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Research : Molecular Epidemiology of Inflammatory Bowel Disease (IBD)
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Hypothesis and Specific Aims This is a prospective case control study involving longitudinal assessment of biomarkers of genetic damage in relation to disease activity and specific therapies (Aim 1), folate deficiency and effect of repletion (Aim 2), and urinary phenol excretion before and one month after initiating therapy for active inflammatory bowel disease (IBD) (Aim 3). The following hypotheses will be tested:
Background and Significance Inflammatory Bowel Diseaase (IBD), characterized by acute and chronic inflammation of the bowel, includes two primary diagnoses: Crohn's disease (CD) and ulcerative colitis (UC). Both types of IBD have major similarities, including inflammatory infiltrates in the gastrointestinal tract, folate deficiency, and increased risk of cancer. The prevalence of IBD varies by study and by country. Typical frequencies range between 12 and 40 per 100,000. The risk of colorectal cancer in patients with IBD is increased 4 to 20-fold compared to the general population, and some malignancies can develop in apparently uninvolved sites. Patients with UC and CD have been reported to develop leukemia, suggesting a potential relationship between IBD and leukemia. While the pathological mechanism of IBD remains unsolved, one typical characteristic of patients with IBD is folate deficiency. Moreover, one of the primary therapies for IBD is sulfasalazine (SASP), which also increases the risk of folate deficiency. Finally, a recent study noted reduced red blood cell folate levels in patients with UC and colorectal cancer compared with a control group that did not have colorectal cancer.Folate deficiency has been studied within a number of populations. A recent study of folate intake among preschool children suggested that 60% consumed less than the RDA. Blount, et al, recently showed that in folate deficiency, synthesis of thymidylic acid from dUMP, its precursor, was inhibited, causing dUMP to accumulate in the cell. This condition increases the chance of misincorporation of uracil into DNA in place of thymidine. When the DNA repair system removes the uracil, it causes breaks in both DNA and chromosomes. In a group of folate deficient volunteers, misincorporation of uracil into DNA and an increased frequency of micronucleated erythrocytes, presumably resulting from chromosome breaks, were observed. Thus, uracil misincorporation has been suggested as an important consequence of folate deficiency. Folate supplementation is recommended for IBD patients on SASP to compensate for potential folate deficiency, but adequacy or consistency of the treatment is poorly evaluated. Hanauer has reported that folate levels can be normalized in about 80% of adult patients supplemented with folic acid. However, data pertaining to the effects of supplementation of pediatric patients are lacking. Additionally, little data are available on the molecular or Cytogenetics effects of folate deficiency or SASP treatment or the effects of folate supplementation in any IBD patients. Only one recent report notes increased Cytogenetics damage in adults with CD compared with controls. A possible explanation is that oxidative damage ensues, caused by altered characteristics of inflamed mucosa and generation of oxygen-derived free radicals by phagocytic cells. The impact of folate deficiency, which could potentially increase both oxidative damage and uracil misincorporation, was not investigated. MTHFR, an important enzyme in folate metabolism, reduces the N5, N10 - methylenetetrahydrofolate into 5-methyltetrahydrofolate. N5, N10 - methylenetetrahydrofolate is a folate metabolite that methylates dUMP into dTMP. Increased levels of N5, N10 - methylenetetrahydrofolate, related to decreased MTHFR activity might reduce uracil misincorporation into DNA by decreasing the cellular dUMP/dTMP ratio. Conversely, patients with normal MTHFR activity would demonstrate increased uracil misincorporation and are potentially at increased risk for cancer. Recently, a recessive polymorphism (677 C->T alanine to valine) was identified in the MTHFR gene. This polymorphic allele is common: 40% of a group of male American physicians screened were found to have C/T heterozygous genotype, while 15% were C/C homozygous. Since this genetic polymorphism can decrease the MTHFR enzymatic activity, persons homozygous for this polymorphism will are less likely to misincorporate uracil into their DNA, and thus have a decreased background frequency of MN (a measure of DNA strand breakage) and decreased cancer risk. As part of this project, we will explore an association of MTHFR genetic polymorphism and IBD as reported by Mahmud et al and risk of leukemia or colorectal cancer (elevated biomarker of genetic damage ) in relation to MTHFR. In summary we propose to initiate studies of molecular and genetic damage in pediatric patients with IBD. We will determine the relationship of markers of Cytogenetics damage with disease activity, MTHFR polymorphisms, and with phenol and folate levels. Preliminary Studies We have developed a procedure that improves the MN assay in exfoliated cells by using fluorescent in situ hybridization (FISH) with a pancentromeric probe. The main advantage of this approach is that it allows for the mechanism of MN formation to be readily identified. Micronucleus (MN) frequencies, estimated by the two methods, are comparable. A significant 3-fold increase was observed in the buccal cells of mortuary science students exposed to formaldehyde using the new FISH-based technique, and the MN were found to be primarily induced by chromosome breakage.Preliminary data on exfoliated cells in African-American children of Oakland demonstrate the feasibility of collecting a sufficient number of samples for Cytogenetics analyses not only from adults but also from children. More than 80% of samples had more than 1000 scorable cells per slide, an adequate number for effective analysis. Pilot analysis of the MN frequencies indicate that inner-city African-American children tend to have variable MN levels, including several individuals with rather high MN frequencies, which may reflect either folate deficiency or environmental exposure or both. Preliminary analysis of MN frequency in mononucleated and binucleated lymphocytes from healthy children and adults suggests a comparable level of Cytogenetics damage to that expected from other studies. Currently, analysis of Immunological Biomarkers and Genome Wide Association study are conducted in collaboration with Pediatric IBD Consortium. (make link)
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