Diet, Genetic Factors, and the Gut Microbiome in Crohn's Disease

This manuscript is part of a pilot effort on the part of NIH staff and the Nature publishing group to provide a more convenient archive for "marker papers" to be published. These "marker papers" are designed to provide the users of community resource data sets with information regarding the status and scope of individual community resource projects. For further information see editorial in September 2010 edition of Nature Genetics (Nature Genetics, 42, 729 (2010)), and the Nature Precedings HMP summary page.Project IDs: 46313, 46879Publication moratorium: 1 year We propose to investigate the hypothesis that consistent changes in the human gut microbiome are associated with Crohn’s disease, a form of inflammatory bowel disease, and that altered microbiota contributes to pathogenesis. Analysis of this problem is greatly complicated by the fact that multiple factors influence the composition of the gut microbiota, including diet, host genotype, and disease state. For example, data from us and others document a drastic impact of diet on the composition of the gut microbiome. No amount of sequencing will yield a useful picture of the role of the microbiota in disease if samples are confounded with uncontrolled variables. Our proposed project will take advantage of our experience with deep sequencing to characterize the composition of the gut microbiome, but also control diet, host genotype, and disease state.Our initial study, known as Fecal Storage Methods (FSM), was designed to systematically evaluate methods for surveying bacterial communities in human feces using 454/Roche pyrosequencing of 16S rRNA gene sequences, generating a total of 797,276 tags. We analyzed fecal samples from 10 individuals and compared methods for fecal storage, DNA purification and sequence acquisition. To assess reproducibility, we compared samples one cm apart on a single stool specimen for each individual. To analyze storage methods, we compared 1) immediate freezing at -80oC, 2) storage on ice for 24 hours, 3) on ice for 48 hours, or 4) fresh samples. For DNA purification methods, we tested three commercial kits and prior bead beating in hot phenol. Six 16S rDNA primer sets were also compared. We plan to utilize these data to optimize protocols to collect, process and sequence bacterial 16S rDNA from fecal samples in our subsequent studies.Additional studies that have been initiated include Controlled Feeding Experiments (CaFE) that address the effects of controlled diets on gut microbiome composition in normal human subjects sequestered in the Penn CTRC. CaFE 1 examines the effect of a low fat/high carbohydrate vs. high fat/low carbohydrate diet on gut microbiome composition whereas CaFE 2 examines the effect of caloric intake. Data acquisition also includes metagenomic shotgun sequencing of DNA isolated on the first (day 1) and last (day 10) stool samples from subjects enrolled in the CaFE 1 study.To determine the effect of diet on gut microbiome composition in the outpatient setting, we have also initiated a Cross-sectional Study of Diet and Stool Microbiome Composition (COMBO). Healthy volunteers age 2 to 50 are recruited who provide demographic data, a detailed diet history including measurement of usual diet with the Harvard Food Frequency Questionnaire (FFQ) and recent diet using three 24 hour diet recall interviews in the week immediately preceding sample collection.