Variants That Affect Function of Calcium Channel TRPV6 Are Associated With Early-onset Chronic Pancreatitis

Journal Pre-proof Variants That Affect Function of Calcium Channel TRPV6 Are Associated With Early-onset Chronic Pancreatitis Atsushi Masamune, Hiroshi Kotani, Franziska Lena Sörgel, Jian-Min Chen, Shin Hamada, Reiko Sakaguchi, Emmanuelle Masson, Eriko Nakano, Yoichi Kakuta, Tetsuya Niihori, Ryo Funayama, Matsuyuki Shirota, Tatsuya Hirano, Tetsuya Kawamoto, Atsuki Hosokoshi, Kiyoshi Kume, Lara Unger, Maren Ewers, Helmut Laumen, Peter Bugert, Masayuki X. Mori, Volodymyr Tsvilovskyy, Petra Weißgerber, Ulrich Kriebs, Claudia Fecher-Trost, Marc Freichel, Kalliope N. Diakopoulos, Alexandra Berninger, Marina Lesina, Kentaro Ishii, Takao Itoi, Tsukasa Ikeura, Kazuichi Okazaki, Tom Kaune, Jonas Rosendahl, Masao Nagasaki, Yasuhito Uezono, Hana Algül, Keiko Nakayama, Yoichi Matsubara, Yoko Aoki, Claude Férec, Yasuo Mori, Heiko Witt, Tooru Shimosegawa PII: DOI: Reference:

S0016-5085(20)30017-2 https://doi.org/10.1053/j.gastro.2020.01.005 YGAST 63118

To appear in: Gastroenterology Accepted Date: 2 January 2020 Please cite this article as: Masamune A, Kotani H, Sörgel FL, Chen J-M, Hamada S, Sakaguchi R, Masson E, Nakano E, Kakuta Y, Niihori T, Funayama R, Shirota M, Hirano T, Kawamoto T, Hosokoshi A, Kume K, Unger L, Ewers M, Laumen H, Bugert P, Mori MX, Tsvilovskyy V, Weißgerber P, Kriebs U, Fecher-Trost C, Freichel M, Diakopoulos KN, Berninger A, Lesina M, Ishii K, Itoi T, Ikeura T, Okazaki K, Kaune T, Rosendahl J, Nagasaki M, Uezono Y, Algül H, Nakayama K, Matsubara Y, Aoki Y, Férec C, Mori Y, Witt H, Shimosegawa T, Variants That Affect Function of Calcium Channel TRPV6 Are Associated With Early-onset Chronic Pancreatitis, Gastroenterology (2020), doi: https://doi.org/10.1053/ j.gastro.2020.01.005. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

© 2020 by the AGA Institute

Pancreatitis-related gene mutations

F340/F380

Ca2+-channel coding gene TRPV6: Loss-of function mutations in patients with early-onset CP

Development of chronic pancreatitis

1

Wild-type

Mutant

0.5

PRSS1 SPINK1 CPA1 and others… Increased trypsin activity / or ER stress

0

Exacerbation of caeruleininduced pancreatitis

A novel mechanism of pancreatitis due to Ca2+ dysregulation

1

Variants That Affect Function of Calcium Channel TRPV6 Are Associated With Early-onset Chronic Pancreatitis Short title: TRPV6 and pancreatitis

Atsushi Masamune1*%, Hiroshi Kotani2#, Franziska Lena Sörgel3#, Jian-Min Chen4#, Shin Hamada1#, Reiko Sakaguchi2,5, Emmanuelle Masson4,6, Eriko Nakano1, Yoichi Kakuta1, Tetsuya Niihori7, Ryo Funayama8, Matsuyuki Shirota8, Tatsuya Hirano2, Tetsuya Kawamoto2, Atsuki Hosokoshi2, Kiyoshi Kume1, Lara Unger3, Maren Ewers3, Helmut Laumen3, Peter Bugert9, Masayuki X Mori2, Volodymyr

Tsvilovskyy10,

Petra

Weißgerber11,

Ulrich

Kriebs10,

Claudia

Fecher-Trost11, Marc Freichel10, Kalliope N Diakopoulos12, Alexandra Berninger12, Marina Lesina12, Kentaro Ishii13, Takao Itoi13, Tsukasa Ikeura14, Kazuichi Okazaki14, Tom Kaune15, Jonas Rosendahl15, Masao Nagasaki16, Yasuhito Uezono17, Hana Algül12, Keiko Nakayama8, Yoichi Matsubara18, Yoko Aoki7, Claude Férec4,6, Yasuo Mori2, Heiko Witt3%, and Tooru Shimosegawa1%

1

Division of Gastroenterology, 7Division of Medical Genetics, 8Division of Cell

Proliferation, United Centers for Advanced Research and Translational Medicine, Tohoku University Graduate School of Medicine, Sendai, Japan 2

Laboratory of Molecular Biology, Department of Synthetic Chemistry and

Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto, Japan 3

Else Kröner-Fresenius-Zentrum für Ernährungsmedizin (EKFZ), Paediatric

2

Nutritional Medicine, Technische Universität München (TUM), 85354 Freising, Germany 4

Inserm, Univ Brest, EFS, UMR 1078, GGB, F-29200 Brest, France

5

Institute for Integrated Cell-Material Sciences, Kyoto University, Kyoto, Japan

6

CHU Brest, Service de Génétique, Brest, France

9

Institute of Transfusion Medicine and Immunology, Medical Faculty Mannheim,

Heidelberg University, German Red Cross Blood Service of Baden-WürttembergHessen, Mannheim, Germany 10

Pharmakologisches Institut, Universität Heidelberg, D-69120 Heidelberg; and

DZHK (German Center for Cardiovascular Research), partner site Heidelberg, Germany 11

Experimentelle und Klinische Pharmakologie und Toxikologie, Universität des

Saarlandes, D-66421 Homburg, Germany 12

Mildred-Scheel-Chair of Tumor Metabolism and Comprehensive Cancer Center

Munich at the Klinikum rechts der Isar, Technical University of Munich (TUM), Munich, Germany 13

Department of Gastroenterology and Hepatology, Tokyo Medical University,

Tokyo, Japan 14

Department of Gastroenterology and Hepatology, Kansai Medical University,

Hirakata, Japan 15

Department of Internal Medicine I, Martin Luther University, Halle (Saale),

Germany 16

Department of Integrative Genomics, Tohoku Medical Megabank Organization,

Tohoku University, Sendai, Japan

3

17

Cancer Pathophysiology Division, National Cancer Center Research Institute,

Tokyo, Japan 18

#

National Center for Child Health and Development, Tokyo, Japan

: These authors equally contributed to the work.

%

: Senior authors

Grant support: This work was supported in part by Grant-in-Aid from the Japan Society for the Promotion of Science (16K15421, 17K15916, 19K17450); Pancreas Research– Foundation of Japan; the HIROMI Medical Research Foundation; the Mother and Child Health Foundation; the Smoking Research Foundation, the Ministry of Health, Labour, and Welfare of Japan; 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; and the Else Kröner-Fresenius-Stiftung (EKFS) (2017_A108-EKFZ).

Abbreviations used are: AM, acetoxymethyl ester; CI, confidence interval; CP, chronic pancreatitis; H&E, hematoxylin & eosin; INDELs, insertions/deletions; OR, odds ratio; SNV, single nucleotide variant; TRPV6, transient receptor potential vanilloid subfamily member 6; WES, whole exome sequencing; WT, wild-type

*Corresponding author: Atsushi Masamune, M. D., Ph.D., Division of Gastroenterology, Tohoku University Graduate School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai 980-8574 Japan tel: 81-22-717-7171, fax: 81-22-717-7177, e-mail: [email protected]

4

Competing interests: none declared

Author contributions: A Masamune, K Nakayama, and Y Aoki, H Witt and T Shimosegawa designed and directed the study. This study originally started from the research project of the Research Committee of the Intractable Pancreatic Diseases (Chairman, T Shimosegawa), provided by the Ministry of Health, Labour and Welfare of Japan. A Masamune, J-M Chen, E Masson, S Hamada, E Nakano, K Kume, P Bugert, K Ishii, T Itoi, T Ikeura, K Okazaki, and J Rosendahl recruited the subjects and collected the data. A Masamune, F Sörgel, E Nakano, K Kume, L Unger, M Ewers, and T Kaune conducted DNA preparation and Sanger sequencing. T Niihori, R Funayama, K Nakayama and Y Aoki conducted WES and analyzed the data. H Kotani, R Sakaguchi, T Hirano, T Kawamoto, A Hosokoshi, MX Mori and Y Mori conducted functional assays. Y Kakuta performed statistical analysis. M Shirota conducted structure analysis. Y Uezono prepared the wild-type TRPV6 expression vector and S Hamada conducted in vitro mutagenesis. V Tsvilovskyy, P Weißgerber, U Kriebs, C Fecher-Trost, and M Freichel– generated TRPV6mut/mut mice and conducted preliminary animal experiments. K Diakopoulos and A Berninger were responsible for the mouse lines and conducted the animal experiments. M Lesina analyzed sera and tissue of the mice. H Algül designed the experiments and supervised the conduction as well as analysis of the animal experiments. M Nagasaki provided the variant frequency data of Tohoku Medical Megabank. A. Masamune, H Laumen, Y Matsubara, C Férec, Y Mori, H Witt, and T Shimosegawa supervised the study. A Masamune and H Witt drafted the first and subsequent versions of the report. All authors read, commented, and gave final approval of the report.

5

6

Abstract Background and Aims: Changes in pancreatic calcium levels affect secretion and might be involved in development of chronic pancreatitis (CP). We investigated the association of CP with the transient receptor potential cation channel subfamily V member 6 gene (TRPV6), which encodes a Ca2+-selective ion channel, in an international cohort of patients and in mice. Methods: We performed whole-exome sequencing DNA from a patient with idiopathic CP and from his parents, who did not have CP. We validated our findings by sequencing DNA from 300 patients with CP (not associated with alcohol consumption) and 1070 persons from the general population in Japan (controls). In replication studies, we sequenced DNA from patients with early-onset CP (20 y or younger), not associated with alcohol consumption, from France (n=470) or Germany (n=410). We expressed TRPV6 variants in HEK293 cells and measured their activity using Ca2+ imaging assays. CP was induced by repeated injections of cerulein in TRPV6mut/mut mice. Results: We identified the variants c.629C>T (p.A210V) and c.970G>A (p.D324N) in TRPV6 in the index patient. Variants that affected function of the TRPV6 product were found in 13/300 cases (4.3%) and 1/1070 controls (0.1%) from Japan (OR, 48.4; 95% CI, 6.3 371.7; P=2.4 × 10-8). Twelve of 124 patients (9.7%) with early-onset CP had such variants. In the replication set from Europe, 18 patients with CP (2.0%) carried variants that affected the function of the TRPV6 product compared with 0 controls (P=6.2 × 10-8). Variants that did not affect the function of the TRPV6 product (p.I223T and p.D324N) were overrepresented in Japanese cases vs controls (OR, 10.9; 95% CI, 4.5–25.9; P=7.4 × 10-9 for p.I223T; and P=.01 for p.D324N), whereas the p.L299Q was overrepresented in European cases vs controls (OR, 3.0; 95% CI, 1.9–4.8; P = 1.2 × 10-5). TRPV6mut/mut given cerulein developed more severe pancreatitis than control mice, demonstrated by increased levels of pancreatic enzymes, histologic alterations, and pancreatic fibrosis. Conclusions: We found that patients with early-onset CP not associated with alcohol consumption carry variants in TRPV6 that affect the function of its product, perhaps by altering Ca2+ balance in pancreatic cells. TRPV6 regulates Ca2+ homeostasis and pancreatic inflammation.

7

Introduction Chronic pancreatitis (CP) is an inflammatory disease of the pancreas, characterized by irreversible morphological changes, pain, and/or permanent loss of exocrine and endocrine functions of the gland (1, 2). CP is caused by multiple interactions between genetic and environmental factors (1, 2). Pancreatitis is thought to be an intra-acinar event provoked by premature activation of digestive enzymes (3). Several pancreatitis susceptibility genes have been identified, including cationic trypsinogen (PRSS1) (4), cystic fibrosis transmembrane conductance regulator (CFTR) (5, 6), serine protease inhibitor Kazal type 1 (SPINK1) (7), trypsin-degrading enzyme chymotrypsin C (CTRC) (8), carboxypeptidase A1 (CPA1) (9), carboxyl ester lipase (CEL) (10), and pancreatic lipase (PNLIP) (11). Besides CFTR, these gene products act as components of the pancreatic digestive enzyme/enzyme inhibitor system. The underlying pathogenic mechanisms of mutations in these susceptibility genes include an imbalance of trypsin and its counterparts or endoplasmic reticulum stress in pancreatic acinar cells (4–12). However, in many patients with hereditary or early-onset idiopathic CP, no pathogenic mutation in any of the known pancreatitis susceptibility genes can be detected (13), suggesting the presence of yet unknown inherited factors. Dysregulated Ca2+ homeostasis, such as a sustained global elevation of intracellular Ca2+, plays a critical role in the pathogenesis of pancreatitis (14, 15). Ca2+ is known to stimulate premature trypsin activation (16). Transient receptor potential vanilloid subfamily member 6 (TRPV6) represents a constitutively active Ca2+-selective ion channel, which belongs to the vanilloid subfamily of transient receptor potential channels (17-19). TRPV6 is a highly Ca2+-selective ion channel that regulates apical Ca2+ entry in absorptive and secretory tissues (17-20). TRPV6 plays a central role in the

8

Ca2+ homeostasis including Ca2+ absorption in the intestine (20, 21). In humans, TRPV6 is mainly expressed in the exocrine pancreas, salivary glands, prostate, and placenta, based on the Genotype-Tissue Expression (GTEx) project (version 7, Date of download: April 1st, 2019) (https://gtexportal.org/home/) (22), where it seems to maintain intracellular Ca2+ concentrations (18, 23). In the pancreas, TRPV6 expression is approximately six times higher in ductal cells compared to acinar cells (24). Based on the initial identification by whole exome sequencing (WES), we here report that functionally-defective TRPV6 variants are strongly associated with CP not associated with alcohol consumption (non-alcoholic CP).

9

Materials and Methods Study subjects A 34-year-old male was referred to our hospital for examination of the cause of his CP. He had developed recurrent pancreatitis attacks since the age of 25. He had no history of drinking alcohol or smoking. His parents had no past history of pancreatitis. For the validation study, we enrolled 300 (152 males and 148 females; median age of onset 25 years [range 1–70 years]) Japanese patients with non-alcoholic CP including 42 cases with family history of recurrent pancreatitis or CP. There were two ever smokers but no current smokers. To minimize the possibility that the results are due to population stratification in the Japanese cohort, we investigated two independent populations as replication sets. We investigated unrelated subjects with early-onset non-alcoholic CP (age of onset ≤20 years) originating from France (n=470; 249 males and 221 females; median age of onset 16 years [range, 1–20 years]) and Germany (n=410; 184 males and 226 females; median age of onset 11 years [range, 0–20 years]). In addition, 300 (267 male and 33 female) patients with alcohol-related CP originating from Japan were analyzed. The variant frequencies in the Japanese population (n=1,070) were obtained from the Tohoku Medical Megabank database (25). Control subjects were also recruited from France (n=570) and Germany (n=750). For investigation of p.A18S and p.L299Q, we extended the German cohort by analysis of additional 120 non-alcoholic CP patients recruited in Leipzig, Germany, and additional 2,625 control samples consisting of blood donors recruited in Mannheim, Germany. We also extended the French cohort by analysis of 384 non-alcoholic CP patients and 480 control subjects (additional controls only for p.L299Q). This study was approved by the institutional review board of all participating

10

study centers. All subjects gave written informed consent.

Whole exome sequencing and annotation We prepared genomic DNA from peripheral blood leukocytes. Exon capture was performed using the SureSelectXT Human All Exon v5 capture kit (Agilent Technologies, Santa Clara, CA). We sequenced exon-enriched DNA libraries using the Illumina HiSeq 2500 platform (Illumina, San Diego, CA). We aligned paired-end 101-base pair reads to the reference human genome (UCSC hg19) using the Burrows-Wheeler Alignment tool 0.6.2 (26). We annotated single nucleotide variants (SNVs) and insertions/deletions (INDELs) using ANNOVAR (BIOBASE, Wolfenbüttel, Germany) (27). We excluded the following variants: (i) synonymous or intronic variants (more than 2 base pairs from the exon/intron boundary); (ii) variants with allele frequencies of more than 0.01 in 1000 Genomes (28) or in the Human Genetic Variation Database, which provided frequencies of genetic variations of 1,208 healthy Japanese individuals (29). Visual inspection of the alignment quality was performed using the Integrative Genomics Viewer (30). WES was performed in 130 Japanese patients with non-alcoholic CP in addition to the index patient.

Genotyping All exons and adjacent intronic regions of TRPV6 were amplified by PCR in the Japanese patients using the primer sets and conditions shown in Supplementary Table 1. Following the clean-up using the illustra ExoProStar S (GE Healthcare Life Sciences; Little Chalfont, United Kingdom), the PCR products were sequenced on the ABI3730xl DNA Analyzer (Applied Biosystems). Rare variants detected in patients and

11

in controls (except for Japanese) as well as those detected by WES were verified by re-sequencing the samples from an independent PCR. For analysis of p.A18S and p.L299Q, additional German and French patients and control subjects were investigated by melting curve analysis using simple probes on the LightCycler 480 platform (Roche Diagnostics). Primers and simple probes were designed and synthesized by TIB MOLBIOL (Berlin, Germany) based on the published nucleotide sequence (ENSG00000165125). Sequences of primers and probes as well as reaction conditions are shown in Supplementary Table 2. In addition, we sequenced all exons and adjacent intronic regions of PRSS1, SPINK1, CTRC, and CPA1 as previously reported (4, 7-9, 31). All exons and adjacent intronic regions of CFTR were screened in European patients by high-resolution DNA or FRET probe melting analysis (32, 33).

Expression plasmids and in vitro mutagenesis The construction of the full-length wild-type (WT) TRPV6 and the ancestral haplotype expression vectors in pcDNA3.1(−) was previously described (34). TRPV6 mutant constructs were prepared using the KOD-Plus-Mutagenesis Kit (TOYOBO, Osaka, Japan) according to the manufacturer’s instruction. Successful mutagenesis was confirmed by direct sequencing.

Ca2+ imaging assay HEK293 cells (American Type Culture Collection) were co-transfected with recombinant TRPV6 expression plasmids and pEGFP-N1 or pEGFP-C1 (Clontech Laboratories) as a transfection marker, using the SuperFect Transfection Reagent

12

(Qiagen). After transfection, cells were plated on coverslips and loaded with 1 µM Fura-2-acetoxymethyl ester (AM) (Dojindo, Kumamoto, Japan). Fluorescence images of the cells were recorded and analyzed with a video image analysis system (AQUACOSMOS;

Hamamatsu

Photonics,

Hamamatsu,

Japan).

Fura-2-AM

fluorescence at an emission wavelength of 510 nm was obtained by exciting Fura-2-AM sequentially at 340 and 380 nm. The increase in the fluorescence ratio F340/F380 (F340/F380) evoked by the application of 2 mM Ca2+ was determined. The ∆F340/F380 value in the cells expressing WT TRPV6 was regarded as 100% TRPV6 activity. We defined functionally-defective variants as those in which an increase of [Ca2+]i was significantly diminished compared to the WT.

Western blotting At 48 h after transfection, HEK293 cells expressing pcDNA3.1(−) (mock-transfected), WT, or TRPV6 variants cultured on 6 cm culture dish were lysed in 500 µL of buffer containing 50 mM Tris (pH 8.0), 150 mM NaCl, 1.0 w/v % Nonidet P-40. After denaturing and reduction, protein samples (30 µL of total cell lysates) were fractioned by electrophoresis through 9% SDS polyacrylamide gels and analyzed by Western blotting using rabbit anti-TRPV6 polyclonal antibody (at 1:1000; ACC-036, Alomone Labs, Jerusalem, Israel) followed by anti-rabbit IgG HRP conjugate (at 1:2000; NA9340, GE Healthcare). The anti-TRPV6 antibody recognizes C-terminal region (amino acid residues 752-765 of human TRPV6, Uniprot entry Q9H1D0-1). The chemiluminescence intensities of the bands were measured by Multigauge version 3.0 (Fuji film, Tokyo, Japan). The expression of α-tubulin was examined in a similar manner using mouse anti-α-tubulin monoclonal antibody (at 1:1000; T6074, Sigma

13

Aldrich) followed by anti-mouse IgG HRP conjugate (at 1:2000; NA9310, GE Healthcare).

In vivo model of pancreatitis We used TRPV6mut/mut mice in which the single aspartic acid residue in the pore region critical for Ca2+ permeation, located at position 541 in TRPV6 (D541, corresponding to D581 according to the newer Uniprot entry Q91WD2), is replaced by an alanine. TRPV6 (p.D541A, corresponding to p.D581A in Uniprot entry Q91WD2) mutation blocks the ability of TRPV6 to conduct Ca2+ (35). The first-generation offspring from the mating of 129SvJ and C57Bl6/N mice were used as controls (129B6F1). TRPV6mut/mut and 129B6F1 mice were kept in an animal room (room temperature range between 20 and 22°C) with a light: dark cycle of 12:12 hours (light period: 06:00 am– 06:00 pm) in groups of 2–4 animals in type III cages (Tecniplast, Buguggiate, Italy) with bedding and nesting material. All animals were provided with standard maintenance food for mice (No. 1324 – 10 mm pellets, Altromin, Lage, Germany) and water ad libitum and housed under specific pathogen-free conditions in accordance with the European Directive 2010/63/EU. All animal experiments were approved and conducted in accordance with the federal German guidelines for ethical animal treatment (Regierung von Oberbayern). CP was induced in TRPV6mut/mut mice and 129B6F1 mice, age 8-10 weeks, according to the Jensen protocol using the cholecystokinin analogue cerulein (36). In more detail, mice were injected intraperitoneally each hour for 8 h per day during two consecutive days (d1, d2) with 0,1 µg cerulein/ g mouse body weight. Blood was taken at 0 h, 8 h, 24 h, 32 h, and at sacrifice 96 h (d5) after the first injection to quantify the

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damage-induced pancreatic enzyme release. Serum was diluted 1/10 with 0.9% NaCl. Amylase activity (in units/ l) was quantified by a colorimetric assay according to the IFCC method (AMYL2 Cobas, Roche. Lipase activity (in units/ l) was quantified by DGGR substrate-based assay (LIPC Cobas, Roche). At sacrifice pancreatic tissue was removed, fixed with paraformaldehyde, and embedded in paraffin for histological analyses (see scheme in Figure 5A). Two µm-thick tissue sections were prepared and stained with hematoxylin & eosin (H&E) for morphological examination. Collagen was visualized by Sirius Red staining as previously described (37, 38).

Structural analysis of the mutated residues The three-dimensional structure of human TRPV6 (39) (PDB ID: 6BO8) was obtained from Protein Data Bank Japan2 (40) in mmCIF format. The atomic coordinates and interactions of those residues that were mutated in the CP patients were manually inspected and visualized with PyMOL 1.8 (The PyMOL Molecular Graphics System, Version 1.8 Schrödinger, LLC).

Electrophysiological measurements Electrophysiological measurements were performed as previously reported (41). Briefly, whole-cell currents were recorded at room temperature (22–24 °C) using the conventional whole-cell patch clamp technique with an EPC10 amplifier (HEKA Electronics, Lambrecht, Germany). The patch electrodes were prepared from borosilicate glass capillaries and had a resistance of 2–3 MΩ. Series resistance was compensated (to 50–70%) to minimize voltage errors. Current signals were filtered at 2.9 kHz with a four-pole Bessel filter and digitized at 200 kHz. We used the

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PatchMaster software (HEKA Electronics) for pulse control, data acquisition, and analysis. Currents were measured by applying the first hyperpolarization step from +20 mV to -100 mV and subsequent voltage-steps of 50 ms duration from -100 mV to +110 mV in 15 mV increments, followed by the final -100 mV step at 0.2 Hz (Supplementary Figure 4). The fraction of open channels at the end of each voltage step was assessed by normalizing the initial current amplitude during the final -100 mV step to the steady state current during the first hyperpolarization step. The external solution contained 150 mM NaCl, 10 mM EDTA, 10 mM glucose, and 10 mM HEPES, adjusted to pH 7.4 with NaOH. The pipette internal solution contained 145 mM cesium glutamate, 8 mM NaCl, 3.3 mM CaCl2, 0 or 3 mM MgCl2, 10 mM EGTA, and 10 mM HEPES, adjusted to pH 7.2 with CsOH. The osmolarity of the solutions was ≈ 330 mOsm.

Nomenclature The NM_018646.5 GenBank reference sequence was used in this study. The translation of the TRPV6 initiates at a non-canonical start codon (ACG) that is decoded by methionine located 120 nucleotides upstream of the annotated AUG (42). The A of the ACG start codon was used as nucleotide +1. The mutations were described according to the nomenclature recommended by the Human Genome Variation Society.

Statistical analysis The significance of the differences in variant frequencies between patients and controls was tested by two-sided Fisher’s exact test. For Ca2+ imaging assays, data are shown as mean + SEM of the assays from the sum of two or three independent

16

transfections. The differences between more than two groups in Ca2+ imaging assays were analyzed using the Tukey-Kramer method. The differences between the TRPV6mut/mut and WT groups in amylase and lipase assays, and those between the TRPV6 variants and WT groups in electrophysiological measurements were analyzed using unpaired t-test. A P value of less than 0.05 was considered significant. All statistical analyses were performed using the SPSS version 20.0 statistical analysis software (SPSS Inc., Chicago, IL) and R software (version 3.4.4).

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Results Whole exome sequencing identified p.A210V and de novo p.D324N variants in TRPV6 To identify novel pancreatitis susceptibility genes, we performed WES of a patient with idiopathic CP who developed symptoms at the age of 25 years. We identified 10,278 non-synonymous or splice site variants in this patient. Among them, 421 variants were novel or rare (minor allele frequency <0.01) in the 1000 Genomes (28) or in the Human Genetic Variation Database (29). After comparison of the data with the WES data of his clinically unaffected parents and visual inspection of the alignment quality using the Integrative Genomics Viewer (30), we identified one de novo heterozygous c.970G>A (p.D324N) variant in TRPV6. The patient also carried another rare non-synonymous TRPV6 variant, c.629C>T (p.A210V), inherited from his mother.

Functionally-defective TRPV6 variants are associated with non-alcoholic CP in Japan We next analyzed all exonic and flanking intronic regions of TRPV6 in 300 Japanese patients with non-alcoholic CP including 42 cases with a family history of recurrent pancreatitis or CP, and compared the distribution to that of 1,070 control subjects (25). We identified 33 missense and 2 nonsense variants (Table 1). Three patients had the ancestral-haplotype (p.C197R + p.M418V + p.M721T), which shows a positive selection in human evolution (43, 44). The locations of the non-synonymous TRPV6 variants identified in Japanese patients with CP are shown in Supplementary Figure 1. Six patients were compound heterozygous for TRPV6 missense variants: in

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two patients with p.D324N, we detected the variants p.A210V and p.R672Q, respectively; and in four patients with heterozygous p.I223T, we detected the variants p.D319N, p.R425Q, p.G428R, and p.I580F, respectively. In addition, four patients were homozygous for TRPV6 variants: two subjects for p.I223T, one for p.R174X, and one for p.R646P. Non-synonymous TRPV6 variants were not detected in the two ever smokers. Three non-synonymous variants including p.A210V (P = 0.048), p.I223T (odds ratio [OR] = 10.9, 95% confidence interval [CI] = 4.5–25.9, P = 7.4 × 10-9), and p.D324N (P = 0.01) were significantly overrepresented in patients with CP compared to control subjects (Table 1). To assess the functional impact of these variants, we introduced them into TRPV6 cDNA constructs, expressed them in HEK293 cells, and evaluated the TRPV6 activity by Ca2+ imaging assays. In response to the application of 2 mM Ca2+-containing solutions, a faint increase of the intracellular Ca2+ concentrations ([Ca2+]i) was observed in cells transfected with pcDNA3.1(−) (mock-transfected cells), resulting from endogenously expressed Ca2+ channels (34). A marked increase of [Ca2+]i was observed in cells expressing the WT TRPV6, p.S18A, p.I223T and p.D324N, but this increase was absent in cells expressing here identified TRPV6 variants such as p.R174X and p.A210V (Figure 1). An increase of [Ca2+]i was significantly diminished in 12 out of 25 non-synonymous variants found in patients with non-alcoholic CP compared to the WT TRPV6, suggesting that these variants are functionally-defective (Figure 2). In contrast, only one (p.L392F) out of 12 non-synonymous variants detected in 1,070 Japanese control subjects was functionally-defective. Overall, functionally-defective TRPV6 variants were significantly overrepresented in the non-alcoholic CP group (13/300, 4.3%) as compared to controls (1/1,070, 0.1%) (odds ratio [OR] = 48.4, 95% confidence

19

interval [CI] = 6.3–371.7, P = 2.4 × 10-8) (Table 1). When the data were stratified according to the age of symptom onset, functionally-defective TRPV6 variants were found in 12/124 (9.7%) early-onset (age of symptom onset <20 years) cases, but in only one out of 176 (0.6%) cases that developed symptoms after the age of 20 years (OR = 18.8; 95% CI = 2.4–146.2; P = 0.00014). Among the 42 cases with a family history of recurrent pancreatitis or CP, functionally-defective TRPV6 variants were found in 4 cases (p.R174X, p.A606T, p.L608R, and p.L609F variants). We also analyzed all TRPV6 exons in 300 Japanese subjects with alcohol-related CP. p.I223T was significantly overrepresented in patients with alcohol-related CP compared to control subjects. (OR = 3.6; 95% CI = 1.3–10.0; P = 0.019). One patient was compound heterozygous for p.I223T and p.D324N. Only one out of 300 (0.3%) patients carried a functionally-defective TRPV6 variant p.L392F (Supplementary

Table

3).

These

findings

suggest

a

limited

role

of

functionally-defective TRPV6 in alcoholic CP.

The association of defective TRPV6 variants with non-alcoholic CP was robustly replicated in European cohorts As demonstrated recently, a geographical and ethnic heterogeneity of genetic associations in CP patients has been observed. For example, a CEL hybrid allele was found to increase susceptibility to CP in Europe, but not in Asian populations originating from China, Japan, and India (10, 45). To assess whether TRPV6 variants are globally associated with non-alcoholic CP, we sequenced all TRPV6 exons in 470 non-alcoholic CP cases and 570 controls from France, and in 410 non-alcoholic CP cases and 750 controls from Germany. The locations of the non-synonymous TRPV6

20

variants identified in CP patients in Europe are shown in Supplementary Figure 2. Again, functionally-defective TRPV6 variants were overrepresented in European patients with CP (18/880; 2.0%) compared to the controls (0/1,320; 0%) (P = 6.2 × 10-8) (Table 2, Figure 3). Even if stratified by population, functionally-defective TRPV6 variants were overrepresented in French patients with CP (9/470, 1.9%) compared to the controls (0/570, 0%) (P = 0.00075) (Supplementary Table 4) as well as in German CP cases (9/410, 2.2%) compared to the controls (0/750, 0%) (P = 0.0001) (Supplementary Table 5).

Protein expression of TRPV6 was diminished in some TRPV6 variants We examined the TRPV6 protein expression in HEK293 cells transfected with TRPV6 variants by Western blotting. As shown in Figure 4, TRPV6 expression was decreased in cells expressing many functionally-defective TRPV6 variants including missense, frame-shift, and nonsense ones. The expression of the upper bands around 90 kDa in size, representing glycosylated forms (46, 47), was decreased in some TRPV6 variants. These results suggest that functional deficiency at least in part results from decreased TRPV6 expression.

Cerulein-induced pancreatitis was exacerbated in TRPV6mut/mut mice CP was induced in TRPV6mut/mut and control mice (129B6F1) by serial cerulein injections (Figure 5A). Serum amylase and lipase levels were elevated both in TRPV6mut/mut mice and the control mice at 8, 24, and 32 h after CP induction, but those levels were higher in TRPV6mut/mut mice than the control mice at 24 h (Figure 5B). Interestingly, at 96 h, both enzyme levels were greatly reduced in TRPV6mut/mut mice

21

compared to control mice, indicating the loss of acinar cell tissue and therefore enzyme production. Correspondingly, H&E and Sirius Red stainings revealed increased fibrosis, loss of acinar cells, and acinar-to-ductal metaplasia in TRPV6mut/mut mice compared to control mice (Figure 5C). Thus, functional TRPV6 is important in vivo for protection against CP.

Some functionally non-defective TRPV6 variants were associated with CP In Japanese cohorts, two functionally non-defective variants, p.I223T and p.D324N, were overrepresented in patients with CP compared to control subjects. In European cohorts, p.A18S was less common and p.L299Q was more common in cases, and thus analyzed in additional patients and controls. The c.896T>A (p.L299Q) variant, which was absent in Japanese subjects, was significantly enriched in European patients (33/1,384, 2.4%) compared to controls (36/4,425, 0.8%) (OR = 3.0; 95% CI = 1.9–4.8; P = 1.2 × 10-5) (Table 2). If stratified by population, p.L299Q was significantly overrepresented in both of the two European cohorts (Supplementary Table 4 and Supplementary Table 5). Another variant, c.T52G>T (p.A18S), which was found in all investigated populations, was underrepresented in European CP cases (52/1,379, 3.8%) compared to controls (252/3,712; 6.8%) (OR = 0.5; 95% CI = 0.4–0.7; P = 3.4 × 10-5) (Table 2). However, a significant association was observed for the French cohort only (Table 1, Supplementary Table 4 and Supplementary Table 5).

The p.L299Q and p.D324N variants might constitutively suppress TRPV6 function through the pore blockade mechanism Although a crystal structure analysis (30) suggested that the functionally

22

non-defective p.I223T, p.L299Q, and p.D324N variants were likely to affect the TRPV6 function (Supplementary Figure 3), increases in [Ca2+]i were not abrogated in HEK293 cells expressing these variants. Previous studies have shown that voltage-dependent gating of TRPV6 channel depends on intracellular Mg2+, which acts as a pore blocker suppressing TRPV6 currents (41, 48). We examined whether these variants exhibit voltage-dependent blocking of the TRPV6 channel. When the pipette solution containing 3 mM Mg2+ was intracellularly perfused, HEK293 cells expressing all of these 3 variants as well as WT exhibited slowly activating monovalent currents during the first hyperpolarization step from +20 mV to -100 mV (Supplementary Figure 4B). When Mg2+-free solution was perfused, HEK293 cells expressing WT or p.I223T TRPV6 exhibited accelerated activation kinetics (Supplementary Figure 4C). In contrast, cells expressing p.L299Q or p.D324N maintained slow activation kinetics during the first hyperpolarization step (Supplementary Figure 4C). TRPV6 monovalent currents showed deactivation during the subsequent steps to different depolarizing potentials (-100 mV to +110 mV). The fraction of open channels at the end of each voltage step showed the membrane potential dependency in the presence of 3 mM Mg2+ (Supplementary Figure 4D). Even in the absence of Mg2+ in the pipette solution, the membrane potential dependency remained in cells expressing p.L299Q and p.D324N, but not in cells expressing p.I223T or WT TRPV6 (Supplementary Figure 4E). These results suggest that p.L299Q and p.D324N variants might constitutively suppress the TRPV6 function through the pore blockade mechanism, which is only triggered by Mg2+ in the WT TRPV6 channel.

23

SPINK1

p.N34S

was

found

in

20%

of

the

patients

carrying

the

functionally-defective TRPV6 variants CP is a complex multigenic disease often with mutations in several pancreatitis susceptibility genes in affected subjects (12, 33, 49). We analyzed PRSS1, SPINK1, CTRC, and CPA1 variants in 300 Japanese patients with non-alcoholic CP to clarify the relationship between mutations in these genes and TRPV6 variants. In total, 63/300 (21%) of the cases carried at least one of the previously known pancreatitis-associated mutations (Supplementary Table 6). Among the 13 Japanese CP patients carrying functionally-defective TRPV6 variants, one case was trans-heterozygous for the TRPV6 p.R345C and SPINK1 p.N34S. In addition, we analyzed PRSS1, SPINK1, CTRC, CPA1, and CFTR variants in French and German patients. Two French patients carrying the functionally-defective TRPV6 variants p.R174X and p.G311V, respectively, were trans-heterozygous for SPINK1 p.N34S and one French subject was trans-heterozygous for TRPV6 p.R345C and CFTR p.F508del. Three of 9 German patients with functionally-defective TRPV6 variants p.R342Q, p.R483W and p.C659X, respectively, were

trans-heterozygous

for

SPINK1

p.N34S.

No

German

patient

was

trans-heterozygous for PRSS1, CTRC, CPA1, or CFTR. In total, 6/30 (20%) patients with functionally-defective TRPV6 variants were trans-heterozygous for SPINK1 p.N34S. Regarding the functionally non-defective TRPV6 variants, one Japanese patient was trans-heterozygous for the TRPV6 p.H205Y and SPINK1 p.N34S. One French patient was trans-heterozygous for the TRPV6 p.A18S and SPINK1 p.N34S. Four patients had TRPV6 p.L299Q and SPINK1 p.N34S (three heterozygous and one

24

homozygous). Three German patients with TRPV6 p.L299Q were trans-heterozygous for SPINK1 p.N34S (two heterozygous and one homozygous).

25

Discussion Since the landmark discovery of PRSS1 mutations in patients with hereditary pancreatitis (4), the old concept of Hans Chiari that pancreatic inflammation results from autodigestion (50) became the predominant pathophysiologic model. The identification of mutations in additional susceptibility genes such as SPINK1, CTRC, CPA1, CEL and PNLIP (7-11) further underlined the enzyme-centered concept of a disturbed balance of pancreatic digestive enzymes and their specific inhibitors within the acinar cells as early pathogenic key event. In this model, pancreatitis is thought as consequence of intra-acinar processes such as pathologic enzyme activation, endoplasmic reticulum stress, autophagy and mitochondrial dysfunction (3). In contrast, less attention has been paid to the pathophysiologic role of ductal cells in pancreatic injury. So far, only two genes which are mainly expressed in ductal cells have been linked to pancreatitis: CFTR, the gene responsible for cystic fibrosis (5, 6), and CLDN2, encoding the tight junction protein claudin 2 (51). In the present study, we show that functionally-defective variants in TRPV6, which is predominantly expressed in pancreatic ducts (24), are globally associated with non-alcoholic CP in Japan, France and Germany, identifying TRPV6 as novel pancreatitis susceptibility gene. Functional TRPV6 is important for the protection against CP, as shown by in vivo experiments in which cerulein-induced pancreatitis was exacerbated in TRPV6mut/mut mice compared to control mice. An association was not found in alcohol-related CP in Japan. Smoking is another significant risk factor for pancreatitis (52). We did not evaluate the association of TRPV6 variants with smoking-induced CP, because only two patients with non-alcoholic CP were ever smokers and did not carry non-synonymous TRPV6 variants.

26

The physiologic role of TRPV6 in the pancreas and the pathologic mechanisms by which impaired function predispose to pancreatitis remain elusive. TRPV6 represents a highly selective Ca2+ ion channel that regulates apical Ca2+ entry in absorptive and secretory tissues (17-21). Previous studies have suggested deleterious roles of calcium influx and overload in pancreatitis (14, 15). Functional impairment might decrease Ca2+ entry from pancreatic ducts, resulting in increased Ca2+ concentrations in pancreatic fluids. Similar findings have been reported in Trpv6-/- mice, in which excessively high Ca2+ concentrations in epididymal fluid resulting from impaired Ca2+ uptake in epididymal epithelial cells have been described (53). In our study, TRPV6 expression was decreased in the presence of many functionally-defective TRPV6 variants, suggesting that diminished channel function per se or altered function of other membrane channels predispose to pancreatitis. On the other hand, TRPV6 is also expressed in human pancreatic acinar cells, where the protein can be detected in the apical secretory pole, with both intracellular and apical membrane staining, suggesting TRPV6 expression in zymogen granules (54). TRPV6 might be involved in the clearance of Ca2+ released from the zymogen granules by re-uptake of Ca2+ into the cells, thereby refilling the Ca2+ pool (55). Defective TRPV6 might result in a disturbance of Ca2+ homeostasis in zymogen granules, where Ca2+ concentrations will remain at high levels. However, Ca2+ signaling in pancreatic acinar as well as ductal cells is complex, and the nature of the Ca2+ entry and exit pathways remains uncertain (56). Interestingly,

recent

studies

have

identified

homozygous

and

compound-heterozygous TRPV6 variants in patients with a rare syndrome consisting of transient neonatal hyperparathyroidism, decreased bone mineralization, and dysplasia of

27

the fetal skeleton due to impaired placental calcium transport (57, 58). In these two studies, however, pancreatitis was not reported, neither in the patients nor in the heterozygous parents. It would be interesting to see whether these patients develop pancreatitis later in life. A remarkable finding underlining the complexity of genetic predisposition to CP was, that 6/30 (20%) patients with functionally-defective TRPV6 variants were trans-heterozygous for a well-characterized pancreatitis predisposing variant in SPINK1 (p.N34S). This is a striking percentage considering that trans-heterozygosity has been reported only in approximately 10% of CP patients with an identified genetic defect so far (9). Thus, a cumulative genetic handicap seems to be necessary to develop the disease. One might speculate that only a combination of mutated TRPV6 and genetic alterations in additional gene results in a predisposition to pancreatitis, whereas homozygosity or compound-heterozygosity for severe TRPV6 variants alone drive the phenotype to transient neonatal hyperparathyroidism. The p.I223T and p.D324N variants were overrepresented in Japanese cases, whereas the p.L299Q variant was overrepresented in German and French cases. Although a crystal structure analysis (39, 40) suggested that these variants were likely to affect the TRPV6 function (Supplementary Figure 3), increases in [Ca2+]i were not impaired in HEK293 cells expressing these variants. In agreement with our results, it was reported that the Ca2+ current amplitude was not significantly decreased in HEK293T cells expressing the p.I223T variant in whole-cell patch-clamp recordings (57). We further examined whether these variants affect the voltage-dependent blocking of TRPV6 channel in the presence or absence of intracellular Mg2+, a pore blocker of TRPV6 channel (48). In cells expressing p.L299Q and p.D324N, voltage-dependent blocking was observed even without the intracellular perfusion of Mg2+. These TRPV6

28

variants might constitutively suppress the TRPV6 function through the pore blockade mechanism, which is only triggered by Mg2+ in the WT TRPV6 channel. Interestingly, the spatial and temporal distribution of intracellular Mg2+ is known to affect Ca2+ signals in pancreatic acinar cells (59). Obviously, further studies are warranted to dissect the mechanisms linking these TRPV6 variants and pancreatitis. In conclusion, TRPV6 variants are globally associated with non-alcoholic CP. TRPV6 is a novel pancreatitis-associated gene beyond the pancreatic digestive enzyme/enzyme inhibitor system, and the first one that directly regulates Ca2+ homeostasis. Our findings open a completely new avenue by emphasizing the potential role of ductal cells and especially calcium channels in the pathophysiology of pancreatitis which might lead to the development of personalized medicine targeting TRPV6 channel activity.

29

Acknowledgements The authors are grateful to Makiko Sumii, Yoko Tateda, Miyuki Tsuda, Mami Kikuchi, Makiko Nakagawa and Kiyotaka Kuroda for the excellent technical assistance. We also acknowledge the technical support of the Biomedical Research Core of Tohoku University Graduate School of Medicine.

30

URLs 1,000 Genomes, www.1000genomes.org/; ANNOVAR, http://www.openbioinformatics.org/annovar/; Burrows-Wheeler Alignment tool 0.6.2, http://bio-bwa.sourceforge.net/; Genome Analysis Toolkit v1.6 software, http://www.broadinstitute.org/gatk/; Genotype-Tissue Expression project, https://gtexportal.org/home/; Human Genetic Variation Database, www.genome.med.kyoto-u.ac.jp/SnpDB/; Human Genome Variation Society, http://www.hgvs.org/mutnomen/; Integrative Genomics Viewer, https://www.broadinstitute.org/igv/; Picard program, http://broadinstitute.github.io/picard/; Uniprot, https://www.uniprot.org/ Protein Data Bank Japan2, https://pdbj.org/; PyMOL 1.8, https://pymol.org/2/; R software (version 3.4.4), http://www.r-project.org/

31

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2016;61:547-553. 30. Robinson JT, Thorvaldsdóttir H, Winckler W, et al. Integrative genomics viewer. Nat Biotechnol 2011;29:24-26. 31. Nakano E, Masamune A, Niihori T, et al. Targeted next-generation sequencing effectively analyzed the cystic fibrosis transmembrane conductance regulator gene in pancreatitis. Dig Dis Sci 2015;60:1297-1307. 32. Masson E, Chen JM, Audrézet MP, et al. 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 2013;8:e73522. 33. Rosendahl J, Landt O, Bernadova J, et al. CFTR, SPINK1, CTRC and PRSS1 variants in chronic pancreatitis: is the role of mutated CFTR overestimated? Gut 2013;62:582-592. 34. Sudo Y, Matsuo K, Tetsuo T, et al. Derived (mutated)-types of TRPV6 channels elicit greater Ca²+ influx into the cells than ancestral-types of TRPV6: evidence from Xenopus oocytes and mammalian cell expression system. J Pharmacol Sci 2010;114:281-291. 35. Weissgerber P, Kriebs U, Tsvilovskyy V, et al. Male fertility depends on Ca²+ absorption by TRPV6 in epididymal epithelia. Sci Signal 2011;4:ra27. 36. Jensen JN, Cameron E, Garay MV, et al. Recapitulation of elements of embryonic development

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47. Park EJ, Kim BJ, Kim SH, et al. Altered biochemical properties of transient receptor potential vanilloid 6 calcium channel by peptide tags. Biol Pharm Bull. 2009;32:1790-1794. 48. Voets T, Janssens A, Prenen J, et al. Mg2+-dependent gating and strong inward rectification of the cation channel TRPV6. J Gen Physiol 2003;121:245-260. 49. Zou WB, Tang XY, Zhou DZ, et al. SPINK1, PRSS1, CTRC, and CFTR genotypes influence disease onset and clinical outcomes in chronic pancreatitis. Clin Transl Gastroenterol 2018;9:204. 50. Chiari H. Über die Selbstverdauung des menschlichen Pankreas. Z Heilk. 1896;17:69-96. 51. Whitcomb DC, LaRusch J, Krasinskas AM, et al. Common genetic variants in the CLDN2 and PRSS1-PRSS2 loci alter risk for alcohol-related and sporadic pancreatitis. Nat Genet 2012;44:1349-1354. 52. Aune D, Mahamat-Saleh Y, Norat T, et al. Tobacco smoking and the risk of pancreatitis: A systematic review and meta-analysis of prospective studies. Pancreatology 2019;19:1009-1022. 53. Weissgerber P, Kriebs U, Tsvilovskyy V, et al. Excision of Trpv6 gene leads to severe defects in epididymal Ca2+ absorption and male fertility much like single D541A pore mutation. J Biol Chem 2012:287:17930-17941. 54. Zhuang L, Peng JB, Tou L, et al. Calcium-selective ion channel, CaT1, is apically localized in gastrointestinal tract epithelia and is aberrantly expressed in human malignancies. Lab Invest 2002;82:1755-1764. 55. Peng JB, Brown EM, Hediger MA. Epithelial Ca2+ entry channels: transcellular Ca2+ transport and beyond. J Physiol 2003;551:729-740.

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Author names in bold designate shared co-first authorship.

38

Legends for Figures Figure 1. The increases in the [Ca2+]i evoked by the application of Ca2+ were reduced in cells expressing several TRPV6 variants. HEK293 cells were co-transfected with recombinant TRPV6 expression plasmids and pEGFP-N1 or pEGFP-C1 as a transfection marker. After transfection, cells were plated on coverslips and loaded with 1 µM Fura-2-AM. Fura-2-AM fluorescence at an emission wavelength of 510 nm was obtained by exciting Fura-2-AM sequentially at 340 and 380 nm. Representative Fura-2-AM fluorescence curves recorded from HEK293 cells expressing pcDNA3.1(−) (vector), wild-type TRPV6 and several TRPV6 variants in response to the application of 2 mM Ca2+-containing solutions at 180 seconds (arrows).

Figure 2. Summary of TRPV6 activity in TRPV6 variants found in Japanese subjects. TRPV6 activity in HEK293 cells expressing TRPV6 variants found in Japanese subjects was assessed by Ca2+ imaging assays. Data represent mean + SEM (number of assays=25-126) from the sum of two or three independent transfections. The ∆F340/F380 value in the cells expressing wild-type (WT) TRPV6 was regarded as 100% TRPV6 activity. #The ancestral haplotype ((p.C197R + p.M418V + p.M721T) expression vector was used. **: P<0.01 vs. WT TRPV6.

Figure 3. Summary of TRPV6 activity in TRPV6 variants found in French and German subjects. TRPV6 activity in HEK293 cells expressing TRPV6 variants found in French

39

(A) and German (B) subjects was assessed by Ca2+ imaging assays. Data represent mean + SEM (number of assays=25-158) from the sum of two or three independent transfections. The ∆F340/F380 value in the cells expressing wild-type (WT) TRPV6 was regarded as 100% TRPV6 activity. #The ancestral haplotype ((p.C197R + p.M418V + p.M721T) expression vector was used. **: P<0.01 vs. WT TRPV6. Some variants were also found in Japanese cohorts.

Figure 4. TRPV6 protein expression was decreased in HEK293 cells expressing several TRPV6 variants compared to the WT. Total cell lysates were prepared from HEK293 cells expressing pcDNA3.1(−) (mock-transfected), WT, or TRPV6 variants at 48 h after transfection. The levels of TRPV6 and α-tubulin were determined by Western blotting. The upper panels show the results of functionally-defective variants found in Japanese subjects. Lower panels show the results of functionally-defective variants found in French (left) and German (right) subjects. Of note, the expression of the upper bands around 90 kDa in size, representing glycosylated forms, was decreased in some TRPV6 variants.

Figure 5. Cerulein-induced CP was exacerbated in TRPV6mut/mut mice. (A) TRPV6mut/mut and control mice (129B6F1) were injected intraperitoneally each hour for 8h per day during two consecutive days (d1, d2) with 0.1 mg cerulein/ g mouse body weight. Blood was taken at 0 h, 8 h, 24 h, 32 h, and at sacrifice 96 h (d5) after the first injection. (B) Quantification of serum amylase and lipase (U/l) at 0 h, 8 h, 24 h, 32 h, and 96 h after the first cerulein injection; mean+SEM (n=5), ***P<0.001, **P<0.01, *P<0.05. (C) H&E (morphology) and Sirius Red (fibrosis) staining of

40

TRPV6mut/mut and control tissue sections at sacrifice compared to control mice; scale bar = 100 mm. Lower panels represent enlarged images.

41

Table 1. Non-synonymous TRPV6 variants in Japanese subjects with non-alcoholic CP and controls Exon

Nucleotide change

Amino acid change

rs number

Non-alcoholic CP (%) (n=300)

Controls (%) (n=1,070)

P value

1

c.T52G>T

p.A18S

rs17881456

21 (7.0)

63 (5.9)

0.50

1

c.218G>A

p.R73Q

rs151059940

1 (0.3)

3 (0.3)

1.00

3

c.403G>A

p.A135T

rs111318322

0

1 (0.1)

1.00

4

c.520C>T

p.R174X (hom)

rs1309922604

1 (0.3)

0

0.22

4

c.521G>A

p.R174Q

rs150734746

1 (0.3)

0

0.22

4

c.589T>C

p.C197R

rs4987657

3 (1.0)

14 (1.3)

1.00

5

c.613C>T

p.H205Y

rs142701244

1 (0.3)

0

0.22

5

c.629C>T

p.A210V

2 (0.7)

0

0.048

5

c.668T>C

p.I223T (het)

rs529924080

18 (6.0)

7 (0.7)

7.4 × 10-9

5

c.668T>C

p.I223T (hom)

-

2 (0.7)

0

-

6

c.786C>G

p.Y262X

1 (0.3)

0

0.22

7

c.955G>A

p.D319N

rs779469652

1 (0.3)

0

0.22

7

c.970G>A

p.D324N

rs757050801

3 (1.0)

0

0.01

&

8

c.1033C>T

p.R345C

rs775866936

1 (0.3)

0

0.22

8

c.1088G>A

p.R363Q

rs534600735

0

1 (0.1)

1.00

8

c.1174C>T

p.L392F

0

1 (0.1)

1.00

9

c.1252A>G

p.M418V

rs4987667

3 (1.0)

14 (1.3)

1.00

9

c.1274G>A

p.R425Q

rs1281361203

1 (0.3)

0

0.22

9

c.1282G>A

p.G428R

rs1327315227

1 (0.3)

0

0.22

9

c.1301T>C

p.I434T

rs148077292

3 (1.0)

3 (0.3)

0.12

Odds ratio

95%CI

10.9

4.5–25.9

10 c.1352G>C p.G451A 1 (0.3) 0 Table 2. Non-synonymous TRPV6 variants in European subjects with non-alcoholic CP and controls 11 c.1448G>A p.R483Q 1 (0.3) 0

0.22 0.22

12

c.1610T>C

p.M537T

1 (0.3)

0

0.22

12

c.1618G>T

p.V540F

1 (0.3)

0

0.22

13

c.1646A>G

p.Y549C

rs750624044

0

1 (0.1)

1.00

13

c.1672G>A

p.E558K

rs776041915

3 (1.0)

7 (0.7)

0.46

13

c.1738A>T

p.I580F

1 (0.3)

0

0.22

13

c.1816G>A

p.A606T

1 (0.3)

0

0.22

13

c.1823T>G

p.L608R

1 (0.3)

0

0.22

13

c.1825C>T

p.L609F

1 (0.3)

0

0.22

14

c.1937G>C

p.R646P (hom)

1 (0.3)

0

0.22

14

c.1981C>T

p.R661W

0

1 (0.1)

1.00

14

c.2014C>T

p.R672W

0

1 (0.1)

1.00

14

c.2015G>A

p.R672Q

rs200085165

1 (0.3)

0

0.22

15

c.2083G>A

p.G695S

rs200385286

0

1 (0.1)

1.00

15

c.2162T>C

p.M721T

rs4987682

3 (1.0)

14 (1.3)

1.00

13 (4.3)

1 (0.1)

2.4 × 10-8

rs771971978

rs201887033

Number of subjects carrying functionally-defective TRPV6 variants (%)

48.4

6.3–371.7

CI, confidence interval; het, heterozygous; hom, homozygous. P values were determined by Fisher’s exact test. &

This patient was trans-heterozygous for SPINK1 p.N34S.

Alterations in bold indicate functionally-defective TRPV6 variants. We defined functionally-defective variants if an increase of [Ca2+]i was significantly diminished compared to the controls. Odds ratios are indicated if the distribution was different between the cases and controls.

Exon

Nucleotide change

Amino acid change

rs number

Non-alcoholic CP (%) (n=880)

Controls (%) (n=1,320)

P value

1

c.29C>T

p.P10L

rs541889808

0

2 (0.2)

0.52

1

c.52G>T

p.A18S

rs17881456

35 (4.0)

109 (8.3)

6.6 × 10-5

1

c.67C>A

p.P23T

rs761656516

1 (0.1)

0

0.40

1

c.125G>A

p.G42D

0

1 (0.1)

1.00

1

c.215G>A

p.S72N

0

2 (0.2)

0.52

1

c.217C>G

p.R73G

1 (0.1)

0

0.40

1

c.218G>A

p.R73Q

0

1 (0.1)

1.00

1

c. 245delA

p.K82RfsX10

1 (0.1)

0

0.40

2

c.303delG

p.N102TfsX24

1 (0.1)

0

0.40

rs141313361

rs151059940

&

4

c.520C>T

p.R174X

rs1309922604

1 (0.1)

0

0.40

4

c.535C>T

p.R179C

rs141260669

1 (0.1)

0

0.40

4

c.577C>G

p.R193G

rs148239732

1 (0.1)

0

0.40

4

c. 581G>A

p.R194H

rs189956366

0

1 (0.1)

1.00

4

c.589T>C

p.C197R (het)

rs4987657

142 (16.1)

190 (14.4)

0.33

4

c.589T>C

p.C197R (hom)

-

3 (0.3)

2 (0.2)

5

c.614A>G

p.H205R

rs200186434

2 (0.2)

0

0.16

5

c.668T>C

p.I223T

rs529924080

1 (0.1)

0

0.40

5

c.659G>A

p.R220Q

rs768442389

0

1 (0.1)

1.00

6

c.787G>A

p.D263N

rs150421102

1 (0.1)

0

0.40

7

c.896T>A

p.L299Q

rs151308770

24 (2.7)

13 (1.0)

0.003

7

c.926C>T

p.T309M

rs201899094

3 (0.3)

9 (0.7)

0.38

Odds ratio

95%CI

0.5

0.3-0.7

3.0

1.9-4.8

7

c.926C>A

p.T309K

rs201899094

1 (0.1) &

0

0.40

7

c.932G>T

p.G311V

1 (0.1)

0

0.40

7

c. 967A>G

p.I323V

1 (0.1)

0

0.40

&

7

c.1025G>A

p.R342Q

rs192217657

1 (0.1)

0

0.40

8

c.1033C>T

p.R345C

rs775866936

1 (0.1)

0

0.40

8

c. 1034G>A

p.R345H

rs745408146

1 (0.1)

0

0.40

8

c.1088G>A

p.R363Q

rs534600735

0

1 (0.1)

1.00

8

c. 1094G>A

p.G365E

1 (0.1)

0

0.40

8

c.1141A>G

p.I381V

2 (0.2)

0

0.16

8

c.1210G>A

p.D404N

1 (0.1)

0

0.40

9

c.1252A>G

p.M418V (het)

rs4987667

142 (16.1)

191 (14.5)

0.35

9

c.1252A>G

p.M418V (hom)

-

3 (0.3)

2 (0.2)

9

c.1295C>G

p.T432S

1 (0.1)

0

0.40

4 (0.5)

0

0.0025

rs139508663

rs755916513

&

11

c.1447C>T

p.R483W

11

c.1448G>A

p.R483Q

1 (0.1)

0

0.40

11

c.1465G>A

p.G489R (hom)

1 (0.1)

0

0.40

11

c. 1474G>C

p.V492L

1 (0.1)

0

0.40

11

c.1480A>G

p.M494V

rs200554107

0

1 (0.1)

1.00

11

c.1513G>A

p.V505I

rs369658985

2 (0.2)

0

0.16

13

c.1723G>A

p.E575K

rs1489493030

1 (0.1)

0

0.40

p.H622GfsX20

1 (0.1)

0

0.40

p.A626P

1 (0.1)

0

0.40

13 13

c.1864_1867d elCACT c. 1876G>C

1 (0.1)&

0

0.40

rs201887033

1 (0.1)

1 (0.1)

1.00

p.M721T (het)

rs4987682

142 (16.1)

190 (12.8)

0.33

c.2162T>C

p.M721T (hom)

-

3 (0.3)

2 (0.2)

c.2188C>T

p.R730C

rs1191758873

0

1 (0.1)

1.00

18 (2.0)

0 (0)

P = 6.19 × 10-8

14

c. 1977C>A

p.C659X

14

c.1981C>T

p.R661W

15

c.2162T>C

15 15

Number of subjects carrying functionally-defective TRPV6 variants (%) CI, confidence interval; CP, chronic pancreatitis; het, heterozygous; hom, homozygous. P values were determined by Fisher’s exact test. &

One patient with p.R174X, one with p.G311V, one with p.R342Q, and two with p.R483W were trans-heterozygous for SPINK1 p.N34S.

Alterations in bold indicate functionally-defective TRPV6 variants. We defined functionally-defective variants if an increase of [Ca2+]i was significantly diminished compared to the controls. Odds ratios are indicated if the distribution was different between the cases and controls.

Legends for Supplementary Figures Supplementary Figure 1. Distribution of the non-synonymous TRPV6 variants found in patients with chronic pancreatitis (CP) in Japan. The distributions of non-synonymous TRPV6 variants found in Japanese patients with CP in the cDNA sequences encoding ankyrin repeats (beige boxes), transmembrane domain regions (blue boxes), pore (red box), and the calmodulin binding site (green box) are presented. Note, just 25 variants are depicted, as the three ancestral haplotype variants (p.C197R, p.M418V, and p.M721T) were excluded. Alterations in red indicate functionally-defective variants. We defined functionally-defective variants as those in which an increase of [Ca2+]i was significantly diminished compared to the WT.

Supplementary Figure 2. Distribution of the non-synonymous TRPV6 variants found in patients with chronic pancreatitis (CP) in France and Germany. The distributions of non-synonymous TRPV6 variants found in CP patients in France (a) and Germany (b) in the cDNA sequences encoding ankyrin repeats (beige boxes), transmembrane domain regions (blue boxes), pore (red box), and the calmodulin binding site (green box) are presented. Note, just 21 (a) or 16 (b) variants are depicted, respectively, as the three derived-haplotype variants (p.C197R, p.M418V, and p.M721T) were excluded. Alterations in red indicate functionally-defective variants. We defined functionally-defective variants as those in which an increase of [Ca2+]i was significantly diminished compared to the WT.

Supplementary Figure 3. Location of the mutated residues in the human TRPV6 tetramer. (A) View of human TRPV6 tetramer and the location of the mutated residues which were overrepresented in patients with non-alcoholic CP. (B) Close-up views of 3D structures surrounding the mutated residues. The mutated residues are shown in magenta and the residues interacting with them are shown in sticks. Numbers in black indicate interatomic distances in angstroms. The helix in the neighboring subunit is shown in cyan in the right lower panel. ANK indicates each unit of the ankyrin repeats. The side chain of A210 is buried in the hydrophobic cluster between ankyrin repeats 3 and 4. Since its Cβ atom is in close contact (< 4Å) with the side chains of I168, I218, and L222, the mutation of this residue to a larger valine may cause steric crushes in the hydrophobic core and destabilize the ankyrin repeat domain. I223 is buried

in a hydrophobic pocket formed by the side chains of L207, l229, L259, and Y262 between the ankyrin repeats 4 and 5, and is also in close contact with the main chain carbonyl of A227 at the connecting loop. The substitution of this hydrophobic residue to polar threonine may affect both the hydrophobic core and loop structures. L299 is buried in a hydrophobic pocket formed by the side chains of I244, Y256, F284, and M295 between ankyrin repeats 5 and 6, and the introduction of a polar side chain to this buried moiety by the p.L299Q mutation may destabilize the ankyrin repeat domain. D324, which is in the linker connecting the ankyrin repeat domain to pre-S1 helix, forms salt bridges with K360 and R655, and the loss of the negative charge by p.D324N mutation could interrupt this salt bridge network. Supplementary Figure 4. Electrophysiological analysis of the Mg2+-dependent blockade. (A) The voltage step protocol used in this study. After the first hyperpolarization step from +20 mV to -100 mV, subsequent voltage steps (-100 mV to +110 mV in 15 mV increments, 50 ms duration) were applied followed by the final -100 mV step. (B, C) The representative current traces for HEK293 cells expressing wild-type (WT) or functionally non-defective TRPV6 variants in the divalent-free external solution in response to the voltage step protocol. Monovalent cations such as Na+ (in the extracellular and pipette solution) and Cs+ (in pipette solution) were used as the charge carrier. The pipette solution contained 3 mM (B) or no (C) Mg2+. (D, E) The fraction of open channels at the end of each voltage step was determined by normalizing the initial current amplitude during the final -100 mV step to the steady state current during the first hyperpolarization step. Voltage dependence of the channel open probability was assessed with intracellular perfusion of solutions containing 3 mM (D) or no (E) Mg2+. Data are shown as mean+SEM (n=5, each). In panel E, ***P<0.001, **P<0.01, *P<0.05 vs. WT.

Supplementary Table 1. Primers used for direct sequencing of the TRPV6 gene in Japanese subjects Exon

Forward primer

Reverse primer

Size of PCR product (bp)

1

AACTCACAGCCCTCTCCAAA

CTCAACCCCATCCTCTTTCA

522

2&3

CTGCTGAGGCACACACATCT

ACACGGAAGGGAGACAGAGA

628

4

TGTTCTCCATCCCAGGCTAC

CCCAACAGATACGGCTGAAG

351

5&6

GTGGGGGAAGATAAGGCTGT

GCGTTCACCTCTGTTTCTCC

607

7

TGCCCTCTAAAGGCTGTCAT

CAGATATATGGCACCCAGCA

465

8

GGACGTATGGACCACTGACC

CCCTGCTGATCTGCTCCTAC

428

9&10

CGCCCACAAAAACACACATA

CAGGAATCCAAGGAGTGGAA

498

11

TATGACCCTGGGCATTGAGT

CTGCTGACTGTGGCTCTG

351

12

CTGGTCCACAGGCCATAGAT

GGACGTCCCATCCTCTGATA

348

13

GCACTGCAGAGCTGGTGATA

TGCCACAACTGTCCAAACAT

504

14

TCATGGATGTTGGGTCATTG

CCTCACCAGCTCAATCAACA

400

15

GGACCAGGGGATAACAGACA

ACGTACATTCCTTGGCGTTC

510

bp: base pairs The PCR conditions were as follows: preheating at 95 °C for 5 min, followed by 40 cycles of 95 °C for 30 sec, 60 °C for 30 sec, and 72 °C for 30 sec; and then a final extension at 72 °C for 5 min. DNA sequencing was performed using both forward and reverse primers.

Supplementary Table 2. Primers and probes used for melting curve analysis in German subjects Target

Forward primer

Reverse primer

Probe

p.S18A

TGGCTCCTCCTGCTCACTCC

CAGACCCTGACGGGACTCA

GCTGAXITGTGTCCCCAAGGCTGAGT

p.L299Q

CCTATCCACACTCTGTGACCG

CCACTGGGTGTGCTTCCG

TTCAGCACCAGAXITGCAGAAGC-phosphate

The PCR conditions were as follows: preheating at 95 °C for 1 min, followed by 45 cycles of 95 °C for 15 sec, 62 °C (for p.S18A) or 56 °C (for p:L299Q) for 15 sec, and 72 °C for 15 sec. Melting curve analysis conditions for both variants were as follows: initial denaturation for 30 sec at 95 °C (ramp: 4.6 °C/s), cooling down to 40 °C for 2 min (ramp: 2 °C/sec), melting curve analysis while increasing temperature up to 80 °C (ramp: 0.29 °C/sec) and cooling down to 40 °C for 30 sec.

Supplementary Table 3. Non-synonymous TRPV6 variants in Japanese subjects with alcohol-related CP

Alcoholic CP (%) Controls (%) Nucleotide Exon Supplementary TableAmino acid change TRPV6 rs number P value 4. Non-synonymous variants in French subjects with non-alcoholic CP and controls change (n=300) (n=1070) 1 4 5 7 8 9 9 13 15

Exon 1 1 1 1 2 4

c.52G>T Nucleotide change c.589T>C

c.668T>C c.29C>T c.970G>A c.52G>T c.125G>A c.1174C>T c.1252A>G c. 245delA c.1301T>C c.303delG c.1672G>A c.520C>T c.2162T>C

p.A18S rs17881456 15 (5.0) Non-alcoholic 63 (5.9) CP Amino acid change rs number (%) (n=470) p.C197R rs4987657 4 (1.3) 14 (1.3) p.I223T p.P10L rs529924080 rs541889808 7 (2.3) 0 7 (0.7) p.D324N p.A18S rs757050801 rs17881456 1 (0.3) 16 (3.4) 0 p.G42D 0 (0)1 (0.1) p.L392F 1 (0.3) p.M418V 4 (1.3) 14 (1.3) p.K82RfsX10 rs4987667 1 (0.2) p.I434T 1 (0.3) 3 (0.3) p.N102TfsX24 rs148077292 1 (0.2) & rs13099226041 (0.3) p.E558K rs776041915 p.R174X 1 (0.2)7 (0.7) p.M721T rs4987682 4 (1.3) 14 (1.3)

Number of subjects carrying functionally-defective TRPV6 variants (%)

1 (0.3)

1 (0.1)

0.67 (%) Controls (n=570) 1.00 0.019 2 (0.4) 48 0.22 (8.4) 10.39 (0.2) 1.00 0 1.00 0 1.00 0 1.00

Odds ratio

95%CI

P value 3.6

0.50 0.001 1.00 0.45 0.45 0.45

1.3-10.0

0.39

CP, chronic pancreatitis; CI, confidence interval. P values were determined by Fisher’s exact test. Alterations in bold indicate functionally-defective TRPV6 variants. We defined functionally-defective variants as those in which an increase of [Ca2+]i was significantly diminished compared to the WT.

4 4 4 5 5 6 7 7 7 7 8 8 8 8 9 9 11 11 11 11 13 14 15 15

c.535C>T c.577C>G c.589T>C c.614A>G c.659G>A c.787G>A c.896T>A c.926C>T c.926C>A c.932G>T c.1033C>T c.1088G>A c.1141A>G c.1210G>A c.1252A>G c.1295C>G c.1447C>T c.1465G>A c.1480A>G c.1513G>A c.1723G>A c.1981C>T c.2162T>C c.2188C>T

p.R179C p.R193G p.C197R p.H205R p.R220Q p.D263N p.L299Q p.T309M p.T309K p.G311V p.R345C p.R363Q p.I381V p.D404N p.M418V p.T432S p.R483W p.G489R (hom) p.M494V p.V505I p.E575K p.R661W p.M721T p.R730C

rs141260669 rs148239732 rs4987657 rs200186434 rs768442389 rs150421102 rs151308770 rs201899094 rs201899094 rs775866936 rs534600735 rs139508663 rs4987667 rs755916513 rs374296899 rs200554107 rs369658985 rs1489493030 rs201887033 rs4987682 rs1191758873

Number of subjects carrying functionally-defective TRPV6 variants (%)

1 (0.2) 1 (0.2) 86 (18.3) 2 (0.4) 0 1 (0.2) 13 (2.8) 2 (0.4) 1 (0.2) & 1 (0.2) 1 (0.2) 0 2 (0.4) 1 (0.2) 86 (18.3) 1 (0.2) 2 (0.4) 1 (0.2) 0 2 (0.4) 1 (0.2) 1 (0.2) 86 (18.3) 0

0 0 94 (16.5) 0 1 (0.2) 0 7 (1.2) 4 (0.7) 0 0 0 1 (0.2) 0 0 95 (16.7) 0 0 0 1 (0.2) 0 0 1 (0.2) 94 (16.5) 1 (0.2)

0.45 0.45 0.46 0.20 1.00 0.45 0.11 0.70 0.45 0.45 0.45 1.00 0.20 0.45 0.51 0.45 0.20 0.45 1.00 0.20 0.45 1.00 0.46 1.00

9 (1.9)

0 (0)

0.00075

hom, homozygous. P values were determined by Fisher’s exact test. &

These 2 patients were trans-heterozygous for SPINK1 Supplementary Table 5. Non-synonymous TRPV6 variantsp.N34S. in German subjects with non-alcoholic CP and controls Alterations in bold indicate functionally-defective TRPV6 variants. We defined functionally-defective variants as those in which an increase of [Ca2+]i was significantly diminished compared to the WT.

Exon

Nucleotide change

Amino acid change

1 1 1 1 1 4 4 4 5 7 7 7 7 8 8 9 9 11 11 11 13 13 14 15

c.52G>T c.67C>A c.215G>A c.217C>G c.218G>A c. 581G>A c.589T>C c.589T>C c.668T>C c.896T>A c.926C>T c. 967A>G c.1025G>A c. 1034G>A c. 1094G>A c.1252A>G c.1252A>G c.1447C>T c.1448G>A c. 1474G>C c.1864_1867delCACT c. 1876G>C c. 1977C>A c.2162T>C

p.A18S p.P23T p.S72N p.R73G p.R73Q p.R194H p.C197R (het) p.C197R (hom) p.I223T p.L299Q p.T309M p.I323V p.R342Q p.R345H p.G365E p.M418V (het) p.M418V (hom) p.R483W p.R483Q p.V492L p.H622GfsX20 p.A626P p.C659X p.M721T (het)

rs number rs17881456 rs761656516 rs141313361 rs151059940 rs189956366 rs4987657 rs529924080 rs151308770 rs201899094 rs192217657 rs745408146 rs4987667 rs755916513

rs4987682

Non-alcoholic CP (%) (n=410)

Controls (%) (n=750)

19 (4.6) 1 (0.2) 0 1 (0.2) 0 0 56 (13.7) 3 (0.7) 1 (0.2) 11 (2.7) 1 (0.2) 1 (0.2) 1 (0.2)& 1 (0.2) 1 (0.2) 56 (13.7) 3 (0.7) 2 (0.5)& 1 (0.2) 1 (0.2) 1 (0.2) 1 (0.2) & 1 (0.2) 56 (13.7)

61 (8.1) 0 2 (0.3) 0 1 (0.1) 1 (0.1) 96 (12.8) 2 (0.3) 0 6 (0.8) 5 (0.7) 0 0 0 0 96 (12.8) 2 (0.3) 0 0 0 0 0 0 96 (12.8)

P value 0.03 0.35 0.54 0.35 1.00 1.0 0.45 0.35 0.018 0.67 0.35 0.35 0.35 0.35 0.45 0.13 0.35 0.35 0.35 0.35 0.35 0.45

15 c.2162T>C p.M721T (hom) Number of subjects carrying functionally-defective TRPV6 variants

-

3 (0.7)

2 (0.3)

9 (2.2)

0 (0)

0.0001

CP, chronic pancreatitis; het, heterozygous; hom, homozygous. P values were determined by Fisher’s exact test. & These 3 patients were trans-heterozygous for SPINK1 p.N34S. Alterations in bold indicate functionally-defective TRPV6 variants. We defined functionally-defective variants as those in which an increase of [Ca2+]i was significantly diminished compared to the WT.

Supplementary Table 6. Mutations in pancreatitis susceptibility genes found in Japanese patients with non-alcoholic CP. Number of patients 9 1 2 19 1 1 6 5 9 5 2 1 1 1 Hm: homozygous.

PRSS1

SPINK1

p.R122H p.R122H p.N29I

p.N34S

CTRC

CPA1

p.N34S p.N34S p.N34S p.N34S/c.194+2T>C p.N34S (hm) c.194+2T>C c.194+2T>C (hm) p.P45S

TRPV6

p.R345C p.H205Y

p.R29Q p.V251M p.T368_Y369ins20

Alterations in bold indicate a functionally-defective TRPV6 variant. We defined functionally-defective variants as those in which an increase of [Ca2+]i was significantly diminished compared to the WT. Other mutations were heterozygous.

What You Need to Know BACKGROUND AND CONTEXT: Changes in pancreatic calcium levels affect secretion and might be involved in development of chronic pancreatitis (CP).

NEW FINDINGS: Patients with early-onset CP not associated with alcohol consumption carry variants in TRPV6, which encodes a Ca2+-selective ion channel. Higher proportions of patients than controls carried variants that affect TRPV6 function, and mutation of this gene increased the severity of pancreatitis in mice.

LIMITATIONS: Further studies are needed to determine how these variants and loss of TRPV6 function contributes to development of pancreatitis.

IMPACT: Patients with CP not associated with alcohol consumption should be screened for TRPV6 variants. Strategies to restore TRPV6 function might be developed for treatment of CP.

Lay Summary: We analyzed genetic features of patients with chronic pancreatitis not associated with alcohol consumption, and found that they more frequently carry variants in a gene that encodes a calcium channel, compared with controls. Studies are needed to determine how these alterations contribute to pancreatic inflammation.