John Walker Fellowship Mentoring

Crohn's disease (CD) is a chronic fonn ofinfIammatorybowei disease (IBD). This disease predominantly affects people between the ages of 20 and 40 years old with an incidence of35 to 45 per lQO,OOO. Crohn's disease (CD) commonly involves the tenninal ileum, the ileocaecal region, or the colon at distinct segments (called skip lesions). The disease is characterized by acute episodes of abdominal distress, diarrhea, rectal bleeding, anorexia, and weight loss followed by periods of remissions. Although uncommon, the disease can spread from the initial intestinal sites to the mouth, esophagus, and stomach. This condition can also have extraintestinal manifestations in the liver, bile ducts (sclerosing cholangitis), joints (arthralgia of the peripheral joints and spine), skin (erythema nodosum orpyodenna gangrenosum), and even the eyes (anterior uveitis, episcleritis, and conjunctivitis). Women who have had a fistula.,as a result of CD, may leakage from the intestine enter the pelvis resulting in a reduced ability to conceive and a higher chance of ectopic pregnancy. The etiology ofCrahn's disease is unknown but thought to be multifactorial. Genetic factors have been suggested because 10-20% of patients with CD have affected family members. The search for a genetic factor in the etiology of CD has utilized two methods. One method is candidate gene studies. This approach tests for associations between inflanunatory bowel disease and polymorphisms in genes that play a role in inunune regulation and inflammation. The second approach is the systemic genome approach. This involves doing genome scans and replication studies in a large number offamilies with multiple IBD-affected individuals. The individuals are genotyped and genetic linkage analysis is perfonned. The linkage analysis looks for chromosoma110cations of disease susceptibility genes by identifying the loci of polymorphic genetic markers that are transmitted within the families of multiple ffiD patients. Parametric methods are used to locate these genes. Parametric methods are only good for diseases inherited in a mendelian mode of inheritance. Crohn's disease is not inherited in this classic sense so non-parametric methods of analysis are necessary. Nonparametric methods are based on the premise that affected pairs of relatives tend to share genetic marker alleles if the suspected marker is linked closely to a disease susceptibility locus. Multiple affected relatives sharing the suspected genetic marker strongly suggest a link between the marker and the locus. These two approaches have led to the discovery of the IBDllocus and subsequently the NOD2 gene - both of which are associated with Crohn's disease. The locus linked to increased susceptibility of CD development, IBD1, is found in the peri-centromere region of chromosome 16. The possible gene within this locus is called nucleotide oligomerization domain-2 (NOD2). It is expressed in peripheral monocytes and has a leucine rich domain in its last portion necessary for lipopolysaccharide-induced activation ofNF-1CB,a transcription factor activating a number of genes including those that mediate the inflammatory cascade. Mutations of NOD2 result in a decreased LPS-induced NF-1d3activation. Although this would seem to cause a decreased.inflammatory reaction, the opposite is true. Mutations ofNOD2 cause an increased inflammatory response because the monocytes release pro-inflammatory agents like TNF -ex.IL-l. and INF-y. Heterozygotes have a 1.5 to 3~foldincrease for developing CD. Homozygotes have an 18 to 44-fold increase for developing CD. The C) CJ familial risk of developing CD is 15-20% higher than that of the general population. This presents strong evidence of a complex genetic etiology for Crohn's disease, although the disease is not inherited in a classical mendelian manner. This project is a retrospective study involving the genetics and epidemiology of Crohn's disease. We will study a patient population of300 subjects. The patients for the study will be Crohn's disease patients seen in the gastrointestinal clinic at the University of Kentucky Medical Center. Diagnoses of these patients have been confirmed with clinical, radiological, and endoscopic analysis. The site of the CD was detennined by colonoscopy confirmed by a barium follow-through study. All patients will give informed consent before being included in the study. We will obtain the patient's venous blood sample from which genomic DNA will be isolated using a standard protocol. We will use a standard PCR protocol and specific polymerase chain reaction (PCR) primers to screen for four types of mutations of the NOD2 gene. The mutations we will look for are 3020insC (a frameshift C-msertion), P268S, R702W, and G908R (aUthree of these are missense mutations). The PCR data will be analyzed to look for the presence and prevalence of these polymorphisms as well as the presence and prevalence of homozygous (both alleles with the wild type NOD2 or any combination of the four mutations ofNOD2 on either allele) and heterozygotes (wild type on one allele and any of the four m.utationson the other allele) within our study population. Demographic data will include gender, age, smoking history, duration and location of the disease, surgical history, and use of specific medications. This data will be gathered from the medical chart of the study subjects. The genotype data will then be used to determine a genotypephenotype relationship (the location and presentation of the Crohn's disease) in the study subjects.The demographicandgenotypedata willbe analyzedto look for linksbetween a genotype, demographic factor, and development of certain phenotypic manifestations of Crohn's disease. The data from the medical charts and the results of the PCR will be entered into a database created fIom MicrosoftTMACCESS. The data will then be statistically analyzed. This study will provide valuable data on genetic aspects ofCrobn's disease in the Kentucky population. The infonnation obtained wj]] help healthcare workers understand which individuals may be susceptible to the disease as well as provide useful therapeutic knowledge.