Mismatch repair polymorphisms and risk of colon cancer, tumour microsatellite instability and interactions with lifestyle factors.

BACKGROUND: Germline mutations in DNA mismatch repair (MMR) genes cause Lynch syndrome colon cancers. Less understood is the risk of colon cancer associated with common polymorphisms in MMR genes and the potential interacting role of lifestyle factors known to damage DNA. METHODS: A study was conducted to examine whether MLH1 (-93G>A and Ile219Val) and MSH6 (Gly39Glu) polymorphisms were associated with risk of colon cancer in data from 1609 colon cancer cases and 1972 controls. Genotype data were further stratified by microsatellite instability status, smoking, alcohol, Western diet, alcohol and obesity, to investigate potential heterogeneity. RESULTS: The MSH6 39Glu allele was associated with increased risk of colon cancer among men (Gly/Glu or Glu/Glu vs Gly/Gly, OR 1.27; 95% CI 1.04 to 1.54). Neither MLH1 polymorphism was associated with colon cancer risk overall. When stratified by microsatellite stability status, however, the MLH1 -93A allele was associated with a more than doubling in microsatellite instability (MSI)-positive colon cancer risk (AA vs GG, OR 2.47; 95% CI 1.48 to 4.11); no associations were observed between the MMR polymorphisms examined and MSI-negative colon cancer. Statistically significant interactions were observed between: MLH1 -93G>A and smoking (MSI-negative colon cancer only, p value interaction: 0.005); and MLH1 Ile219Val and Western diet (p value interaction: 0.03). CONCLUSIONS: The MSH6 Gly39Glu and MLH1 -93G>A polymorphisms were associated with risk of overall colon and MSI-positive colon cancers, respectively. Risk for colon cancer, stratified by MMR genotype, was further modified by smoking and Western diet.

Identifying rarer genetic variants for common complex diseases: diseased versus neutral discovery panels.

The power of genetic association studies to identify disease susceptibility alleles fundamentally relies on the variants studied. The standard approach is to determine a set of tagging-SNPs (tSNPs) that capture the majority of genomic variation in regions of interest by exploiting local correlation structures. Typically, tSNPs are selected from neutral discovery panels - collections of individuals comprehensively genotyped across a region. We investigated the implications of discovery panel design on tSNP performance in association studies using realistically-simulated sequence data. We found that discovery panels of 24 sequenced 'neutral' individuals (similar to NIEHS or HapMap ENCODE data) were sufficient to select well-powered tSNPs to identify common susceptibility alleles. For less common alleles (0.01-0.05 frequency) we found neutral panels of this size inadequate, particularly if low-frequency variants were removed prior to tSNP selection; superior tSNPs were found using panels of diseased individuals. Only large neutral panels (200 individuals) matched diseased panel performance in selecting well-powered tSNPs to detect both common and rarer alleles. The 1000 Genomes Project initiative may provide larger neutral panels necessary to identify rarer susceptibility alleles in association studies. In the interim, our results suggest investigators can boost power to detect such alleles by sequencing diseased individuals for tSNP selection.

PER protein interactions and temperature compensation of a circadian clock in Drosophila.

The periods of circadian clocks are relatively temperature-insensitive. Indeed, the perL mutation in the Drosophila melanogaster period gene, a central component of the clock, affects temperature compensation as well as period length. The per protein (PER) contains a dimerization domain (PAS) within which the perL mutation is located. Amino acid substitutions at the perL position rendered PER dimerization temperature-sensitive. In addition, another region of PER interacted with PAS, and the perL mutation enhanced this putative intramolecular interaction, which may compete with PAS-PAS intermolecular interactions. Therefore, temperature compensation of circadian period in Drosophila may be due in part to temperature-independent PER activity, which is based on competition between inter- and intramolecular interactions with similar temperature coefficients.

Temporally regulated nuclear entry of the Drosophila period protein contributes to the circadian clock.

The Drosophila period protein (PER) is a predominantly nuclear protein and a likely component of a circadian clock. PER is required for daily oscillations in the transcription of its own gene and thus participates in a circadian feedback loop. In this study, key pacemaker neurons of the Drosophila brain were examined to determine whether the subcellular distribution of PER changes with the time of day. Indeed, PER was found to accumulate in the cytoplasm for several hours before entering the nucleus during a narrow time window. Three long-period mutations (perL) cause a delay in the timing of nuclear translocation and a further delay at elevated temperature. The data indicate that regulation of PER nuclear entry is critical for circadian oscillations by providing a necessary temporal delay between PER synthesis and its effect on transcription.

The timSL mutant of the Drosophila rhythm gene timeless manifests allele-specific interactions with period gene mutants.

To identify new components of the Drosophila circadian clock, we screened chemically mutagenized flies for suppressors or enhancers of the long periods characteristic of the period (per) mutant allele perL. We isolated a novel mutant that maps to the rhythm gene timeless (tim). This novel allele, timSL, alters the temporal pattern of perL protein nuclear localization and restores temperature compensation to perL flies. timSL more generally manifests specific interactions with different per alleles. The identification of this first period-altering tim allele provides further evidence that TIM is a major component of the clock, and the allele-specific interactions with PER provide evidence that the PER/TIM heterodimer is a unit of circadian function. Although timSL fails to restore PER-L/TIM temperature insensitivity in yeast, it alters the TIM phosphorylation pattern during the late night. The effects on phosphorylation suggest that timSL functions as a partial bypass suppressor of perL and provide evidence that the TIM phosphorylation program contributes to the circadian timekeeping mechanism.

Associations between cigarette smoking, lifestyle factors, and microsatellite instability in colon tumors.

BACKGROUND: Microsatellite instability (MSI) has been reported to occur in approximately 10%-15% of colon tumors. MSI is characterized by the presence of mutations in tandemly repeated DNA sequences known as microsatellites. Some individuals with unstable tumors have inherited mutations in mismatch repair genes, but MSI is also observed in sporadic colon cancer. It is unknown whether lifestyle factors associated with colon cancer, such as physical activity, body size, cigarette smoking, or use of aspirin and/or nonsteroidal anti-inflammatory drugs, contribute to MSI in sporadic tumors. METHODS: Data from a population-based, case-control study of colon cancer were used. Case subjects were between 30 and 79 years of age at the time of diagnosis and included both men and women. Questionnaire data were used to obtain information on lifestyle factors. Tumor MSI was determined with the use of a panel of 10 tetranucleotide repeats and two mononucleotide repeats. A total of 1510 case subjects had valid questionnaire data and tumor DNA from which we were able to obtain MSI status. Questionnaire data were compared with lifestyle factors reported by 2410 population-based control subjects. All statistical tests were two-sided. RESULTS: MSI-positive (MSI(+)) tumors were most common in older people and women and in the proximal colon. Patients with MSI(+) tumors were more likely to smoke 20 or more cigarettes a day than case subjects with MSI-negative (MSI(-)) tumors (odds ratio for being a smoker = 1.6 [95% confidence interval = 1.0-2.5] for men and 2.2 [95% confidence interval = 1.4-3.5] for women). The association between MSI(+) tumors and cigarette smoking was strongest among case subjects who started to smoke at a young age, smoked for 35 or more years, and were either current smokers or had stopped fewer than 15 years before diagnosis. A statistically significant linear trend of increased risk of MSI(+) tumors was observed with increasing amount smoked (P<.01). CONCLUSIONS: This study suggests that smoking cigarettes statistically significantly contributes to MSI in colon tumors. We estimate that approximately 21% of MSI in colon tumors may be attributable to cigarette smoking.

Associations between dietary intake and Ki-ras mutations in colon tumors: a population-based study.

Ki-ras mutations are thought to be early events in the carcinogenic process leading to colon tumors. Dietary factors associated with colon cancer may be associated with these mutations. Data from a population-based, multicenter, case-control study of colon cancer were used to determine whether dietary factors are associated with Ki-ras mutations. Ki-ras mutations were detected by direct sequencing of codons 12 and 13 of the Ki-ras gene on exon 1 from DNA obtained from archival tissue. Ki-ras data were available for 1428 cases with valid interview data; data from 2410 controls were available for comparison with cases positive and negative for Ki-ras mutations. Mutations in the Ki-ras gene were detected in 32% of tumors. Of these mutations, 32.8% were G-->A transitions in the second base of codon 12 (2G-->A). Other than cruciferous vegetables, there were no nutrients or foods associated specifically with Ki-ras mutations [odds ratio (OR) for high intake relative to low intake, 0.7; 95% confidence interval (CI), 0.5-1.0]. However, evaluation of specific types of Ki-ras mutations revealed that for each of the most common types of mutation, dietary associations existed. Dietary factors involved in DNA methylation pathways were associated with 2G-->A mutations. Comparison of individuals with and without Ki-ras mutations revealed that individuals with low levels of dietary folate (OR, 0.7; 95% CI, 0.4-1.3), vitamin B6 (OR, 0.5; 95% CI, 0.3-1.0), vitamin B12 (OR, 0.6; 95% CI, 0.3-1.1), and high levels of alcohol (OR, 0.7; 95% CI, 0.4-1.1) were less likely to have a 2G-->A mutation. Individuals with high levels of dietary carbohydrate (OR, 2.0; 95% CI, 0.9-4.4) and a high glycemic index (OR, 1.9; 95% CI, 0.8-4.6) were more likely to have a G-->A transition mutation in the second base of codon 13 (5G-->A). Individuals with high levels of dietary fat (OR, 1.6; 95% CI, 0.8-3.2), saturated fat (OR, 1.7; 95% CI, 0.8-3.5), and monounsaturated fat (OR, 1.9; 95% CI, 1.0-3.7) were more likely to harbor a 2G-->T mutation. Low levels of cruciferous vegetable intake and high levels of processed meat intake also were associated with fewer 5G-->A, as reflected by the ORs (OR, 0.4; 95% CI, 0.2-1.0 and OR, 0.4; 95% CI 0.2-0.8, respectively). These data suggest that diet may be involved in disease pathways represented by specific Ki-ras mutations. However, given the limited information currently available on associations between specific genetic mutations in colon tumors and diet, these findings also should be viewed as hypothesis generating.

Estrogens reduce and withdrawal of estrogens increase risk of microsatellite instability-positive colon cancer.

There are sex differences in the occurrence of microsatellite instability (MSI) in colon tumors. Taken together with the epidemiological evidence that hormone replacement therapy (HRT) and, less consistently, parity, are inversely associated with colon cancer, it has been hypothesized that estrogens are associated with MSI. The purpose of this study was to evaluate sex-specific differences in the prevalence of MSI in colon tumors and to determine whether reproductive history and hormonal exposures are associated with MSI. Using data from a population-based case-control study of 1836 cases with MSI data and 2410 population-based controls, we evaluated sex, reproductive factors, and hormone exposure in relation to the presence or absence of MSI in tumors. MSI was evaluated by a panel of 10 tetranucleotide repeats, the noncoding mononucleotide repeat BAT-26, and the coding mononucleotide repeat in transforming growth factor beta receptor type II (TGFbetaRII). Exposure data on reproduction, hormone use, obesity, and physical activity were obtained from an interviewer-administered questionnaire. Women were less likely then men to have MSI+ tumors at a young age and more likely to have unstable tumors at an older age; we observed a significant interaction (P < 0.01) between age, sex, and MSI. Evaluation of reproductive factors showed that women who had ever been pregnant had half the risk of MSI+ tumors compared with women who had never been pregnant. In complementary fashion, total ovulatory months were associated with an increased risk of MSI+ tumors [odds ratio (OR), 2.1; 95% confidence interval (CI), 1.1-4.0 comparing MSI+ versus MSI- tumors]. Age at first and last pregnancy did not influence the association. The observed associations were strongest among women <60 years of age at the time of diagnosis. Having used oral contraceptives was associated with a lower risk of MSI+ tumors (OR, 0.7; 95% CI, 0.4-1.2); recent users of HRT were at a reduced risk of MSI+ tumors (OR, 0.8; 95% CI, 0.5-1.4); and women who were former HRT users were at an increased risk of MSI+ tumors (OR, 1.8; 95% CI, 1.1-3.0). Obesity and lack of physical activity were associated with an elevated risk of both MSI+ (OR, 1.7; 95% CI, 0.7-3.3) and MSI- (OR, 2.2; 95% CI, 1.7-3.) tumors in men, but only with MSI- (OR, 1.5; 95% CI, 1.1-2.2) tumors in women. The excess of MSI+ tumors in women is explained by the excess of MSI+ tumors at older ages. Our data suggest that estrogen exposure in women protects against MSI, whereas the lack of estrogen in older women increases risk of instability. HRT in these older women may, again, reduce the risk of unstable tumors. A model for the way in which estrogens (endogenous, exogenous, and obesity-associated) modify the risk of MSI+ tumors is proposed.

Familial colorectal cancer risk by subsite of primary cancer: a population-based study in Utah.

BACKGROUND: Familial occurrence is common in colorectal cancer (CRC), but whether this increased familial risk differs by colonic subsite of the index patients CRC is not well understood. AIM: To quantify the risk of CRC in first-degree (FDR), second-degree (SDR) and first cousin (FC) relatives of individuals with CRC, stratified by subsite in the colorectum and age at diagnosis. METHODS: Colorectal cancers diagnosed between 1980 and 2010 were identified from the Utah Cancer Registry and linked to pedigrees from the Utah Population Database. Age and gender-matched CRC-free controls were selected to form the comparison group for determining CRC risk in relatives using Cox regression analysis. RESULTS: Of the 18,208 index patients diagnosed with CRC, 6584 (36.2%) were located in the proximal colon, 5986 (32.9%) in the distal colon and 5638 (31%) in the rectum. The elevated risk of CRC in relatives was similar in analysis stratified for CRC colorectal subsites in the index cases. FDR had similarly elevated risk of all site CRC, whether the index patient had cancer in the proximal colon [hazards ratio (HR): 1.85; 95% CI: 1.70-2.02], distal colon (HR: 1.90; 95% CI: 1.73-2.08) or rectum (HR: 1.83; 95% CI: 1.66-2.02) compared to relatives of controls. This risk was consistently greater for FDR when cases developed CRC below the age of 60 years. CONCLUSIONS: Relatives of CRC patients have a similarly elevated risk of CRC regardless of colonic tumour subsite in the index patient, and it is greatest for relatives of younger age index cases.

Drosophila has several genes for gap junction proteins.

The Innexin gene family forms gap junctions in invertebrates. Many genes in this family have been identified in Caenorhabditis elegans, but only two in Drosophila. We have used PCR techniques to identify three new members of this family from Drosophila. These are designated pas-related proteins (prp) 6, 7, and 33. The putative proteins coded by these new genes show 25-35% identity and 39-66% similarity to other Drosophila innexins and share a similar hydrophobicity profile. The genes form two small clusters on the X-chromosome, with three of the genes sitting within 10kb of each other. The closeness in sequence and location suggests an evolutionary origin of these genes via local duplication. In situ hybridization shows expression in the CNS, gut and epidermis. Each gene has a distinct pattern of expression in different tissues at different developmental times. However, parts of the expression patterns overlap, especially for prp33 and ogre which may be expressed from the same transcriptional enhancers. This suggest that the Prp33 and Ogre proteins may join in forming heteromeric gap junction channels.

Carotenoids and colon cancer.

BACKGROUND: Carotenoids have numerous biological properties that may underpin a role for them as chemopreventive agents. However, except for beta-carotene, little is known about how dietary carotenoids are associated with common cancers, including colon cancer. OBJECTIVE: The objective of this study was to evaluate associations between dietary alpha-carotene, beta-carotene, lycopene, lutein, zeaxanthin, and beta-cryptoxanthin and the risk of colon cancer. DESIGN: Data were collected from 1993 case subjects with first primary incident adenocarcinoma of the colon and from 2410 population-based control subjects. Dietary data were collected from a detailed diet-history questionnaire and nutrient values for dietary carotenoids were obtained from the US Department of Agriculture-Nutrition Coordinating Center carotenoid database (1998 updated version). RESULTS: Lutein was inversely associated with colon cancer in both men and women [odds ratio (OR) for upper quintile of intake relative to lowest quintile of intake: 0.83; 95% CI: 0.66, 1.04; P = 0.04 for linear trend]. The greatest inverse association was observed among subjects in whom colon cancer was diagnosed when they were young (OR: 0.66; 95% CI: 0.48, 0.92; P = 0.02 for linear trend) and among those with tumors located in the proximal segment of the colon (OR: 0.65; 95% CI: 0.51, 0.91; P < 0.01 for linear trend). The associations with other carotenoids were unremarkable. CONCLUSION: The major dietary sources of lutein in subjects with colon cancer and in control subjects were spinach, broccoli, lettuce, tomatoes, oranges and orange juice, carrots, celery, and greens. These data suggest that incorporating these foods into the diet may help reduce the risk of developing colon cancer.

Relationship of Ki-ras mutations in colon cancers to tumor location, stage, and survival: a population-based study.

Some previous studies have demonstrated significant results between Ki-ras mutations and tumor stage, survival, and/or other clinical variables, whereas others have not. We therefore evaluated the significance of codons 12 and 13 Ki-ras mutations in a large population-based study of 1413 individuals with colon cancer. Ki-ras mutations were identified in approximately 32% of tumors. Codon 12 mutations were significantly more common in proximal than distal tumors (29.1% versus 20.5%; P < 0.01) and in tumors of advanced stage. Tumors from men were more likely to have transition mutations and codon 12 G-->A mutations. After adjusting for age and stage, the codon 13 G-->A mutation was associated with a 40% (95% confidence interval, 0.95-2.0) increase in short-term mortality from colon cancer. In conclusion, this population-based study demonstrates important relationships between Ki-ras mutations and stage, survival, tumor location, and gender.

Microsatellite instability in sporadic colon cancer is associated with an improved prognosis at the population level.

Some previous studies have reported an improved prognosis in sporadic colon cancers with microsatellite instability, whereas others have not. In addition, relatively few of those reporting an improved prognosis controlled for tumor stage or were population-based. Therefore, we evaluated the relationship between microsatellite instability and prognosis, tumor stage, and other clinical variables in a population-based study of 1026 individuals. Microsatellite instability was determined by the noncoding mononucleotide repeat BAT-26 and the coding mononucleotide repeat in transforming growth factor-beta receptor type II. Significant relationships were seen between microsatellite instability and proximal tumor location, female gender, young and old age at diagnosis, poor histological differentiation, and low tumor stage (P < 0.01). There was a significant relationship between microsatellite instability and improved prognosis, even after adjusting for stage, with a reduction in the risk of death attributable to colon cancer of approximately 60%. Most of this risk reduction occurred in individuals with American Joint Committee on Cancer stage III tumors, although transforming growth factor-beta receptor type II mutations were associated with a significant reduction in colon cancer death in tumors with distant metastases. We conclude that microsatellite instability in sporadic colon cancer is associated with an improved prognosis at the population level.

Choosing quality of care measures based on the expected impact of improved care on health.

Consumers, payers, and policymakers are demanding to know more about the quality of the services they are purchasing or might purchase. The information provided, however, is often driven by data availability rather than by epidemiologic and clinical considerations. In this article, we present an approach for selecting topics for measuring technical quality of care, based on the expected impact on health of improved quality. This approach employs data or estimates on disease burden, efficacy of available treatments, and the current quality of care being provided. We use this model to select measures that could be used to measure the quality of care in health plans, but the proposed framework could also be used to select quality of care measures for other purposes or in other contexts (for example, to select measures for hospitals). Given the limited resources available for quality assessment and the policy consequences of better information on provider quality, priorities for assessment efforts should focus on those areas where better quality translates into improved health.

Lifestyle factors and Ki-ras mutations in colon cancer tumors.

Heterogeneity in colon tumors implies that environmental, lifestyle, or genetic factors influence the type of mutations seen in tumors. In this study we evaluate the association between previously identified risk factors for colon cancer and Kirsten-ras (Ki-ras) mutations in tumors. The presence of Ki-ras mutations in codons 12 and 13 were determined in a population-based case-control study of colon cancer. Participants were between 30 and 79 years of age at time of diagnosis and include both men and women. Questionnaire data were used to obtain information on lifestyle factors. Valid study data and Ki-ras mutational status were available from 1428 cases of colon cancer, data from 2410 controls were available for comparative purposes. Participants with Ki-ras mutations were more likely to have proximal rather than distal tumors. Cigarette smoking, use of aspirin and/or NSAIDs, use of vitamin/mineral supplements, and consumption of caffeine were associated with both Ki-ras+ and Ki-ras- tumors; the associations were not confounded by dietary intake or other lifestyle factors. Among men, but not among women, those with low levels of physical activity were more likely to have a tumor with a Ki-ras mutation than one without a Ki-ras mutation. However, among women, those with a larger BMI were more likely to have a Ki-ras mutation in their tumor. Given the limited information available on what causes Ki-ras mutations, the information generated from this study indicates that these factors previously associated with colon cancer work through other disease pathways.

Use of archival tissue in epidemiologic studies: collection procedures and assessment of potential sources of bias.

Collection and analyses of archival tumor tissue as a means to increase our understanding of disease pathways is becoming an important avenue of epidemiologic research. In this paper, we present methods of collection and processing of archival tissue and assess the population characteristics of those for whom we were able to and unable to obtain tumor DNA. Cases of colon cancer diagnosed between September, 1991 and October, 1994 living in Utah, Northern California, or the Twin Cities Metropolitan area of Minnesota were targeted for this study. Of the 2477 people for whom we had permission to obtain tumor blocks, we were able to collect blocks and extract DNA for 2117 (85.5%). There were no differences in age, tumor site, or diet and lifestyle characteristics between those with and without DNA extracted. However, we were less likely to be able to extract DNA if the case was diagnosed at a more advanced disease stage or at the earliest disease. Potential bias from exclusion of those with the most advanced disease stage is discussed.

Detection of occult esophageal narrowing with a barium tablet during chest radiography.

Dysphagia is a delayed symptom of esophageal constriction and often appears after the luminal diameter is reduced to less than 10 mm. Earlier diagnosis of benign or malignant strictures while clinically silent would be desirable; hence we investigated the detectability of occult esophageal lesions with the aid of an ingested barium tablet during routine chest radiography. We prospectively examined 300 patients older than 40 years, who were referred for chest films because of indications unrelated to the upper gastrointestinal tract. Each patient was instructed to swallow a 12.5-mm barium tablet with 100 mL of water immediately prior to the exposure of posteroanterior and lateral chest films. Radiographs of 17 patients (5.6%) revealed intraesophageal retention of the tablet, and their prompt evaluation with double-contrast esophagrams confirmed various structural or functional abnormalities in 15 patients. Therefore, the oral administration of a barium tablet during routine chest radiography is a simple efficacious method to assess esophageal patency and detect occult narrowings from structural or functional causes.

MTHFR variants reduce the risk of G:C->A:T transition mutations within the p53 tumor suppressor gene in colon tumors.

5,10-Methylene-tetrahydrofolate reductase (MTHFR) is a key enzyme in folate-mediated 1-carbon metabolism. Reduced MTHFR activity has been associated with genomic DNA hypomethylation. Methylated cytosines at CpG sites are easily mutated and have been implicated in G:C-->A:T transitions in the p53 tumor suppressor gene. We investigated 2 polymorphisms in the MTHFR gene (C677T and A1298C) and their associations with colon tumor characteristics, including acquired mutations in Ki-ras and p53 genes and microsatellite instability (MSI). The study population comprised 1248 colon cancer cases and 1972 controls, who participated in a population-based case-control study and had been analyzed previously for MSI, acquired mutations in Ki-ras, p53, and germline MTHFR polymorphisms. Multivariable-adjusted odds ratios are presented. Overall, MTHFR genotypes were not associated with MSI status or the presence of any p53 or Ki-ras mutation. Individuals with homozygous variant MTHFR genotypes had a significantly reduced risk of G:C-->A:T transition mutations within the p53 gene, yet, as hypothesized, only at CpG-associated sites [677TT vs. 677CC (referent group) OR = 0.4 (95% CI: 0.1-0.8) for CpG-associated sites; OR = 1.5 (0.7-3.6) for non-CpG associated sites]. Genotypes conferring reduced MTHFR activity were associated with a decreased risk of acquired G:C-->A:T mutations within the p53 gene occurring at CpG sites. Consistent with evidence on the phenotypic effect of the MTHFR C677T variant, we hypothesize that this relation may be explained by modestly reduced genomic DNA methylation, resulting in a lower probability of spontaneous deamination of methylated cytosine to thymidine. These results suggest a novel mechanism by which MTHFR polymorphisms can affect the risk of colon cancer.

Intervention in elder abuse: a swift blade, or a dull-edged saw?

In spite of a geriatric team's offer to intervene in what was perceived to be a case of psychologic and financial abuse, an elderly patient who was being discharged from hospital chose to remain in her home. Frustration and concern about the victim prompted medical student Kim Curtin to write this essay that considers the issues of judgement, beneficence and medical intervention.