Through the discovery and characterization of a new form of pancreatitis

Rajput Bhatti, Memoona [UCL] Helaers, Raphaël [UCL] Loriot, Axelle [UCL] Achouri, Younes [UCL] Scheers, Isabelle [UCL] Jacquemin, Patrick [UCL]

The causes of chronic pancreatitis in children are partly different from those found in adults and remain incompletely understood. Thus, the presence of genetic mutations predisposing to pancreatitis is suspected to be a more frequent cause in children. In this context, among pediatric patients with precursor signs of pancreatitis, we identified a category of patients with symptoms typically associated with ciliopathies, a group of genetic disorders related to primary cilium dysfunction. In this work, we therefore aimed to identify mutations in ciliary genes in these patients, and to establish a link between these mutations and the development of pancreatitis. Via Whole Exome Sequencing of genomic DNA from 43 pediatric patients with idiopathic chronic pancreatitis, we first identified in three of them a mutation in the ciliary/ciliogenic genes PKHD1, HNF1β, and NPHP3. We then focused on the NPHP3 gene and generated by CRISPR/Cas9 technique two new transgenic mouse models; one replicates the NPHP3 gene mutations present in the corresponding patient (NPHP3mut1/mut2 model), while the other is a conditional inactivation of NPHP3 (NPHP3f/f model). Phenotypic analysis of these two models allowed us to conclude that loss of function of NPHP3 in pancreatic ductal cells led to inflammation and mild fibrosis associated with significant acinar atrophy and severe lipomatosis. This suggests a new form of pancreatitis in these murine models, characterized by a ciliopathic origin. In order to confirm the presence of pancreatic lipomatosis in patients with HNF1β and NPHP3 mutations, initial MRI analyses were performed and revealed a significant percentage of adipose tissue within the pancreas of these patients. Deeper analysis of mouse models, in particular to characterize the molecular mechanisms involved in the observed phenotypes, and the recruitment of a larger number of patients and control subjects, should allow us to confirm the existence of this new form of pancreatitis of ciliopathic origin. Lay abstract (English) The causes of chronic pancreatitis in children are partially different than in adults and are still incompletely understood. We found that a subset of pediatric patients with signs of early pancreatitis have clinical symptoms typically associated with ciliopathies. The objectives were to identify mutated ciliary genes in these patients and establish a link between mutations, ciliary phenotype, and pancreatitis development. Using a Whole Exome Sequencing strategy, we analyzed the genomic DNA of 43 children with idiopathic chronic pancreatitis. We generated with the CRISPR/Cas9 technique, two new transgenic mouse models to determine in vivo how ciliary gene mutations cause pancreatitis. We identified 3 patients, each with mutations in a ciliary gene: PKHD1, HNF1β and NPHP3. In both murine models, either the one reproducing the mutations found in one patient or the one with a specific deletion of NPHP3 in pancreatic ductal cells, an atypic pancreatitis characterized by the presence of lipomatosis was observed. In order to confirm the presence of pancreatic lipomatosis in patients suffering from NPHP3 or HNF1β mutations, MRI analysis was performed and revealed an important percentage of adipose tissue inside their pancreas. We validated some steps in our project with the discovery of three ciliary gene mutations in three patients. Analysis of mouse models reproducing these mutations confirms that the renal and pancreatic phenotypes found in a patient are indeed due to mutations of this gene. Lipomatosis development in the pancreas following a ciliary gene defect could be a sign of a new form of pancreatitis.