Congenital pancreatic lipase deficiency (CPLD, OMIM #614338) is a rare exocrine pancreatic disorder presenting in late infancy with steatorrhea, fat-soluble vitamin deficiency, and low pancreatic lipase activity. Variants of the pancreatic triglyceride lipase (PNLIP) gene have been linked to CPLD. Six children from four Amish families exhibited CPLD symptoms, and two had decreased fecal elastase levels when tested. A novel homozygous PNLIP variant, c.869G>A (p.S290N), was identified in these children. This study aimed to characterize the PNLIP variant to understand its mechanism underlying CPLD. The variant impact was first evaluated using computational modeling. Functional analyses included activity assays, cellular PNLIP partition assessments, and endoplasmic reticulum (ER) stress evaluation in transfected cells. Computational modeling showed that p.Ser290 is highly conserved across species and the variant causes steric hindrance, resulting in protein misfolding. Functional assays revealed that the PNLIP variant had a complete loss of activity compared to the wild type (WT), with defects in catalytic function and secretion. Immunoblotting showed reduced PNLIP variant in the medium and increased accumulation in the detergent-insoluble fraction, consistent with protein misfolding. Variant-expressing cells had elevated levels of BiP, an ER stress marker, and increased Xbp1 mRNA splicing, suggesting an elevated ER stress and unfolded protein response (UPR). In conclusion, the PNLIP p.S290N variant causes CPLD through a loss-of-function mechanism, characterized by loss of enzymatic activity and defective secretion due to protein misfolding. Further studies are needed to determine whether the misfolding variant protein induces proteotoxicity, potentially increasing the risk of pancreatic injury, including chronic pancreatitis.

Studies of a rare homozygous missense mutation identified in two brothers diagnosed with congenital pancreatic lipase deficiency (CPLD) provided the first definitive evidence linking CPLD with missense mutations in the gene of PNLIP. Herein, we investigated the molecular basis for the loss-of-function in the three novel PNLIP variants (c.305G > A, p.(W102∗); c.562C > T, p.(R188C); and c.1257G > A, p.(W419∗)) associated with CPLD. We characterized three novel PNLIP variants in transfected cells by assessing their secretion, intracellular distribution, and markers of endoplasmic reticulum (ER) stress. All three variants had secretion defects. Notably, the p.R188C and p.W419∗ variants induced misfolding of PNLIP and accumulated as detergent-insoluble aggregates resulting in elevated BiP at both protein and mRNA levels indicating increased ER stress. All three novel PNLIP variants cause a loss-of-function through impaired secretion. Additionally, the p.R188C and p.W419∗ variants may induce proteotoxicity through misfolding and potentially increase the risk for pancreatic acinar cell injury.

Congenital pancreatic triglyceride lipase (PNLIP) deficiency is a rare disorder with uncertain genetic background as most cases were described before gene sequencing was readily available. Recently, two brothers with PNLIP deficiency were found to carry a homozygous missense mutation, c.662C>T (p.T221M) in the PNLIP gene (J. Lipid Res. 2014. 55:307-312). Molecular modeling suggested the substitution would change the orientation of residues in the catalytic site and disrupt the function of p.T221M PNLIP. To test the effect of the p.T221M mutation on PNLIP function, we expressed wild-type and p.T221M PNLIP in human embryonic kidney (HEK) 293A cells and dexamethasone-differentiated AR42J rat acinar cells. In both cellular models, wild-type PNLIP was secreted into the conditioned medium where it was readily detectable by protein staining, immunoblot or lipase activity assays. In contrast, mutant p.T221M was not secreted into the medium, but it was present in cell lysates where it accumulated in the insoluble fraction. Intracellular retention of mutant p.T221M resulted in endoplasmic reticulum (ER) stress as measured by elevated XBP1 splicing and increased levels of ER chaperones. Our results demonstrate that the presence of methionine at position 221 in the PNLIP protein sequence causes misfolding and aggregation of the p.T221M mutant inside the cell. The consequent loss of enzyme secretion adequately explains the clinical phenotype of PNLIP deficiency reported for homozygous carriers of p.T221M. Furthermore, the ability of mutant p.T221M to induce ER stress suggests that this form of PNLIP deficiency might cause acinar cell damage as well.