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Letter
Novel p.K374E variant of CPA1 causes misfolding-induced hereditary pancreatitis with autosomal dominant inheritance
  1. Balázs Csaba Németh1,2,
  2. Anna Orekhova3,
  3. Wenying Zhang4,5,
  4. Shannon A Nortman5,
  5. Tyler Thompson6,
  6. Péter Hegyi2,7,
  7. Maisam Abu-El-Haija4,6
  1. 1First Department of Medicine, University of Szeged, Szeged, Hungary
  2. 2Momentum Gastroenterology Multidisciplinary Research Group, Hungarian Academy of Sciences and University of Szeged, Szeged, Hungary
  3. 3Department of Molecular and Cell Biology, Henry M Goldman School of Dental Medicine, Boston University, Boston, Massachusetts, USA
  4. 4Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, Ohio, USA
  5. 5Division of Human Genetics, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, USA
  6. 6Division of Pediatric Gastroenterology, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, USA
  7. 7Institute for Translational Medicine and First Department of Medicine, Medical School, University of Pécs, Pécs, Hungary
  1. Correspondence to Dr Balázs Csaba Németh, University of Szeged First Department of Medicine, Szeged, Korányi fasor 8-10., H-6720, Hungary; nemeth.balazs{at}med.u-szeged.hu

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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 293 T 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.

Acknowledgments

The authors acknowledge the help of Professor Miklós Sahin-Tóth for providing laboratory space for functional analysis, helpful discussions and advice.

References

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Footnotes

  • Contributors Study concept and design: BCN and MAEH. Acquisition, analysis or interpretation of data: all authors. Drafting of the manuscript: all authors. Critical revision of the manuscript for important intellectual content: all authors. Obtained funding: BCN, PH, MAEH, and funds by the Molecular Genetics Laboratory at Cincinnati Children’s Hospital Medical Center. Administrative, technical or material support: all authors. Study supervision: BCN and MAEH. Final approval of manuscript as submitted: all authors. Guarantors of the article: BCN and MAEH.

  • Funding Hungarian National Research, Development and Innovation Fund (FK124632 to BCN and K116634 to PH); Momentum Grant of the Hungarian Academy of Sciences (LP2014-10/2014 to PH); Economic Development and Innovation Operative Programme Grant of the National Research, Development and Innovation Office (GINOP 2.3.2-15-2016-00048 to PH); National Institutes of Health (R43 DK 105640-01 to MAEH).

  • Disclaimer The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

  • Competing interests None declared.

  • Ethics approval This study was reviewed and approved by the Cincinnati Children’s Hospital Medical Center (CCHMC) Institutional Review Board (IRB number 2018-7699).

  • Provenance and peer review Not commissioned; internally peer reviewed.

  • Patient consent for publication Obtained.

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