Results and discussion

The p.A121T variant does not segregate with pancreatitis in the published pedigrees

In the pedigree presented by Liu et al, only the index patient was verified to carry the p.A121T variant and suffer from chronic pancreatitis.19 The index patient's son was an unaffected carrier, whereas the father, who apparently suffered from chronic pancreatitis, had died and was not available for genetic testing. A remarkably similar pedigree was presented by Felderbauer et al.20 Again, only the index patient was confirmed to carry the p.A121T variant and have chronic pancreatitis. The index patient's brother and first-degree cousin both carried the p.A121T variant and suffered from recurrent abdominal pain apparently associated with cholelithiasis, whereas his niece and her mother were asymptomatic carriers. The index patient's deceased father and both grandparents had a history of abdominal pain and gallstone disease, but no clinically diagnosed pancreatitis. Thus, the p.A121T variant seemed to segregate with cholelithiasis rather than chronic pancreatitis in this family. Whether or not abdominal pain in some of these patients was due to pancreatitis but was erroneously attributed to cholelithiasis cannot be ascertained from the available clinical information.

The p.A121T variant is functionally identical to wild-type cationic trypsinogen

The study by Felderbauer et al20 presented functional experiments, in which bovine trypsin was used to digest model peptides corresponding to human cationic trypsinogen between Val118 and Leu128. The authors found that tryptic cleavage of the p.A121T containing peptide was somewhat faster than digestion of the wild-type peptide, whereas, as expected, the p.R122H containing peptide was not cleaved. The authors concluded that the p.A121T variant might enhance trypsin degradation and thereby cause pancreatitis, whereas the p.R122H mutation would cause the same disease through inhibition of trypsin degradation.

To test the functional effect of p.A121T in the context of the entire human cationic trypsinogen molecule, we expressed and purified this variant and compared its properties to wild-type cationic trypsinogen. As shown in supplementary figure S1, autoactivation of wild-type and p.A121T trypsinogens was identical at pH 5.0 or at pH 8.0. This result stands in contrast to the documented increase in autoactivation caused by PRSS1 mutants p.D19A, p.D22G, p.K23R, p.N29I, p.N29T, and p.R122H (Sahin-Tóth11and references therein). During autoactivation, some of cationic trypsinogen becomes cleaved at the Arg122-Val123 peptide bond, which results in two characteristic bands on polyacrylamide gels. When wild-type and p.A121T trypsinogens were compared, no difference was detectable in the kinetics of appearance of these two bands or their intensity, indicating that the Arg122-Val123 peptide bond was cleaved at similar rates (supplementary figure S1B, lower panel). Although data are not shown, there was no difference in the activation of wild-type and p.A121T trypsinogens by human enteropeptidase at pH 8.0 or by human cathepsin B at pH 4.0.

To test whether degradation of cationic trypsin was affected by the p.A121T variant, first we followed autolysis of wild-type and p.A121T trypsins. Because autolysis of cationic trypsin is a very slow process, these experiments were performed in the absence of added calcium and in the presence of 10 mM K-EDTA. Autolysis was followed up to 5 h, during which time approximately 50% of the initial trypsin activity was lost (supplementary figure S2A). However, no difference was observed between the autolysis kinetics of wild-type and p.A121T trypsins. We recently demonstrated that human cationic trypsin is specifically degraded by chymotrypsin C by a mechanism that involves cleavage of the Leu81-Glu82 peptide bond followed by tryptic cleavage of the Arg122-Val123 peptide bond.12 Mutation p.R122H protects against chymotrypsin C mediated cleavage.12 In contrast, variant p.A121T had no such effect and chymotrypsin C degraded wild-type and p.A121T trypsins at identical rates (supplementary figure S2B).

We also compared the enzyme kinetic parameters of wild-type and p.A121T cationic trypsins and found no appreciable difference (supplementary table S1). Similarly, both trypsins digested bovine β casein with comparable efficiency (supplementary figure S3). Finally, both wild-type and p.A121T trypsins were inhibited by human pancreatic secretory trypsin inhibitor (SPINK1) stoichiometrically and with equal affinity (not shown).

We recently found that PRSS1 variants p.R116C and p.C139S cause trypsinogen misfolding and induce endoplasmic reticulum stress.15 As a consequence of misfolding and intracellular retention, these mutants are secreted poorly from transfected mammalian cells. To test for this possibility, we transfected HEK 293T cells with wild-type and p.A121T trypsinogens and measured trypsinogen secretion up to 48 h. Supplementary figure S4 demonstrates that the two trypsinogens were secreted at similar rates and accumulated to comparable levels in the conditioned medium.

Concluding remarks

Taken together, the inconclusive genetic evidence and our new experimental results indicate that PRSS1 variant p.A121T might not be associated with hereditary pancreatitis and exerts no effect whatsoever on the functional properties of human cationic trypsinogen and trypsin. Variants identified in the PRSS1 gene are often assigned clinical relevance solely based on a perceived analogy with the genuine disease causing PRSS1 mutations. Because misclassification of clinically harmless genetic variants can directly affect the lives of the carriers and their relatives, further studies are required before rare PRSS1 variants are declared pancreatitis associated.