identification of possible predisposing genetic factors is difficult, because of their low penetrance. We took advantage of murine strains that are genetically susceptible or resistant to lung tumour development, to map murine genes associated with susceptibility to lung carcinogenesis. An F2 population of urethan-treated A/J x C3H/He mice was scored with 83 genetic markers. A chromosome 6 distal region, spanning mice was scored with 83 genetic markers. A chromosome 6 distal region, spanning 35 centiMorgans, contained a major lung tumour susceptibility locus. No other chromosomal region was significantly associated with lung tumour development.

Host immune genetic variations influence the risk of developing acute myeloid leukaemia: results from the NuCLEAR consortium.

The purpose of this study was to conduct a two-stage case control association study including 654 acute myeloid leukaemia (AML) patients and 3477 controls ascertained through the NuCLEAR consortium to evaluate the effect of 27 immune-related single nucleotide polymorphisms (SNPs) on AML risk. In a pooled analysis of cohort studies, we found that carriers of the IL13rs1295686A/A genotype had an increased risk of AML (PCorr = 0.0144) whereas carriers of the VEGFArs25648T allele had a decreased risk of developing the disease (PCorr = 0.00086). In addition, we found an association of the IL8rs2227307 SNP with a decreased risk of developing AML that remained marginally significant after multiple testing (PCorr = 0.072). Functional experiments suggested that the effect of the IL13rs1295686 SNP on AML risk might be explained by its role in regulating IL1Ra secretion that modulates AML blast proliferation. Likewise, the protective effect of the IL8rs2227307 SNP might be mediated by TLR2-mediated immune responses that affect AML blast viability, proliferation and chemorresistance. Despite the potential interest of these results, additional functional studies are still warranted to unravel the mechanisms by which these variants modulate the risk of AML. These findings suggested that IL13, VEGFA and IL8 SNPs play a role in modulating AML risk.

Cytokine gene polymorphisms and the risk of adenocarcinoma of the stomach in the European prospective investigation into cancer and nutrition (EPIC-EURGAST).

BACKGROUND: The relative contribution to gastric cancer (GC) risk of variants in genes that determine the inflammatory response remains mostly unknown and results from genotyping studies are inconsistent. PATIENTS AND METHODS: A nested case-control study within the prospective European Prospective Investigation into Cancer and Nutrition cohort was carried out, including 248 gastric adenocarcinomas and 770 matched controls. Twenty common polymorphisms at cytokine genes [interleukin (IL)1A, IL1B, IL1RN, IL4, IL4R, IL6, IL8, IL10, IL12A, IL12B, lymphotoxin alpha and tumor necrosis factor (TNF)] were analyzed. Antibodies against Helicobacter pylori (Hp) and CagA were measured. RESULTS: IL1RN 2R/2R genotype [odds ratio (OR) 2.43; 95% confidence interval (CI) 1.19-4.96] and allele IL1RN Ex5-35C were associated with an increased risk of Hp(+) non-cardia GC. IL8 -251AA genotype was associated with a decreased risk of Hp(+) non-cardia GC (OR 0.51; 95% CI 0.32-0.81), mainly of the intestinal type. These associations were not modified by CagA status. Carriers of IL1B -580C and TNF -487A alleles did not associate with an increased risk. A moderately increased risk of Hp(+) non-cardia GC for IL4R -29429T variant was observed (OR 1.74; 95% CI 1.15-2.63). CONCLUSION: This prospective study confirms the association of IL1RN polymorphisms with the risk of non-cardia GC and indicates that IL8 -251T>A may modify the risk for GC.

Instability of microsatellites in rat colon tumors induced by heterocyclic amines.

Microsatellite instability in rat colon tumors induced by heterocyclic amines was examined by studies on the lengths of 85 microsatellite sequences, covering most of the rat chromosomes in tumors and normal tissues. Seven of eight colon tumors induced by 2-amino-1-methyl-6- phenylimidazo-[4,5-b]pyridine showed alterations at least at one locus of microsatellite sequences, whereas no mutations were observed in colon tumors induced by 2-amino-3-methylimidazo[4,5-f]quinoline. Three 2-amino-1-methyl-6-phenylimidazo-[4,5-b]pyridine-induced colon tumors had mutations in more than one microsatellite, their mutation rates being 2 of 85, 2 of 85, and 3 of 85 allele/mircrosatellite sequence, respectively. These data suggest that rat colon adenocarcinomas induced by 2-amino-1-methyl-6- phenylimidazo-[4,5-b]pyridine but not 2-amino-3-methylimidazo[4,5-f] quinoline show a trait of microsatellite instability. This is the first systematic study of microsatellite instability in experimental animal models of carcinogenesis.

Chromosome mapping of murine susceptibility loci to liver carcinogenesis.

The validity of mouse liver tumors is controversial in the risk assessment of carcinogenicity of chemicals in humans, because mice used in carcinogenicity bioassays are genetically predisposed to liver tumors. The argument could be resolved once liver tumor susceptibility genes have been cloned and their role in liver tumor development elucidated. We performed a genetic linkage analysis to map murine liver tumor susceptibility genes, as a first step toward their identification. An F2 population of 87 urethane-treated male A/J x C3H/He mice was scored with 83 genetic markers. Three regions, localized on chromosomes 7, 8, and 12, were found to contain putative liver tumor susceptibility genes.

Semiautomated assessment of loss of heterozygosity and replication error in tumors.

Loss of heterozygosity (LOH) and replication error (RER) are important phenomena in tumor development, with diagnostic and prognostic relevance. Therefore, screening for LOH and RER is a desirable first step in the molecular analysis of tumors. We used semiautomated procedures based on multicolor fluorescently labeled microsatellite markers and an automated sequencer for PCR amplification, electrophoresis of PCR products, and allele detection with a set of 16 microsatellites in 56 colorectal tumors. We improved existing software for computer-assisted assessment of LOH and RER. A comparison of these results with those of a conventional, radioactive technique and visual interpretation shows a high degree of correlation between the two methods. The detection rates of LOH and RER are similar to those reported previously. The main advantages of the semiautomated fluorescence-based typing are in the objective, observer-unrelated, easy, and rapid computer-based scoring, and the resulting quantitative assessment of RER.

Identification of genetic loci controlling hepatocarcinogenesis on rat chromosomes 7 and 10.

Neoplastic liver nodules and hepatocellular carcinomas (HCCs) were induced, by "resistant hepatocyte" model, 32 and 70 weeks after initiation with diethylnitrosamine, respectively, in F344 Brown Norway (BN), and (BNxF344)F1 rats. Nodule number/liver (N) did not significantly differ among rat strains, whereas nodule mean volume (V) and nodule volume fraction (VF) were higher in susceptible F344 than in resistant BN and BFF1 strains and were predictive of subsequent development of HCCs. Genomic scanning of 157 backcross BFF1xF344 rats with 190 polymorphic microsatellites, and linkage analysis, revealed two quantitative trait loci (QTL) on chromosomes 7 and 10, which showed significant linkage with VF, and two QTL on chromosomes 4 and 8, which showed suggestive linkage with V and VF. On the basis of phenotypic patterns of homozygous and heterozygous backcross progeny and of allelic distribution pattern, QTL on chromosomes 10, 8, and 4 were tentatively identified as resistance loci, and QTL on chromosome 7 was identified as susceptibility locus for rat hepatocarcinogenesis. An analysis of interactions allowed us to identify additional putative QTL on chromosomes 5 and 8 and suggested an additive effect of loci on chromosomes 10, 8, and 4 for VF and V. These data are the first to identify chromosomal regions containing putative susceptibility/resistance loci for rat hepatocarcinogenesis, which seems to be highly complex in terms of the number of genetic factors involved.

Data mining: Efficiency of using sequence databases for polymorphism discovery.

An open question in research on Single Nucleotide Polymorphisms (SNPs) is, what is the percentage of true SNPs found by in silico pre-screening? To this end, we selected 13 genes, and determined the complete collection of "true" polymorphisms, or polymorphisms experimentally detected, existing in these genes in our laboratory using Denaturing High Performance Liquid Chromatography (DHPLC) and fluorescent sequencing, or in other laboratories using comparable methods. The genes studied by our group were PTGS2, IGFBP1, IGFBP3, and CYP19. GenBank sequence information was then aligned using two methods, and sequence differences termed "candidate" polymorphisms. We then compared the series of SNPs obtained experimentally and in silico and we have found that in silico methods are relatively specific (up to 55% of candidate SNPs found by SNPFinder have been discovered by experimental procedure) but have low sensitivity (not more than 27% of true SNPs are found by in silico methods).

Genetic heterogeneity in familial nonmedullary thyroid carcinoma: exclusion of linkage to RET, MNG1, and TCO in 56 families. NMTC Consortium.

Epidemiological studies show a very high relative risk for first degree relatives of probands with thyroid cancer. The familial form of nonmedullary thyroid carcinoma (NMTC) gives a more severe phenotype and appears earlier than its sporadic counterpart. Moreover, benign thyroid pathologies are often observed in NMTC kindreds. Little is known about the genetic risk factors of the disease. To study them, an international consortium has been organized at the International Agency for Research on Cancer over the past 2 yr to collect biological samples from NMTC families. The only genes known to be directly involved in susceptibility to NMTC are MNG1 on chromosome 14q32 and TCO on chromosome 19q13.2, previously localized by us and others. In addition to those two genes, the genes for Cowden's syndrome and familial adenomatous polyposis are associated with thyroid cancer, but not as an indicative phenotype. Another important gene in thyroid carcinogenesis is RET, which is mutated in the majority of cases of hereditary medullary thyroid cancer and rearranged in an important fraction of sporadic cases of NMTC. Here we report the result of a linkage analysis performed on the 56 more informative kindreds we have collected through the international consortium. Linkage analysis using both parametric and nonparametric methods excluded MNG1, TCO, and RET as major genes of susceptibility to NMTC and demonstrated that this trait is characterized by genetic heterogeneity.

Genetic polymorphisms and susceptibility to cancer development.

Humans show heterogeneous susceptibility to cancer development, suggesting the involvement of various genetic backgrounds in control of the production of endogenous carcinogens, the metabolism of carcinogens, the repair of DNA damage, cell proliferation and defence mechanisms including immune reactions. Gastric cancer is the major cancer in Japan. However, little is known about the genes linked with its development. In 1967, we found that N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) induced gastric cancers in Wistar rats. Subsequently the Buffalo strain of rats was reported to be resistant to MNNG stomach carcinogenesis, while ACI rats were very sensitive. In a carcinogenesis study using F1 and F2 rats, we suggested that this trait of MNNG stomach carcinogenesis-resistance was regulated by a single autosomal dominant allele. The O6-methylguanine adduct levels in gastric mucosa induced by MNNG were the same in Buffalo and ACI rats, but cell proliferation induced by MNNG was much higher in ACI than Buffalo animals. Chromosome mapping of the gene responsible for susceptibility to MNNG-induced carcinogenesis is now in progress and its identification will hopefully give us clues to the involvement of genetic traits in susceptibility to gastric cancer in humans. In addition, the genetic background of susceptibility to breast cancer is also being studied. In Japan, about 5% of all cases of breast cancer are familial. We have studied BRCA1, the breast cancer susceptibility gene, as a determinant of susceptibility to breast cancer by linkage analyses in 11 families, but our results indicate that BRCA1 may not be important for development of familial breast cancer in Japanese.(ABSTRACT TRUNCATED AT 250 WORDS)

At least three genes account for familial papillary thyroid carcinoma: TCO and MNG1 excluded as susceptibility loci from a large Tasmanian family.

Recent studies have mapped two susceptibility loci which appear to account for familial multinodular goitre (MNG1) and a variant of familial papillary thyroid cancer (PTC), with associated multinodular goitre (TCO). A Tasmanian family (Tas1) has been identified with an autosomal dominant form of PTC. This study has examined the MNG1 and TCO loci to determine if they are similarly predisposing the Tas1 family to PTC. Linkage analysis using identical microsatellite markers described in the two previous studies was used to determine the significance of these loci in the Tasmanian family. The resultant LOD scores were sufficiently negative using multipoint parametric analysis to exclude these two loci from involvement in the Tasmanian family. In addition, six candidate genes, RET, TRK, MET, TSHR, APC and PTEN were also excluded as susceptibility genes in Tas1 by using microsatellites that are positioned in or in close proximity to these genes. These results suggest that there are at least three susceptibility genes that predispose families to familial PTC.

Mapping of a gene predisposing to familial thyroid tumors with cell oxyphilia to chromosome 19 and exclusion of JUN B as a candidate gene.

BACKGROUND: Familial nonmedullary thyroid carcinoma (FNMTC) is a clinical entity characterized by a more aggressive phenotype than the sporadic counterpart. The transmission of susceptibility of FNMTC is compatible with autosomal dominant inheritance. We report the identification of a new entity of FNMTC and the mapping of the responsible gene named TCO (for thyroid tumor with cell oxyphilia). METHODS: In one family, multinodular goiters were diagnosed in six individuals and papillary thyroid carcinoma was diagnosed in three. Eight patients were operated on. Blood samples were collected from the nine affected patients and from eight unaffected relatives. The gene was mapped by linkage analysis with a whole-genome panel of microsatellite markers. RESULTS: The neoplastic cells from all lesions showed characteristic faint to marked cytoplasmic oxyphilia. We found a logarithm of odd ratio (LOD) score of 2.41 at theta = 0 for marker D19S586. Additional markers were typed in the region and were found to be in linkage, with LOD scores peaking at markers D19S916 (Zmax = 3.01 at theta = 0) and D19S413 (Zmax = 2.95 at theta = 0). All these markers have been physically mapped to 19p13.2. CONCLUSIONS: TCO was mapped to chromosome 19p13.2. Interestingly, both the benign and malignant thyroid tumors in this family exhibit some degree of oxyphilia, which has not been described until now in the familial forms of NMTC.

Hepatocarcinogenesis: a polygenic model of inherited predisposition to cancer.

The murine inbred strain C3H provides an experimental model of inherited predisposition to hepatocellular cancer. Hepatocellular neoplastic lesions induced by chemical carcinogens reach a volume 10-100-fold greater in C3H mice than in genetically resistant strains. However, the huge strain differences in tumor size are explained by relatively small differences (10%-30%) in tumor cell kinetics. Genetic linkage experiments in different crosses demonstrated that six unlinked hepatocarcinogen sensitivity (Hcs) and two hepatocarcinogen resistance (Hcr) loci determined quantitative variations in susceptibility to hepatocarcinogenesis. Such results provide the genetic basis for the strain variations in susceptibility to hepatocarcinogenesis and demonstrate a new model of polygenic inheritance of predisposition to cancer.

Expression of nucleoside-metabolizing enzymes in myelodysplastic syndromes and modulation of response to azacitidine.

The nucleoside analog azacitidine (AZA) is used in the treatment of myelodysplastic syndromes (MDS), but 30-40% of patients fail to respond or relapse after treatment. Hence, to identify new molecular alterations that allow for identification of patients unlikely to respond to AZA could impact the utility of this therapy. We determined the expression levels of genes involved in AZA metabolism: UCK1, UCK2, DCK, hENT1, RRM1 and RRM2 using quantitative PCR in samples from 57 patients with MDS who received AZA. Lower expression of UCK1 was seen in patients without a response to AZA (median 0.2 vs 0.49 for patients with response to AZA, P=0.07). This difference in UCK1 expression was not influenced by aberrant methylation of the UCK1 promoter. In addition, the seven polymorphic loci found in the coding sequence were not associated with UCK1 gene expression nor AZA response. Silencing of UCK1 by siRNA leads to blunted response to AZA in vitro. The univariate analysis revealed that patients expressing lower than median levels of UCK1 had a shorter overall survival (P=0.049). Our results suggest that expression level of UCK1 is correlated with clinical outcome and may influence the clinical response to AZA treatment in patients with MDS.

Alcohol dehydrogenase and aldehyde dehydrogenase gene polymorphisms, alcohol intake and the risk of colorectal cancer in the European Prospective Investigation into Cancer and Nutrition study.

BACKGROUND/OBJECTIVES: Heavy alcohol drinking is a risk factor of colorectal cancer (CRC), but little is known on the effect of polymorphisms in the alcohol-metabolizing enzymes, alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) on the alcohol-related risk of CRC in Caucasian populations. SUBJECTS/METHODS: A nested case-control study (1269 cases matched to 2107 controls by sex, age, study centre and date of blood collection) was conducted within the European Prospective Investigation into Cancer and Nutrition (EPIC) to evaluate the impact of rs1229984 (ADH1B), rs1573496 (ADH7) and rs441 (ALDH2) polymorphisms on CRC risk. Using the wild-type variant of each polymorphism as reference category, CRC risk estimates were calculated using conditional logistic regression, with adjustment for matching factors. RESULTS: Individuals carrying one copy of the rs1229984(A) (ADH1B) allele (fast metabolizers) showed an average daily alcohol intake of 4.3 g per day lower than subjects with two copies of the rs1229984(G) allele (slow metabolizers) (P(diff)<0.01). None of the polymorphisms was associated with risk of CRC or cancers of the colon or rectum. Heavy alcohol intake was more strongly associated with CRC risk among carriers of the rs1573496(C) allele, with odds ratio equal to 2.13 (95% confidence interval: 1.26-3.59) compared with wild-type subjects with low alcohol consumption (P(interaction)=0.07). CONCLUSIONS: The rs1229984(A) (ADH1B) allele was associated with a reduction in alcohol consumption. The rs1229984 (ADH1B), rs1573496 (ADH7) and rs441 (ALDH2) polymorphisms were not associated with CRC risk overall in Western-European populations. However, the relationship between alcohol and CRC risk might be modulated by the rs1573496 (ADH7) polymorphism.

Polymorphisms of genes coding for insulin-like growth factor 1 and its major binding proteins, circulating levels of IGF-I and IGFBP-3 and breast cancer risk: results from the EPIC study.

Insulin-like growth factor I (IGF-I) stimulates cell proliferation and can enhance the development of tumours in different organs. Epidemiological studies have shown that an elevated level of circulating IGF-I is associated with increased risk of breast cancer, as well as of other cancers. Most of circulating IGF-I is bound to an acid-labile subunit and to one of six insulin-like growth factor binding proteins (IGFBPs), among which the most important are IGFBP-3 and IGFBP-1. Polymorphisms of the IGF1 gene and of genes encoding for the major IGF-I carriers may predict circulating levels of IGF-I and have an impact on cancer risk. We tested this hypothesis with a case-control study of 807 breast cancer patients and 1588 matched control subjects, nested within the European Prospective Investigation into Cancer and Nutrition. We genotyped 23 common single nucleotide polymorphisms in IGF1, IGFBP1, IGFBP3 and IGFALS, and measured serum levels of IGF-I and IGFBP-3 in samples of cases and controls. We found a weak but significant association of polymorphisms at the 5' end of the IGF1 gene with breast cancer risk, particularly among women younger than 55 years, and a strong association of polymorphisms located in the 5' end of IGFBP3 with circulating levels of IGFBP-3, which confirms previous findings. Common genetic variation in these candidate genes does not play a major role in altering breast cancer risk in Caucasians.

Phylogenetics of the laboratory rat Rattus norvegicus.

A genealogic tree was constructed for inbred strains of the laboratory rat, including 63 strains and 214 of their substrains. Information on genetic and biochemical marker typings of these lines was collected from the literature and from the World Wide Web. Data on 995 polymorphisms were processed into a phylogenetic distance matrix, and a tree was obtained by the Fitch-Margoliash distance matrix method. The inbred strains of the laboratory rat showed an average polymorphism for pairwise comparison of 53%. Strain BN showed the highest genetic divergence from all the other ones. Comparison with the mouse phylogenetic tree indicated that laboratory rats possess a higher diversity than inbred strains of mice not derived from wild species. These results provide a phylogenetic basis in the choice of rat strains for genetic linkage experiments.

Genotype transposer: automated genotype manipulation for linkage disequilibrium analysis.

UNLABELLED: The purpose of this work is to provide the modern molecular geneticist with tools to perform more efficient and more accurate analysis of the genotype data they produce. By using Microsoft Excel macros written in Visual Basic, we can translate genotype data into a form readable by the versatile software 'Arlequin', read the Arlequin output, calculate statistics of linkage disequilibrium, and put the results in a format for viewing with the software 'GOLD'. AVAILABILITY: The software is available by FTP at: ftp://xcsg.iarc.fr/cox/Genotype_Transposer/. SUPPLEMENTARY INFORMATION: Detailed instruction and examples are available at: ftp://xcsg.iarc.fr/cox/Genotype&_Transposer/. Arlequin is available at: http://lgb.unige.ch/arlequin/. GOLD is available at: http://www.well.ox.ac.uk/asthma/GOLD/.