Abstract
The pancreas produces vital digestive enzymes. When these are produced in inadequate amounts patients experience gastrointestinal distress and malabsorption, termed exocrine pancreatic insufficiency. This PhD is focused on a local whānau (te reo Māori for extended family) with likely pathogenic variants in a gene which is exclusively expressed in the pancreas along with its highly homologous paralogue (gene names redacted); two siblings are affected by exocrine pancreatic insufficiency which manifested as low stool elastase-1 levels, fat soluble vitamin deficiencies, along with abdominal bloating and constipation, and failure to thrive in infancy. The affected children demonstrate compound heterozygosity at the specific pancreatic locus; a rare maternal missense mutation, which seemingly confers a hypofunctional enzyme product, in addition to a paternal deletion of one allele. As the minor allelic frequency of the gene deletion is relatively common in the general population (minor allelic frequency 0.04), and if the pancreatic enzyme deficiency is inherited in an autosomal recessive manner, then this genetic enzyme deficiency could affect 1 in 2500 people, comparable to the prevalence of cystic fibrosis, the commonest cause of exocrine pancreatic insufficiency in childhood.
Cases of isolated forms of exocrine pancreatic insufficiency have been described in the literature, such as congenital co-lipase deficiency and amylase deficiency. Hereditary deficiency at this locus has not previously been reported as a form of monoenzymatic pancreatic insufficiency, despite the significant contribution of the protein to the pancreatic proteome. The literature suggests that one, or perhaps both, of the paralogues form macromolecular complexes in the gut with cholesterol and bile acids, though it remains unclear whether the paralogues enact differing functions, or whether gene dosage is of greater importance.
It was hypothesised that there may be a subset of patients currently diagnosed with a functional bowel disorder such as irritable bowel syndrome, who instead have an unrecognized monoenzymatic form of exocrine pancreatic insufficiency due to deficiency of the pancreatic proteases, with consequences for gastrointestinal health, and cholesterol and bile acid related metabolic disease over the lifespan. A phylogenetic analysis demonstrated that only humans, primates, and mice retain the gene duplication, whilst other mammals only retain a single copy. This is of potential therapeutic import as current mainstays of pancreatic enzyme replacement therapy are derived from crude porcine pancreatic extracts.
An exploratory cross-sectional matched genetic epidemiology study utilizing data from the UK Biobank study demonstrated statistically significant associations (unadjusted p < 0.05) between homozygous deletions at this locus, and increased odds of cholelithiasis (OR 6.2, 95% CI 1.2–31.6), cholecystitis (OR 6.6, 95% CI 1.47-29.98), type 2 diabetes mellitus (OR 2.02, 95% CI 1.05–3.88), hypovitaminosis B2 OR 7.3, 95% CI 1.94–27.3), and non-ulcer dyspepsia (OR 2.5, 95% CI 1–1.59), as well as a non-statistically significant association with non-alcoholic steatohepatitis (NASH) (OR 5.1, 95% CI 0.92-28.4). The last disease association was specifically tested in a sub analysis after the association of a single nucleotide variant (SNV) in one of the gene paralogues which leads to improvement in NASH outcomes was published during the PhD. GTex expression data suggests that the presence of the SNV increases expression of the pancreatic enzyme. The above disease associations were further supported in biochemical indices in which individuals with homozygous deletions demonstrated raised odds compared to homozygous reference controls for increased fasting glucose > 6.6 mmol/L (OR 2.16, 95% CI 1.1-4.0), raised GGT > 30 IU/L in females (OR 1.78, 95% CI 1.05-3.04), and raised ALP >128 U/L in males (OR 3.4, 95% CI 1.2-9.6). There was also a positive association with HbA1c > 50 mmol/L (RRR 2.59 compared to 0.71, 95% CI 1.19-5.59).
A comprehensive allelic series was generated and assessed in a CRISPR-Cas9 engineered knock-out mouse model. Knock-out mice lacking both copies of the paralogues demonstrated reduced early growth in males, and a plateau in weight-gain from 16 weeks in female mice. Serum biochemistry results showed statistically significant increases in serum cholesterol in knock-out mice, paired with increased serum Vitamin D, and reduced Vitamin A levels. Results of a random non-fasting glucose were visually suggestive of raised glycaemic indices and require formal testing in follow-up studies.
The exploratory results of this thesis might suggest that these pancreatic proteins could enact a hitherto unrecognized role in gastrointestinal physiology, potentially influencing several common metabolic diseases, and represent a currently unrecognised form of exocrine pancreatic insufficiency with ramifications for long-term health in children and adults mediated through cholesterol and bile acid homeostasis in the gut. The role of bile acids in glucose homeostasis and metabolism is a growing area of interest, particularly for the development of novel therapeutics which target specific bile acid receptors such as Farnesoid X (FXR) and Takeda G protein 5 (TGR5). Future studies will seek to confirm the results from the human epidemiology study in a replication cohort, and the mouse model will be expanded to confirm the preliminary results, as well as to assess formal glucose tolerance testing, features of NASH, and to interrogate signalling of bile acid receptors involved and protease activated receptors in the homeostasis of the enterohepatic circulation. Future in vitro binding experiments are required to determine whether these proteins act as natural bile acid sequestrants with preference for specific bile acid species. A preclinical study utilising this protein as an oral protein therapy could be designed to assess whether these pancreatic proteins may ameliorate metabolic disease in murine models of hypercholesterolaemia, NASH and impaired glucose tolerance.