CHARACTERIZATION OF FOUR NOVEL, DENOVO ACTIVIATING GUCY2C VARIANTS CAUSING CONGENITAL SODIUM DIARRHEA

Mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) chloride (Cl^-) and bicarbonate (HCO₃^-) channel lead to Cystic Fibrosis (CF) and multiple intestinal defects including increased acidity linked to inspissated mucus, malabsorption, obstruction, infection, inflammation, and cancer that increases morbidity and reduces quality of life for CF patients. CFTR High Expresser Cells (CHEs), a subpopulation of small intestinal enterocytes, express unusually high levels of CFTR compared to neighboring villus enterocytes. How CHEs contribute to intestinal disease in CF is unknown. CHEs are present in human and rat duodenum and jejunum. Recently, fluorescence in situ hybridization (FISH) identified CHEs as the major source of uroguanylin (UGN) in the human duodenum. UGN, a peptide hormone that is secreted into the intestinal lumen, regulates HCO₃^- secretion through CFTR in response to an acidic milieu. To characterize UGN protein expression in CHEs and understand the role of CHEs and UGN in intestinal CF pathophysiology, intestinal tissue from rats harboring the common dF508 mutation and control littermates were employed. Double-labeling immunofluorescence (IF) of CFTR and UGN or CFTR and guanylate cyclase C (GC-C), the UGN receptor, were performed along the rostrocaudal axis of rat intestine. CFTR subcellular distribution in CHEs was examined after low luminal pH and CHE abundance was quantified. Single-labeling FISH of guanylate cyclase activator 2B (GUCAB) or IF double-labeling of GC-C and MEIS1, a transcription factor specific to CHEs, were performed in dF508 jejunum samples and respective control. Consistent with published findings by ISH, UGN protein was highest in the duodenum and decreased along the rostrocaudal axis of the small intestine. All CHEs in rat duodenum were UGN+, but not all UGN+ cells were CHEs. In rat jejunum, CHEs displayed varying levels of UGN expression and some CHEs displayed minimal or no UGN label. CHEs in duodenum and jejunum expressed high levels of the GC-C receptor. Apical GC-C label was identified in Brunner’s gland of rat proximal duodenum. Luminal exposure to pH 2.0 for 30 min robustly increased apical CFTR in duodenal villus CHEs compared to PBS (pH 7.4), and increased CHE abundance 3-fold. dF508 rat jejunum displayed villus atrophy and intraepithelial lymphocytes compatible with human CF. dF508 jejunum displayed increased number of GUCA2B+ cells and GC-C/MEIS1+ CHEs compared to control jejunum. CHEs in dF508 jejunum displayed higher levels of GC-C compared to neighboring enterocytes. Conclusions: Our findings suggest an important role for CHEs in sensing and regulating pH and fluid secretion in the small intestine. Ongoing studies will characterize the role of UGN/GC-C and CHEs in the pathophysiology of CF intestinal disease.

Background: Gastrointestinal (GI) polyposis is one of the most penetrant features in adults with PTEN hamartoma tumor syndrome (PHTS). PHTS, regardless of phenotype, is characterized by germline PTEN mutations, confers a high risk of specific cancers, and shown to be the most common genetic cause of autism spectrum disorder (ASD). Endoscopic evaluation in children with PHTS is typically symptom-driven, as polyps are thought to be adult-onset. GI phenotypes, especially in children, are not well studied. Thus, our aim was to characterize the GI and hepatic manifestations in children with PHTS and to investigate genotype-phenotype associations. Methods: We performed a retrospective chart review of prospectively accrued children with PHTS followed at the Cleveland Clinic and Children’s Hospital of Philadelphia. Demographic data, PTEN mutation data, laboratory findings, PHTS-related clinical features, and prevalence of GI phenotypes were collected. Wilcoxon Rank-Sum, Chi-squared, Fisher’s exact tests, and multivariable logistic regression were utilized to explore associations between variables. Results: In this series, 80 patients (60% males) with confirmed PTEN mutations were included with a median age at consent of 9 years (range 1-21 years). GI manifestations included constipation in 41 (51%), feeding issues in 31 (39%), polyps in 22 (28%), gastrointestinal reflux disease (GERD) in 15 (19%), and hematochezia in 14 (18%). We observed lymphoid hyperplasia in 9 (11%) and eosinophilic gastrointestinal disorders (EGIDs) in 5 (6%). EGIDs are associated with comorbid atopy (4/5, 80%), and autoimmune conditions (3/5, 60%), including type I diabetes, Hashimoto’s thyroiditis, and celiac disease. Intussusception and hepatic steatosis were reported twice each. Crohn’s disease and protein-losing enteropathy were observed once each. Despite high prevalence of feeding issues, the median BMI was in the 85^th percentile. Of the 28 patients who underwent colonoscopy, 22 (79%) had polyps, with all histological types being observed. Polyps were associated with the absence of ASD (P = 0.032). EGIDs were exclusively seen in those without ASD (P = 0.024), with the majority having nonsense mutations (4/5, 80%). Large PTEN deletions/duplications (P = 0.043) and nonsense mutations (P = 0.042) were associated with polyps. Conclusion: Overall, we found that functional constipation and feeding issues are common in children with PHTS. Importantly, polyps are more prevalent in children with PHTS than previously described. Our findings suggest that children without ASD may represent a distinct patient subset with a predisposition to polyps and EGIDs that are associated with large PTEN deletions/duplications and/or nonsense mutations. Endoscopic evaluation should continue to be performed in symptomatic children with PHTS, with consideration of closer follow-up in those without ASD.

Background: We diagnosed an infant with congenital sodium diarrhea (CSD), after uncovering a novel variant in GUCY2C, which produces cGMP and stimulates enterocyte chloride secretion and inhibits sodium absorption. This genetic cause of CSD is the least reported and lacks targeted treatments. We hypothesized in-vitro characterization of the novel, de-novo GUCY2C variants of four individuals with CSD would reveal activating variants that quantitatively correlated with the severity of illness.

Methods: After recruiting our initial patient, three families heard of our study via a support group and contacted us to enroll their children. All parents and participants underwent a combination of next generation and sanger sequencing via our lab, commercial sequencing panels, or both.
Our novel GUCY2C variants were reproduced via site-directed mutagenesis, and we transiently transfected all variants into HEK293T cells to measure the production of cGMP under baseline and enterotoxin-stimulated conditions via ELISA. To ultimately study the effects of target therapeutics on the chloride secretion of these variants, we used lentivirus transduction to make stable T84 cells expressing our variants of interest.

Results: All participants had a history of premature birth, polyhydramnios, and often dilated bowel loops on prenatal ultrasound. They experienced high output secretory diarrhea notable for elevated stool chloride and sodium, which produced a metabolic acidosis and required IV nutrition and hydration, which all participants required for at least the first three years of life if not indefinitely.
Sequencing revealed four de novo, novel GUCY2C variants, half of which were found in GUCY2C’s guanylate cyclase domain: c.2732A>T p.H911L, c.2356T>C p.Y786H, c.2306A>C p.Q769P, c.2536G>A p.V846M.
All four variants as expressed in HEK293T cells showed cGMP concentrations that when stimulated with enterotoxin were increased three-fold or greater when compared to wild-type (WT) GUCY2C. Interestingly, higher increases in cGMP concentration correlated with severity of clinic course. Stably transduced T84 cell lines have been created and are being used in ongoing studies.

Conclusions and ongoing studies: We showed four novel GUCY2C variants in patients with CSD led to over-activation of GUCY2C. Moreover, the functional impact of these variants correlates with clinical outcomes. In fact, the most severely affected participant’s variant has shown higher cGMP concentrations than the most activating mutation reported in the literature. Ongoing studies center around already created in-vitro and planned ex-vivo systems that will enable us to investigate targeted therapeutics already approved for use in humans in a novel, off-label format that holds the potential to not only improve our participants' quality of life but also advance the treatment of secretory diarrhea in general.