Document Detail

Common genetic variants in the CLDN2 and PRSS1-PRSS2 loci alter risk for alcohol-related and sporadic pancreatitis.
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MedLine Citation:
PMID:  23143602     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Pancreatitis is a complex, progressively destructive inflammatory disorder. Alcohol was long thought to be the primary causative agent, but genetic contributions have been of interest since the discovery that rare PRSS1, CFTR and SPINK1 variants were associated with pancreatitis risk. We now report two associations at genome-wide significance identified and replicated at PRSS1-PRSS2 (P < 1 × 10(-12)) and X-linked CLDN2 (P < 1 × 10(-21)) through a two-stage genome-wide study (stage 1: 676 cases and 4,507 controls; stage 2: 910 cases and 4,170 controls). The PRSS1 variant likely affects disease susceptibility by altering expression of the primary trypsinogen gene. The CLDN2 risk allele is associated with atypical localization of claudin-2 in pancreatic acinar cells. The homozygous (or hemizygous in males) CLDN2 genotype confers the greatest risk, and its alleles interact with alcohol consumption to amplify risk. These results could partially explain the high frequency of alcohol-related pancreatitis in men (male hemizygote frequency is 0.26, whereas female homozygote frequency is 0.07).
Authors:
David C Whitcomb; Jessica LaRusch; Alyssa M Krasinskas; Lambertus Klei; Jill P Smith; Randall E Brand; John P Neoptolemos; Markus M Lerch; Matt Tector; Bimaljit S Sandhu; Nalini M Guda; Lidiya Orlichenko; ; Samer Alkaade; Stephen T Amann; Michelle A Anderson; John Baillie; Peter A Banks; Darwin Conwell; Gregory A Coté; Peter B Cotton; James DiSario; Lindsay A Farrer; Chris E Forsmark; Marianne Johnstone; Timothy B Gardner; Andres Gelrud; William Greenhalf; Jonathan L Haines; Douglas J Hartman; Robert A Hawes; Christopher Lawrence; Michele Lewis; Julia Mayerle; Richard Mayeux; Nadine M Melhem; Mary E Money; Thiruvengadam Muniraj; Georgios I Papachristou; Margaret A Pericak-Vance; Joseph Romagnuolo; Gerard D Schellenberg; Stuart Sherman; Peter Simon; Vijay P Singh; Adam Slivka; Donna Stolz; Robert Sutton; Frank Ulrich Weiss; C Mel Wilcox; Narcis Octavian Zarnescu; Stephen R Wisniewski; Michael R O'Connell; Michelle L Kienholz; Kathryn Roeder; M Michael Barmada; Dhiraj Yadav; Bernie Devlin
Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't     Date:  2012-11-11
Journal Detail:
Title:  Nature genetics     Volume:  44     ISSN:  1546-1718     ISO Abbreviation:  Nat. Genet.     Publication Date:  2012 Dec 
Date Detail:
Created Date:  2012-11-29     Completed Date:  2013-02-13     Revised Date:  2014-09-24    
Medline Journal Info:
Nlm Unique ID:  9216904     Medline TA:  Nat Genet     Country:  United States    
Other Details:
Languages:  eng     Pagination:  1349-54     Citation Subset:  IM    
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MeSH Terms
Descriptor/Qualifier:
Claudins / genetics*
Female
Gene Frequency
Genetic Predisposition to Disease
Genetic Variation*
Genome-Wide Association Study
Homozygote
Humans
Male
Mutation
Pancreatitis, Alcoholic / genetics*,  pathology
Sex Factors
Trypsin / genetics*
Trypsinogen / genetics*
Grant Support
ID/Acronym/Agency:
AG010491/AG/NIA NIH HHS; AG019757/AG/NIA NIH HHS; AG021547/AG/NIA NIH HHS; AG025688/AG/NIA NIH HHS; AG027944/AG/NIA NIH HHS; AG030653/AG/NIA NIH HHS; AG05128/AG/NIA NIH HHS; AG05144/AG/NIA NIH HHS; HHSN268200782096C//PHS HHS; K23 AG034550/AG/NIA NIH HHS; M01RR00096/RR/NCRR NIH HHS; MH60451/MH/NIMH NIH HHS; NS39764/NS/NINDS NIH HHS; P01AG002219/AG/NIA NIH HHS; P01AG019724/AG/NIA NIH HHS; P01AG03991/AG/NIA NIH HHS; P30 AG008017/AG/NIA NIH HHS; P30 AG008051/AG/NIA NIH HHS; P30 AG010124/AG/NIA NIH HHS; P30 AG010129/AG/NIA NIH HHS; P30AG008017/AG/NIA NIH HHS; P30AG010124/AG/NIA NIH HHS; P30AG010129/AG/NIA NIH HHS; P30AG010161/AG/NIA NIH HHS; P30AG012300/AG/NIA NIH HHS; P30AG013846/AG/NIA NIH HHS; P30AG013854/AG/NIA NIH HHS; P30AG019610/AG/NIA NIH HHS; P30AG028377/AG/NIA NIH HHS; P30AG028383/AG/NIA NIH HHS; P30AG08051/AG/NIA NIH HHS; P30AG10133/AG/NIA NIH HHS; P30CA047904/CA/NCI NIH HHS; P50 AG016573/AG/NIA NIH HHS; P50 AG016574/AG/NIA NIH HHS; P50AG005131/AG/NIA NIH HHS; P50AG005133/AG/NIA NIH HHS; P50AG005134/AG/NIA NIH HHS; P50AG005136/AG/NIA NIH HHS; P50AG005138/AG/NIA NIH HHS; P50AG005142/AG/NIA NIH HHS; P50AG005146/AG/NIA NIH HHS; P50AG005681/AG/NIA NIH HHS; P50AG008671/AG/NIA NIH HHS; P50AG008702/AG/NIA NIH HHS; P50AG016570/AG/NIA NIH HHS; P50AG016573/AG/NIA NIH HHS; P50AG016574/AG/NIA NIH HHS; P50AG016575/AG/NIA NIH HHS; P50AG016576/AG/NIA NIH HHS; P50AG016577/AG/NIA NIH HHS; P50AG016582/AG/NIA NIH HHS; P50AG023501/AG/NIA NIH HHS; R01 AG015819/AG/NIA NIH HHS; R01 AG022018/AG/NIA NIH HHS; R01 AG032990/AG/NIA NIH HHS; R01 AG040770/AG/NIA NIH HHS; R01 MH057881/MH/NIMH NIH HHS; R01AG017173/AG/NIA NIH HHS; R01AG019085/AG/NIA NIH HHS; R01AG020688/AG/NIA NIH HHS; R01AG025259/AG/NIA NIH HHS; R01AG026916/AG/NIA NIH HHS; R01AG027944/AG/NIA NIH HHS; R01AG031581/AG/NIA NIH HHS; R01AG15819/AG/NIA NIH HHS; R01AG17917/AG/NIA NIH HHS; R01AG30146/AG/NIA NIH HHS; R01AG33193/AG/NIA NIH HHS; R01CA117926/CA/NCI NIH HHS; R01CA129769/CA/NCI NIH HHS; R01DK054709/DK/NIDDK NIH HHS; R01DK077906/DK/NIDDK NIH HHS; R01MH057881/MH/NIMH NIH HHS; R01MH080295/MH/NIMH NIH HHS; R01NS36960/NS/NINDS NIH HHS; R37AG015473/AG/NIA NIH HHS; R56DK061451/DK/NIDDK NIH HHS; RC2AG036528/AG/NIA NIH HHS; T32DK063922/DK/NIDDK NIH HHS; U01 AG016976/AG/NIA NIH HHS; U01 HG006375/HG/NHGRI NIH HHS; U01AG016976/AG/NIA NIH HHS; U01AG032984/AG/NIA NIH HHS; U01AG10483/AG/NIA NIH HHS; U24AG021886/AG/NIA NIH HHS; UL1 RR024153/RR/NCRR NIH HHS; UL1 RR029893/RR/NCRR NIH HHS; UL1 TR000005/TR/NCATS NIH HHS; UL1RR024153/RR/NCRR NIH HHS; UL1RR02777/RR/NCRR NIH HHS; UL1RR029893/RR/NCRR NIH HHS; UL1TR000005/TR/NCATS NIH HHS
Chemical
Reg. No./Substance:
0/CLDN2 protein, human; 0/Claudins; 103964-84-7/PRSS2 protein, human; 9002-08-8/Trypsinogen; EC 3.4.21.4/PRSS1 protein, human; EC 3.4.21.4/Trypsin
Investigator
Investigator/Affiliation:
Marilyn S Albert / ; Roger L Albin / ; Liana G Apostolova / ; Steven E Arnold / ; Clinton T Baldwin / ; Robert Barber / ; Lisa L Barnes / ; Thomas G Beach / ; Gary W Beecham / ; Duane Beekly / ; David A Bennett / ; Eileen H Bigio / ; Thomas D Bird / ; Deborah Blacker / ; Adam Boxer / ; James R Burke / ; Joseph D Buxbaum / ; Nigel J Cairns / ; Laura B Cantwell / ; Chuanhai Cao / ; Regina M Carney / ; Steven L Carroll / ; Helena C Chui / ; David G Clark / ; David H Cribbs / ; Elizabeth A Crocco / ; Carlos Cruchaga / ; Charles DeCarli / ; F Yesim Demirci / ; Malcolm Dick / ; Dennis W Dickson / ; Ranjan Duara / ; Nilufer Ertekin-Taner / ; Kelley M Faber / ; Kenneth B Fallon / ; Martin R Farlow / ; Steven Ferris / ; Tatiana M Foroud / ; Matthew P Frosch / ; Douglas R Galasko / ; Mary Ganguli / ; Marla Gearing / ; Daniel H Geschwind / ; Bernardino Ghetti / ; John R Gilbert / ; Sid Gilman / ; Jonathan D Glass / ; Alison M Goate / ; Neill R Graff-Radford / ; Robert C Green / ; John H Growdon / ; Hakon Hakonarson / ; Kara L Hamilton-Nelson / ; Ronald L Hamilton / ; Lindy E Harrell / ; Elizabeth Head / ; Lawrence S Honig / ; Christine M Hulette / ; Bradley T Hyman / ; Gregory A Jicha / ; Lee-Way Jin / ; Gyungah Jun / ; M Ilyas Kamboh / ; Anna Karydas / ; Jeffrey A Kaye / ; Ronald Kim / ; Edward H Koo / ; Neil W Kowall / ; Joel H Kramer / ; Patricia Kramer / ; Walter A Kukull / ; Frank M LaFerla / ; James J Lah / ; James B Leverenz / ; Allan I Levey / ; Ge Li / ; Chiao-Feng Lin / ; Andrew P Lieberman / ; Oscar L Lopez / ; Kathryn L Lunetta / ; Constantine G Lyketsos / ; Wendy J Mack / ; Daniel C Marson / ; Eden R Martin / ; Frank Martiniuk / ; Deborah C Mash / ; Eliezer Masliah / ; Ann C McKee / ; Marsel Mesulam / ; Bruce L Miller / ; Carol A Miller / ; Joshua W Miller / ; Thomas J Montine / ; John C Morris / ; Jill R Murrell / ; Adam C Naj / ; John M Olichney / ; Joseph E Parisi / ; Elaine Peskind / ; Ronald C Petersen / ; Aimee Pierce / ; Wayne W Poon / ; Huntington Potter / ; Joseph F Quinn / ; Ashok Raj / ; Murray Raskind / ; Eric M Reiman / ; Barry Reisberg / ; Christiane Reitz / ; John M Ringman / ; Erik D Roberson / ; Howard J Rosen / ; Roger N Rosenberg / ; Mary Sano / ; Andrew J Saykin / ; Julie A Schneider / ; Lon S Schneider / ; William W Seeley / ; Amanda G Smith / ; Joshua A Sonnen / ; Salvatore Spina / ; Robert A Stern / ; Rudolph E Tanzi / ; John Q Trojanowski / ; Juan C Troncoso / ; Debby W Tsuang / ; Otto Valladares / ; Vivianna M Van Deerlin / ; Linda J Van Eldik / ; Badri N Vardarajan / ; Harry V Vinters / ; Jean Paul Vonsatte / ; Li-San Wang / ; Sandra Weintraub / ; Kathleen A Welsh-Bohmer / ; Jennifer Williamson / ; Randall L Woltjer / ; Clinton B Wright / ; Steven G Younkin / ; Chang-En Yu / ; Lei Yu /
Comments/Corrections

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine

Full Text
Journal Information
Journal ID (nlm-journal-id): 9216904
Journal ID (pubmed-jr-id): 2419
Journal ID (nlm-ta): Nat Genet
Journal ID (iso-abbrev): Nat. Genet.
ISSN: 1061-4036
ISSN: 1546-1718
Article Information
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License:
nihms-submitted publication date: Day: 16 Month: 10 Year: 2012
Electronic publication date: Day: 11 Month: 11 Year: 2012
Print publication date: Month: 12 Year: 2012
pmc-release publication date: Day: 01 Month: 6 Year: 2013
Volume: 44 Issue: 12
First Page: 1349 Last Page: 1354
PubMed Id: 23143602
ID: 3510344
DOI: 10.1038/ng.2466
ID: NIHMS414572

Common genetic variants in the CLDN2 and PRSS1-PRSS2 loci alter risk for alcohol-related and sporadic pancreatitis
David C. Whitcomb123128
Jessica LaRusch1
Alyssa M. Krasinskas4
Lambertus Klei5
Jill P. Smith6
Randall E. Brand1
John P. Neoptolemos7
Markus M. Lerch8
Matt Tector9
Bimaljit S. Sandhu10
Nalini M. Guda9
Lidiya Orlichenko1
Alzheimer's Disease Genetics Consortium
Samer Alkaade12
Stephen T. Amann13
Michelle A. Anderson14
John Baillie15
Peter A. Banks16
Darwin Conwell16
Gregory A. Coté17
Peter B. Cotton18
James DiSario19
Lindsay A. Farrer1120
Chris E. Forsmark21
Marianne Johnstone7
Timothy B. Gardner22
Andres Gelrud1
William Greenhalf7
Jonathan L. Haines1123
Douglas J. Hartman4
Robert A. Hawes18
Christopher Lawrence18
Michele Lewis24
Julia Mayerle8
Richard Mayeux1125
Nadine M. Melhem5
Mary E. Money26
Thiruvengadam Muniraj27
Georgios I. Papachristou1
Margaret A. Pericak-Vance1128
Joseph Romagnuolo18
Gerard D. Schellenberg1129
Stuart Sherman17
Peter Simon8
Vijay K. Singh1
Adam Slivka1
Donna Stolz2
Robert Sutton7
Frank Ulrich Weiss8
C. Mel Wilcox30
Narcis Octavian Zarnescu1
Stephen R. Wisniewski31
Michael R. O'Connell1
Michelle L. Kienholz1
Kathryn Roeder32
M. Michael Barmada3
Dhiraj Yadav1
Bernie Devlin35128
Marilyn S. Albert33
Roger L. Albin3435
Liana G. Apostolova36
Steven E. Arnold37
Clinton T. Baldwin38
Robert Barber39
Lisa L. Barnes4041
Thomas G. Beach42
Gary W. Beecham28
Duane Beekly43
David A. Bennett4044
Eileen H. Bigio45
Thomas D. Bird46
Deborah Blacker4748
Adam Boxer49
James R. Burke15
Joseph D. Buxbaum505152
Nigel J. Cairns53
Laura B. Cantwell29
Chuanhai Cao54
Regina M. Carney55
Steven L. Carroll56
Helena C. Chui57
David G. Clark58
David H. Cribbs59
Elizabeth A. Crocco60
Carlos Cruchaga61
Charles DeCarli62
F. Yesim Demirci3
Malcolm Dick63
Dennis W. Dickson64
Ranjan Duara65
Nilufer Ertekin-Taner6466
Kelley M. Faber67
Kenneth B. Fallon56
Martin R. Farlow68
Steven Ferris69
Tatiana M. Foroud67
Matthew P. Frosch70
Douglas R. Galasko71
Mary Ganguli5
Marla Gearing7273
Daniel H. Geschwind74
Bernardino Ghetti75
John R. Gilbert28
Sid Gilman34
Jonathan D. Glass76
Alison M. Goate61
Neill R. Graff-Radford6466
Robert C. Green77
John H. Growdon78
Hakon Hakonarson79
Kara L. Hamilton-Nelson28
Ronald L. Hamilton80
Lindy E. Harrell58
Elizabeth Head81
Lawrence S. Honig82
Christine M. Hulette83
Bradley T. Hyman78
Gregory A. Jicha84
Lee-Way Jin85
Gyungah Jun388687
M. Ilyas Kamboh388
Anna Karydas49
Jeffrey A. Kaye8990
Ronald Kim91
Edward H. Koo71
Neil W. Kowall9293
Joel H. Kramer94
Patricia Kramer8995
Walter A. Kukull96
Frank M. LaFerla97
James J. Lah76
James B. Leverenz98
Allan I. Levey76
Ge Li99
Chiao-Feng Lin29
Andrew P. Lieberman100
Oscar L. Lopez89
Kathryn L. Lunetta87
Constantine G. Lyketsos101
Wendy J. Mack102
Daniel C. Marson58
Eden R. Martin28
Frank Martiniuk103
Deborah C. Mash104
Eliezer Masliah71105
Ann C. McKee9293
Marsel Mesulam106
Bruce L. Miller49
Carol A. Miller107
Joshua W. Miller86
Thomas J. Montine98
John C. Morris53108
Jill R. Murrell6775
Adam C. Naj28
John M. Olichney62
Joseph E. Parisi109110
Elaine Peskind99
Ronald C. Petersen85
Aimee Pierce59
Wayne W. Poon63
Huntington Potter54
Joseph F. Quinn89
Ashok Raj54
Murray Raskind99
Eric M. Reiman112113114
Barry Reisberg69115
Christiane Reitz2582
John M. Ringman36
Erik D. Roberson58
Howard J. Rosen49
Roger N. Rosenberg116
Mary Sano51
Andrew J. Saykin67117
Julie A. Schneider40118
Lon S. Schneider57119
William W. Seeley49
Amanda G. Smith54
Joshua A. Sonnen98
Salvatore Spina75
Robert A. Stern92
Rudolph E. Tanzi78
John Q. Trojanowski29
Juan C. Troncoso120
Debby W. Tsuang99
Otto Valladares29
Vivianna M. Van Deerlin29
Linda J. Van Eldik121
Badri N. Vardarajan38
Harry V. Vinters36122
Jean Paul Vonsattel123
Li-San Wang29
Sandra Weintraub106
Kathleen A. Welsh-Bohmer15124
Jennifer Williamson82
Randall L. Woltjer125
Clinton B. Wright126
Steven G. Younkin64
Chang-En Yu127
Lei Yu40
1Division of Gastroenterology, Hepatology and Nutrition, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.
2Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.
3Department of Human Genetics, University of Pittsburgh Graduate School of Public Health, Pittsburgh, Pennsylvania, USA.
4Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.
5Department of Psychiatry, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.
6Gastroenterology and Liver Diseases, Pennsylvania State University, Hershey, Pennsylvania, USA.
7Department of Molecular and Clinical Cancer Medicine, National Institute for Health Research Liverpool Pancreas Biomedical Research Unit, Institute of Translational Medicine, University of Liverpool, Liverpool, UK.
8Department of Medicine A, University Medicine Greifswald, Greifswald, Germany.
9Aurora Health Care System, St. Luke's Medical Center, Milwaukee, Wisconsin, USA.
10Division of Gastroenterology, Hepatology and Nutrition, Virginia Commonwealth University Medical Center, Richmond, Virginia, USA.
11A full list of members and affiliations appears at the end of this paper, Alzheimer Disease Genetics Consortium.
12Department of Medicine, Saint Louis University, St. Louis, Missouri, USA.
13North Mississippi Medical Center, Tupelo, Mississippi, USA.
14Division of Gastroenterology, University of Michigan, Ann Arbor, Michigan, USA.
15Department of Medicine, Duke University Medical Center, Durham, North Carolina, USA.
16Division of Gastroenterology, Brigham and Women's Hospital, Boston, Massachusetts, USA.
17Department of Medicine, Indiana University Medical Center, Indianapolis, Indiana, USA.
18Digestive Disease Center, Medical University of South Carolina, Charleston, South Carolina, USA.
19Department of Medicine, University of Utah Health Science Center, Salt Lake City, Utah, USA.
20Department of Medicine/Biomedical Genetics Section, Boston University School of Medicine, Boston, Massachusetts, USA.
21Department of Medicine, University of Florida, Gainesville, Florida, USA.
22Dartmouth-Hitchcock Medical Center, Hanover, New Hampshire, USA.
23Department of Molecular Physiology and Biophysics and Vanderbilt Center for Human Genetics Research, Vanderbilt University, Nashville, Tennessee, USA.
24Division of Gastroenterology and Hepatology, Mayo Clinic, Jacksonville, Florida, USA.
25Gertrude H. Sergievsky Center and Taub Institute on Alzheimer's Disease and the Aging Brain, Department of Neurology, Columbia University, New York, New York, USA.
26Washington Hospital, Hagerstown, Maryland, USA.
27Department of Internal Medicine, Yale University and Griffin Hospital, Derby, Connecticut, USA.
28The John P. Hussman Institute for Human Genomics and Dr. John T. Macdonald Foundation Department of Human Genetics, University of Miami, Miami, Florida, USA.
29Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA.
30University of Alabama at Birmingham, Birmingham, Alabama, USA.
31Department of Epidemiology, Epidemiology Data Center, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, PA USA.
32Department of Statistics, Carnegie Mellon University, Pittsburgh, Pennsylvania, USA.
33Department of Neurology, Johns Hopkins University, Baltimore, Maryland, USA.
34Department of Neurology, University of Michigan, Ann Arbor, Michigan, USA.
35Geriatric Research, Education and Clinical Center (GRECC), VA Ann Arbor Healthcare System (VAAAHS), Ann Arbor, Michigan, USA.
36Department of Neurology, University of California Los Angeles, Los Angeles, California, USA.
37Department of Psychiatry, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania, USA.
38Department of Medicine (Genetics Program), Boston University, Boston, Massachusetts, USA.
39Department of Pharmacology and Neuroscience, University of North Texas Health Science Center, Fort Worth, Texas, USA.
40Department of Neurological Sciences, Rush University Medical Center, Chicago, Illinois, USA.
41Department of Behavioral Sciences, Rush University Medical Center, Chicago, Illinois, USA.
42Civin Laboratory for Neuropathology, Banner Sun Health Research Institute, Phoenix, Arizona, USA.
43National Alzheimer's Coordinating Center, University of Washington, Seattle, Washington, USA.
44Rush Alzheimer's Disease Center, Rush University Medical Center, Chicago, Illinois, USA.
45Department of Pathology, Northwestern University, Chicago, Illinois, USA.
46Department of Neurology, University of Washington, Seattle, Washington, USA.
47Department of Epidemiology, Harvard School of Public Health, Boston, Massachusetts, USA.
48Department of Psychiatry, Massachusetts General Hospital/Harvard Medical School, Boston, Massachusetts, USA.
49Department of Neurology, University of California San Francisco, San Francisco, California, USA.
50Department of Neuroscience, Mount Sinai School of Medicine, New York, New York, USA.
51Department of Psychiatry, Mount Sinai School of Medicine, New York, New York, USA.
52Departments of Genetics and Genomic Sciences, Mount Sinai School of Medicine, New York, New York, USA.
53Department of Pathology and Immunology, Washington University, St. Louis, Missouri, USA.
54USF Health Byrd Alzheimer's Institute, University of South Florida, Tampa, Florida, USA.
55Department of Psychiatry, Vanderbilt University, Nashville, Tennessee, USA.
56Department of Pathology, University of Alabama at Birmingham, Birmingham, Alabama, USA.
57Department of Neurology, University of Southern California, Los Angeles, California, USA.
58Department of Neurology, University of Alabama at Birmingham, Birmingham, Alabama, USA.
59Department of Neurology, University of California Irvine, Irvine, California, USA.
60Department of Psychiatry and Behavioral Sciences, Miller School of Medicine, University of Miami, Miami, Florida, USA.
61Department of Psychiatry and Hope Center Program on Protein Aggregation and Neurodegeneration, Washington University School of Medicine, St. Louis, Missouri, USA.
62Department of Neurology, University of California Davis, Sacramento, California, USA.
63Institute for Memory Impairments and Neurological Disorders, University of California Irvine, Irvine, California, USA.
64Department of Neuroscience, Mayo Clinic, Jacksonville, Florida, USA.
65Wien Center for Alzheimer's Disease and Memory Disorders, Mount Sinai Medical Center, Miami Beach, Florida, USA.
66Department of Neurology, Mayo Clinic, Jacksonville, Florida, USA.
67Department of Medical and Molecular Genetics, Indiana University, Indianapolis, Indiana, USA.
68Department of Neurology, Indiana University, Indianapolis, Indiana, USA.
69Department of Psychiatry, New York University, New York, New York, USA.
70C.S. Kubik Laboratory for Neuropathology, Massachusetts General Hospital, Charlestown, Massachusetts, USA.
71Department of Neurosciences, University of California San Diego, La Jolla, California, USA.
72Department of Pathology and Laboratory Medicine, Emory University, Atlanta, Georgia, USA.
73Emory Alzheimer's Disease Center, Emory University, Atlanta, Georgia, USA.
74Neurogenetics Program, University of California Los Angeles, Los Angeles, California, USA.
75Department of Pathology and Laboratory Medicine, Indiana University, Indianapolis, Indian, USA.
76Department of Neurology, Emory University, Atlanta, Georgia, USA.
77Division of Genetics, Department of Medicine and Partners Center for Personalized Genetic Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts, USA.
78Department of Neurology, Massachusetts General Hospital/Harvard Medical School, Boston, Massachusetts, USA.
79Center for Applied Genomics, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, USA.
80Department of Pathology (Neuropathology), University of Pittsburgh, Pittsburgh, Pennsylvania, USA.
81Sanders-Brown Center on Aging, Department of Molecular and Biomedical Pharmacology, University of Kentucky, Lexington, Kentucky, USA.
82Taub Institute on Alzheimer's Disease and the Aging Brain, Department of Neurology, Columbia University, New York, New York, USA.
83Department of Pathology, Duke University, Durham, North Carolina, USA.
84Sanders-Brown Center on Aging, Department Neurology, University of Kentucky, Lexington, Kentucky, USA.
85Department of Pathology and Laboratory Medicine, University of California Davis, Sacramento, California, USA.
86Department of Biostatistics, Boston University, Boston, Massachusetts, USA.
87Department of Ophthalmology, Boston University, Boston, Massachusetts, USA.
88University of Pittsburgh Alzheimer's Disease Research Center, Pittsburgh, Pennsylvania, USA.
89Department of Neurology, Oregon Health & Science University, Portland, Oregon, USA.
90Department of Neurology, Portland Veterans Affairs Medical Center, Portland, Oregon, USA.
91Department of Pathology and Laboratory Medicine, University of California Irvine, Irvine, California, USA.
92Department of Neurology, Boston University, Boston, Massachusetts, USA.
93Department of Pathology, Boston University, Boston, Massachusetts, USA.
94Department of Neuropsychology, University of California San Francisco, San Francisco, California, USA.
95Department of Molecular & Medical Genetics, Oregon Health & Science University, Portland, Oregon, USA.
96Department of Epidemiology, University of Washington, Seattle, Washington, USA.
97Department of Neurobiology and Behavior, University of California Irvine, Irvine, California, USA.
98Department of Pathology, University of Washington, Seattle, Washington, USA.
99Department of Psychiatry and Behavioral Sciences, University of Washington, Seattle, Washington, USA.
100Department of Pathology, University of Michigan, Ann Arbor, Michigan, USA.
101Department of Psychiatry, Johns Hopkins University, Baltimore, Maryland, USA.
102Department of Preventive Medicine, University of Southern California, Los Angeles, California, USA.
103Department of Medicine - Pulmonary, New York University, New York, New York, USA.
104Department of Neurology, University of Miami, Miami, Florida, USA.
105Department of Pathology, University of California San Diego, La Jolla, California, USA.
106Cognitive Neurology and Alzheimer's Disease Center, Northwestern University, Chicago, Illinois, USA.
107Department of Pathology, University of Southern California, Los Angeles, California, USA.
108Department of Neurology, Washington University, St. Louis, Missouri, USA.
109Department of Anatomic Pathology, Mayo Clinic, Rochester, Minnesota, USA.
110Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota, USA.
111Department of Neurology, Mayo Clinic, Rochester, Minnesota, USA.
112Arizona Alzheimer's Consortium, Phoenix, Arizona, USA.
113Banner Alzheimer's Institute, Phoenix, Arizona, USA.
114Neurogenomics Division, Translational Genomics Research Institute, Phoenix, Arizona, USA.
115Alzheimer's Disease Center, New York University, New York, New York, USA.
116Department of Neurology, University of Texas Southwestern, Dallas, Texas, USA.
117Department of Radiology and Imaging Sciences, Indiana University, Indianapolis, Indiana, USA.
118Department of Pathology (Neuropathology), Rush University Medical Center, Chicago, Illinois, USA.
119Department of Psychiatry, University of Southern California, Los Angeles, California, USA.
120Department of Pathology, Johns Hopkins University, Baltimore, Maryland, USA.
121Sanders-Brown Center on Aging, Department of Anatomy and Neurobiology, University of Kentucky, Lexington, Kentucky
122Department of Pathology & Laboratory Medicine, University of California Los Angeles, Los Angeles, California, USA.
123Taub Institute on Alzheimer's Disease and the Aging Brain, Department of Pathology, Columbia University, New York, New York, USA.
124Department of Psychiatry & Behavioral Sciences, Duke University, Durham, North Carolina, USA.
125Department of Pathology, Oregon Health & Science University, Portland, Oregon, USA.
126Evelyn F. McKnight Brain Institute, Department of Neurology, Miller School of Medicine, University of Miami, Miami, Florida, USA.
127Department of Medicine, University of Washington, Seattle, Washington, USA.
128Correspondence should be addressed to D.C.W. (whitcomb@pitt.edu) or B.D. (devlinbj@upmc.edu).

The exocrine pancreas is a simple digestive gland of only two primary cell types, each with a single function (Supplementary Figure 1). Recurrent acute pancreatic inflammation can, but does not always, progress to irreversible damage of the gland, including fibrosis, atrophy, pain, and exocrine and endocrine insufficiency,1-3 known as chronic pancreatitis Different genetic and environmental factors produce the same clinical phenotype4.

We collected biological samples and phenotypic data from 1000 patients with recurrent acute pancreatitis and chronic pancreatitis plus controls in the North American Pancreatitis Study 2 (NAPS2)5. The primary environmental risk factor identified was heavy alcohol drinking when symptoms of pancreatitis began, based on the assessment of the study physician, called here alcohol-related pancreatitis.

To further define genetic risk, we conducted a two-stage (discovery/replication) genomewide association study (GWAS). The final data set for the Stage 1 cohort included 676 chronic pancreatitis cases and 4507controls of European ancestry (Supplementary Figs. 2-3) genotyped at 625,739 SNPs (Table 1; Supplementary Table 1). Genomewide significant associations (p-value < 5 × 10-8) were identified at two loci. The most highly associated SNP fell in Xq23.3, dubbed the CLDN2 locus, the other in 7q34, the PRSS1-PRSS2 locus (Fig. 1; Table 2; Supplementary Figs. 4-5, Supplementary Table 2). CLDN2 encodes the protein claudin-2, while PRSS1 encodes cationic trypsinogen, and PRSS2 encodes anionic trypsinogen.

The Stage 2 cohort included 910 cases (331 chronic pancreatitis, 579 recurrent acute pancreatitis; Table 1, Supplementary Table 1), again genotyped at 625,739 SNPs, and 4170 controls, most genotyped previously on the Illumina 1M. All subjects were of European ancestry as determined by genetic analyses. Recurrent acute pancreatitis and chronic pancreatitis were modeled as having common susceptibilities, with chronic pancreatitis occurring over time in the presence of additional disease-modifying factors.6 It is possible that this assumption reduces power relative to a study comprising solely chronic pancreatitis or recurrent acute pancreatitis cases. Our primary targets in Stage 2 were the PRSS1-PRSS2 and CLDN2 loci, although we also conducted a joint analysis7 of Stage 1 and Stage 2 data to uncover any new risk loci. After controlling for ancestry, these data demonstrated significant effects for the CLDN2 and PRSS1-PRSS2 loci (Figure 1; Supplementary Table 2-3; Supplementary Figs. 6-7). Quality of SNP genotypes supported the association (Supplementary Fig. 8). The frequencies of the putative risk alleles at these 2 loci were 0.57 for the C allele at rs10273639 (PRSS1-PRSS2 locus), with the minor T allele reducing risk, and 0.26 for the T allele at rs12688220 (CLDN2 locus). No other locus shows association after accounting for SNP genotype quality (Supplementary Figs. 6-8).

PRSS1 gain-of-function mutations, such as p.R122H, increase risk for recurrent acute pancreatitis and chronic pancreatitis8, as do increased copy number9,10. Rare loss-of-function mutations in PRSS2 are protective11. However, rs10273639 is in the 5′ promoter region of PRSS1. Because it is the only highly associated SNP in the locus, we validated its genotypes by independent TaqMan genotyping and also genotyped two SNPs in linkage disequilibrium with it (footnote, Supplementary Table 4)12, 13. We screened PRSS1 for rare variants in 1138 subjects: 418 chronic pancreatitis, 350 recurrent acute pancreatitis, and 379 controls. Three known disease-associated variants (A16V, N29I, R122H) were identified in 23 subjects (Supplementary Table 4). These gain-of-function variants occur almost solely in cases (22 out of 23), and two of them, A16V and R122H, likely fall on the C or risk haplotype of this locus (Supplementary Table 4). Nonetheless, with only 19 A16V and R122H events in cases, these rare alleles cannot account for the association observed at this locus.

Sixty-nine control pancreas tissue samples from three sources were genotyped at rs10273639, and cDNA was used to quantify PRSS1 and control gene expression (Supplementary Table 5). For all three sets of quantitative PCR data, the slope relating count of genotype C allele to PRSS1 expression level was positive; together, the samples provide evidence (p = 0.01) that alleles at rs10273639 affect expression of PRSS1: expression levels were highest in patients with two C alleles at rs10273639, intermediate in heterozygotes, and lowest in subjects with two T alleles. Based on this evidence, we posit that reduced trypsinogen production protects the pancreas from injury, as has been observed in genetic mouse models14.

CLDN2 is considered the primary candidate gene within our CLDN2 locus. Claudin-2 is attractive because it serves as a highly regulated tight junction protein forming low-resistance, cation-selective ion and water channels between endothelial cells15,16 and is normally expressed at low levels between cells of the pancreatic ducts and in pancreatic islets17,18. The CLDN2 promoter includes an NFκB binding site19, and gene expression is enhanced in other cells under conditions associated with injury or stress20-22. Claudin-2 can also be expressed by acinar cells when stressed, as reported in porcine models of acute pancreatitis23. Other genes within the CLND2 locus include MORC4, RIPPLY1, and TBC1D8B. MORC4 is expressed at low levels in most tissues, including the pancreas, with higher levels in the placenta and testis24. The MORC4 protein contains a CW four-cysteine zinc-finger motif, nuclear localization signal, and nuclear matrix-binding domain, suggesting that it may be a transcription factor24, but its expression does not appear to correlate with pancreatitis (Supplementary Fig. 9). RIPPLY1 and TBC1D8B are not known to be expressed in the pancreas.

To our knowledge, genetic variations in CLDN2 have never been associated with disease in humans. We assessed DNA sequence variants around CLDN2, RNA, and protein expression for claudin-2 in control tissue classified by histology and genotype (Supplementary Table 6, Supplementary Fig. 10). Evaluating 1000 Genomes data, no exonic variation was identified that could explain the association signal. Using materials and methods described previously for PRSS1 expression, CLDN2 expression levels in control tissues did not correlate with the CLDN2 locus risk genotype (p-value = 0.32). Protein was extracted from the tissue, and only one protein band of the appropriate size was observed with anti-claudin-2 antibodies on Western blot, which correlated with tissue inflammation as determined by systematic grading of histology in adjacent tissue (Fig. 2A, Supplementary Fig. 10). Immunohistochemical staining with anti-claudin-2 antibodies was verified in normal tissue (Fig. 2B), with kidney, duodenum, and bile ducts serving as additional positive controls (not shown). Protein localization was assessed in 12 GWAS cases who underwent pancreatic surgery: 6 with the CLDN2-containing high-risk genotype and 6 without. Claudin-2 cytoplasmic granular staining was markedly increased in both duct and acinar cells in chronic pancreatitis cases (Fig. 2C-E). Only chronic pancreatitis cases with the high-risk CLDN2 genotype demonstrated moderate-to-strong claudin-2 staining along the basolateral membrane of acinar cells (Fig. 2D, 2E, Supplementary Table 6). Claudin-2 was also expressed in macrophages, which could contribute to the pathologic inflammatory process25 (Fig. 2C, F).

Most studies report excessive alcohol consumption as the major risk factor for adult-onset chronic pancreatitis26-29. However, only 3% of patients who are alcoholics develop chronic pancreatitis30, suggesting a pancreas-targeting risk factor. We compared genotypes based on whether pancreatitis was alcohol-related (yes/no) 5,31. Setting control genotypes counts as the baseline category to be compared with case genotypes, the jointly estimated odds ratios for cases with a positive alcohol-related pancreatitis was greater for both rs10273639 (PRSS1-PRSS2 locus) and rs12688220 (CLDN2 locus) than those estimated for cases with a negative alcohol-related pancreatitis (Table 3). Thus, the effects of both loci appeared to be amplified by alcohol consumption. In a case-only analysis, both loci appear to interact with alcohol-related pancreatitis (Table 3), the CLDN2 locus most prominently (p-value = 4×10-7).

We conclude that a common allele in the PRSS1-PRSS2 locus is associated with lower PRSS1 gene expression and that this effect is independent of the previously reported rare gain-of-function PRSS1 variants that increase susceptibility to both recurrent acute pancreatitis and chronic pancreatitis8. For this reason, and because risk variants at the PRSS1-PRSS2 locus exert a similar effect in patients with recurrent acute pancreatitis or chronic pancreatitis, it is reasonable to conjecture that variation at rs10273639 or variation in linkage disequilibrium with it directly affects risk for chronic pancreatitis and recurrent acute pancreatitis through its impact on trypsinogen expression. Variation at the CLDN2 locus, however, is much more strongly associated with chronic pancreatitis than recurrent acute pancreatitis, suggesting that it likely acts as a disease modifier to accelerate transition from recurrent acute pancreatitis to chronic pancreatitis. The significant association of the CLDN2 locus with alcohol suggests that the high-risk allele in the CLDN2 locus may modify risk through a non-trypsin-dependent process. Thus, we have characterized two common genetic risk modifiers for sporadic and alcohol-related chronic pancreatitis.


Online Methods
Subject recruitment

Details of recruitment of cases and controls are reported in Supplementary Table 1. All studies were conducted under institutional review board-approved protocols.

Stage 1 samples

All N = 758 Stage 1 case samples were from the North American Acute Pancreatitis Study (NAPS2 5) were diagnosed with chronic pancreatitis, and were characterized for alcohol-related pancreatitis (Table 1). chronic pancreatitis occurs in less than 0.05% of the population, so a convenience sample provides essentially identical power as a same-sized sample of controls selected for the absence of chronic pancreatitis 32. For controls, we used genotypes from 4076 cases and controls from the Alzheimer Disease Genetics Consortium (ADGC) and 493 NAPS2 subjects, all genotyped on the same platform as the chronic pancreatitis samples.

Stage 2 samples

The Stage 2 samples consisted of N=343 chronic pancreatitis and N=627 recurrent acute pancreatitis cases (Table 1, Supplementary Table 1) as well as 4191 control subjects (3986 from the NeuroGenetics Research Consortium, NGRC, and 205 NAPS2 controls).

Genotypes

All cases and NAPS2 controls were genotyped by the University of Pittsburgh Genomics and Proteomics Core Laboratories using the Illumina HumanOmniExpress Beadchip. Samples were processed and scanned using the manufacturer's recommended protocols with no modifications. ADGC samples33 were also genotyped using Illumina HumanOmniExpress Beadchips, whereas NGRC samples 34 were genotyped on the Illumina Human1M-Duo DNA Analysis BeadChip.

Quality Control (QC) for Stage 1

QC was performed for individuals and then SNPs to determine which samples and SNPs should not be included in the analysis (“dropped’). Assessing sex miscalls based on × chromosome genotypes using Plink35, 7 chronic pancreatitis cases and 20 controls (10 NAPS2; 10 ADGC) were dropped. Based on the requirement for ≥ 95% complete genotypes per individual, 40 cases and 27 controls (20 NAPS2 controls and 7 ADGC controls) were dropped. Searching duplicate or highly related samples based on genotype and using GCTA software 36 (Genetic Relationship Matrix score GRM > 0.4), 35 cases and 78 controls (2 NAPS2, 76 ADGC) were dropped. After these QC filters, 676 cases and 4507 controls remained for association analysis.

SNP QC was first performed using NAPS2 and ADGC samples separately.. Ancestry was estimated using dacGem37 based on 9700 SNPs that had a genotype completion rate of ≥99.9%, a minor allele frequency MAF ≥ 0.05, and were separated by at least 500Kb. Analysis of genotypes from NAPS2 subjects identified 1 significant dimensions of ancestry and clustered subjects into 3 groups (Supplementary Fig. 1). Groups A and B, illustrated in Supplementary Figure 1, delineate 764 and 282 subjects, respectively, of European ancestry (self-identified); SNP QC for MAF and Hardy Weinberg Equilibrium (HWE) were performed on data from these subjects. Of 731,442 SNPs received, 633,790 passed QC filters. SNPs were dropped for the following reasons: 3165 for map location; 11,977 for call rate; 77,300 for MAF < 0.01; and 5219 failed HWE (p-value < 0.005).

ADGC data were received in three waves of 1763, 1110, and 1266 subjects. In the first wave, 659,224 SNPs were received, while in waves two and three, 730,525 SNPs were received. After QC as described for the chronic pancreatitis cohort, including harmonization with SNPs passing QC in the chronic pancreatitis cohort, 604,059, 632,761, and 633,023 SNPs remained, respectively. After merging cohorts, 30 related subjects were dropped, leaving 4046 ADGC subjects. Of the 633,615 unique SNPs in this ADGC, QC filters dropped 5 for low MAF and 5316 for HWE, leaving 628,294 SNPs. Combining ADGC and chronic pancreatitis cohorts and performing another round of QC yielded 625,739 SNPs for analysis.

QC for Stage 2

QC for individuals was performed as described for Stage 1. These individual-specific QC filters removed 60 cases, leaving 331 chronic pancreatitis and 579 recurrent acute pancreatitis cases for analysis; 14 controls were also removed, leaving 4177 controls for analysis. We analyzed all SNPs passing QC at Stage 1.

Association analysis

To control confounding due to ancestry, the first 10 major eigenvectors from the spectral decomposition were used as covariates in Stage 1 and Stage 2 analyses38, although only one was significant. We contrasted the genotypes of case subjects and controls via logistic regression and a log-additive (logit) model using Plink35. Genotypes for any SNPs showing association p-value < 5 × 10-7 were manually inspected for valid genotype clustering. SNPs showing poor-quality clustering were excluded. Following Skol et al. 7 and others, we take an overall significance level of 5 × 10-8 and 5 × 10-7 for strongly suggestive association.

To determine whether alcohol interacts with genetic variation to alter risk of pancreatitis, data from cases were fit to a general linear model in which count of alleles or genotypes predicted alcohol etiology (yes/no). The test statistic was obtained as a likelihood ratio chi-square. Note that in these analyses and any analyses other than genomewide association, we model the male genotypes as 0 and 2 39,40. For the genomewide association, Plink encodes the count of minor alleles in males as 0 and 1 and includes a sex effect, but the 0/2 encoding for males is a more powerful approach39,40 .

DNA extraction

DNA was obtained using standard methods41.

Pancreatic tissue processing

Tissue was obtained from two sources [Pitt and Pancreatic Adenocarcinoma Gene-Environment Risk (PAGER) from the University of Pittsburgh and PSU from Pennsylvania State University] and processed in three batches: banked (Pitt) and prospectively collected (PAGER) surgical waste from uninvolved pancreas and normal pancreas specimens from the Gift of Life Program that were not used for transplantation (PSU). PAGER samples were snap-frozen, placed in RNAlater solution (Ambion), and stored at –80°C. PSU pancreas samples were also snap frozen and preserved in formalin or placed in RNAlater solution. RNA was isolated using Trizol reagent (Invitrogen), and its quality examined in 1% agarose gel stained with ethidium bromide. cDNA was transcribed using oligo dt primers and the Superscript II reverse transcriptase kit (Invitrogen).

Gene Expression

Relative expression of PRSS1, PRSS2, CTRC, and 18S was determined by analyzing cDNA using Taqman®-based rtPCR assays (Applied Biosystems). Raw absolute quantitation results were analyzed and converted to relative expression results by software packages SDS V2.3 and DataAssist V1.0 (Applied Biosystems). Assays were repeated in triplicate or quadruplicate. Three sets of samples were assessed, two from Pitt (N=10 and 22) and one from PSU (N=37). PSU results were normalized against 18S, Pitt against CTRC. From each of these three data sets, mean gene expression per sample was regressed against allele count to obtain an estimated slope, standard error, and z-score. We then calculated an overall z-score as a weighted average of the individual z-scores, with weights determined by sample size.

Antibodies

Antibodies against claudin proteins (Invitrogen) were assessed using Western blot for mouse anti-claudin-2 (Catalog No. 32-5600), mouse anti-claudin-4 (Catalog No. 32-9400), and mouse alpha-tubulin antibody (Catalog no. AA12.1 The Developmental Studies Hybridoma Bank at the University of Iowa, http://dshb.biology.uiowa.edu/Antibody-list). Immunohistochemistry was performed using monoclonal antibodies for claudin-2 (Catalog #32-5600,1:1,000 dilution). Immunoflourescence was performed using mouse anti-claudin-2 (Catalog No. 32-5600) and goat anti-human CD68 (Catalog #sc-7082, Santa Cruz Biotechnology Inc.). The secondary antibodies for Immunofluorescence were goat anti-mouse CY3 and anti goat Cy5 from Jackson Immunoresearch.

SDS-PAGE and WESTERN Blotting

Protein homogenates for Western blotting were obtained from snap-frozen tissue that was homogenized and sonicated in lysis buffer supplemented with protease inhibitors. Protein concentration was determined by the Bradford method using a kit from Bio-Rad. Proteins were separated on 12% SDS-PAGE42 followed by transfer to polyvinylidene difluoride (PVDF) membranes43, for Western blotting44. Immunodetection of bound antibodies on PVDF membrane was performed using ECL reagents (Amersham Biosciences). All procedures were carried out according to manufacturer instructions.

Immunohistochemistry

Standard automated immunohistochemistry was performed for claudin 2 (antibodies listed above) on formalin-fixed, paraffin-embedded, 5 micron-thick tissue sections. Following deparaffinization in xylene and rehydration in ethanol, antigen retrieval was performed using EDTA pH8 buffer. The Dako Autostainer Plus was used; the slides were incubated for 30 minutes with the primary antibodies, followed by incubation with the secondary reagent (Mach 2 Mouse HRP Polymer from Biocare Medical) for 30 minutes. The chromogen was developed (Dako DAB+) for 10 minutes. The immunohistochemical stains were reviewed by one of the authors (A.M.K.). Cytoplasmic, granular, and membranous staining, predominantly in the lateral cell membranes, were graded on an intensity scale of 0-4 (0, negative; 1, weak; 2, moderate; 3, strong). The staining intensity was very patchy from lobule to lobule in most cases.

Immunofluorescence

Cryostat sections (5 micron) of pancreas were washed 3 times with phosphate-buffered saline (PBS), followed by 3 washes with solution of .5% BSA in PBS. Sections were blocked with 2% BSA solution for 30 minutes. The slides were incubated for 1 hour at room temperature with primary antibody for claudin-2 1:100 and goat anti-human CD68 in 0.5% BSA solution. Slides were washed 3 times with BSA solution and incubated for 1 hour at 20°C with 1:500 dilution anti-goat CY5 and 1:1000 dilution goat anti-mouse CY3 secondary antibodies in BSA solution. Nuclei were stained with Hoeschts dye (bisbenzamide 1mg/100ml water) for 30 seconds. After 3 rinses with PBS, sections were cover slipped with Gelvatol mounting media. Fluorescent images were captured with an Olympus Fluoview 1000 confocal microscope (software version 1.7a). The Cy5 signal (CD68) was pseudocolored as green to show colocalization with the red Claudin signal as yellow.


Supplementary Material 1

Notes

FN2AUTHOR CONTRIBUTIONS

Writing Group: A.M.K., B.D., D.C.W., J.L., L.K., M.L.K.

Project design, management, and coordination: B.D., D.C.W., D.Y., J.P.N., M.M.B., M.M.L., N.M.M., S.R.W.

Sample collection and phenotyping: A.B., A.C.M., A.C.N., A.G., A.G.S., A.I.L., A.J.S., A.K., A.M.G., A.P., A.P.L., A.R., A.S., B.G., B.L.M., B.N.V., B.R., B.S.S., B.T.H., C.A.M., C.B.W., C.Cao, C.Cruchaga, C.D., C.E.F., C.E.Y., C.F.L., C.G.L., C.L., C.M.H., C.M.W., C.R., C.T.B., D.A.B., D.Beekly, D.Blacker, D.C., D.C.Marson, D.C.Mash, D.C.W., D.G.C., D.H.C., D.H.G., D.R.G., D.W.D., D.W.T., D.Y., E.A.C., E.D.R., E.H., E.H.B., E.H.K., E.M., E.M.R., E.P., E.R.M., F.M., F.M.L., F.U.W., F.Y.D., G.A.C., G.A.J., G.I.P., G.J., G.L., G.W.B., H.C.C., H.H., H.J.R., H.P., H.V.V., J.A.K., J.A.Schneider, J.A.Sonnen, J.B., J.B.L., J.C.M., J.C.T., J.D., J.D.B., J.D.G., J.E.P., J.F.Q., J.H.G., J.H.K., J.J.L., J.M., J.M.O., J.M.R., J.P.N., J.P.S., J.P.V., J.Q.T., J.R., J.R.B., J.R.G., J.R.M., J.W., J.W.M., K.A.W.B., K.B.F., K.L.H.N., K.L.L., K.M.F., L.B.C., L.E.H., L.G.A., L.J.V.E., L.L.B., L.S.H., L.S.S., L.S.W., L.W.J., L.Y., M.A.A., M.D., M.E.M., M.Ganguli, M.Gearing, M.I.K., M.J., M.L., M.M., M.M.B., M.M.L., M.P.F., M.R., M.R.F., M.S., M.S.A., M.T., N.E.T., N.J.C., N.M.G., N.R.G.R., N.W.K., O.L.L., O.V., P.A.B., P.B.C., P.K., P.S., R.A.H., R.A.S., R.B., R.C.G., R.C.P., R.D., R.E.B., R.E.T., R.K., R.L.A., R.L.H., R.L.W., R.M.C., R.N.R., R.S., S.A., S.E.A., S.F., S.G., S.G.Y., S.L.C., S.S., S.Spina, S.T.A., S.Weintrub, T.B.G., T.D.B., T.G.B., T.J.M., T.M., T.M.F., V.M.V.D., W.A.K., W.G., W.J.M., W.W.P., W.W.S.

Genotyping and expression studies: A.M.K., D.J.H., D.S., G.D.S., J.L., J.L.H., J.P.S., L.A.F., L.O., M.A.P-V., N.O.Z., R.M., V.K.S.

Statistical analysis: B.D., D.Y., K.R., L.K., M.M.B., M.R.O

FN3COMPETING FINANCIAL INTEREST

The authors declare no competing financial interests*.

*Note to Editor: Dr Whitcomb notes that he owns stock in Ambry Genetics (a genetic testing company) and also the U.S. patent 6406846 entitled “ Method for determining whether a human patient is susceptible to hereditary pancreatitis, and primers therefore ”. Neither of these interest are directly related to the current manuscript.

ACKNOWLEDGMENTS

Technical support was provided by Kimberly Stello, Siddhartha Das, Danielle Dwyer, Alexander Rowland, Paul Alexander Blake, Mark Ross, Liz Kish PhD, and Haq Nawaz MD, Shiela Solomon MS CGC and Sharon Boggiano RN from the University of Pittsburgh; Rachel Ostroff, Meredith Goss and Jan Timm from SomaLogic Inc (Boulder CO). Data management by Laurie Silfies and Darina Protivnak, Epidemiology Data Center, Graduate School of Public Health, University of Pittsburgh. Clinical support: Sharon Boggaino RN, Megan Hendricks RN, Beth Elinoff RN MPH CCRC, University of Pittsburgh, Lee McHenry MD, Glen Lehman MD, James Watkins MD, Evan Fogel MD, Laura Lazzell-Pannell, BSN, RN, CCRC, Indiana University, Indianapolis, IN. Additional samples were provided by Frank Burton MD (deceased) Department of Internal Medicine, St. Louis University School of Medicine, St Louis, MO, Simon Lo MD, Department of Medicine, Cedars-Sinai Medical Center, University of California, Los Angeles; Mark T. DeMeo MD, Department of Medicine, Rush University Medical Center, Chicago, IL; William M. Steinberg MD, Washington Hospital Center, Washington DC; Michael L. Kochman MD, Department of Medicine, University of Pennsylvania, Philadelphia, PA; Babak Etemad MD, Department of Gastroenterology and Hepatology, Ochsner Medical Center, New Orleans, LA. Herbert Zeh MD, Arthur James Moser MD, K. Kenneth Lee MD, Department of Surgery, University of Pittsburgh, Pittsburgh PA.

This publication was made possible by Grant Numbers DK061451 (DCW), DK054709 (DCW), DK063922 (DCW), MH057881 (BD/KR), CA117926 (JPS), UL1 RR024153 and UL1TR000005. Its contents are solely the responsibility of the authors and do not necessarily represent the official view of the NIH. This project used the University of Pittsburgh Genomics and Proteomics Core Laboratories (UL1 RR024153) and the UPCI Clinical Genomics Immunoproteomics and Sequencing Facility (NIH P30CA047904). Jessica LaRusch was supported by Digestive Disease Training Program T32DK063922 (DCW), Narsis Zarnescu MD was supported by the American Gastroenterology Association John I Isenberg MD International Scholar Award. The Liverpool cohort was supported by NIHR Biomedical Research Unit award. Additional support was provided by National Pancreas Foundation (DCW), the Frieda G. and Saul F. Shapira BRCA Cancer Research Program (DCW) and the Wayne Fusaro Pancreatic Cancer Research Fund (DCW) and Gift of Life Foundation

Support for control cohort 1. Alzheimer's Disease Genetics Consortium: The National Institutes of Health, National Institute on Aging (NIH-NIA) supported this work through the following grants: ADGC, U01 AG032984, RC2 AG036528; NACC, U01 AG016976; NCRAD, U24 AG021886; Banner Sun Health Research Institute P30 AG019610; Boston University, P30 AG013846, U01 AG10483, R01 CA129769, R01 MH080295, R01 AG017173, R01 AG025259, R01AG33193; Columbia University, P50 AG008702, R37 AG015473; Duke University, P30 AG028377, AG05128; Emory University, AG025688; Indiana University, P30 AG10133; Johns Hopkins University, P50 AG005146, R01 AG020688; Massachusetts General Hospital, P50 AG005134; Mayo Clinic, P50 AG016574; Mount Sinai School of Medicine, P50 AG005138, P01 AG002219; New York University, P30 AG08051, MO1RR00096, and UL1 RR029893; Northwestern University, P30 AG013854; Oregon Health & Science University, P30 AG008017, R01 AG026916; Rush University, P30 AG010161, R01 AG019085, R01 AG15819, R01 AG17917, R01 AG30146; University of Alabama at Birmingham, P50 AG016582, UL1RR02777; University of Arizona, R01 AG031581; University of California, Davis, P30 AG010129; University of California, Irvine, P50 AG016573, P50, P50 AG016575, P50 AG016576, P50 AG016577; University of California, Los Angeles, P50 AG016570; University of California, San Diego, P50 AG005131; University of California, San Francisco, P50 AG023501, P01 AG019724; University of Kentucky, P30 AG028383, AG05144; University of Michigan, P50 AG008671; University of Pennsylvania, P30 AG010124; University of Pittsburgh, P50 AG005133, AG030653; University of Southern California, P50 AG005142; University of Texas Southwestern, P30 AG012300; University of Miami, R01 AG027944, AG010491, AG027944, AG021547, AG019757; University of Washington, P50 AG005136; Vanderbilt University, R01 AG019085; and Washington University, P50 AG005681, P01 AG03991. The Kathleen Price Bryan Brain Bank at Duke University Medical Center is funded by NINDS grant # NS39764, NIMH MH60451 and by Glaxo Smith Kline. We thank Drs. D. Stephen Snyder and Marilyn Miller from NIA who are ex-officio ADGC members.

Support for control group 2. Data for control group 2 genotypes were obtained from Genome-Wide Association Study of Parkinson Disease: Genes and Environment, dbGaP Study Accession phs000196.v2.p1. This NeuroGenetics Research Consortium (NGRC) is a gene-environment study of Parkinson's disease. Principal Investigator is Haydeh Payami, PhD, New York State Department of Health Wadsworth Center, Albany, NY, USA. Co-investigators are: John Nutt, MD., Oregon Health & Sciences University; Cyrus Zabetian, MS, MD. University of Washington and Puget Sound Veterans Medical Center, Seattle, WA, USA; Stewart Factor, DO. Emory University, Atlanta, GA, USA; Eric Molho, MD. Albany Medical Center, Albany, NY, USA; and Donald Higgins, MD. Albany Medical Center and Albany Veterans Medical Center Albany, NY, USA. Funding Source: 5R01NS36960-10. Genotyping provided by Genotyping Center, Johns Hopkins University Center for Inherited Disease Research (CIDR), Baltimore, MD, USA. Funding Source for Genotyping: HHSN268200782096C. NIH contract “High throughput genotyping for studying the genetic contributions to human disease” from the National Institutes of Health, Bethesda, MD, USA.


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