BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) J Med Genet Dilated cardiomyopathy caused by truncating titin variants – Long-term outcomes, arrhythmias, response to treatment and sex differences SUPPLEMENTARY MATERIAL Christoffer Rasmus Vissing1*, MD; Torsten Bloch Rasmussen2, MD, PhD; Anne Mette Dybro2, MD; Morten Salling Olesen3,4, MSc, PhD; Lisbeth Nørum Pedersen5; MSc, PhD; Morten Jensen, MD, PhD2; Henning Bundgaard1, MD, DMSc; Alex Hørby Christensen1,6 MD, PhD 1The Capital Region’s Unit for Inherited Cardiac Diseases, Department of Cardiology, Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark 2Department of Cardiology, Aarhus University Hospital, Aarhus, Denmark 3Laboratory for Molecular Cardiology, University of Copenhagen, Copenhagen, Denmark 4Department of Biomedical Sciences, University of Copenhagen, Copenhagen, 2200 N, Denmark 5Department of Molecular Medicine, Aarhus University Hospital, Denmark. 6Department of Cardiology, Herlev-Gentofte Hospital, Copenhagen University Hospital, Copenhagen, Denmark *Corresponding author: Christoffer Rasmus Vissing, MD The Capital Region’s Unit for Inherited Cardiac Diseases, Department of Cardiology, The Heart Center, Rigshospitalet, Copenhagen University Hospital, Blegdamsvej 9, DK-2100 Copenhagen, Denmark, E-mail: [email protected]; Phone +45 3545 5045 1 Vissing CR, et al. J Med Genet 2020;0:1–10. doi: 10.1136/jmedgenet-2020-107178 BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) J Med Genet Supplementary Materials Contents Dilated cardiomyopathy caused by truncating titin variants – Long-term outcomes, arrhythmias, response to treatment and sex differences ...................................................................................................................... 1 SUPPLEMENTARY METHODS ............................................................................................................................. 3 Definitions ..................................................................................................................................................... 3 Genetic Screening ......................................................................................................................................... 3 Statistical analyses ........................................................................................................................................ 4 INFORMATION ON PATIENTS WITH MULTIPLE VARIANTS: ............................................................................... 5 FIGURES ............................................................................................................................................................. 6 Supplementary Figure 1: Forest-plots depicting hazard ratios in Cox proportional hazards models for the three studied cardiac outcomes .................................................................................................................... 6 Supplementary Figure 2: Forest-plots depicting univariable hazard ratios of 6 clinical variables in the three studied cardiac outcomes .................................................................................................................... 7 Supplementary Figure 3: Outcomes according to site of truncation in titin ................................................. 8 Supplementary Figure 4: Outcomes in probands vs relatives ....................................................................... 9 SUPPLEMENTARY TABLES ................................................................................................................................ 10 Supplementary Table 1. Identified truncating TTN variants. All variants are annotated in relation to the titin-metatranscript (NM_001267550.1) ..................................................................................................... 10 Supplementary Table 2: Association of clinical variables with combined end-point (implantation of LVAD, heart transplantation or death) in univariable and multivariable cox regression analysis ......................... 21 Supplementary Table 3: Association of clinical variables with ventricular arrhythmia in univariable and multivariable cox regression analysis. ......................................................................................................... 23 Supplementary Table 4: Association of clinical variables with atrial fibrillation in univariable cox regression analysis. ...................................................................................................................................... 25 2 Vissing CR, et al. J Med Genet 2020;0:1–10. doi: 10.1136/jmedgenet-2020-107178 BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) J Med Genet SUPPLEMENTARY METHODS Definitions Severe systemic hypertension We defined the exclusion criteria severe systemic hypertension as: an untreated systolic blood pressure > 180mmHg, and/or an untreated diastolic blood pressure > 110 mmHg, and/or a systolic blood pressure > 160 mmHg on antihypertensive treatment and/or a diastolic blood pressure > 100 mmHg on antihypertensive treatment. Diabetes mellitus Patients were registered as having diabetes if they had a clinical diagnosis of diabetes mellitus or if they were prescribed antidiabetic medications. Severely dysregulated diabetes mellitus was defined as patients with neuropathy, nephropathy or retinopathy. Metabolic, infectious or inflammatory cardiomyopathies We defined the exclusion criteria for the above terms to encompass myocarditis caused by viral, bacterial, fungal or parasitic infections. Autoimmune diseases including giant cell myocarditis, non-infectious myocarditis, polymyositis/dermatomyositis, Churg-Strauss syndrome, Wegener’s granulomatosis, systemic lupus erythematosus or sarcoidosis. Nutritional deficiencies or hemochromatosis. Uncontrolled hypothyroidism or hyperthyroidism, Cushing’s disease, Addison disease, pheochromocytoma, acromegaly, fatty acid oxidation disorders, carnitine deficiency, glycogen storage diseases, organic acidurias and disorders of oxidative phosphorylation. Genetic Screening Old genetic panel The first genetic panel used for screening cardiomyopathy patients at our institutions included the following 14 genes: ACTC, CRSP3, LAMP2, LMNA, MYL2, MYL3, MYH7, MYBPC3, PRKAG2, (RBM20), SCN5A, TNNI3, TNNT2 and TPM. RBM20 is in brackets since it was not a part of the panel originally, but all patients have been sequenced in RBM20 after the initial screening. This panel was implemented in 2006 and was in 3 Vissing CR, et al. J Med Genet 2020;0:1–10. doi: 10.1136/jmedgenet-2020-107178 BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) J Med Genet clinical use until late 2018, although most cardiomyopathy patients were screened with additional genetic panels. New genetic panel (Copenhagen) The current genetic panel in Copenhagen consists of the following 62 genes: ACTC, ACTN2, ANKRD1, BAG3, CALR3, CASQ2, CAV3, CRYAB, CSRP3, CTF1, CTNNA3, DES, DMD, DNAJC19, DSC2, DSG2, DSP, DTNA, EMD, EYA4, FHL1, FHL2, FLNC, GATAD1, GLA, HCN4, ILK, JPH2, JUP, LAMA4, LAMP2, LDB3, LMNA, MIB1, MYBPC3, MYH6, MYH7, MYL2, MYL3, MYLK2, MYOZ2, MYPN, NEBL, NEXN, OBSCN, PKP2, PLN, PRDM16, PRKAG2, PSEN1, PSEN2, RBM20, SCN5A, SGCB, SGCD, SLC22A5, TAZ, TCAP, TMEM43, TMPO, TNNC1, TNNI3, TNNI3K, TNNT2, TPM1, TTN, TTR, VCL New genetic panel (Aarhus) The current genetic panel in Aarhus consists of the following 102 genes: ABCC9, ACTC1, ACTN2, AKAP9, ANK2, ANKRD1, ANO1, APOB, BAG3, BEST3, CACNA1C, CACNA2D1, CACNB2, CALM1, CALM2, CALM3, CASQ2, CAV3, CDH2, CRYAB, CSRP3, CTNNA3, DES, DMD, DNAJC19, DSC2, DSG2, DSP, DTNA, EMD, EYA4, FHL1, FHL2, FKTN, FLNC, FXN, GATA4, GLA, GPD1L, GAA, HCN4, JPH2, JUP, KCND3, KCNE1M KCNE2M KCNE3, KCNE5, KCNH2, KCNJ5, KCNJ8, KCNQ1, LAMA4, LAMP2, LDB3, LDLR, LMNA, MYBPC3, MYH6, MYH7, MYL2, MYL3, MYOZ2, MYPBC3, MYPN, NEBL, NEXN, PCSK9, PKP2, PLN, PRDM16, PRKAG2, PSEN1, PSEN2, PTPN11, RAF1, RANGRF, RBM20, RYR2, SCN10A, SCN1B, SCN2B, SCN3B, SCN4B, SCN5A, SGCD, SLC4AE, SNTA1, TAZ, TCAP, TMEM43, TMPO, TNNC1, TNNI3, TNNT2, TPM1, TRDN, TRPM4, TTN, TTR, VCL and ZBTB17. Statistical analyses Cox proportional hazards models were used to examine the relationship between baseline variables and the three studied outcomes which included 1) A composite outcome of occurrence of implantation of left ventricular assist device, heart transplantation or death (the combined outcome), 2) The occurrence of sustained ventricular tachycardia, ventricular fibrillation, sudden cardiac death, aborted sudden cardiac death or appropriate shock by implantable cardioverter defibrillator (the ventricular arrhythmia outcome), and 3) The occurrence of atrial fibrillation or fluttering. The timing to outcomes was defined as follow-up time from time of dilated cardiomyopathy diagnosis. Multivariable Cox proportional-hazards regression analyses were created by including pre-defined variables known to be clinically