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A recombined allele of the lipase gene CEL and its pseudogene CELP confers susceptibility to chronic pancreatitis

Abstract

Carboxyl ester lipase is a digestive pancreatic enzyme encoded by the CEL gene1. Mutations in CEL cause maturity-onset diabetes of the young as well as pancreatic exocrine dysfunction2. Here we describe a hybrid allele (CEL-HYB) originating from a crossover between CEL and its neighboring pseudogene, CELP. In a discovery series of familial chronic pancreatitis cases, we observed CEL-HYB in 14.1% (10/71) of cases compared to 1.0% (5/478) of controls (odds ratio (OR) = 15.5; 95% confidence interval (CI) = 5.1–46.9; P = 1.3 × 10−6 by two-tailed Fisher's exact test). In three replication studies of nonalcoholic chronic pancreatitis, we identified CEL-HYB in a total of 3.7% (42/1,122) cases and 0.7% (30/4,152) controls (OR = 5.2; 95% CI = 3.2–8.5; P = 1.2 × 10−11; formal meta-analysis). The allele was also enriched in alcoholic chronic pancreatitis. Expression of CEL-HYB in cellular models showed reduced lipolytic activity, impaired secretion, prominent intracellular accumulation and induced autophagy. These findings implicate a new pathway distinct from the protease-antiprotease system of pancreatic acinar cells in chronic pancreatitis.

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Figure 1: Copy-number variants of the human CEL gene.
Figure 2: Regional plot showing proxy SNPs of the CEL-HYB variant.
Figure 3: Altered structure, expression and enzyme activity of the CEL-HYB protein compared with wild-type CEL (CEL-WT).
Figure 4: Intracellular properties of CEL-HYB.

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References

  1. Lombardo, D. Bile salt-dependent lipase: its pathophysiological implications. Biochim. Biophys. Acta 1533, 1–28 (2001).

    Article  CAS  Google Scholar 

  2. Ræder, H. et al. Mutations in the CEL VNTR cause a syndrome of diabetes and pancreatic exocrine dysfunction. Nat. Genet. 38, 54–62 (2006).

    Article  Google Scholar 

  3. Nilsson, J. et al. cDNA cloning of human-milk bile-salt-stimulated lipase and evidence for its identity to pancreatic carboxylic ester hydrolase. Eur. J. Biochem. 192, 543–550 (1990).

    Article  CAS  Google Scholar 

  4. Lidberg, U. et al. Genomic organization, sequence analysis, and chromosomal localization of the human carboxyl ester lipase (CEL) gene and a CEL-like (CELL) gene. Genomics 13, 630–640 (1992).

    Article  CAS  Google Scholar 

  5. Madeyski, K., Lidberg, U., Bjursell, G. & Nilsson, J. Structure and organization of the human carboxyl ester lipase locus. Mamm. Genome 9, 334–338 (1998).

    Article  CAS  Google Scholar 

  6. Lindquist, S., Blackberg, L. & Hernell, O. Human bile salt-stimulated lipase has a high frequency of size variation due to a hypervariable region in exon 11. Eur. J. Biochem. 269, 759–767 (2002).

    Article  CAS  Google Scholar 

  7. Higuchi, S., Nakamura, Y. & Saito, S. Characterization of a VNTR polymorphism in the coding region of the CEL gene. J. Hum. Genet. 47, 213–215 (2002).

    Article  CAS  Google Scholar 

  8. Bengtsson-Ellmark, S.H. et al. Association between a polymorphism in the carboxyl ester lipase gene and serum cholesterol profile. Eur. J. Hum. Genet. 12, 627–632 (2004).

    Article  CAS  Google Scholar 

  9. Torsvik, J. et al. Mutations in the VNTR of the carboxyl-ester lipase gene (CEL) are a rare cause of monogenic diabetes. Hum. Genet. 127, 55–64 (2010).

    Article  CAS  Google Scholar 

  10. Ragvin, A. et al. The number of tandem repeats in the carboxyl-ester lipase (CEL) gene as a risk factor in alcoholic and idiopathic chronic pancreatitis. Pancreatology 13, 29–32 (2013).

    Article  CAS  Google Scholar 

  11. Ræder, H. et al. Carboxyl-ester lipase maturity-onset diabetes of the young is associated with development of pancreatic cysts and upregulated MAPK signaling in secretin-stimulated duodenal fluid. Diabetes 63, 259–269 (2014).

    Article  Google Scholar 

  12. Lupski, J.R. Genomic disorders: structural features of the genome can lead to DNA rearrangements and human disease traits. Trends Genet. 14, 417–422 (1998).

    Article  CAS  Google Scholar 

  13. McCarroll, S.A. et al. Integrated detection and population-genetic analysis of SNPs and copy number variation. Nat. Genet. 40, 1166–1174 (2008).

    Article  CAS  Google Scholar 

  14. Johansson, B.B. et al. Diabetes and pancreatic exocrine dysfunction due to mutations in the carboxyl ester lipase gene-maturity onset diabetes of the young (CEL-MODY): a protein misfolding disease. J. Biol. Chem. 286, 34593–34605 (2011).

    Article  CAS  Google Scholar 

  15. Torsvik, J. et al. Endocytosis of secreted carboxyl-ester lipase in a syndrome of diabetes and pancreatic exocrine dysfunction. J. Biol. Chem. 289, 29097–29111 (2014).

    Article  CAS  Google Scholar 

  16. Hansson, L. et al. Recombinant human milk bile salt-stimulated lipase: catalytic activity is retained in the absence of glycosylation and the unique proline-rich repeats. J. Biol. Chem. 268, 26692–26698 (1993).

    CAS  PubMed  Google Scholar 

  17. Downs, D., Xu, Y.Y., Tang, J. & Wang, C.S. Proline-rich domain and glycosylation are not essential for the enzymic activity of bile salt-activated lipase: kinetic studies of T-BAL, a truncated form of the enzyme, expressed in Escherichia coli. Biochemistry 33, 7979–7985 (1994).

    Article  CAS  Google Scholar 

  18. Gukovskaya, A.S. & Gukovsky, I. Autophagy and pancreatitis. Am. J. Physiol. Gastrointest. Liver Physiol. 303, G993–G1003 (2012).

    Article  CAS  Google Scholar 

  19. Whitcomb, D.C. et al. Hereditary pancreatitis is caused by a mutation in the cationic trypsinogen gene. Nat. Genet. 14, 141–145 (1996).

    Article  CAS  Google Scholar 

  20. Le Maréchal, C. et al. Hereditary pancreatitis caused by triplication of the trypsinogen locus. Nat. Genet. 38, 1372–1374 (2006).

    Article  Google Scholar 

  21. Witt, H. et al. Mutations in the gene encoding the serine protease inhibitor, Kazal type 1 are associated with chronic pancreatitis. Nat. Genet. 25, 213–216 (2000).

    Article  CAS  Google Scholar 

  22. Rosendahl, J. et al. Chymotrypsin C (CTRC) variants that diminish activity or secretion are associated with chronic pancreatitis. Nat. Genet. 40, 78–82 (2008).

    Article  CAS  Google Scholar 

  23. Masson, E., Chen, J.M., Scotet, V., Le Marechal, C. & Ferec, C. Association of rare chymotrypsinogen C (CTRC) gene variations in patients with idiopathic chronic pancreatitis. Hum. Genet. 123, 83–91 (2008).

    Article  CAS  Google Scholar 

  24. Witt, H. et al. Variants in CPA1 are strongly associated with early onset chronic pancreatitis. Nat. Genet. 45, 1216–1220 (2013).

    Article  CAS  Google Scholar 

  25. Witt, H. et al. A degradation-sensitive anionic trypsinogen (PRSS2) variant protects against chronic pancreatitis. Nat. Genet. 38, 668–673 (2006).

    Article  CAS  Google Scholar 

  26. Sharer, N. et al. Mutations of the cystic fibrosis gene in patients with chronic pancreatitis. N. Engl. J. Med. 339, 645–652 (1998).

    Article  CAS  Google Scholar 

  27. Cohn, J.A. et al. Relation between mutations of the cystic fibrosis gene and idiopathic pancreatitis. N. Engl. J. Med. 339, 653–658 (1998).

    Article  CAS  Google Scholar 

  28. Whitcomb, D.C. et al. Common genetic variants in the CLDN2 and PRSS1-PRSS2 loci alter risk for alcohol-related and sporadic pancreatitis. Nat. Genet. 44, 1349–1354 (2012).

    Article  CAS  Google Scholar 

  29. Derikx, M.H. et al. Polymorphisms at PRSS1-PRSS2 and CLDN2-MORC4 loci associate with alcoholic and non-alcoholic chronic pancreatitis in a European replication study. Gut doi:10.1136/gutjnl-2014-307453 (24 September 2014).

  30. Weiss, F.U. et al. Fucosyltransferase 2 (FUT2) non-secretor status and blood group B are associated with elevated serum lipase activity in asymptomatic subjects, and an increased risk for chronic pancreatitis: a genetic association study. Gut doi:10.1136/gutjnl-2014-306930 (15 July 2014).

  31. Rosendahl, J. et al. CFTR, SPINK1, CTRC and PRSS1 variants in chronic pancreatitis: is the role of mutated CFTR overestimated? Gut 62, 582–592 (2013).

    Article  CAS  Google Scholar 

  32. Masson, E., Chen, J.M., Audrezet, M.P., Cooper, D.N. & Ferec, C. A conservative assessment of the major genetic causes of idiopathic chronic pancreatitis: data from a comprehensive analysis of PRSS1, SPINK1, CTRC and CFTR genes in 253 young French patients. PLoS ONE 8, e73522 (2013).

    Article  CAS  Google Scholar 

  33. Witt, H., Apte, M.V., Keim, V. & Wilson, J.S. Chronic pancreatitis: challenges and advances in pathogenesis, genetics, diagnosis, and therapy. Gastroenterology 132, 1557–1573 (2007).

    Article  CAS  Google Scholar 

  34. Witt, H. et al. Mutation in the SPINK1 trypsin inhibitor gene, alcohol use, and chronic pancreatitis. J. Am. Med. Assoc. 285, 2716–2717 (2001).

    Article  CAS  Google Scholar 

  35. Kirby, A. et al. Mutations causing medullary cystic kidney disease type 1 lie in a large VNTR in MUC1 missed by massively parallel sequencing. Nat. Genet. 45, 299–303 (2013).

    Article  CAS  Google Scholar 

  36. Scholz, M. & Hasenclever, D. Comparison of estimators for measures of linkage disequilibrium. Int. J. Biostat. 6 (Article 1) (2010).

  37. Sannerud, R., Marie, M., Hansen, B.B. & Saraste, J. Use of polarized PC12 cells to monitor protein localization in the early biosynthetic pathway. Methods Mol. Biol. 457, 253–265 (2008).

    Article  CAS  Google Scholar 

  38. Castino, R., Fiorentino, I., Cagnin, M., Giovia, A. & Isidoro, C. Chelation of lysosomal iron protects dopaminergic SH-SY5Y neuroblastoma cells from hydrogen peroxide toxicity by precluding autophagy and Akt dephosphorylation. Toxicol. Sci. 123, 523–541 (2011).

    Article  CAS  Google Scholar 

  39. Hou, X. et al. Proteome of the porosome complex in human airway epithelia: interaction with the cystic fibrosis transmembrane conductance regulator (CFTR). J. Proteomics 96, 82–91 (2014).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

The authors thank all study participants and the members of the Gesellschaft für Pädiatrische Gastroenterologie und Ernährung (GPGE) for providing clinical data and blood samples. The confocal imaging was performed at the Molecular Imaging Center, Department of Biomedicine, University of Bergen. We are also grateful to G. Bjørkøy for commenting on the manuscript. This work was supported by grants and fellowships to P.R.N. and A.M. from the Translational Fund of Bergen Medical Research Foundation, KG Jebsen Foundation, University of Bergen, Research Council of Norway and Western Norway Regional Health Authority (Helse Vest), and to P.R.N. from the European Research Council. Work performed in the German, French and Belgian laboratories was supported by grants from the German Federal Ministry of Education and Research (BMBF GANI-MED 03152061A and BMBF 0314107), European Union Framework Programme 7 (EPC-TM, REGPOT-2010-1 and BetaBat), Europäische Fonds für regionale Entwicklung, State Ministry of Economics Mecklenburg-Vorpommern (V-630-S-150-2012/132/133), Deutsche Forschungsgemeinschaft (RO 3929/1-1, RO 3939/2-1 1, Wi 2036/2-3, SFB 1052 C01 and SPP 1629 TO 718/2-1), Colora Stiftung gGmbH, Leipzig Interdisciplinary Research Cluster of Genetic Factors, Clinical Phenotypes and Environment (LIFE Center, Universität Leipzig), INSERM, French Association des Pancréatites Chroniques Héréditaires (APCH), Actions de Recherche Concertée de la Communauté Française (ARC) and Fonds National de la Recherche Scientifique (FNRS, Belgium). We thank O. Hernell (Umeå University, Sweden) for providing antibodies.

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Contributions

K.F., M.M.L., P.R.N., S.J. and A.M. conceived, designed and directed the study. K.F., F.U.W., D.L., J.R., J.-M.C., C.F., H.K., M. Scholz, H.W., S.J. and A.M. designed, performed and interpreted genetic analyses, with substantial contributions from C.R., E.M., S.J.S., M.R. and P. Sztromwasser. K.F., B.B.J., M.C., J.T. and E.T. carried out functional analyses of CEL-HYB. K.F. and A.M. wrote the manuscript, with substantial contributions from F.U.W., J.R., J.-M.C., H.K., H.W., M.M.L., P.R.N. and S.J. P.B., R.G., J. Mayerle, J. Mössner, H.-U.S., M. Sendler and P. Simon recruited study subjects, collected clinical data and/or provided genomic DNA samples. All authors approved the final manuscript and contributed critical revisions to its intellectual content.

Corresponding author

Correspondence to Anders Molven.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Screening strategy for CEL-HYB.

(a) Schematic outline of the long-range duplex PCR assay (one common forward primer, two reverse primers) developed to detect CEL-HYB. The allele was amplified by primers L11F and CELP-VNTR-R, which bind to CEL- and CELP-specific sequence, respectively. For the CEL-CELP wild-type allele, the PCR product that would be generated by these two primers is too long (19 kb) to be detected by the assay. Therefore, amplification of CEL-WT by the primers L11F and IAR was included as internal control. (b) PCR products from the assay visualized by agarose gel electrophoresis. The sizes of the CEL-WT- and CEL-HYB-specific amplification products are 4.2 kb and 3.2 kb, respectively. For CEL-HYB-negative samples, only the upper CEL-WT band is seen. For CEL-HYB-positive samples, two bands are detected. The CEL-WT product was designed to be larger than the CEL-HYB-specific product in order to verify the quality of the DNA sample. The gel picture shows screening of six samples (lanes 1-6). B, blank; N, negative control; P, positive control.

Supplementary Figure 2 Breakpoint region and haplotypes of the CEL-HYB allele.

(a) The region of CEL-HYB where CEL has been joined to CELP. The grey-shaded area indicates the breakpoint region, which contains all of intron 10 as well as the adjacent exon boundaries. Upon sequencing, the region was found to span 548 bp with the proximal breakpoint located between positions 13389-13936 in CEL and the distal breakpoint between positions 29161-29707 in CELP (GenBank accession AF072711.1). The premature exon 11’ stop codon of CEL-HYB is indicated. SNPs in exons 10 and 11’ are shown with their rs number, chromosomal position and predicted amino acid change (Ensembl Variation database: CEL, ENSG00000170835; CELP, ENSG00000170827). All CEL-HYB-positive subjects carried the gene/protein variants highlighted in red, which therefore were included in the construct employed in functional testing. In CEL-HYB carriers, two new SNPs (green) were detected in the breakpoint region. (b) SNP analysis and haplotypes of the CEL-HYB breakpoint region. When including also the two newly discovered SNPs, five different haplotypes could be distinguished. For each SNP, the most frequent allele is shown in a white box. Values to the right are haplotype frequencies.

Supplementary Figure 3 Examples of pedigrees carrying CEL-HYB.

The figure shows the two CEL-HYB-positive families of the German discovery cohort for which additional members were available for genotyping. The pedigrees have been simplified and adjusted to protect their identities. DNA samples were provided for those subjects indicated by small circles.

Supplementary Figure 4 Expression of CEL protein variants in vitro and in acinar cells.

(a) In vitro transcription and translation of the CEL-WT and CEL-HYB expression vectors employed in cellular studies. Both protein variants exhibited their predicted unglycosylated molecular sizes (100 kDa and 65 kDa, respectively) when detected by western blotting. (b) Expression of CEL-WT and CEL-HYB in acinar cells. Mouse 266-6 cells were transiently transfected with the CEL-WT or CEL-HYB expression vectors for 24 hours, and cell lysates (L) and media (M) were analyzed by western blotting. Transfection of empty vector (EV) was included as negative control. The actin bands indicate the amount of loaded protein in the lysate lanes only. In both a and b, the blots are representative of three independent experiments.

Supplementary Figure 5 Representative example of results from the LightCycler assay used for primary CEL-HYB screening.

The image shows the melting curves of 96 samples, including six no-template controls. The melting point of the amplified CEL-HYB allele is about 8 °C higher than for the CEL-WT (58 °C vs. 50 °C). Three samples were scored as CEL-HYB-positive and 87 as negative.

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Fjeld, K., Weiss, F., Lasher, D. et al. A recombined allele of the lipase gene CEL and its pseudogene CELP confers susceptibility to chronic pancreatitis. Nat Genet 47, 518–522 (2015). https://doi.org/10.1038/ng.3249

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