(P21-024-24) Isolation of gDNA From Fecal Samples of Healthy Subjects for Assessing the Influence of Dietary Pulse Consumption on Tumor Suppressor Gene Expression and Methylation Status

Objectives: Colorectal cancer risk is strongly influenced by dietary and lifestyle factors that affect the epigenetic regulation of tumor suppressor genes. The potential for diet and bioactive dietary components to target epigenetic pathways in colorectal cancer is now widely appreciated, but we must first be able to distinguish a “healthy” epigenetic pattern before the utility of dietary epigenetic intervention strategies can be fully realized. Pulses are fiber- and phytochemical-rich food sources for which substantial chemo-preventative evidence exists, yet more research is needed to understand how their influence on epigenetic gene expression protects against colorectal cancer development. The goal of this study was to determine if sufficient quantities of quality human gDNA could be extracted from fecal samples of healthy subjects to assess how daily pulse consumption might protect against colorectal cancer.

Methods: Fecal samples were obtained from twenty-three (23) healthy menopausal women (45-70 yrs), prior to, and following the consumption of 100 g pulses daily for 9 months. Total gDNA was extracted from fecal samples using the Quick-DNA Fecal/Soil Microbe kit (Zymo Research). The concentration and integrity of the gDNA were determined by UV spectrophotometry and agarose gel electrophoresis, respectively. The human fraction contributing to the total fecal gDNA content was determined using a Femto DNA Quantification Kit (Zymo Research). To determine if the quantity and quality of the human gDNA fraction isolated was sufficient to assess diet-mediated changes in tumor suppressor gene expression, the mRNA expression of N-Myc Downstream-Regulated Gene 4 Protein (NDRG4) was determined by qPCR.

Results: Consistent with previous findings, the amount of human DNA present in the gDNA extracted from the individual fecal samples was ≤ 1% of the total, but this was a sufficient quantity to assess the effects of pulse consumption on NDRG4 expression. Ongoing studies are aimed at assessing the effects of pulse consumption on NDRG4 methylation status.

Conclusions: Fecal samples may represent a non-invasive means of assessing the efficacy of dietary chemoprevention studies.

Funding Sources: This study was funded by the USDA-PCHI.