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A novel p.Ser282Pro CPA1 variant is associated with autosomal dominant hereditary pancreatitis
  1. Aleksandra A Kujko1,
  2. Dorottya M. Berki2,
  3. Grzegorz Oracz3,
  4. Karolina Wejnarska3,
  5. Justyna Antoniuk1,
  6. Katarzyna Wertheim-Tysarowska1,
  7. Elwira Kołodziejczyk3,
  8. Jerzy Bal1,
  9. Miklós Sahin-Tóth2,
  10. Agnieszka M Rygiel1
  1. 1Department of Medical Genetics, Institute of Mother and Child, Warsaw, Poland
  2. 2Department of Molecular and Cell Biology, Center for Exocrine Disorders, Boston University Henry M. Goldman School of Dental Medicine, Boston, Massachusetts, USA
  3. 3Department of Gastroenterology, Hepatology and Feeding Disorders and Pediatrics, The Children's Memorial Health Institute, Warsaw, Poland
  1. Correspondence to Dr Agnieszka M Rygiel, Institute of Mother and Child, Kasprzaka 17a, Warsaw 01-211, Poland; agnieszka.rygiel{at}

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We read with great interest the recent publication by Németh et al.1 in which the authors demonstrate that a misfolding human cationic trypsinogen (serine protease 1, PRSS1) variant causes autosomal dominant hereditary chronic pancreatitis (CP) by inducing endoplasmic reticulum (ER) stress. Previously, the same mechanism was proposed for carboxypeptidase A1 (CPA1) gene variants strongly associated with early onset sporadic CP;2 however, the association of misfolding CPA1 variants with familial or hereditary CP has not been demonstrated so far.

Here, we report two Polish families with hereditary CP carrying a novel heterozygous c.844T>C (p.Ser282Pro) CPA1 variant. In Family 1, the index patient, her mother and uncle (half-brother of the mother) developed CP (figure 1). The age of diagnosis was 17 years for the index patient and 51 and 31 years (with earlier abdominal pain) for the mother and the uncle, respectively. In Family 2, three members were affected by CP (figure 1): the index patient, her mother and her father with ages of onset of 12, 32 and 34 years, respectively. In both families, the diagnosis of CP was confirmed by imaging methods. CP risk factors such as alcohol abuse, smoking, injury, anatomical defects, metabolic and bile duct disorders were excluded.

Figure 1

Pedigrees of Polish families with hereditary chronic pancreatitis (CP) associated with the p.Ser282Pro carboxypeptidase A1 (CPA1) variant. The arrow points to the index patient. Solid black symbols indicate family members with CP. Open symbol with a dot designates unaffected carrier. Crossed symbol indicates deceased family member. nd, genotype not determined. Known carriers of the p.Gly60= CTRC variant are also indicated.

Each person gave informed consent for genetic testing. All exons of CPA1, PRSS1, SPINK1, CTRC and exons 4, 9, 10 and 11 of CFTR were analysed by Sanger sequencing in the index patients from both families. Large unbalanced rearrangements in PRSS1 and SPINK1 were excluded by Multiplex Ligation-Dependent Probe Amplification (MRC Holland). In the rest of the affected and unaffected family members, sequencing of exon 8 of CPA1 and exon 3 of CTRC was performed. In Family 1, all three affected individuals and the unaffected grandfather of the index patient (age 83 years) were heterozygous for the p.Ser282Pro CPA1 variant (figure 1). All other unaffected individuals available for testing did not carry the CPA1 variant. In Family 2, the index patient inherited the p.Ser282Pro CPA1 variant from the affected mother and the heterozygous c.180C>T (p.Gly60=) CTRC variant from the affected father who was negative for the CPA1 variant (figure 1). The heterozygous p.Gly60= CTRC variant is a weak CP risk factor increasing the disease risk about twofold. No other pathogenic variants were identified in the susceptibility genes tested in the index patients of the two families.

The functional effects of the p.Ser282Pro CPA1 variant were compared with those of the pathogenic variant p.Asn256Lys previously shown to cause misfolding and ER stress.2 Human embryonic kidney (HEK) 293T cells transfected with wild-type and mutant CPA1 constructs only secreted the wild-type proenzyme (figure 2A), while both mutants were retained intracellularly and suffered degradation, indicative of misfolding (figure 2B). Importantly, variants p.Ser282Pro and p.Asn256Lys induced ER stress to a comparable extent as judged by elevated mRNA levels of the chaperone BiP and increased splicing of the XBP1 mRNA relative to wild type (figure 2C).

Figure 2

Effects of p.Ser282Pro (S282P) and p.Asn256Lys (N256K) variants on carboxypeptidase A1 (CPA1) secretion, intracellular CPA1 levels and endoplasmic reticulum stress markers in transfected HEK 293T cells. (A) Secretion of CPA1 to the growth medium was analysed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and Coomassie Blue staining. Samples were loaded in duplicates. CPA1 activity in the conditioned medium is also indicated in milliOD/min units. (B) Intracellular and secreted levels of CPA1 were evaluated by western blotting. Twenty micrograms of total cell lysate protein and 4 μL of 2 mL conditioned media were loaded in duplicates. (C) Expression of BiP mRNA was measured by quantitative reverse-transcription (RT)-PCR; levels of spliced XBP1 (sXBP1) were assessed by RT-PCR, agarose gel electrophoresis and densitometry of the spliced and unspliced bands. Average of three experiments with SD is shown. For experimental details, see Witt et al.2

In conclusion, we demonstrated that the novel p.Ser282Pro CPA1 variant causes hereditary CP by inducing ER stress in a similar manner to the previously reported misfolding p.Leu104Pro PRSS1 variant. The observations indicate that misfolding PRSS1 and CPA1 variants are similarly strong risk factors and ER stress is a highly relevant pathological mechanism in CP as suggested by Németh et al.1 and Witt et al.2


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  • AAK and DB are equally contributing first authors.

  • MS-T and AMR are equally contributing senior authors.

  • Contributors Study concept, design and supervision: AMR and MS-T. Acquisition and analysis of genetic data: AAK, AMR, JA, KW-T and JB. Functional analysis: DB and MS-T. Patient enrolment, clinical data collection and interpretation: GO, KW and EK. Drafting the manuscript: AMR, AAK, MS-T and DB. Final approval of manuscript as submitted: all authors. AMR and MS-T contributed equally to this study. The first authors AAK and DB contributed equally to the laboratory experiments.

  • Funding This study was supported by the National Science Centre, Poland, grant 2015/19/B/NZ5/02224 (to AMR) and National Institutes of Health grant R01 DK058088 (to MS-T).

  • Competing interests None declared.

  • Ethics approval The study was approved by the Committee on Bioethics at Institute of Mother and Child (approval 28/2016).

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

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