Novel p.K374E variant of CPA1 causes misfolding-induced hereditary pancreatitis with autosomal dominant inheritance

We read the publication written by Lin et al¹ with great interest. The authors suggest that the majority of functionally defective carboxypeptidase A1 (CPA1) mutations can elicit reduced expression due to nonsense-mediated decay (NMD) and therefore, only a small subset of the earlier reported CPA1 variants² will predispose to chronic pancreatitis (CP) via the misfolding-dependent pathway.³ This paper seemingly offered an explanation for the earlier finding of Wu et al⁴ where the authors reported no association with CP of rare functionally defective CPA1 variants in a large Chinese cohort.

However, it was earlier reported that functionally impaired variants of the CPA1 gene were strongly associated with sporadic cases of non-alcoholic, early-onset CP in European, Indian and Japanese populations.⁵ In addition, members of two Polish families carrying the p.S282P variant of the CPA1 gene developed hereditary CP.⁶ Protein misfolding and consequent endoplasmic reticulum (ER) stress were described as a potential disease mechanism of hereditary pancreatitis caused by loss-of-function variants of CPA1 and PRSS1 genes.^3,6,7 Recently, the finding that a knock-in mouse model carrying the misfolding-causing p.N256K CPA1 variant developed CP confirmed the significance of the misfolding-dependent pathway in the disease mechanism⁸ and revealed a novel opportunity for in vivo investigation of pancreatitis.⁹

As independent confirmation of the significance of the misfolding-dependent pathway, we report here the discovery of a novel missense variant c.1120A>G (p.K374E) in the CPA1 gene associated with hereditary pancreatitis in a family of US origin.

The index patient is a 16-year-old female patient with idiopathic acute recurrent pancreatitis (ARP). She developed the first attack of acute pancreatitis (AP) at the age of 15 with a total of three documented attacks. Her brother developed the first episode of AP at the age of 12 with a total of three documented attacks. The index patient’s mother was diagnosed with diabetes in her late 20s and had a single episode of AP aged 40. Next-generation sequencing of the known disease-associated genes (CASR, CEL, CFTR, CLDN2, CPA1, CTRC, PRSS1, SBDS, SPINK1 and UBR1) was carried out in the index patient, and the novel variant c.1120A>G (p.K374E) in exon 10 of the CPA1 gene was found in heterozygous form. No other pathogenic variant was found in the index patient. Targeted analyses for the presence of CPA1 p.K374E variant were performed in all family members. Each family member who developed pancreatitis carried the p.K374E variant of the CPA1 gene in heterozygous form (figure 1). The index patient’s older sister also carried the novel variant but without any episode of AP. No smoking and/or alcohol consumption was reported as a potential aetiology for ARP in any family member.

Figure 1.

(A) Pedigree of a family with hereditary pancreatitis associated with the CPA1 c.1120A>G (p.K374E) variant. The arrow points to the index patient with acute recurrent pancreatitis. Solid black symbols indicate affected family members with acute recurrent pancreatitis; the solid grey symbol indicates the subject with one acute pancreatitis attack. The open symbol with a dot designates unaffected carrier. (B) Electropherogram of a heterozygous carrier of the c.1120A>G CPA1 variant.

Functional analysis of the CPA1 p.K374E variant was carried out in transiently transfected HEK293T cells as described elsewhere.⁵ Our in vitro experimental findings were summarised in figure 2. Briefly, by using sodium dodecyl sulfate polyacrylamide gel electrophoresis and western blot analysis of the cell lysate and growth medium, we detected significant secretion defect of the novel CPA1 variant. The p.K374E and p.N256K variants caused elevation of ER stress markers compared with the wild-type CPA1 in transiently transfected HEK293T cells measured by reverse transcription PCR.

Figure 2.

Effect of the p.Asn256Lys (N256K) and p.Lys374Glu (K374E) variants on CPA1 secretion, intracellular CPA1 levels and endoplasmic reticulum (ER) stress markers in transiently transfected HEK 293T cells. (A) Secretion of CPA1. Duplicate samples of 400 μL growth medium were analysed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie Blue staining. (B) Intracellular and secreted levels of CPA1 were evaluated by western blotting. Twenty micrograms total cell lysate protein and 2 μL of 2 mL conditioned media were loaded in duplicates. Two gels of cell lysate and two gels of growth medium were run with the same set of samples. The membranes were probed separately for CPA1 and actin. (C) Expression of BiP (HSPA5) mRNA was measured by quantitative reverse transcription (RT) PCR. Average of three independent experiments with SD is shown. (D) RT-PCR of XBP1 splicing was visualised by 2% agarose gel electrophoresis with ethidium bromide staining. A representative gel of three independent experiments is shown. For further experimental details see Witt et al.⁵

In summary, we identified and functionally characterised the novel c.1120A>G (p.K374E) variant in the CPA1 gene. We confirmed that ER stress-related missense CPA1 variants can be responsible for hereditary pancreatitis. Therefore, without disputing the role of NMD in cases of non-sense CPA1 variants, our results underline the significance of the misfolding-dependent pathway in the pathogenesis of pancreatitis.