Methylenetetrahydrofolate reductase and thymidylate synthase genotypes modify oral mucositis severity following hematopoietic stem cell transplantation.
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Alleles↗
Biomedical subjects
J Bigler
Publications and source records attributed to J Bigler.
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.
Adult↗
Pharmacogenetics of methotrexate: toxicity among marrow transplantation patients varies with the methylenetetrahydrofolate reductase C677T polymorphism.
This study investigated whether a polymorphism in the 5,10-methylenetetrahydrofolate reductase (MTHFR) gene (C677T) modifies responses to methotrexate (MTX) in patients undergoing bone marrow transplantation. About 10% to 12% of the population carry the MTHFR TT genotype (enzyme activity, 30% of wild type [CC]). Patients (n = 220) with chronic myelogenous leukemia underwent marrow allografts and were given a short course of MTX. MTX toxicity measures included the oral mucositis index (OMI), speed of engraftment (platelet and granulocyte counts), and bilirubin. Patients with lower MTHFR activity (TT genotype) had 36% higher mean OMI during days 1 to 18 (+5.7, P =.046) and 20% higher OMI between days 6 and 12 (+3.8, P =.27). Platelet counts recovered more slowly among patients with the TT genotype compared to wild type (24% slower recovery to 10 000 platelets/microL, P =.23; 34% slower to 20 000/microL, P =.08). Patients with decreased MTHFR activity appear at risk of higher MTX toxicity. Because of the high prevalence of the TT genotype, these results may have implications for MTX dosage.
Adult↗
CYP2C9 and UGT1A6 genotypes modulate the protective effect of aspirin on colon adenoma risk.
Regular use of nonsteroidal anti-inflammatory drugs (NSAIDs) has a protective effect on the incidence of colon neoplasia. However, polymorphisms in NSAID-metabolizing enzymes may alter this effect. NSAIDs, particularly aspirin, are glucuronidated by UGT1A6 and some classes of NSAIDs are also metabolized by cytochrome P450 (CYP) 2C9. Both of these enzymes have slow-metabolizing, variant forms. We tested the hypothesis that the slow alleles of these enzymes can modify the inverse association between NSAIDs and colon neoplasia in the Minnesota Cancer Prevention Research Unit (CPRU) adenomatous polyp case-control study. CYP2C9 and UGT1A6 genotypes were determined for 474 adenoma cases and 563 controls. NSAID use was inversely associated with adenoma risk [odds ratio (OR), 0.63; 95% confidence interval (CI), 0.44-0.90 for aspirin; and OR, 0.50; 95% CI, 0.31-0.82 for nonaspirin NSAID]. However, this association was absent in aspirin users who carried the CYP2C9 variant alleles (OR, 0.88; 95% CI, 0.51-1.53) or who were homozygous wild-type UGT1A6 (OR, 0.86; 95% CI, 0.50-1.50). Carriers of both of these alleles who use aspirin were also not at reduced risk of adenomatous polyps (OR, 1.59; 95% CI, 0.68-3.73). The variants of these enzymes did not influence the association between nonaspirin NSAIDs and adenoma risk. These data indicate that the effectiveness of chemopreventive drugs can be modulated by the genotype of metabolizing enzymes.
Adenoma↗
Vitamin D receptor polymorphism and the risk of colorectal adenomas: evidence of interaction with dietary vitamin D and calcium.
Laboratory studies and epidemiological investigations suggest that vitamin D plays a role in the etiology of colorectal adenomas, possibly through a mechanism mediated by the vitamin D receptor (VDR). We conducted a clinic-based case-control study to examine the association between VDR polymorphisms and colorectal adenomas. We selectively identified a random subset of 393 cases of colorectal adenomas and 406 colonoscopy-negative controls from a clinic-based case-control study conducted in the metropolitan Minneapolis/St. Paul area during 1991-1994. A self-administered questionnaire was used to collect data on dietary and supplement intake of vitamin D and calcium, as well as on demographics, physical activity, medical information, lifestyle factors, reproductive history, and anthropometry. DNA was extracted from whole blood and assayed for the BsmI VDR polymorphism using an ABI 7700 TaqMan assay. Adjusted odds ratios (OR) and 95% confidence intervals (CIs) were evaluated using logistic regression. Compared with the bb genotype (33% of controls), neither the Bb (48.8% of controls) nor the BB (18.2% of controls) genotypes was strongly associated with risk of colorectal adenomas (OR = 0.86, CI = 0.63-1.19 and OR = 0.77, CI = 0.50-1.18, respectively). However, those with the lowest tertile of vitamin D intake and the BB genotype had a lower risk of colorectal adenoma (OR = 0.24, CI = 0.08-0.76) than those with the highest tertile of intake and the bb genotype. Similarly, those with the lowest tertile of calcium intake and the BB genotype had a reduced risk of colorectal adenoma (OR = 0.34, CI = 0.11-1.06). Although it has generally been shown that higher calcium and vitamin D intake are associated with a modestly reduced risk of colorectal neoplasia, our data suggest that those with the BB BsmI VDR genotype may be at reduced risk of colorectal adenoma in the presence of lower calcium and vitamin D intake.
Adenoma↗
Epoxide hydrolase Tyr113His polymorphism is associated with elevated risk of colorectal polyps in the presence of smoking and high meat intake.
Microsomal epoxide hydrolase (mEH) metabolizes polycyclic aromatic hydrocarbons, carcinogens found in cigarette smoke and cooked meat. Polymorphisms in exon 3 and exon 4 of the mEH gene have been found to alter mEH activity. We investigated the association between these polymorphisms and colorectal polyps within the Minnesota Cancer Prevention Research Unit case-control study. Cases were diagnosed with colonoscopically confirmed adenomas (n = 530) or hyperplastic polyps (n = 202); controls (n = 649) were polyp-free at colonoscopy. Smoking history and meat consumption were obtained from self-administered questionnaires before colonoscopy. mEH genotypes were determined by PCR/RFLP or oligonucleotide ligation assay. The overall risks associated with exon 3 or exon 4 polymorphisms for both adenomas and hyperplastic polyps were not statistically different from 1.0. Compared with exon 3 Tyr/Tyr, 0 pack-years, risk was highest among those with the exon 3 His/His genotype and >25 pack-years of smoking [adenoma, odds ratio (OR) = 4.9 (1.9-12.8); hyperplastic, OR = 7.7 (2.5-24.0)]. Risks were not elevated among exon 4 homozygous variants, even in the presence of heavy smoking. Fried, baked, or broiled meat intake of > or =two servings/week (high) compared with < or =one serving/week was associated with a 2-fold increase in risk of adenoma. The highest risks were seen for those with the exon 3 His/His genotype and high cooked meat intake [OR = 3.3 (1.4-7.9); reference group: Tyr/Tyr, < or = 1 serving/week). Although mEH polymorphisms are not associated with an increased risk of colorectal polyps overall, genotypes that produce a slow phenotype appear to be associated with an increased risk in the presence of smoking and high intakes of cooked meat.
Adult↗
Prevalence of polymorphisms in the human UDP-glucuronosyltransferase 2B family: UGT2B4(D458E), UGT2B7(H268Y), and UGT2B15(D85Y).
UDP-glucuronosyltransferases (UGTs) of the UGT2B family conjugate steroid hormones as well as bile acids and xenobiotics. UGT2Bs are expressed in numerous human tissues, such as skin, breast, prostate, adipose, and intestine and are hypothesized to modulate steroid metabolism and excretion. Polymorphisms have been identified that may modify substrate specificities or enzyme activities of UGT2B family isozymes. We determined the prevalence of the UGT2B4(D458E), UGT2B7(H268Y), and UGT2B15(D85Y) polymorphisms in a sample of 233 individuals. The allele frequencies were significantly different (P < 0.02) between individuals of Caucasian and Asian descent for all three polymorphisms. In Asians (n = 32), the frequencies of the UGT2B4(D458), UGT2B7(H268), and UGT2B15(D85) alleles were 1.00, 0.73, and 0.64, respectively, whereas, in Caucasians (n = 202), the frequencies of UGT2B4(D458), UGT2B7(H268), and UGT2B15(D85) were 0.75, 0.46, and 0.45, respectively. The distribution of the UGT2B4(D458E), UGT2B7(H268Y), and UGT2B15(D85Y) genotypes also differed by ethnic group (P < 0.0001, P = 0.002, and P = 0.02, respectively). All Asians were homozygous for UGT2B4(D458) and had a greater than 2-fold higher prevalence of the UGT2B7(H268) and UGT2B15(D85) homozygous genotypes compared with Caucasians: 56.2% versus 21.8%, and 46.9% versus 22.3%, respectively. Concomitantly, only 9.4% of Asians were UGT2B7(Y268) homozygous and 18.7% were UGT2B15(Y85) homozygous compared with 29.2% and 32.2%, respectively, of Caucasians. The data suggest that there may be large differences in UGT2B polymorphisms between Asians and Caucasians. This warrants evaluation both in larger, multiethnic cohorts and in relation to known ecological differences in risk of sex hormone-dependent cancers.
Adult↗
Lack of association between the C677T MTHFR polymorphism and colorectal hyperplastic polyps.
Colorectal hyperplastic polyps are benign lesions that share many risk factors with colorectal adenomas and cancers. Low folate intakes are associated with an increased risk of colon cancer. The enzyme 5,10-methylene-tetrahydrofolate reductase (MTHFR) may be linked to DNA methylation and nucleotide synthesis and thus play a role in the etiology of colorectal neoplasia. We investigated an association between the common MTHFR polymorphism (C677T) and colorectal hyperplastic polyps within the Minnesota Cancer Prevention Research Unit case-control study. Cases (n = 200) were diagnosed with colonoscopically confirmed hyperplastic polyps; controls (n = 645) were derived from the same gastroenterology practice and were polyp-free at colonoscopy. Dietary intakes were estimated from a self-administered food-frequency questionnaire prior to colonoscopy. Multivariate adjusted odds ratios (ORs) and 95% confidence intervals for MTHFR status were 0.8 (0.6-1.2; CT versus CC wild-type) and 0.9 (0.5-1.6; TT versus CC). In subgroup analyses stratified on dietary intakes of folate, vitamin B12, vitamin B6, or methionine, those with the TT genotype and either low intakes of folate or vitamin B6 were at increased risk relative to those with normal or high vitamin intake. However, most 95% confidence intervals included 1.0, and no consistent trends were observed. In contrast to our findings on colorectal adenomas, increasing alcohol consumption was associated with an elevated risk of colorectal hyperplastic polyps, regardless of genotype. The MTHFR (C677T) variant genotype does not appear to be related to risk of colorectal hyperplastic polyps, and there is no convincing evidence that MTHFR shows a different relation to risk, dependent on dietary intakes of nutrients related to its pathway.
Adenoma↗
Searching expressed sequence tag databases: discovery and confirmation of a common polymorphism in the thymidylate synthase gene.
Databases of expressed sequence tags (EST) can be used to screen rapidly for potential polymorphisms in candidate proteins. As part of this study, we screened the gene for the enzyme thymidylate synthase (TS). TS is important physiologically because it is essential for the synthesis of deoxythymidylate, a nucleotide required for DNA synthesis and repair. TS is also a major target for cancer chemotherapeutic drugs, especially the widely used 5-fluorouracil. Using sequence alignment of ESTs, we identified a candidate 6-bp variation at bp 1494 in the 3'-untranslated region of the TS mRNA. This sequence variation occurred in 21 of 34 aligned ESTs at this location, including ESTs from various tissue sources. The presence of this polymorphism was confirmed in a Caucasian population (n = 95) by polymerase chain restriction amplification/RFLP analysis. The allele frequency of the 6-bp deletion was found to be 0.29 (wildtype +6 bp/+6 bp, 48%; +6 bp/-6 bp, 44%; -6 bp/-6 bp, 7%). Although the function of this polymorphism has not yet been investigated, the 3'-untranslated region of a gene can play a role in mRNA stability and translation. This study illustrates an approach to polymorphism discovery in candidate enzymes of physiological interest by searches of publicly available sequence data, a rapid and inexpensive method. The potential functional relevance of the common 6-bp deletion in the TS gene needs to be investigated, because this enzyme is plausibly of major importance not only in cancer treatment but also in cancer prevention.
Databases, Factual↗
Negative transcriptional regulation mediated by thyroid hormone response element 144 requires binding of the multivalent factor CTCF to a novel target DNA sequence.
DNA target sites for a "multivalent" 11-zinc-finger CCTC-binding factor (CTCF) are unusually long ( approximately 50 base pairs) and remarkably different. In conjunction with the thyroid receptor (TR), CTCF binding to the lysozyme gene transcriptional silencer mediates the thyroid hormone response element (TRE)-dependent transcriptional repression. We tested whether other TREs, which in addition to the presence of a TR binding site require neighboring sequences for transcriptional function, might also contain a previously unrecognized binding site(s) for CTCF. One such candidate DNA region, previously isolated by Bigler and Eisenman (Bigler, J., and Eisenman, R. N. (1995) EMBO J. 14, 5710-5723), is the TRE-containing genomic element 144. We have identified a new CTCF target sequence that is adjacent to the TR binding site within the 144 fragment. Comparison of CTCF recognition nucleotides in the lysozyme silencer and in the 144 sequences revealed both similarities and differences. Several C-terminal CTCF zinc fingers contribute differently to binding each of these sequences. Mutations that eliminate CTCF binding impair 144-mediated negative transcriptional regulation. Thus, the 144 element provides an additional example of a functionally significant composite "TRE plus CTCF binding site" regulatory element suggesting an important role for CTCF in cooperation with the steroid/thyroid superfamily of nuclear receptors to mediate TRE-dependent transcriptional repression.
Animals↗
UDP-glucuronosyltransferase (UGT1A1*28 and UGT1A6*2) polymorphisms in Caucasians and Asians: relationships to serum bilirubin concentrations.
Polymorphisms that alter UDP-glucuronosyltransferase (UGT) activities have been identified. Mutations in the promoter of the UGT1A1 gene (UGT1A1*28), resulting in 5, 7 or 8, instead of 6 thymine-adenine (TA) repeats, alter bilirubin conjugation. Two missense mutations on one allele of UGT1A6 (UGT1A6*2) result in T181A and R184S amino acid substitutions and reduced activity against phenolics, such as 4-nitrophenol, 4-hydroxycoumarin and butylated hydroxy anisole. We determined the frequency of these polymorphisms in 245 healthy men and women, aged 20-40 years and examined the relationship between TA repeat number and serum bilirubin concentrations in a subset of 24 Asians and 169 Caucasians. The frequencies of the UGT1A1*28 genotypes were 0.537, 0.348, 0.098, 0.008 and 0.008 for promoter TA repeats 6/6, 6/7, 7/7, 5/6 and 6/8, respectively. Both allele and genotype frequencies varied by race (P < 0.02), with 11% of the Caucasians and none of the Asians having the 7/7 genotype. Within both ethnic groups, serum bilirubin increased with increased numbers of UGT1A1 promoter TA repeats (P = 0.0001). However, a strong ethnic group-by-UGT1A1 genotype interaction suggests that additional ethnic differences in bilirubin metabolism contribute to observed bilirubin concentrations. Genotype frequencies for UGT1A6*2 were 0.478, 0.392, 0.029, 0.090, 0.012 for wild-type (wt)/wt, wt/T181A + R184S, wt/R184S, T181A + R184S/T181A + R184S and T181A + R184S/R184S, respectively. The co-occurrence of polymorphisms in UGT1A1 and UGT1A6 differed from that expected (P < 0.0001): individuals homozygous wild-type for UGT1A1 and UGT1A6 were observed at twice the expected frequency; individuals homozygous variant for both genes were ten-fold more frequent and individuals homozygous wild-type for one gene and homozygous variant for the other were ten-fold less frequent than expected. Overall, 8% were homozygous variant for both UGT1 polymorphisms and 43% had at least one variant allele for both UGT1A1*28 and UGT1A6*2. These highly prevalent polymorphisms, which result in modified expression and activity of UGTs, may influence susceptibility to cancers associated with altered metabolism of endogenous and xenobiotic compounds.
Asian People↗
Colorectal adenomas and the C677T MTHFR polymorphism: evidence for gene-environment interaction?
5,10-Methylene-tetrahydrofolate reductase (MTHFR), an enzyme in folate metabolism, may play a role in the etiology of colorectal adenomas via effects on DNA methylation and nucleotide synthesis. We investigated the association between a common polymorphism (C677T, reduced MTHFR activity) and colorectal adenomas within the Minnesota CPRU case-control study. Cases (n = 527) were diagnosed with colonoscopically confirmed adenomas; controls (n = 645) were derived from the same gastroenterology practice and were polyp free at colonoscopy. Dietary intakes were obtained from a self-administered food-frequency questionnaire prior to colonoscopy. Age- and sex-adjusted odds ratios (ORs) and 95% confidence intervals for the MTHFR genotype were 0.9 (0.7-1.2; CT versus CC wild-type) and 0.8 (0.6-1.3; TT versus CC). The associations between dietary intakes of folate, vitamin B12, vitamin B6, or methionine and risk of adenomas showed consistent patterns dependent upon MTHFR genotype. Individuals with the TT genotype and intakes of any of these nutrients in the lowest tertile were at elevated risk for adenomas (about 2-3-fold when compared with TT genotype with high intakes). These trends were more pronounced among individuals over age 60, resulting in a 3-6-fold increase for low intakes of folate, B12, and B6. An increased risk with increasing alcohol consumption was observed only among those with the CC genotype (P-trend = 0.005); among those with the TT genotype, those with moderate alcohol consumption were at lowest risk (P for interaction P = 0.02). In conclusion, nutrients involved in the MTHFR metabolic pathway may modify the relationship between the MTHFR C677T polymorphism and colorectal adenomas. Low intakes of folate, vitamin B12, and vitamin B6 increase risk among those (particularly the elderly) with the MTHFR TT genotype.
Adenoma↗
Meat consumption, genetic susceptibility, and colon cancer risk: a United States multicenter case-control study.
Meat consumption may especially increase risk of colon cancer when the meat is prepared at high temperatures and consumed by subjects with an inherited susceptibility to well-done meat. In this United States case-control study, the association between meat consumption, genetic susceptibility, and colon cancer risk was studied. Meat consumption data were available from a detailed diet history questionnaire and from questions about methods of preparation. Molecular variants in the carcinogen-metabolizing genes NAT2 and GSTM1 were determined in DNA extracted from WBCs. A total of 1542 cases and 1860 population-based controls were included in these analyses. The amount of red and white meat consumed was not associated with overall colon cancer risk. Processed meat consumption was weakly positively associated with colon cancer risk in men only (odds ratio for highest versus lowest quintile of intake = 1.4, 95% confidence interval = 1.0-1.9). The frequency of fried, broiled, baked, or barbecued meat, use of drippings, and doneness of meat were not significantly associated with risk. The Mutagen Index, as an estimate for exposure to mutagenic or carcinogenic substances, was slightly positively associated with colon cancer risk in men (odds ratio = 1.3, 95% confidence interval = 1.0-1.7). No significant associations with colon cancer risk were observed for different NAT2 and GSTM1 gene variants. The observed associations with processed meat and the Mutagen Index were strongest for those with the intermediate or rapid NAT2 acetylator phenotype. Associations were not markedly influenced by lack of the GSTM1 gene. This study provides little support for an association between meat consumption and colon cancer risk but does provide some, albeit not strong, evidence for a modifying effect of molecular variants of the NAT2 gene.
Adult↗
Colorectal adenomatous and hyperplastic polyps: smoking and N-acetyltransferase 2 polymorphisms.
Arylamine N-acetyltransferase 2 (NAT2) is involved in both the detoxification and bioactivation of carcinogenic arylamines and other mutagens. This enzyme is polymorphic, and the fast and slow phenotypes are thought to be risk factors for colon and bladder cancer, respectively. Here, we report on a case-control study of adenomatous and hyperplastic polyps, with particular attention to tobacco smoking, a known risk factor for adenomas, and polymorphisms of NAT2. All participants underwent complete colonoscopy and were subsequently divided into case and control groups on the basis of pathology. Cases were diagnosed with confirmed adenomas (n = 527) or hyperplastic polyps (n = 200); controls (n = 633) had no history of colonic neoplasia and no polyps at colonoscopy. NAT2 genotype was determined using an oligonucleotide ligation assay and fast, intermediate, or slow phenotype imputed. Multivariate-adjusted odds ratios (ORs) and 95% confidence intervals were computed using logistic regression adjusting for age, sex, nonsteroidal anti-inflammatory drug use, and hormone replacement therapy use. Smoking was associated with an increased risk of adenomas [current versus never smoking OR = 2.0 (95% confidence interval, 1.4-2.9)] and hyperplastic polyps [current versus never smoking OR = 4.1 (2.6-6.5)]. NAT2 status among adenomatous polyp patients and hyperplastic polyp patients, respectively, showed ORs of 1.1 (0.8-1.4) and 1.2 (0.8-1.6; intermediate versus slow) and 1.1 (0.6-1.9) and 0.9 (0.4-1.9; fast versus slow). There were no differences in risk when adenoma patients were stratified on multiplicity, size, or histopathological subtype of polyps. Never-smokers showed no variation in risk across acetylator status for either species of polyp, whereas current smokers showed ORs of 2.0 (1.2-3.2) and 2.3 (1.4-3.9) for adenomas and 3.9 (2.1-7.1) and 4.9 (2.6-9.4) for hyperplastic polyps for slow and intermediate/fast NAT2, respectively, compared with slow-NAT2 never-smokers. Risks of both multiple [OR = 4.3 (2.1-8.8)] and large [OR = 3.8 (1.9-7.5)] adenomas were somewhat elevated in current smokers with an intermediate/fast phenotype compared with smokers with a slow NAT2 phenotype, but the interaction was not statistically significant. Risk of hyperplastic polyps and adenomatous polyps is strongly related to smoking. There is little suggestion of interaction between NAT2 status and smoking and no relationship with NAT2 genotype alone.
Adenomatous Polyps↗
NAT2, GSTM-1, cigarette smoking, and risk of colon cancer.
Cigarette smoking has been associated inconsistently with colon cancer. The extent to which genetic profile influences susceptibility to the inducement of colon cancer by cigarette smoking is not known. In this study, we evaluated the associations between smoking cigarettes and polymorphisms of the NAT2 and GSTM-1 genes using data obtained from an incident case-control study of 1993 cases of colon cancer and 2410 age- and sex- matched controls. Neither NAT2 nor GSTM-1 polymorphisms were significantly associated with colon cancer, except among older women, in whom the intermediate/rapid imputed phenotype was associated with increased risk of colon cancer [odds ratio (OR) = 1.4, 95% confidence interval (CI) = 1.0-1.81. Using several indicators of cigarette smoking, we observed no significant interaction between these genotypes and cigarette smoking and colon cancer. The major variation in association with colon cancer was from the amount of cigarette exposure, with those smoking a pack or more of cigarettes per day being at an approximately 40% increased risk of colon cancer; this association did not vary by genotype. However, those who stopped smoking 5-14 years prior to diagnosis and who where intermediate/rapid acetylators were at a slightly greater risk than those who were slow acetylators (for men, OR = 1.6, 95% CI = 1.0-2.4; for women, OR = 2.5, 95% CI = 1.4-4.4). Associations were similar when proximal and distal tumors were examined and separated for age at the time of diagnosis. The lack of an association does not rule out the possibility of other genetic polymorphisms interacting with cigarette smoke to cause colon cancer, nor does it take into account individual phenotypic variability.
Adult↗
Determination of human NAT2 acetylator genotype by oligonucleotide ligation assay.
The oligonucleotide ligation assay (OLA) was adapted to the genotyping of the N-arylamine-acetyltransferase (NAT2) gene. This assay allows the use of 96-well microplates and robotic workstations for high sample throughput. We found this assay to be accurate, efficient and reliable. Another advantage for epidemiological studies where the DNA supply is limited is the small amount of genomic DNA required. A single PCR with an input of 50-100 ng of genomic DNA provides sufficient amounts of amplified NAT2 fragment to analyze five missense mutations.
Alleles↗
Novel location and function of a thyroid hormone response element.
We describe a novel thyroid hormone response element (TRE)-containing sequence, clone 144, isolated by immunoprecipitation of nuclear thyroid hormone receptor (TR)-DNA complexes from the rat pituitary tumor cell line GH4. These cells express several mRNAs of approximately 10 kb that hybridize to the TRE-containing genomic clone 144. These mRNAs are up-regulated at the transcriptional level in the absence of thyroid hormone (T3) and repressed in its presence. The sequence protected from DNase I digestion by TR in clone 144 contains two consensus TRE half-sites arranged as inverted palindromes. The clone 144 TRE is located in the 3' untranslated region (UTR) of several related mRNAs. A reporter construct transfected into 293 cells was responsive to TR regulation when the clone 144 TRE was inserted in the 3' UTR but not when inserted upstream of the promoter. As found for the endogenous 144 mRNAs, the 144 TRE reporter construct is activated by TR in the absence of T3, but not in its presence. Deletion analysis showed that clone 144 sequences flanking the TRE were necessary for TR-mediated regulation, suggesting that the mechanism by which TR regulates transcription through a TRE in the 3' UTR is different from that through the TREs located in the promoter region.
Animals↗
Isolation of a thyroid hormone-responsive gene by immunoprecipitation of thyroid hormone receptor-DNA complexes.
Thyroid hormone (T3) receptor (TR) is a ligand-dependent transcription factor that acts through specific binding sites in the promoter region of target genes. In order to identify new genes that are regulated by T3, we used anti-TR antiserum to immunoprecipitate TR-DNA complexes from GH4 cell nuclei that had previously been treated with a restriction enzyme. Screening of the immunopurified, cloned DNA for TR binding sites by electrophoretic mobility shift assay yielded 53 positive clones. A subset of these clones was specifically immunoprecipitated with anti-TR antiserum and may therefore represent biologically significant binding sites. One of these clones, clone 122, was characterized in detail. It includes sequences highly related to the NICER long terminal repeat-like element and contains three TR binding sites as determined by DNase I footprinting. Two of the clone 122 TR binding sites are located upstream of the TATA box, and one is located downstream. The TR binding site downstream from the promoter was necessary and sufficient to confer T3-dependent regulation in transient transfection experiments. Expression of a reporter construct under the control of the clone 122 promoter region was activated by TR in the absence of ligand and returned to basal levels after T3 addition. Clone 122 sequences hybridize to at least two different mRNAs of approximately 6 and 10 kb from GH4 cells. The levels of both of these mRNAs increased upon removal of T3. Our studies suggest that specific immunoprecipitation of chromatin allows identification of binding sites and target genes for transcription factors.
Animals↗