Article Text

Original research
Genome-wide analysis of 944 133 individuals provides insights into the etiology of haemorrhoidal disease
  1. Tenghao Zheng1,2,
  2. David Ellinghaus3,4,
  3. Simonas Juzenas3,5,
  4. François Cossais6,
  5. Greta Burmeister7,
  6. Gabriele Mayr3,
  7. Isabella Friis Jørgensen4,
  8. Maris Teder-Laving8,
  9. Anne Heidi Skogholt9,
  10. Sisi Chen10,
  11. Peter R Strege10,
  12. Go Ito3,11,
  13. Karina Banasik4,
  14. Thomas Becker12,
  15. Frank Bokelmann13,
  16. Søren Brunak4,
  17. Stephan Buch14,
  18. Hartmut Clausnitzer15,
  19. Christian Datz16,
  20. DBDS Consortium,
  21. Frauke Degenhardt3,
  22. Marek Doniec17,
  23. Christian Erikstrup18,
  24. Tõnu Esko8,
  25. Michael Forster3,
  26. Norbert Frey19,20,21,
  27. Lars G Fritsche22,
  28. Maiken Elvestad Gabrielsen9,
  29. Tobias Gräßle23,24,
  30. Andrea Gsur25,
  31. Justus Gross7,
  32. Jochen Hampe14,26,
  33. Alexander Hendricks7,
  34. Sebastian Hinz7,
  35. Kristian Hveem9,
  36. Johannes Jongen27,28,
  37. Ralf Junker15,
  38. Tom Hemming Karlsen29,
  39. Georg Hemmrich-Stanisak3,
  40. Wolfgang Kruis30,
  41. Juozas Kupcinskas31,
  42. Tilman Laubert27,28,32,
  43. Philip C Rosenstiel3,33,
  44. Christoph Röcken34,
  45. Matthias Laudes35,
  46. Fabian H Leendertz23,24,
  47. Wolfgang Lieb36,
  48. Verena Limperger15,
  49. Nikolaos Margetis37,
  50. Kerstin Mätz-Rensing38,
  51. Christopher Georg Németh12,39,
  52. Eivind Ness-Jensen9,40,41,
  53. Ulrike Nowak-Göttl15,
  54. Anita Pandit22,
  55. Ole Birger Pedersen42,
  56. Hans Günter Peleikis27,28,
  57. Kenneth Peuker14,26,
  58. Cristina Leal Rodriguez4,
  59. Malte Christoph Rühlemann3,
  60. Bodo Schniewind43,
  61. Martin Schulzky3,
  62. Jurgita Skieceviciene31,
  63. Jürgen Tepel44,
  64. Laurent Thomas9,45,46,47,
  65. Florian Uellendahl-Werth3,
  66. Henrik Ullum48,
  67. Ilka Vogel49,
  68. Henry Volzke50,
  69. Lorenzo von Fersen51,
  70. Witigo von Schönfels12,
  71. Brett Vanderwerff22,
  72. Julia Wilking7,
  73. Michael Wittig3,
  74. Sebastian Zeissig14,26,
  75. Myrko Zobel52,
  76. Matthew Zawistowski22,
  77. Vladimir Vacic53,
  78. Olga Sazonova53,
  79. Elizabeth S Noblin53,
  80. The 23andMe Research Team,
  81. Gianrico Farrugia10,
  82. Arthur Beyder10,
  83. Thilo Wedel6,
  84. Volker Kahlke27,28,54,
  85. Clemens Schafmayer7,
  86. Mauro D'Amato1,2,55,56,
  87. Andre Franke3,33
  1. 1 School of Biological Sciences, Monash University, Clayton, Victoria, Australia
  2. 2 Unit of Clinical Epidemiology, Department of Medicine Solna, Karolinska Institutet, Stockholm, Sweden
  3. 3 Institute of Clinical Molecular Biology, Christian-Albrechts-University of Kiel, Kiel, Germany
  4. 4 Novo Nordisk Foundation Center for Protein Research, Disease Systems Biology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
  5. 5 Institute of Biotechnology, Life Science Centre, Vilnius University, Vilnius, Lithuania
  6. 6 Institute of Anatomy, Christian-Albrechts-University of Kiel, Kiel, Germany
  7. 7 Department for General, Visceral, Vascular and Transplantation Surgery, University Medical Center Rostock, Rostock, Germany
  8. 8 Institute of Genomics, University of Tartu, Tartu, Estonia
  9. 9 K.G. Jebsen Center for Genetic Epidemiology, Department of Public Health and Nursing, NTNU, Norwegian University of Science and Technology, Trondheim, Norway
  10. 10 Enteric NeuroScience Program, Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester, MN, USA
  11. 11 Institute of Advanced Research, Tokyo Medical and Dental University, Tokyo, Japan
  12. 12 Department of General-, Visceral- Transplant-, Thoracic and Pediatric Surgery, Kiel University, Kiel, Germany
  13. 13 Medical Office for Surgery Preetz, Preetz, Germany
  14. 14 Medical Department 1, University Hospital Dresden, Technische Universität Dresden (TU Dresden), Dresden, Germany
  15. 15 University Hospital Schleswig-Holstein, Institute of Clinical Chemistry, Thrombosis & Hemostasis Treatment Center, Campus Kiel & Lübeck, Kiel, Germany
  16. 16 Department of Internal Medicine, Hospital Oberndorf, Teaching Hospital of the Paracelsus Private Medical University of Salzburg, Oberndorf, Austria
  17. 17 Medical office for Colo-Proctology Kiel, Kiel, Germany
  18. 18 Department of Clinical Immunology, Aarhus University Hospital, Aarhus, Denmark
  19. 19 Department of Internal Medicine III, University Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany
  20. 20 Department of Internal Medicine III, University Hospital Heidelberg, Heidelberg, Germany
  21. 21 DZHK (German Centre for Cardiovascular Research), Partner Site Hamburg/Kiel/Lübeck, Kiel, Germany
  22. 22 Department of Biostatistics, University of Michigan, Ann Arbor, MI, USA
  23. 23 Epidemiology of highly pathogenic microorganisms, Robert Koch-Institute, Berlin, Germany
  24. 24 Department of Primatology, Max Planck Institute for Evolutionary Anthropology, Leipzig, Germany
  25. 25 Department of Medicine I, Institute of Cancer Research, Medical University Vienna, Vienna, Austria
  26. 26 Center for Regenerative Therapies Dresden (CRTD), Technische Universität (TU) Dresden, Dresden, Germany
  27. 27 Department of Proctological Surgery Park Klinik Kiel, Kiel, Germany
  28. 28 Proctological Office Kiel, Kiel, Germany
  29. 29 Research Institute for Internal Medicine, Division of Surgery, Inflammatory Diseases and Transplantation, Oslo University Hospital Rikshospitalet and University of Oslo, Oslo, Norway
  30. 30 Faculty of Medicine, University of Cologne, Cologne, Germany
  31. 31 Department of Gastroenterology, Institute for Digestive Research, Lithuanian University of Health Sciences, Kaunas, Lithuania
  32. 32 University of Lübeck, Lübeck, Germany
  33. 33 University Hospital of Schleswig-Holstein (UKSH), Kiel Campus, Kiel, Germany
  34. 34 Department of Pathology, University Medical Center Schleswig-Holstein, Kiel, Germany
  35. 35 Division of Endocrinology, Diabetes and Clinical Nutrition, Department of Medicine 1, University of Kiel, Kiel, Germany
  36. 36 Institute of Epidemiology, Christian-Albrechts-University of Kiel, Kiel, Germany
  37. 37 University of Athens, Athens Euroclinic, Athens, Greece
  38. 38 Pathology Unit, German Primate Center, Leibniz Institute for Primatology, Göttingen, Germany
  39. 39 Department of Ophthalmology, Hospital Frankfurt Hoechst, Frankfurt, Germany
  40. 40 Upper Gastrointestinal Surgery, Department of Molecular Medicine and Surgery, Karolinska Institutet, Stockholm, Sweden
  41. 41 Department of Medicine, Levanger Hospital, Nord-Trøndelag Hospital Trust, Levanger, Norway
  42. 42 Department of Clinical Immunology, Naestved Hospital, Naestved, Denmark
  43. 43 General Hospital Lüneburg, Lüneburg, Germany
  44. 44 Department of General and Thoracic Surgery, Hospital Osnabrück, Osnabrück, Germany
  45. 45 Department of Clinical and Molecular Medicine, Norwegian University of Science and Technology, Trondheim, Norway
  46. 46 BioCore – Bioinformatics Core Facility, Norwegian University of Science and Technology, Trondheim, Norway
  47. 47 Clinic of Laboratory Medicine, St.Olavs Hospital, Trondheim University Hospital, Trondheim, Norway
  48. 48 Department of Clinical Immunology, Copenhagen University Hospital, Copenhagen, Denmark
  49. 49 Department of Surgery, Community Hospital Kiel, Kiel, Germany
  50. 50 Institute for Community Medicine, University Medicine Greifswald, Greifswald, Germany
  51. 51 Department Research & Conservation, Zoo Nuremberg, Nuremberg, Germany
  52. 52 Department of Gastroenterology, Helios Hospital Weißeritztal, Freital, Germany
  53. 53 23andMe Inc, Sunnyvale, CA, USA
  54. 54 Christian-Albrechts-University of Kiel, Kiel, Germany
  55. 55 Gastrointestinal Genetics Lab, CIC bioGUNE - BRTA, Derio, Spain
  56. 56 Ikerbasque, Basque Foundation for Science, Bilbao, Spain
  1. Correspondence to Prof Mauro D'Amato, Gastrointestinal Genetics Lab, CIC bioGUNE - BRTA, Bizkaia Science and Technology Park, Building 801A, 48160 Derio, Spain; mdamato{at}cicbiogune.es; Prof Andre Franke, Institute of Clinical Molecular Biology (IKMB) & University Hospital Schleswig-Holstein (UKSH), Christian-Albrechts-University of Kiel (CAU), Rosalind-Franklin-Str. 12, D-24105 Kiel, Germany; a.franke{at}mucosa.de

Abstract

Objective Haemorrhoidal disease (HEM) affects a large and silently suffering fraction of the population but its aetiology, including suspected genetic predisposition, is poorly understood. We report the first genome-wide association study (GWAS) meta-analysis to identify genetic risk factors for HEM to date.

Design We conducted a GWAS meta-analysis of 218 920 patients with HEM and 725 213 controls of European ancestry. Using GWAS summary statistics, we performed multiple genetic correlation analyses between HEM and other traits as well as calculated HEM polygenic risk scores (PRS) and evaluated their translational potential in independent datasets. Using functional annotation of GWAS results, we identified HEM candidate genes, which differential expression and coexpression in HEM tissues were evaluated employing RNA-seq analyses. The localisation of expressed proteins at selected loci was investigated by immunohistochemistry.

Results We demonstrate modest heritability and genetic correlation of HEM with several other diseases from the GI, neuroaffective and cardiovascular domains. HEM PRS validated in 180 435 individuals from independent datasets allowed the identification of those at risk and correlated with younger age of onset and recurrent surgery. We identified 102 independent HEM risk loci harbouring genes whose expression is enriched in blood vessels and GI tissues, and in pathways associated with smooth muscles, epithelial and endothelial development and morphogenesis. Network transcriptomic analyses highlighted HEM gene coexpression modules that are relevant to the development and integrity of the musculoskeletal and epidermal systems, and the organisation of the extracellular matrix.

Conclusion HEM has a genetic component that predisposes to smooth muscle, epithelial and connective tissue dysfunction.

  • anorectal disorders
  • genetics
  • anal canal histopathology

Data availability statement

Data are available in a public, open access repository. Genome-wide summary statistics of our analyses are publicly available through our web browser (http://hemorrhoids.online) and have been submitted to the European Bioinformatics Institute (www.ebi.ac.uk/gwas) under the accession number GCST90014033. RNA-seq data have been deposited at NCBI Gene Expression Omnibus (GEO) under the accession number GSE154650.

http://creativecommons.org/licenses/by-nc/4.0/

This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/.

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Data availability statement

Data are available in a public, open access repository. Genome-wide summary statistics of our analyses are publicly available through our web browser (http://hemorrhoids.online) and have been submitted to the European Bioinformatics Institute (www.ebi.ac.uk/gwas) under the accession number GCST90014033. RNA-seq data have been deposited at NCBI Gene Expression Omnibus (GEO) under the accession number GSE154650.

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Footnotes

  • Twitter @mruehlemann, @beyderlab, @damato_mauro

  • TZ, DE and SJ contributed equally.

  • CS, MD and AF contributed equally.

  • Contributors AF, CS and MD’A were responsible for the concept and the design of the study. CS and GB coordinated the recruitment of the German clinical cohort. VK, JG, IV, BS, HGP, AH, JJ, JW, TB, MD, FB, SH, JK, JS, WK, MZo, CGN, TL, JT and WS recruited and phenotyped patients of the clinical cohorts. VK collected and phenotyped the biopsy samples that were used in the immunohistochemistry and transcriptome analyses. Animal samples for histology were taken and provided by KP, SZ, LF, TG, FHL and KM-R. Immunohistochemistry and histology analyses were performed by FC and TW with support of JW and CR. TZ, DE and FD performed genotype and phenotype data collection. TZ, DE, and FD performed GWAS data quality control and imputation. SJ performed the transcriptome (RNA-Seq) analysis with help from GI and PR. FU-W, SBu, and MF helped with bioinformatic analyses. IFJ, KB, SBr, CLR, CE, OBP, and HU conducted analyses in DBDS and DNPR. HC, VL, RJ, NF, WL, ML, and UN-G contributed German control data. SBu, CD, HV, AG and JH contributed the GWAS data on diverticular disease. Norwegian HUNT data were contributed by AHS, THK, MEG, LT, EN-J and KH. The Estonian dataset was provided by TE and MT-L. Protein models and analyses were performed by GM. The analysis of the MGI data set was performed by MZa, BV, AP and LGF. The 23andMe data set was analyzed and provided by OS, ESN, and VV. MW implemented ABO blood-group inference. SC, PRS, GF and AB performed site-directed ANO1 mutagenesis and whole-cell electrophysiology experiments. TZ, DE implemented statistical models and performed the (meta-)analysis. TZ, DE, and SJ curated and interpreted results. MS designed online supplementary figure S2 with scientific input from NM and AF. The GWAS data browser was implemented by MR and set up by GH-S. AF, MD’A and DE wrote the manuscript draft with substantial contributions from TZ and SJ. All authors reviewed, edited and approved the final manuscript.

  • Funding This project was funded by Andre Franke’s and Clemens Schafmayer’s DFG grant “Discovery of risk factors for hemorrhoids“ (ID: FR 2821/19-1). The study received infrastructure support from the DFG Cluster of Excellence 2167 “Precision Medicine in Chronic Inflammation (PMI)” (DFG Grant: “EXC2167”). The project was supported by grants from the Swedish Research Council to MD (VR 2017-02403), the Novo Nordisk Foundation (grants NNF17OC0027594 and NNF14CC0001) and BigTempHealth (grant 5153-00002B). We are indebted to the valuable assistance by Tanja Wesse (Genotyping of the German samples) and Petra Röthgen (German DZHK control data set). We thank Clemens Franke (Institute of Anatomy, Christian-Albrechts-University of Kiel, Kiel, Germany) for graphic assistance (figure 6, online supplementary figure S1 and S13). This research has been conducted using the UK Biobank Resource under Application Number 31435. The work on cross-trait analysis for diverticular disease presented in this manuscript was supported by the German Research Council (DFG, ID: Ha3091/9-1) and the Austrian Science Fund (FWF, ID: I1542-B13). Data access to the UK biobank data for diverticular disease was granted under project numbers 22691. EGCUT work has also been supported by the European Regional Development Fund and grants SP1GI18045T, No. 2014-2020.4.01.15-0012 GENTRANSMED and 2014-2020.4.01.16-0125 This study was also funded by EU H2020 grant 692145, Estonian Research Council Grant PUT1660. Data analyzes with Estonian datasets were carried out in part in the High-Performance Computing Center of University of Tartu. The work on site-directed mutagenesis of ANO1 and whole-cell electrophysiology was funded by NIH DK057061. We would like to thank the research participants and employees of 23andMe for making this work possible. The following members of the 23andMe Research Team contributed to this study: Michelle Agee, Adam Auton, Robert K. Bell, Katarzyna Bryc, Sarah L. Elson, Pierre Fontanillas, Nicholas A. Furlotte, David A. Hinds, Karen E. Huber, Aaron Kleinman, Nadia K. Litterman, Jennifer C. McCreight, Matthew H. McIntyre, Joanna L. Mountain, Elizabeth S. Noblin, Carrie A.M. Northover, Steven J. Pitts, J. Fah Sathirapongsasuti, Olga V. Sazonova, Janie F. Shelton, Suyash Shringarpure, Chao Tian, Joyce Y. Tung, and Vladimir Vacic.

  • Competing interests Vladimir Vacic and Olga V. Sazonova are/were employed by and hold stock or stock options in 23andMe, Inc. All other authors have nothing to declare.

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

  • Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.

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