Abstract
Objectives.
Premature activation of the digestive protease trypsin within the pancreatic parenchyma is a critical factor in the pathogenesis of pancreatitis. Alterations in genes that affect intra-pancreatic trypsin activity are associated with chronic pancreatitis (CP). Recently, carboxyl ester lipase (CEL) emerged as a trypsin-independent risk gene. Here, we evaluated PNLIP encoding pancreatic lipase as a potential novel susceptibility gene for CP.
Methods.
We analyzed all 13 PNLIP exons in 429 German non-alcoholic CP patients and in 600 German control subjects, in 632 patients and 957 controls from France, and in 223 patients and 1070 controls from Japan by DNA sequencing. Additionally, we analyzed selected exons in further 545 CP patients and 1849 controls originating from Germany, USA and India. We assessed the cellular secretion, lipase activity and proteolytic stability of recombinant PNLIP variants.
Results.
In the German discovery cohort, 8/429 (1.9%) patients and 2/600 (0.3%) controls carried a PNLIP missense variant (P=0.02, OR=5.7, 95% CI=1.1–38.9). Variants detected in patients were prone to proteolytic degradation by trypsin and chymotrypsin. In the French replication cohort, protease-sensitive variants were also enriched in patients with early-onset CP (5/632 [0.8%]) versus controls (1/957 [0.1%]) (P=0.04, OR=7.6, 95% CI=0.9–172.9). In contrast, we detected no protease-sensitive variants in the non-European populations. In the combined European data, protease-sensitive variants were found in 13/1163 cases (1.1%) and in 3/3000 controls (0.1%) (OR=11.3, 95% CI=3.0–49.9, P<0.0001).
Conclusions.
Our data indicate that protease-sensitive PNLIP variants are novel genetic risk factors for the development of chronic pancreatitis.
Keywords: chronic pancreatitis, pancreatic lipase, hereditary pancreatitis, digestive enzyme mutations
INTRODUCTION
Chronic pancreatitis (CP) is a continuing or relapsing inflammatory disorder characterized by progressive destruction of the pancreatic parenchyma. As a consequence, in advanced stages of the disease, maldigestion and diabetes mellitus develop due to exocrine and endocrine insufficiency of the gland [1]. Development and progression of a single episode of acute pancreatitis to recurrent acute pancreatitis (RAP) and CP is frequently driven by alcohol abuse or genetic risk factors, especially in early-onset disease.
Studies on the genetic basis of non-alcoholic CP identified variants mostly in digestive proteases or their inhibitor. Some of these mutations were primarily detected in patients with a strong family history of pancreatitis (hereditary CP), whereas other variants were mainly found in patients without any prominent etiological factors including alcohol and heredity (idiopathic CP). So far, genetic alterations in cationic trypsinogen (serine protease 1, PRSS1), anionic trypsinogen (serine protease 2, PRSS2), serine protease inhibitor Kazal type 1 (SPINK1), chymotrypsinogen C (CTRC), carboxypeptidase A1 (CPA1), and chymotrypsinogen B1 and B2 (CTRB1-CTRB2) have been found to predispose to or protect from CP [2–7]. In addition to protease and protease-inhibitor genes, mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, in the claudin 2 (CLDN2) locus and a hybrid allele of carboxyl ester lipase (CEL) were found significantly enriched in patients with CP [8–11].
A substantial fraction of patients with non-alcoholic CP, even in the presence of a strong family history, do not carry mutations in the above-mentioned genes, indicating that defects in additional genes contribute to disease pathogenesis. In the present study, we investigated the role of pancreatic lipase (PNLIP) in the development of CP. The PNLIP gene is located on chromosome 10, contains 13 exons and spans approximately 22 kB [12]. We considered PNLIP a likely candidate for a pancreatitis risk gene because it is expressed solely and abundantly in the exocrine pancreas. Furthermore, the discovery that genetic alterations in CEL increase CP risk highlighted the potential role of pancreatic lipases in CP susceptibility [11, 13].
METHODS
Study population.
The medical ethical review committees of all participating study centers approved this study. All study subjects gave informed consent. The non-alcoholic CP study cohorts included patients with a history of RAP and/or pathological imaging findings consistent with CP. We enrolled 531 unrelated German individuals with non-alcoholic CP (429 recruited in Munich and 102 recruited in Halle; 289 females; mean age ± S.D. 13.0 ± 6.8 years; age range: 0–30 years). In the replication study, we investigated 632 unrelated non-alcoholic CP patients originating from France (294 females; mean age ± S.D. 13.7 ± 5.7 years; age range: 0–20 years). We also investigated unrelated subjects affected with non-alcoholic CP from the United States (n = 143), India (n = 300) and Japan (n = 223). Control subjects were recruited from Germany (n = 2043), France (n = 957), the United States (n = 168), India (n = 238), and Japan (n = 1070) [14]. In addition, we compared the genetic data with the Exome Aggregation Consortium (ExAC, http://exac.broadinstitute.org) and the 1000 Genomes Project (http://www.internationalgenome.org).
Mutation screening.
Oligonucleotide sequences, PCR and cycle sequencing conditions are described in the Supplementary material.
Plasmid construction and mutagenesis.
The expression plasmid pcDNA3.1(–) PNLIP was described earlier [15]. For this study, we engineered a 10-His tag to the C terminus of PNLIP. PNLIP mutations were generated by overlap extension PCR mutagenesis and cloned into the expression vector using EcoRI and HindIII restriction sites. The pTrapT7 PRSS1 plasmid carrying the coding DNA for human cationic trypsinogen, the pcDNA3.1(–) CTRB2 plasmid harboring the coding DNA for human chymotrypsinogen B2 with a C-terminal 10-His tag and the pcDNA3.1(–) CTRC plasmid encoding human chymotrypsinogen C with a C-terminal 10-His tag were described previously [16–18].
Expression and purification of PNLIP.
Recombinant PNLIP was expressed in human embryonic kidney (HEK) 293T cells. Cells were grown in 75 cm2 tissue culture flasks in 20 mL DMEM with 4.5 g/L glucose, 4 mM L-glutamine, 10% fetal bovine serum and 100 U/mL penicillin-streptomycin (final concentrations) in 5% CO2 water jacketed incubator at 37 °C. At 70–90% confluence the cells were transfected with 30 μg plasmid DNA and 75 μL Lipofectamine 2000 in 20 mL DMEM medium with supplements. The cells were rinsed with 5 mL Opti-MEM after 16–20 h incubation, and covered with 20 mL Opti-MEM containing 100 U/mL penicillin-streptomycin. After 48 h incubation, the conditioned medium was harvested and the cells were covered with fresh Opti-MEM containing antibiotics and the medium was collected again after additional 48 h incubation. A typical protein expression experiment was performed with 5 tissue culture flasks. The harvested media were pooled and filtered with Steritop bottle top filters (Merck) to remove debris. PNLIP were purified from the conditioned media using a 5 mL Ni-NTA superflow affinity cartridge (Qiagen) attached to an Akta Purifier FPLC system. After sample loading, the cartridge was washed with 100 mL of 50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole (pH 8.0) to remove weakly binding proteins. PNLIP proteins were eluted with 50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole (pH 8.0) and 5 mL fractions were collected. Fractions containing pure PNLIP were pooled and dialyzed against 3 × 3 L of 50 mM Tris-HCl (pH 8.0), 100 mM NaCl with a 10,000 Da molecular weight cutoff (MWCO) Spectra/Por Float-A-Lyzer Dialysis Device (Spectrum Labs). PNLIP samples were concentrated with a Vivaspin 2 Centrifugal Concentrator (MWCO 10,000) to a final volume of 1.5–2.0 mL. The concentration of PNLIP samples was determined from their ultraviolet absorbance at 280 nm using the molar extinction coefficient value 61,725 M−1 cm−1 with the exception of PNLIP mutants p.H42Y, p.D72Y and p.D264Y (63,215 M−1 cm−1), p.Y58H (60,235 M−1 cm−1), p.C198Y (63,090 M−1 cm−1) and p.C254R (61,600 M−1 cm−1). The concentration of PNLIP samples was typically 5.0–8.0 μM.
Expression, purification and activation of human pancreatic proteases.
Human cationic trypsinogen was expressed in E. coli BL21(DE3) and purified with ecotin affinity chromatography [19]. Trypsinogen was activated with recombinant human enteropeptidase and the active trypsin concentration was determined by titration against ecotin.
CTRB2 and CTRC were expressed in HEK 293T cells, as described for PNLIP above. The His-tagged protease precursors were purified with a 5 mL Ni-NTA superflow cartridge on an Akta Purifier FPLC system. The protease precursors were eluted with 50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole (pH 8.0) and 5 mL fractions were collected. Fractions containing pure target proteins were pooled and dialyzed against 3 × 3 L of 100 mM Tris-HCl (pH 8.0), 150 mM NaCl; and concentrated with a Vivaspin 2 Centrifugal Concentrator (MWCO 10,000). Protease precursors were activated with trypsin beads, which were later removed by centrifugation. The concentration of active CTRB2 and CTRC was determined by titration against ecotin.
Digestion of PNLIP with pancreatic proteases.
Purified wild-type and mutant PNLIP (2 μM) were incubated at 37 °C with 200 nM human PRSS1, CTRC or CTRB2 in 0.1 M Tris-HCl (pH 8.0) and 1 mM CaCl2 (final concentrations). At the indicated times, aliquots (100 μL) were precipitated with trichloroacetic acid (10%) and analyzed by SDS-PAGE, Coomassie Blue staining and densitometry.
SDS-PAGE and densitometry.
PNLIP samples were precipitated with 10% trichloroacetic acid (final concentration) and incubated for 5–10 min on ice. The PNLIP precipitate was recovered with centrifugation at 13,000 rpm for 10 min in a tabletop microcentrifuge and solubilized in 15 μL Laemmli sample buffer supplemented with 100 mM dithiotreitol. After heat denaturation at 95 °C for 5 min, proteins were electrophoresed on 15% polyacrylamide minigels followed by Coomassie Blue R250 staining. After destaining with 30% methanol and 10% acetic acid solution, the gels were dried between two layers of cellophane and scanned. Quantitation of bands was performed with the Quantity One 4.6.9 (Bio-Rad) software. Rectangles were drawn around the bands and isometric rectangles placed within the same lanes were used for background subtraction.
Enzyme activity measurement of PNLIP.
Lipase activities of wild-type PNLIP and variant proteins were assayed at room temperature with the standard 5-min pH-stat method as previously described [20, 21]. The assays included an emulsion of tributyrin (114 mM), trioctanoin (27 mM), or triolein (6.9 mM) with 4 mM sodium taurodeoxycholate, in a total volume of 15 mL. Two μg of lipase was added (2.6 nM final concentration) with or without a 5-fold molar excess (2 μg, 13 nM) of purified recombinant human colipase [20]. The lipolytic activities are expressed in international lipase units per mg of enzyme. One unit corresponds to one μmol of fatty acids released per min.
Statistics.
The significance of the differences between mutation frequencies in affected individuals and controls was tested by two-tailed Fisher’s Exact Test. Additional odds ratios (OR) were calculated using SAS/STAT software (v 9.1) and GraphPad Prism (v 4.03).
RESULTS
European discovery and replication cohorts.
In the German discovery cohort, we sequenced all coding exons and the flanking intronic regions in 429 cases with non-alcoholic CP and in 600 control subjects. We found heterozygous missense variants in 8/429 (1.9%) cases and in 2/600 (0.3%) controls (Table 1). All cases with missense variants had early-onset disease (mean age 12.3 years). The most frequently found variant was p.F300L (rs890551695), which was present in four cases but not in controls. To replicate the findings in an independent European cohort, we sequenced PNLIP in 632 early-onset (≤20 years) non-alcoholic CP cases and 957 controls of French origin (Table 2). Heterozygous missense variants were present in 10/632 (1.6%) cases and in 8/957 (0.8%) controls. The only variant that was detected in both cases and controls was p.I265R (rs377358755). Other variants were detected only once with the exception of p.F300L, which was present in three cases but not in controls.
Table 1.
Heterozygous PNLIP missense variants in German subjects with non-alcoholic chronic pancreatitis and healthy controls. Protease-sensitive variants are marked in bold. The enrichment of p.F300L (P=0.03), of all variants (P=0.02, OR=5.7, 95% CI=1. 1–38.9), and of all protease-sensitive variants (P=0.001) in cases is significant.
| Exon | Nucleotide change | Amino-acid change | Cases(%) | Controls (%) | P |
|---|---|---|---|---|---|
| 3 | c.124C>T | p.H42Y | 0/429 (0) | 1/600 (0.2) | |
| 8 | c.733C>G | p.P245A | 1/429 (0.2) | 0/600 (0) | |
| 8 | c.794T>G | p.I265R | 2/429 (0.5) | 0/600 (0) | |
| 9 | c.900C>A | p.F300L | 4/429 (0.9) | 0/600 (0) | 0.03§ |
| 9 | c.911C>T | p.S304F | 1/429 (0.2) | 0/600 (0) | |
| 13 | c.1360G>T | p.V454F | 0/429 (0) | 1/600 (0.2) | |
| All variants | 8/429 (1.9) | 2/600 (0.3) | 0.02$ | ||
| All protease-sensitive variants | 8/429 (1.9) | 0/600 (0) | 0.001& | ||
P=0.03; OR= Inf. (95% CI = 0.9-Inf.)
P=0.02; OR= 5.7 (95% CI = 1.1–38.9)
P=0.001; OR= Inf. (95% CI = 2.1-Inf.)
Table 2.
Heterozygous PNLIP missense variants in French subjects with non-alcoholic chronic pancreatitis and healthy controls. The enrichment of all protease-sensitive variants (p.I265R, p.F300L and p.F314L; marked in bold) in cases is significant (OR 7.6, 95% CI 0.9–172.9, P=0.04).
| Exon | Nucleotide change | Amino-acid change | Cases(%) | Controls (%) | P |
|---|---|---|---|---|---|
| 4 | c.214G>T | p.D72Y | 1/632 (0.2) | 0/957 (0) | |
| 4 | c.274C>A | p.H92N | 0/632 (0) | 1/957 (0.1) | |
| 5 | c.427G>T | p.A143S | 0/632 (0) | 1/957 (0.1) | |
| 6 | c.487G>A | p.V163M | 0/632 (0) | 1/957 (0.1) | |
| 6 | c.520G>C | p.A174P | 0/632 (0) | 1/957 (0.1) | |
| 7 | c.593G>A | p.C198Y | 0/632 (0) | 1/957 (0.1) | |
| 8 | c.698G>A | p.G233E | 1/632 (0.2) | 0/957 (0) | |
| 8 | c.760T>C | p.C254R | 1/632 (0.2) | 0/957 (0) | |
| 8 | c.790G>T | p.D264Y | 0/632 (0) | 1/957 (0.1) | |
| 8 | c.794T>G | p.I265R | 1/632 (0.2) | 1/957 (0.1) | |
| 9 | c.900C>A | p.F300L | 3/632 (0.5) | 0/957 (0) | 0.06 |
| 9 | c.919G>A | p.V307I | 0/632 (0) | 1/957 (0.1) | |
| 10 | c.942C>G | p.F314L | 1/632 (0.2) | 0/957 (0) | |
| 11 | c.1102G>A | p.V368I | 1/632 (0.2) | 0/957 (0) | |
| 12 | c.1241T>A | p.M414K | 1/632 (0.2) | 0/957 (0) | |
| All variants | 10/632 (1.6) | 8/957 (0.8) | 0.17 | ||
| All protease-sensitive variants | 5/632 (0.8) | 1/957 (0.1) | 0.04& | ||
P=0.04; OR= 7.6 (95% CI = 0.9–172.9)
When the two European cohorts were combined, missense variants were found in 18/1061 cases (1.7%) versus in 10/1557 controls (0.6%), indicating a slight enrichment in the patient population (P=0.01, OR=2.7, 95% CI=1.2–6.2). Remarkably, however, most variants were unique to either the patient or the control cohorts, suggesting that variants associated with pancreatitis might be functionally damaging whereas variants found in controls may be functionally innocuous. The exclusive occurrence of the p.F300L variant among early-onset cases (7/1061, 0.7%, P=0.002) was particularly striking in this regard.
Non-European cohorts.
We also sequenced PNLIP in 223 subjects with non-alcoholic CP from Japan (Supplementary Table 2). As controls, we used data from the Tohoku Medical Megabank Organization (ToMMo) database, which contains exome sequencing results for 1070 healthy Japanese individuals. In contrast to the European cohorts, all missense variants found in the Japanese cases were also detected among controls without a significant enrichment in either cohort. If we excluded the relatively common p.I186T variant (rs78536862), missense variants were detected in 4/223 (1.8%) cases and in 35/1070 (3.3%) controls (P=0.29). Only one missense variant, p.V307I (rs773774916), was present in both the Japanese and European cohorts. Notably, the p.F300L variant was absent in the Japanese subjects analyzed. To investigate the frequency of the p.F300L variant in additional cohorts, we sequenced exons 7, 8, 9 and the flanking intronic regions in PNLIP in 300 cases and 238 controls of Indian origin and 143 cases and 168 controls in a cohort from the United States. No missense variants were found in these two cohorts.
Functional effects of PNLIP variants.
To characterize the functional properties of the PNLIP variants identified in the European cohorts, we first determined their secretion from transiently transfected HEK 293T cells. The amount of secreted lipase in the conditioned medium was measured by SDS-PAGE and Coomassie Blue staining followed by densitometry of the lipase bands. As shown in Figure 1, the large majority (19 of 23) variants were secreted normally (>75% of wild-type levels) and only four variants exhibited markedly reduced secretion (<20% of wild-type). Secretion-defective variants were equally present in cases and controls. Thus, variants p.G233E (no rs#) and p.C254R (rs750709623) were each found once in French cases, whereas variants p.A174P (no rs#) and p.V454F (rs148560679) were found in a French and a German control, respectively (Tables 1, 2).
Figure 1.
Secretion of human pancreatic lipase (PNLIP) variants. HEK 293T cells were transiently transfected with the indicated constructs and conditioned media was collected after 48 h. Aliquots (200 μL) of conditioned media were precipitated with trichloroacetic acid and analyzed by SDS-PAGE and Coomassie Blue staining. (A) Representative gels. (B) Densitometric evaluation of PNLIP band intensities. The mean and S.D. of three experiments are shown.
To test the effect of PNLIP variants on lipase activity, 20 of the 23 variants were purified from the conditioned medium of transfected HEK 293T cells and their activity was tested on three different triglyceride substrates with increasing chain length: tributyrin, trioctanoin, and triolein (Figure 2). Only variant p.H92N (rs368162708) exhibited impaired catalytic activity on all substrates. Variants p.C198Y (no rs#) and p.D264Y (rs755039876) readily hydrolyzed tributyrin and trioctanoin but showed diminished activity against triolein. All other variants exhibited high activity (≥50% of wild-type) on all three substrates. None showed significantly increased activity relative to wild-type PNLIP. Considering the three variants with low activity, all three were found once in French controls, indicating that loss of PNLIP activity does not alter pancreatitis risk. Since the variants are heterozygous, clinical lipase deficiency should not occur in these carriers.
Figure 2.
Enzymatic activity of human pancreatic lipase (PNLIP) variants. The indicated lipase variants were purified and their activity was tested against three different triglyceride substrates; (A) tributyrin, (B) trioctanoin, (C) triolein. Mean activity values ± SD (n=3) were plotted. See Methods for experimental details. ND, not determined, because these mutants could not be purified due to their low expression levels.
PNLIP undergoes proteolytic degradation and loss of activity during intestinal transit [22, 23]. To test whether the mutations alter the proteolytic stability of PNLIP, we first incubated wild-type PNLIP and 19 variants with human cationic trypsin and followed their degradation by SDS-PAGE and Coomassie Blue staining (Figure 3). For these experiments, we used lower protease-to-lipase concentration ratios than those present in the intestinal tract. Surprisingly, wild-type PNLIP was unaffected by trypsin while variants p.P245A (no rs#), p.I265R, p.F300L, p.S304F (no rs#), and p.F314L (no rs#) suffered proteolytic cleavage. The 14 other variants tested were as resistant to trypsin as wild-type PNLIP (Supplementary Figure 1). Strikingly, the same pattern emerged when PNLIP variants were digested with human chymotrypsin C (Figure 4) or human chymotrypsin B2 (Figure 5). The five trypsin-sensitive variants were readily cleaved by the human chymotrypsins whereas wild-type PNLIP and the other 14 variants were resistant or showed minimal digestion (Supplementary Figures 2 and 3).
Figure 3.
Effect of human cationic trypsin (PRSS1) on human pancreatic lipase (PNLIP) variants. Digestion experiments were carried out as detailed in Methods. (A) Representative gels from two experiments. N-term, N-terminal PNLIP cleavage fragment. C-term, C-terminal PNLIP cleavage fragment. The asterisk indicates the PRSS1 band. (B) Densitometric evaluation of PNLIP band intensities. The average of two experiments is indicated. The figure shows the five PNLIP variants where significant degradation was observed. The effect of PRSS1 on all other PNLIP variants is shown in Supplementary Figure 1.
Figure 4.
The effect of human chymotrypsin C (CTRC) on human pancreatic lipase (PNLIP) variants. Digestion experiments were performed as described in Methods. (A) Representative gels from two experiments. C-term, C-terminal PNLIP cleavage fragment. The asterisk indicates the CTRC band. (B) Densitometric evaluation of PNLIP band intensities. The average of two experiments is indicated. The figure shows the five PNLIP variants where significant degradation was observed. The effect of CTRC on all other PNLIP variants is shown in Supplementary Figure 2.
Figure 5.
The effect of human chymotrypsin B2 (CTRB2) on human pancreatic lipase (PNLIP) variants. Digestion experiments were carried out as detailed in Methods. (A) Representative gels from two experiments. C-term, C-terminal PNLIP cleavage fragment. Note that human CTRB2 migrates on reducing gels as two smaller bands due to autolytic cleavage of the Tyr164- Asn165 peptide bond. The N-terminal CTRB2 chain co-migrates with the PNLIP C-terminal fragment and the C-terminal CTRB2 chain is indicated by the asterisk. (B) Densitometric evaluation of PNLIP band intensities. The average of two experiments is indicated. The figure shows the five PNLIP variants where significant degradation was observed. The effect of CTRB2 on all other PNLIP variants is shown in Supplementary Figure 3.
To identify the proteolytic sites involved, the cleavage products of variants p.P245A and p.F300L were subjected to N-terminal sequencing by Edman degradation. Figure 6 demonstrates the cleavage sites deduced from the N-terminal sequencing data, which were identical in the two variants analyzed. Trypsin and chymotrypsin cleave PNLIP variants within the peptide segment composed of amino-acids 341–355, which is located in the N-terminal domain near the junction with the C-terminal domain of PNLIP (Figure 6). The five PNLIP variants seem to destabilize this region and render PNLIP sensitive to proteolytic attack. Cleavage generates a stable C-terminal fragment whereas the N-terminal fragment suffers further degradation by chymotrypsin (Figures 3, 4, 5).
Figure 6.
Location of proteolytic cleavage sites in degradation-sensitive human pancreatic lipase (PNLIP) variants. Ribbon model of PNLIP (Protein Data Bank file 1LPB). Amino acids affected by mutations that promote PNLIP degradation are highlighted in red. Labels on orange background indicate the proteolytic cleavage sites identified in PNLIP variants p.F300L and p.P245A by N-terminal sequencing of cleavage fragments. The amino-acid sequence of PNLIP between positions 341 and 355 is also shown with human cationic trypsin (PRSS1), human chymotrypsin C (CTRC) and human chymotrypsin B2 (CTRB2) cleavage sites indicated. Solid (versus dashed) arrows denote stronger cleavage. See text for further details.
Protease-sensitive PNLIP variants are associated with early onset chronic pancreatitis.
Variants p.P245A, p.I265R, p.F300L, p.S304F, and p.F314L that were prone to degradation by trypsin and chymotrypsin were all identified in the European cohorts (Tables 1 and 2). We extended the genetic analysis of exons 8 to 10 by sequencing additional 102 patients and 1443 control subjects originating from Germany. We found no new variants in patients but detected the p.I265R variant in two more controls. Taken together, Table 3 demonstrates that protease-sensitive variants were found in 13/1163 cases (1.1%) and in 3/3000 controls (0.1%). Enrichment of these variants among cases was more than 10-fold (OR 11.3, 95% CI 3.0 – 49.9, P<0.0001), indicating that protease-sensitive PNLIP variants are strong risk factors for CP.
Table 3.
Protease-sensitive PNLIP variants in European subjects with non-alcoholic chronic pancreatitis and healthy controls. The enrichment of p.F300L (P=0.0001) and of all proteasesensitive variants in cases is significant (P<0.0001, OR=11.3, 95% CI=3.0–49.9).
| Exon | Nucleotide change | Amino-acid change | Cases(%) | Controls (%) | P |
| 8 | c.733C>G | p.P245A | 1/1163 (0.1) | 0/3000 (0) | 0.3 |
| 8 | c.794T>G | p.I265R | 3/1163 (0.3) | 3/3000 (0.1) | 0.4 |
| 9 | c.900C>A | p.F300L | 7/1163 (0.6) | 0/3000 (0) | 0.0001 |
| 9 | c.911C>T | p.S304F | 1/1163 (0.1) | 0/3000 (0) | 0.3 |
| 10 | c.942C>G | p.F314L | 1/1163 (0.1) | 0/3000 (0) | 0.3 |
| All protease-sensitive variants | 13/1163 (1.1) | 3/3000 (0.1) | <0.0001 | ||
Characteristics of the eight German and five French patients with protease-sensitive PNLIP variants are listed in Table 4. Remarkably, all carriers were children or young adults and all presented with RAP. None were diagnosed with CP yet and in three cases the referring physician indicated “mild disease” in their charts. With respect to family history of pancreatitis, one patient had a sister who carried a PNLIP variant and exhibited a similarly mild disease course. However, in the other 12 patients no family history was noted, indicating that PNLIP variants typically do not cause hereditary pancreatitis. Two patients carried mutations in other risk genes (CFTR and SPINK1) as well. As expected with pediatric patients, no history of smoking or alcohol abuse was documented.
Table 4.
Characteristics of cases with protease-sensitive PNLIP variants identified in the German and French cohorts. All genetic variants indicated are heterozygous. The patient with the SPINK1 variant also had autosomal dominant polycystic kidney disease. The patient with family history had a sister with the same PNLIP variant and similarly mild disease course. RAP, recurrent acute pancreatitis. Mild disease course denotes the overall clinical impression of the referring physician.
| PNLIP variant | Other variants | Age, years (study entry) |
Age, years (diagnosis) |
Gender | Family history |
Smoking | Alcohol | Disease course |
| German cohort | ||||||||
| p.P245A | CFTR p.F508del | 7 | 4.5 | male | no | no | no | RAP |
| p.I265R | - | 25 | unknown | female | no | unknown | unknown | RAP |
| p.I265R | SPINK1 p.N34S | 16 | 8 | female | no | no | no | RAP, mild |
| p.F300L | - | 12 | 11 | female | no | no | no | RAP |
| p.F300L | - | 12 | 12 | female | sister | no | no | RAP, mild |
| p.F300L | - | 11 | 7 | male | no | no | no | RAP, mild |
| p.F300L | - | 8 | 7 | female | no | no | no | RAP |
| p.S304F | 7 | 6 | female | no | no | no | RAP | |
| French cohort | ||||||||
| p.I265R | - | 15 | 15 | female | no | no | no | RAP |
| p.F300L | - | 17 | 16 | female | no | no | no | RAP |
| p.F300L | - | 19 | 19 | female | no | unknown | unknown | RAP |
| p.F300L | - | 23 | 16 | female | no | unknown | unknown | RAP |
| p.F314L | - | 4 | 3 | male | no | no | no | RAP |
DISCUSSION
The pancreas synthesizes and secretes several lipases including phospholipase A2, carboxyl ester lipase (CEL), PNLIP and PNLIP related protein 2 (PNLIPRP2) [24]. PNLIP is critical for efficient digestion of dietary triglycerides. Because common constituents of the duodenum such as bile salts inhibit PNLIP, it requires another pancreatic protein, colipase, for activity [25]. The role of CEL and PNLIPRP2 in dietary fat digestion in humans is less clear. Animal data demonstrate that both contribute to dietary fat digestion in suckling newborns, but similar data in humans is scarce [26, 27]. Similarly, convincing data describing the function of CEL and PNLIPRP2 in adult animals or humans is minimal. CEL likely contributes to the digestion of cholesterol esters and fat-soluble vitamin esters [28]. Both may contribute to digestion of galactolipids based on in vitro activity [29].
To date, among the pancreatic lipases, only genetic variants in CEL have been demonstrated to alter risk for CP. Human CEL contains a domain with a variable number of tandem repeats at the carboxyl terminus. While the number of repeats does not influence risk for pancreatic disease, single base pair deletions in the repeat region cause CP with exocrine and endocrine insufficiency [13, 30]. Additionally, a recombined allele of CEL and its pseudogene CELP was found to confer risk to CP in Europeans while a different hybrid allele in Asians showed no association with CP [11, 31]. Although the role of PNLIP in CP has not been analyzed prior to this study, a homozygous missense variant was reported in two brothers with steatorrhea caused by PNLIP deficiency [32]. In cell culture experiments, the variant caused PNLIP misfolding, loss of secretion and endoplasmic reticulum (ER) stress [15]. Interestingly, abnormal pancreatic function tests raised the possibility that the brothers may have had CP, suggesting a pathogenic role for PNLIP misfolding in CP.
In the present study, we extended our investigations into the potential role of lipases in CP and analyzed PNLIP by targeted DNA sequencing in European, Japanese, Indian and US cohorts. In the German discovery cohort, PNLIP missense variants, the p.F300L variant in particular, were significantly overrepresented in cases, however, a similar enrichment of missense variants and p.F300L did not reach significance in the French replication cohort. Missense PNLIP variants were found with higher frequency in controls versus patients in the Japanese replication cohort, but the difference was not significant. No missense variants were found in the Indian and US cohorts. All missense variants detected were subsequently subjected to functional analysis to identify alterations in folding, secretion, enzymatic activity and proteolytic degradation. Because PNLIP variants were rare and genetic data alone has been suggestive but inconclusive, we filtered the variants using various functional criteria, which might explain pathogenicity.
Mutations in digestive enzymes can alter their folding and result in reduced secretion with intracellular retention, degradation and resultant endoplasmic reticulum (ER) stress. This mechanism has been observed with a subset of PRSS1 variants and in pathogenic CPA1 variants [33]. Only four of 23 PNLIP variants showed reduced secretion consistent with the possibility of protein misfolding. Modeling these missense mutations in the structure of PNLIP suggests they significantly alter the structure. The Cys254 residue forms a disulfide bond with Cys278 at the base of the lid domain and loss of that bond would disrupt the structure of that domain [34]. Both p.G233E and p.A174P are near the active site and create steric hindrance affecting Asp193 and Ser169, respectively. These residues contribute to the catalytic triad. Finally, p.V454F could potentially alter the structure of the C-terminal domain including the β5’- loop, which contributes to substrate binding [35]. The distribution of these four variants was similar in patients (two variants) and controls (two variants) suggesting that the impact of PNLIP misfolding caused by heterozygous variants may not be sufficient to cause disease. This is likely due to the lower expression levels of PNLIP relative to CPA1 or PRSS1 in the pancreas [36].
We also speculated that increased lipase activity might contribute to CP onset. This does not seem to be the case, as only three PNLIP variants showed impaired catalytic activity and these were all found in control subjects. Two of these, p.C198Y and p.D264Y likely alter the structure of predicted acyl chain binding sites, which can explain why activity against triolein, a long-chain triglyceride was most affected. Mutation p.H92N, which showed impaired activity against all substrates, is predicted to alter the structure of the β5’-loop. The result confirms the importance of this region in the function of PNLIP. In a recent study of pancreatic cancer, the p.H92N variant was found in two affected subjects (0.1%) but not in healthy controls [37]. This represents a 17-fold enrichment over the ExAc database allele frequency. It was not reported if these two subjects had CP, a known risk factor for pancreatic cancer. Still, the findings suggest that variant with p.H92N may increase risk for pancreatic disease.
Finally, we considered the possibility that increased resistance to proteolytic degradation might lead to increased lipase activity. Such mechanism was described for the clinically most frequent PRSS1 variants such as p.R122H [17]. In contrast to our expectations, we found that five variants exhibited accelerated degradation by trypsin and chymotrypsin and these proteolytically unstable variants were almost exclusively detected in young European cases, yielding a respectable OR of about 11. Thus, protease-sensitive PNLIP variants are strong susceptibility factors for early-onset CP. These variants were absent in the Japanese, Indian and US cohorts. This resembles the distribution of the pathogenic CEL hybrid allele, which is frequent in Europeans but absent in Asian populations [11, 31].
The mechanism by which protease-sensitive PNLIP variants increase risk for CP is not readily apparent. One possibility is that increased proteolytic sensitivity is a marker for some other functional defect, e.g. altered protein dynamics resulting in a tendency to aggregate or form a complex with some other protein, perhaps other digestive enzymes. This, in turn, may result in trypsin activation or ER stress, depending on which pathway is activated. Another intriguing scenario is that premature degradation of lipases might trigger regulatory mechanisms resulting in unwanted pancreatic stimulation. Finally, we note that the C-terminal colipase-binding domain of PNLIP remains intact after degradation with trypsin and chymotrypsin. This fragment can bind colipase and inhibit intestinal lipolysis in animals, but this function does not provide an apparent explanation why protease-sensitive PNLIP variants increase risk for CP [38, 39]. We found no evidence that the C-terminal domain can cause acinar cell injury in preliminary cell culture experiments.
Patients with protease-sensitive PNLIP variants were mostly children or young adults with RAP, which is the typical clinical presentation in this age group. In several cases, the referring physician described the disease course as mild. Based on these observations and the well- documented role of lipase activity in disease severity [40, 41], it is intriguing to speculate that even though protease-sensitive PNLIP variants increase risk for pancreatitis onset, they also limit disease severity due to lipase degradation.
In summary, our study identified PNLIP as a novel pancreatitis-associated gene. Proteasesensitive PNLIP variants were significantly enriched in patients with non-alcoholic, early-onset CP. The mechanism by which these PNLIP variants drive the development of CP, however, remains enigmatic.
Supplementary Material
STUDY HIGHLIGHTS.
WHAT IS CURRENT KNOWLEDGE:
Genetic risk for chronic pancreatitis is mainly associated with genes that encode pancreatic digestive proteases.
Studies to date highlighted the central role of trypsin in disease onset and progression.
WHAT IS NEW HERE:
Variants in pancreatic lipase that render the enzyme protease sensitive are highly enriched in chronic pancreatitis.
The finding indicates that mechanisms unrelated to trypsin activity can underlie pancreatitis pathogenesis.
ACKNOWLEDGEMENTS
The authors thank all study participants for providing clinical data and blood samples. This work was supported by the Else Kröner-Fresenius-Foundation (EKFS) (to HW), the Deutsche Forschungsgemeinschaft (DFG) grants Wi 2036/2–3 (to HW); National Institutes of Health grants R01DK058088 and R01DK095753 (to MST), R01DK808820 and R01DK097241 (to MEL), R01DK061451 (to DCW); the Hungarian Scientific Research Fund grant PD120960 (to AS); Grants-in-Aid for Scientific Research (KAKENHI) 16K15421 (to AM), 17K15916 (to EN), the Smoking Research Foundation (to AM); Conseil Régional de Bretagne, The Association des Pancréatites Chroniques Héréditaires, The Association de Transfusion Sanguine et de Biogénétique Gaetan Saleun, The Institut National de la Santé et de la Recherche Médicale (INSERM), France (to CF); the Council of Scientific and Industrial Research, Govt of India, India (to GRC); and the OeNB Jubiläumsfonds 16678 (to TM).
Abbreviations:
- CP
chronic pancreatitis
- OR
odds ratio
- PNLIP
pancreatic lipase
- RAP
recurrent acute pancreatitis
Footnotes
ACCESSION CODES
The genomic sequence of pancreatic lipase (PNLIP) used was ENSG00000175535 (GRCh38:CM000672.2) (Ensemble).
COMPETING FINANCIAL INTERESTS
The authors declare no competing financial interests.
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