D239Y germline variant of NTHL1 glycosylase increases susceptibility to colorectal cancer in mice

Introduction: Persistent genotoxic stress leads to spontaneous DNA damage, which occurs on the order of 104−105 events per cell per day. The base excision repair is the first responder in the repair mechanisms after DNA damage, with DNA glycosylase being the first step in the repair process by binding to the DNA, cleaving the N-glycosidic bond and removing the oxidized pyrimidine DNA lesions. NTHL1 is a pivotal bifunctional DNA glycosylase enzyme involved in base excision repair mechanism. NTHL1 tumor syndrome is characterized by an increased lifetime risk for colorectal cancer (CRC), breast cancer, and colorectal polyposis. A rare rs3087468 polymorphism (D239Y) exhibits no glycosylase activity and may act as a dominant-negative mutation. Hypothesis: NTHL1-D239Y variant conforms susceptibility to inflammation-associated colorectal cancer. Methods: 6-8 weeks/old male and female C57BL/6 wild-type (WT, NTHL1WT/WT), heterozygous (NTHL1WT/D239Y), or homozygous (NTHL1D239Y/D239Y) knock-in mice were injected with 7.5mg/kg azoxymethane (AOM) followed by three cycles of alternating 2.5% dextran sulfate sodium (DSS, 5 days) and normal drinking water (14 days). We monitored body weight, collected fecal samples for microbial 16S rRNA amplicon sequencing, and analyzed colon length and number/size of polyps at euthanasia. The histology score was assessed by an unbiased pathologist to evaluate inflammation and tumor development. Blood samples were used to evaluate micronuclei formation. Results: NTHL1WT/D239Y and NTHL1D239Y/D239Y mice showed more pronounced weight loss starting from the 2nd DSS cycle. The number and size of polyps in the distal colon of NTHL1WT/D239Y and NTHL1D239Y/D239Y male mice were increased compared to WT littermates. NTHL1D239Y/D239Y females, but not males, showed increased polyp number compared to NTHL1WT/D239Y mice. No statistical difference was observed in the polyps’ size. Tumors extending into the proximal colon were observed uniquely in NTHL1D239Y/D239Y females. On the other hand, only NTHL1WT/D239Y males developed high adenomas in the distal colon compared to WT males. Interestingly, only NTHL1D239Y/D239Y males showed statistical difference in micronucleated normochromatic erythrocytes compared to WT mice. NTHL1 genotype did not significantly affect fecal microbial α- or β-diversity or colitis score and colon length in either gender. Conclusion: Our data suggest that NTHL1-D239Y variant confers a complex, zygosity- and sex-dependent susceptibility risk for inflammation-associated colorectal cancer that may be intrinsic and independent of changes in the gut microbiota. In females, it promotes more proximal tumor location, while in males, it promotes the size and tumor progression. 5P30CA023074 (JBS), Phoenix Women's Board of the Steele Children's Research Center (FKG). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.