Doctor of Medical Science Thesis by Christina Ellervik MD, PhD HEMOCHROMATOSIS GENOTYPES AND ELEVATED TRANSFERRIN SATURATION - risk of diabetes mellitus, hypertension,cancer, and total mortality Aected Unaected Carrier Carrier Carrier Father Mother Aected Carrier Carrier Unaected Son Daughter Son Daughter Hemochromatosis genotypes and elevated transferrin saturation - risk of diabetes mellitus, hypertension,cancer, and total mortality Doctor of Medical Science Thesis by Christina Ellervik MD,PhD PhD The Faculty of Health and Medical Sciences at the University of Copenhagen has accepted this dissertation, which consists of the already published dissertations listed below, for pub- lic defence for the doctoral degree in medicine. Copenhagen, October 11th 2015 Ulla M. Wewer Head of Faculty Place and time for defence: St. Auditorium at Herlev Hospital, June 22nd 2016 at 2pm Table of Contents • Papers on which the thesis is based............................................................. 2 • Preface ............................................................................................................. 3 • Scope and delimitation of the thesis ...................................................... 3 - 4 • Introduction ............................................................................................ 4 - 14 Hereditary hemochromatosis ............................................................................................ 4 - 7 Diabetes mellitus (paper 1 and 2) ...................................................................................... 7 - 8 Hypertension (paper 3)............................................................................................................. 8 Cancer (paper 4) .................................................................................................................... 8 - 9 Mortality (paper 5, 6, and 7) ............................................................................................. 9 - 10 Other diseases associated with hereditary hemochromatosis, but not part of the thesis ................................................................................................................... 10 - 11 Classification of diabetes mellitus ................................................................................ 11 - 13 Danish registries for ascertainment of diagnoses used in the thesis ........................... 13 • Objectives .............................................................................................. 13 - 14 • Methods ................................................................................................ 14 - 00 Study populations .................................................................... ....................................... 14 - 16 Genotyping ............................................................................................................................... 16 Measurement of transferrin saturation ...................................................................... 16 - 18 Ascertainment of diagnoses ................................................................................................. 18 Statistics ............................................................................................................................ 18 - 22 • Results ................................................................................................... 22 - 41 Total mortality and incident diabetes, hypertension, cancer, and ischemic heart disease in non-participants vs. participants .................................. 22 - 27 Risk of diabetes mellitus (paper 1 and 2) .................................................................... 28 - 29 Risk of hypertension (paper 3) ..................................................................................... 29 - 31 Risk of cancer (paper 4) .................................................................................................. 31 - 33 Risk of total mortality (paper 5, 6 and 7) .................................................................... 33 - 40 Results on hemochromatosis genotypes C282Y/H63D and C282Y/ wild type in paper 1, 3, 4, 6 and 7 ......................................................................................... 40 Key findings ...................................................................................................................... 40 - 41 • Discussion – methodological considerations .................................... 41 - 47 Errors in sampling ........................................................................................................... 41 - 44 Health examinations .............................................................................................................. 44 Other factors influencing study validity .................................................................... 44 - 45 Patient audit: classification of diabetes mellitus ..................................................... 45 - 46 Ascertainment and misclassification of diagnoses ................................................. 46 - 47 • Discussion – results in perspective ....................................................47 - 62 Diabetes mellitus (paper 1 and 2) ................................................................................. 47 - 51 Hypertension (paper 3) .................................................................................................. 51 - 52 Cancer (paper 4) ............................................................................................................... 52 - 53 Total mortality (paper 5, 6, and 7) ................................................................................ 53 - 56 Discussion of hemochromatosis genotypes C282Y/H63D and C282Y/wild type (paper 1, 3, 4, 6 and 7)...................................................................... 56 -57 Screening for iron overload .......................................................................................... 57 - 58 Strengths and limitations ............................................................................................. 58 - 59 Biological plausibility ..................................................................................................... 59 - 60 Penetrance ....................................................................................................................... 60 - 61 Expressivity ..................................................................................................................... 61 - 62 • Future perspectives and unresolved questions .............................. 62 - 63 • Summary in English .................................................................................... 63 • Summary in Danish ............................................................................. 63 - 64 • Abbreviation list .......................................................................................... 64 • References ............................................................................................ 64 - 85 1 1 Papers on which the thesis is based (According to PubMed style) 1. Ellervik C, Mandrup-Poulsen T, Nordestgaard BG, Larsen LE, Appleyard M, Frandsen M, Petersen P, Schlichting P, Saermark T, Tybjærg-Hansen A, Birgens H. Prevalence of hereditary haemochromatosis in late-onset type 1 diabetes mellitus: a retrospective study. Lancet 2001; 358:1405-9. 2. Ellervik C, Mandrup-Poulsen T, Andersen HU, Tybjærg-Hansen A, Frandsen M, Birgens H, Nordestgaard BG. Elevated transferrin saturation and risk of diabetes: three population-based studies. Diabetes Care 2011; 34:2256-8. 3. Ellervik C, Tybjærg-Hansen A, Appleyard M, Ibsen H, Nordestgaard BG. Haemochromatosis genotype and iron overload: association with hypertension and left ventricular hypertrophy. J Intern Med 2010; 268:252-64 4. Ellervik C, Tybjærg-Hansen A, Nordestgaard BG. Risk of cancer by transferrin satura- tion levels and haemochromatosis genotype: population-based study and meta- analysis. J Intern Med 2012; 271:51-63. 5. Ellervik C, Tybjærg-Hansen A, Nordestgaard BG. Total mortality by transferrin saturation levels: two general population studies and a metaanalysis. Clin Chem 2011; 57:459-66. 6. Ellervik C, Andersen HU, Tybjærg-Hansen A, Frandsen M, Birgens H, Nordestgaard BG, Mandrup-Poulsen T. Total Mortality by elevated transferrin saturation in patients with diabetes. Diabetes Care 2013; 36: 2646-2654. 7. Ellervik C, Mandrup-Poulsen T, Tybjærg-Hansen A, Nordestgaard BG. Total and cause- specific mortality by elevated transferrin saturation and hemochromatosis genotype in individuals with diabetes – two general population studies. Diabetes Care 2014; 37: 444-452. 2 2 Preface My interest in hemochromatosis began as a medical student, when I met Henrik Birgens, Thomas Mandrup-Poulsen, Børge Nordestgaard and Anne Tybjærg-Hansen. Since then, they have encouraged me to continue research within this field. Their contribution to my scien- tific education has been very valuable and has lead to a fruitful and rewarding collaboration during the years. The present thesis is based on studies carried out within this collaboration over the past 10 years. I owe thanks to all the collaborators for always constructive com- ments and criticism. I am also grateful to other co-authors of the presented works, from whom I have learnt much. The assistance of the technical staff and medical doctors at Steno Diabetes Centre (Sonja Bigand, Merete Frandsen, Henrik Ullits Andersen) and at the departments of