Clinical and molecular effects of guanylate cyclase C-activation

We studied a large Norwegian family who had been affected by chronic diarrhoea and other symptoms from the digestive tract throughout several generations. Our genetic studies showed that family members with diarrhoea shared markers on the short arm of chromosome 12. In this part of the genome we discovered a new genetic variant in GUCY2C. This variant was present in 32 family members with diarrhoea, but not in the healthy family members. We performed functional assays of the GUCY2C variant in a cell-model. These studies showed that the genetic variant encodes an increased activity of guanylate cyclase C (GC-C), a protein known to be involved in infectious diarrhoea. Apart from diarrhoea many family members had also experienced acute intestinal obstructions and/or inflammatory bowel disease (IBD) categorized as Crohn’s disease (CD). To our knowledge the constellation of symptoms experienced by our patients had not been reported previously and we assumed that we were dealing with a new disorder. We called this disorder Familial GUCY2C diarrhoea syndrome (FGDS). The publication in 2012 of our research constituted the first report of a human disorder caused by a change in the GUCY2C gene. The further work on this thesis aimed at discovering factors involved in the pathogenesis of FGDS with a main focus on its link to IBD. In parallel with our project multinational case-control studies were charting the genetic basis for IBD, and by 2015 more than 200 genetic associations had been identified. While GUCY2C has not been flagged by these population-based studies we hypothesised that common genetic variants within the GC-C pathway may still contribute to the risk of developing IBD. Rather than assessing single genes, we tested whether an aggregation of genes within the GC-C pathway was associated with IBD. For this analysis we used genetic association statistics made publicly available from the largest IBD case-control studies. The GC-C gene list did indeed show significant enrichment of association in IBD. We then looked at common IBD genetic risk variants in FGDS patients and found that genetic variants in the NOD2 gene distinguished FGDS patients affected by IBD from those not developing IBD. NOD2 encodes a protein involved in sensing and removal of bacteria in the intestinal wall. We studied which genes were differentially expressed in the distal small bowel of FGDS compared to healthy controls as well as patients with CD. Down regulation of metallothionein genes was found in FGDS patients regardless of concomitant IBD when compared to healthy but not when compared to CD. Metallothioneins may serve as signal transducers in the interplay between the human host and its resident bacteria (microbiota). Paucity of these antioxidant proteins may perturb epithelial sensing and clearance of microbes and has been reported in IBD. We hypothesised that GUCY2C related changes of the gut hydration could also impact on its bacterial composition. Using sequencing of the 16S ribosomal RNA gene we analysed the microbial composition in stool from adult FGDS patients, related and unrelated healthy controls as well as patients with IBD. Overall microbiota composition of FGDS patients was different from the other groups, but similar between healthy relatives and unrelated healthy controls. The microbiota of the FGDS patients displayed increased abundance of Enterobacteriaceae and loss of Faecalibacterium, findings that may have a pro-inflammatory potential and are found in IBD. In conclusion we here identified activating mutations of the GUCY2C gene as the cause of familial diarrhoea and defined a new disorder, Familial GUCY2C diarrhoea syndrome. Our work indicates that genetic testing for mutations in GUCY2C should be considered in patients presenting with early onset secretory diarrhoea. Apart from diarrhoea we also highlighted clinical complications potentially linked to hyperactivation of GUCY2C, such as IBD, intestinal obstruction and dysfunctional gut peristalsis. Our subsequent studies suggested that GUCY2C is involved in the crosstalk between the gut mucosa and the adjacent microbiota, providing a potential clue to the link between GUCY2C and IBD