Manuscript Details

Manuscript number IJC_2019_2491_R1

Title Filamin C variants are associated with a distinctive clinical and immunohistochemical arrhythmogenic cardiomyopathy phenotype

Article type Original clinical research study

Abstract Background: Pathogenic variants in the filamin C (FLNC) gene are associated with inherited cardiomyopathies including dilated cardiomyopathy with an arrhythmogenic phenotype. We evaluated FLNC variants in arrhythmogenic cardiomyopathy (ACM) and investigated the disease mechanism at a molecular level. Methods: 120 gene-elusive ACM patients who fulfilled diagnostic criteria for arrhythmogenic right ventricular cardiomyopathy (ARVC) were screened by whole exome sequencing. Fixed cardiac tissue from FLNC variant carriers who had died suddenly was investigated by histology and immunohistochemistry. Results: Novel or rare FLNC variants, four null and five variants of unknown significance, were identified in nine ACM probands (7.5%). In FLNC null variant carriers (including family members, n=16) Task Force diagnostic electrocardiogram repolarization/depolarization abnormalities were uncommon (19%), echocardiography was normal in 69%, while 56% had >500 ventricular ectopics/24 hours or ventricular tachycardia on Holter and 67% had late gadolinium enhancement (LGE) on cardiac magnetic resonance imaging (CMRI). Ten gene positive individuals (63%) had abnormalities on ECG or CMRI that are not included in the current diagnostic criteria for ARVC. Immunohistochemistry showed altered key protein distribution, distinctive from that observed in ARVC, predominantly in the left ventricle. Conclusions: ACM associated with FLNC variants presents with a distinctive phenotype characterized by Holter arrhythmia and LGE on CMRI with unremarkable ECG and echocardiographic findings. Clinical presentation in asymptomatic mutation carriers at risk of sudden death may include abnormalities which are currently non-diagnostic for ARVC. At the molecular level, the pathogenic mechanism related to FLNC appears different to classic forms of ARVC caused by desmosomal mutations.

Keywords Arrhythmogenic cardiomyopathy; ARVC; filamin C variants; immunohistochemistry; late gadolinium enhancement

Taxonomy Myocardial Disease, Genetics, Arrhythmia

Manuscript category Original clinical research studies, basic science/translational research papers

Manuscript region of origin Europe

Corresponding Author PETROS SYRRIS

Corresponding Author's UNIVERSITY COLLEGE LONDON Institution

Order of Authors Charlotte Hall, Mohammed Akhtar, Maria Sabater-Molina, Marta Futema, Angeliki Asimaki, Alexandros Protonotarios, Chrysoula Dalageorgou, Alan Pittman, Mari Paz Suarez, Beatriz Aguilera, Pilar Molina, ESTHER ZORIO, Juan Pedro Hernandez del Rincon, francisco pastor quirante, Juan Gimeno, PETROS SYRRIS, William McKenna

Suggested reviewers Kalliopi Pilichou, Lorenzo Monserrat Highlights

 FLNC-linked ACM presents with LV involvement, ventricular arrhythmia, sudden

death

 Late gadolinium enhancement on cardiac MRI may be the only structural abnormality

 Mutation carriers have abnormalities on ECG or cardiac MRI non-diagnostic for ARVC

 FLNC disease mechanism different to classic ARVC caused by desmosomal mutations ABSTRACT

Background: Pathogenic variants in the filamin C (FLNC) gene are associated with inherited cardiomyopathies including dilated cardiomyopathy with an arrhythmogenic phenotype. We evaluated FLNC variants in arrhythmogenic cardiomyopathy (ACM) and investigated the disease mechanism at a molecular level.

Methods: 120 gene-elusive ACM patients who fulfilled diagnostic criteria for arrhythmogenic right ventricular cardiomyopathy (ARVC) were screened by whole exome sequencing. Fixed cardiac tissue from FLNC variant carriers who had died suddenly was investigated by histology and immunohistochemistry.

Results: Novel or rare FLNC variants, four null and five variants of unknown significance, were identified in nine ACM probands (7.5%). In FLNC null variant carriers (including family members, n=16) Task Force diagnostic electrocardiogram repolarization/depolarization abnormalities were uncommon (19%), echocardiography was normal in 69%, while 56% had

>500 ventricular ectopics/24 hours or ventricular tachycardia on Holter and 67% had late gadolinium enhancement (LGE) on cardiac magnetic resonance imaging (CMRI). Ten gene positive individuals (63%) had abnormalities on ECG or CMRI that are not included in the current diagnostic criteria for ARVC. Immunohistochemistry showed altered key protein distribution, distinctive from that observed in ARVC, predominantly in the left ventricle.

Conclusions: ACM associated with FLNC variants presents with a distinctive phenotype characterized by Holter arrhythmia and LGE on CMRI with unremarkable ECG and echocardiographic findings. Clinical presentation in asymptomatic mutation carriers at risk of sudden death may include abnormalities which are currently non-diagnostic for ARVC. At the molecular level, the pathogenic mechanism related to FLNC appears different to classic forms of ARVC caused by desmosomal mutations. Filamin C variants are associated with a distinctive clinical and

immunohistochemical arrhythmogenic cardiomyopathy phenotype

Charlotte L. Halla, Mohammed M. Akhtara, Maria Sabater-Molinab, Marta Futemaa, Angeliki

Asimakic, Alexandros Protonotariosa, Chrysoula Dalageorgoua, Alan M. Pittmand, Mari Paz

Suareze, Beatriz Aguilerae, Pilar Molinaf, Esther Zoriog, Juan Pedro Hernándezh, Francisco

Pastori, Juan R. Gimenoj, Petros Syrrisa*, William J. McKennaa

a Centre for Heart Muscle Disease, Institute of Cardiovascular Science, University College

London, London, UK b Laboratorio de Cardiogenética. Instituto Murciano de Investigación Biosanitaria and

Universidad de Murcia, Murcia, Spain c Cardiology Clinical Academic Group, Molecular and Clinical Sciences Research Institute,

St Georges University of London, London, UK d Molecular and Clinical Sciences Research Institute, St Georges University of London,

London, UK e Instituto Nacional de Toxicologia y Ciencias Forenses de Madrid (INTCF), Madrid, Spain f Department of Pathology at the Instituto de Medicina Legal y Ciencias Forenses de Valencia

(IMLCF-Valencia), Histology Unit at the Universitat de València, and Research Group on

Inherited Heart Diseases, Sudden Death and Mechanisms of Disease (CaFaMuSMe) from the

Instituto de Investigación Sanitaria (IIS) La Fe, Valencia, Spain g Cardiology Department at Hospital Universitario y Politécnico La Fe and Research Group on

Inherited Heart Diseases, Sudden Death and Mechanisms of Disease (CaFaMuSMe) from the

Instituto de Investigación Sanitaria (IIS) La Fe, Valencia, Spain h Instituto de Medicina Legal de Murcia (IML-Murcia), Murcia, Spain i Servicio de Cardiologia del Hospital Universitario Virgen de la Arrixaca and Departamento de Medicina Interna de la Universidad de Murcia, Murcia, Spain j Hospital Universitario Virgen de la Arrixaca, Murcia, Spain

*Corresponding author:

Petros Syrris, PhD

Centre for Heart Muscle Disease, Institute of Cardiovascular Science, University College

London, London, WC1E 6DD, UK

Tel: +44 207 679 6464; Fax: +44 207 679 6463. Email: [email protected] 1 2 3 4 Filamin C variants are associated with a distinctive clinical and 5 6 immunohistochemical arrhythmogenic cardiomyopathy phenotype 7 8 9 10 11 Charlotte L. Halla, Mohammed M. Akhtara, Maria Sabater-Molinab, Marta Futemaa, Angeliki 12 13 Asimakic, Alexandros Protonotariosa, Chrysoula Dalageorgoua, Alan M. Pittmand, Mari Paz 14 15 Suareze, Beatriz Aguilerae, Pilar Molinaf, Esther Zoriog, Juan Pedro Hernándezh, Francisco 16 17 i j a a 18 Pastor , Juan R. Gimeno , Petros Syrris *, William J. McKenna 19 20 21 22 23 a Centre for Heart Muscle Disease, Institute of Cardiovascular Science, University College 24 25 London, London, UK 26 27 28 “The author takes responsibility for all aspects of the reliability and freedom from bias of the 29 30 data presented and their discussed interpretation” 31 32 b 33 Laboratorio de Cardiogenética. Instituto Murciano de Investigación Biosanitaria and 34 35 Universidad de Murcia, Murcia, Spain 36 37 38 “The author takes responsibility for all aspects of the reliability and freedom from bias of the 39 40 data presented and their discussed interpretation” 41 42 c Cardiology Clinical Academic Group, Molecular and Clinical Sciences Research Institute, 43 44 45 St Georges University of London, London, UK 46 47 48 “The author takes responsibility for all aspects of the reliability and freedom from bias of the 49 50 data presented and their discussed interpretation” 51 52 53 54 55 56 57 58 1 59 60 61 62 d Molecular and Clinical Sciences Research Institute, St Georges University of London, 63 64 65 London, UK 66 67 “The author takes responsibility for all aspects of the reliability and freedom from bias of the 68 69 data presented and their discussed interpretation” 70 71 72 e Instituto Nacional de Toxicologia y Ciencias Forenses de Madrid (INTCF), Madrid, Spain 73 74 75 “The author takes responsibility for all aspects of the reliability and freedom from bias of the 76 77 data presented and their discussed interpretation” 78 79 80 f Department of Pathology at the Instituto de Medicina Legal y Ciencias Forenses de Valencia 81 82 (IMLCF-Valencia), Histology Unit at the Universitat de València, and Research Group on 83 84 Inherited Heart Diseases, Sudden Death and Mechanisms of Disease (CaFaMuSMe) from the 85 86 Instituto de Investigación Sanitaria (IIS) La Fe, Valencia, Spain 87 88 89 “The author takes responsibility for all aspects of the reliability and freedom from bias of the 90 91 data presented and their discussed interpretation” 92 93 94 g Cardiology Department at Hospital Universitario y Politécnico La Fe and Research Group on 95 96 Inherited Heart Diseases, Sudden Death and Mechanisms of Disease (CaFaMuSMe) from the 97 98 Instituto de Investigación Sanitaria (IIS) La Fe, Valencia, Spain 99 100 101 “The author takes responsibility for all aspects of the reliability and freedom from bias of the 102 103 data presented and their discussed interpretation” 104 105 106 h Instituto de Medicina Legal de Murcia (IML-Murcia), Murcia, Spain 107 108 109 “The author takes responsibility for all aspects of the reliability and freedom from bias of the 110 111 data presented and their discussed interpretation” 112 113 114 115 116 117 2 118 119 120 121 i Servicio de Cardiologia del Hospital Universitario Virgen de la Arrixaca and Departamento 122 123 124 de Medicina Interna de la Universidad de Murcia, Murcia, Spain 125 126 127 “The author takes responsibility for all aspects of the reliability and freedom from bias of the 128 129 data presented and their discussed interpretation” 130 131 132 j Hospital Universitario Virgen de la Arrixaca, Murcia, Spain 133 134 135 “The author takes responsibility for all aspects of the reliability and freedom from bias of the 136 137 data presented and their discussed interpretation” 138 139 140 141 142 143 *Corresponding author: 144 145 146 Petros Syrris, PhD 147 148 Centre for Heart Muscle Disease, Institute of Cardiovascular Science, University College 149 150 151 London, London, WC1E 6DD, UK 152 153 Tel: +44 207 679 6464; Fax: +44 207 679 6463. Email: [email protected] 154 155 156 157 158 159 Funding 160 161 This study was funded by Fondation Leducq Transatlantic Networks of Excellence Program 162 163 164 grant no 14CVD03 and supported by the National Institute for Health Research University 165 166 College London Hospitals Biomedical Research Centre in the UK. In Spain, the study was 167 168 supported by Plan Estatal de I+D+I 2013-2016 – European Regional Development Fund 169 170 (FEDER) “A way of making Europe”, Instituto de Salud Carlos III , Spain [PI14/01477, 171 172 173 174 175 176 3 177 178 179 180 PI18/0158 and La Fe Biobank PT17/0015/0043 to E.Z. and PI14/01676, PI18/01231 and the 181 182 183 BioBank “Biobanco en Red de la Región de Murcia” (PT17/0015/0038) to J.R.G]. 184 185 Declarations of interest: None 186 187 188 Keywords: Arrhythmogenic cardiomyopathy; ARVC; filamin C variants; 189 190 immunohistochemistry; late gadolinium enhancement 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 4 236 237 238 239 ABSTRACT 240 241 242 Background: Pathogenic variants in the filamin C (FLNC) gene are associated with inherited 243 244 cardiomyopathies including dilated cardiomyopathy with an arrhythmogenic phenotype. We 245 246 evaluated FLNC variants in arrhythmogenic cardiomyopathy (ACM) and investigated the 247 248 249 disease mechanism at a molecular level. 250 251 Methods: 120 gene-elusive ACM patients who fulfilled diagnostic criteria for arrhythmogenic 252 253 right ventricular cardiomyopathy (ARVC) were screened by whole exome sequencing. Fixed 254 255 256 cardiac tissue from FLNC variant carriers who had died suddenly was investigated by histology 257 258 and immunohistochemistry. 259 260 Results: Novel or rare FLNC variants, four null and five variants of unknown significance, 261 262 263 were identified in nine ACM probands (7.5%). In FLNC null variant carriers (including family 264 265 members, n=16) Task Force diagnostic electrocardiogram repolarization/depolarization 266 267 abnormalities were uncommon (19%), echocardiography was normal in 69%, while 56% had 268 269 >500 ventricular ectopics/24 hours or ventricular tachycardia on Holter and 67% had late 270 271 gadolinium enhancement (LGE) on cardiac magnetic resonance imaging (CMRI). Ten gene 272 273 positive individuals (63%) had abnormalities on ECG or CMRI that are not included in the 274 275 current diagnostic criteria for ARVC. Immunohistochemistry showed altered key protein 276 277 distribution, distinctive from that observed in ARVC, predominantly in the left ventricle. 278 279 280 Conclusions: ACM associated with FLNC variants presents with a distinctive phenotype 281 282 characterized by Holter arrhythmia and LGE on CMRI with unremarkable ECG and 283 284 echocardiographic findings. Clinical presentation in asymptomatic mutation carriers at risk of 285 286 287 sudden death may include abnormalities which are currently non-diagnostic for ARVC. At the 288 289 molecular level, the pathogenic mechanism related to FLNC appears different to classic forms 290 291 of ARVC caused by desmosomal mutations. 292 293 294 5 295 296 297 298 1. Introduction 299 300 301 Efforts to improve early detection of individuals at risk of life threatening arrhythmia from 302 303 inherited cardiovascular disease are ongoing [1]. Identification of patients who present with 304 305 arrhythmias independent of or not explained by recognised causes of cardiac disease has led to 306 307 308 the proposal for the term arrhythmogenic cardiomyopathy (ACM) [2-4]. The recognition of a 309 310 number of inherited arrhythmogenic cardiomyopathies has led to the recent broader acceptance 311 312 of this term [1]. Incorporated within this classification are patients who present with ventricular 313 314 arrhythmia in association with right, left or biventricular disease. Arrhythmogenic Right 315 316 Ventricular Cardiomyopathy (ARVC) caused by mutations in desmosomal genes is the most 317 318 studied of the ACMs with well established clinical and pathological diagnostic criteria [5, 6]. 319 320 321 ACM with prominent left ventricle involvement can be indistinguishable from 322 323 arrhythmogenic forms of dilated cardiomyopathy (DCM) both clinically and genetically [7]. 324 325 The FLNC gene, encoding the major cardiac structural protein filamin C, has been implicated 326 327 in inherited forms of cardiomyopathy, including DCM [8]. Recent studies have reported 328 329 truncating variants in FLNC as the cause of DCM with life-threatening ventricular arrhythmia 330 331 332 [9, 10]. Clinical evaluation of FLNC mutation carriers and molecular characterization of 333 334 cardiac tissue from a patient carrying a truncating variant have shown a link between DCM and 335 336 ARVC, both at the clinical and cellular level [10]. However, to date, the causal role of FLNC 337 338 variants in ACM cases fulfilling Task Force diagnostic criteria for ARVC has not been directly 339 340 investigated. As a result, the clinical and molecular phenotype of FLNC-associated ACM is 341 342 currently unknown. 343 344 345 In this study, we investigate the clinical characteristics of FLNC mutation carriers in ACM 346 347 pedigrees in which the index cases fulfil current diagnostic criteria for ARVC. We also report 348 349 350 351 352 353 6 354 355 356 357 the distribution of key proteins in myocardial tissue with FLNC variants and compare it with 358 359 360 that observed in classic forms of the disorder. 361 362 2. Methods 363 364 365 2.1 Patient cohort 366 367 368 Genetic screening of 269 ACM patients by targeted next generation sequencing as described 369 370 by Lopes et al [11] identified a group of 120 index cases who were free of potentially 371 372 pathogenic variants in major genes associated with cardiomyopathy, arrhythmia and heart 373 374 failure syndromes. This gene-elusive cohort underwent whole exome sequencing in order to 375 376 identify novel causative ACM genes. 377 378 379 Patients were referred to the Inherited Cardiovascular Disease Unit at the Heart Hospital 380 381 (prior to 2014) and the Barts Cardiac Centre, St Bartholomew’s Hospital with a suspicion of 382 383 ACM or with a premature sudden cardiac death and/or known ACM in their family. Clinical 384 385 diagnosis of arrhythmogenic cardiomyopathy was based on the Task Force diagnostic criteria 386 387 388 [6]. Index cases included in this study had a diagnosis of definite or borderline ARVC based 389 390 on the fulfilment of the current Task Force diagnostic criteria for the disorder [6], or had a 391 392 possible diagnosis on account of a first-degree relative who died suddenly aged less than 35 393 394 years with a post mortem (PM) diagnosis of ARVC. Detailed clinical evaluation included 395 396 medical and family history, 12-lead electrocardiogram (ECG), signal averaged ECG (SAECG), 397 398 24-hour ambulatory ECG, standard 2D transthoracic echocardiogram and cardiac magnetic 399 400 resonance imaging (CMRI). When appropriate clinical phenotyping and genetic testing was 401 402 offered to extended relatives within pedigrees. 403 404 405 This study conforms with the ethical guidelines of the 1975 Declaration of Helsinki and has 406 407 received approval by the National Health Service (NHS) Ethics Committees (REC ID: 408 409 15/LO/0549, UK) and CEIC Hospital Virgen de la Arrixaca (Spain) and CEIC Hospital 410 411 412 7 413 414 415 416 Universitario y Politécnico La Fe (Spain). Informed written consent for inclusion in the study 417 418 419 was obtained from all participants or, in cases of minors or deceased individuals, from first- 420 421 degree family members. 422 423 2.2 Whole exome sequencing (WES) 424 425 426 In this study we performed whole exome sequencing on DNA samples from ACM index cases 427 428 and family members. DNA from whole blood, paraffin-embedded cardiac tissue and saliva 429 430 samples was extracted utilizing kits and protocols from Qiagen and DNA Genotek. Genomic 431 432 433 DNA was subjected to sample preparation as per the protocol recommended by Agilent for the 434 XT 435 SureSelect Target Enrichment for Illumina paired-end multiplexed sequencing method. 436 437 Targeted exonic regions were captured using the Agilent SureSelectXT Human Exon V5 438 439 following the manufacturer’s protocol. Enriched DNA libraries were exome sequenced on the 440 441 Illumina NextSeq500 platform as paired-end 75 base reads at a minimum of 30x coverage. 442 443 Bioinformatic analysis of WES data including copy number variation was based on an in-house 444 445 developed pipeline (Supplementary material, Appendix). 446 447 448 2.3 Immunohistochemistry 449 450 451 As cardiac tissue was not available from British patients screened by WES, in order to 452 453 determine the effects of FLNC mutations at the level of the intercalated disc, post mortem 454 455 cardiac tissue from patients with FLNC variants was sourced from a Spanish clinicopathology 456 457 consortium. Right and left ventricle (RV and LV) specimens from eleven sudden cardiac death 458 459 (SCD) victims with a diagnosis of arrhythmogenic cardiomyopathy carrying potentially 460 461 pathogenic FLNC variants were included in this study. Post-mortem (PM) examination 462 463 464 protocol was in keeping with published guidelines. The patients, all male, (aged 16–52, mean 465 466 age of death 33.5 years) had a diagnosis of ARVC or left dominant arrhythmogenic 467 468 cardiomyopathy at autopsy due to the presence of fibrosis and fat infiltration (nine cases) and 469 470 471 8 472 473 474 475 predominant fibrosis (3 cases). Cardiac samples from those cases were fixed in formalin and 476 477 478 preserved in paraffin blocks. Immunohistochemical analysis of key proteins previously 479 480 implicated in the molecular pathogenesis of classical ARVC in myocardial tissue was carried 481 482 out based on the protocol developed by Asimaki et al [12]. Detailed description of the method 483 484 is provided in the Appendix. Tissue samples from age-matched individuals with no clinical or 485 486 pathological evidence of heart disease were subjected to the same protocol and used as negative 487 488 controls (n=5). In summary, RV and LV specimens from each SCD case were stained for 489 490 filamin C, plakoglobin, desmoplakin, connexin 43, synapse-associated protein SAP97 and 491 492 glycogen synthase kinase 3β, GSK3β. 493 494 495 496 497 498 3. Results 499 500 501 3.1 FLNC variants 502 503 WES of a cohort of 120 gene-elusive ACM index cases identified seven novel and two rare 504 505 506 FLNC variants (7.5%). They include four null variants (three nonsense and one splice site 507 508 variant) which are predicted to be pathogenic based on the American College of Medical 509 510 Genetics and Genomics (ACMG) guidelines and five variants of unknown significance (VUS, 511 512 one in-frame deletion and four missense variants) [13]. Details of these variants are given in 513 514 Appendix Table A1. FLNC variants identified by WES were confirmed by Sanger DNA 515 516 sequencing. There were no FLNC copy number variants identified in the cohort. 517 518 519 520 521 522 3.2 Clinical phenotypes of FLNC variant carriers 523 524 Index cases carrying FLNC variants had a diagnosis of definite (n=4), borderline (n=2) or 525 526 possible (n=3) ARVC based on the Task Force diagnostic criteria [6]. All had at least one 527 528 529 530 9 531 532 533 534 sudden cardiac death victim in their extended families ranging from 20 to 71 years (median 535 536 537 40yrs) and ARVC or arrhythmogenic left ventricular cardiomyopathy (ALVC) was diagnosed 538 539 at PM in six deceased family members (Appendix Table A2). 540 541 Segregation analysis and cascade genetic screening with clinical evaluation of relatives in 542 543 544 pedigrees was feasible in the four families carrying the null FLNC variants (Figure 1). Detailed 545 546 clinical features for FLNC variant carriers in Families A-D are provided in Table 1. For the 547 548 VUS variants, pedigree analysis was possible in only two cases (Families E and F) which 549 550 carried the p.59_62DLQRdel and p.K2260R variants respectively (Appendix Figure A1). No 551 552 family members of index cases G, H and I were available. Clinical characteristics of FLNC 553 554 VUS carriers are given in Appendix Table A3. There was no evidence of skeletal muscle 555 556 abnormalities in ACM index cases or their relatives and serum creatine kinase levels were 557 558 normal in those tested. In addition to ACM index cases, another 26 relatives were clinically 559 560 561 evaluated and genotyped for FLNC variants; fourteen of them were genotype positive. DNA 562 563 from two SCD cases was available and those individuals were also found to be FLNC variants 564 565 carriers (Figures 1 and A1 and Tables 1 and A3). 566 567 568 Due to the limited clinical information on pedigrees with FLNC VUS variants and the 569 570 ambiguity regarding possible pathogenicity of such variants, analysis focused on the ACM 571 572 families with FLNC null variants (index cases and relatives, n=16). In this cohort the presence 573 574 of Task Force diagnostic ECG repolarization and depolarization abnormalities were 575 576 uncommon, n=2 (12.5%) and n=1 (6.25%) respectively. ECG was unremarkable in 5 genotype 577 578 positive individuals (31%), abnormalities in the remaining included low voltage 5/16 (32%) 579 580 and poor R wave progression across anterior chest leads 1/16 (6%). In the majority of cases 581 582 echocardiogram did not reveal overt abnormalities (n=11, 69%). Clinical presentation with 583 584 585 palpitation and/or syncope was uncommon, however, 7 patients (44%) had non sustained VT 586 587 and/or >500 VES/24 hours and 2 (12.5%) presented with sustained VT. Of 15 FLNC null 588 589 10 590 591 592 593 variant carriers who had cardiac MRI, the majority (n=10, 67%) showed late gadolinium 594 595 596 enhancement (LGE). Characteristic ECG and CMR images from a FLNC variant carrier are 597 598 shown in Figure 2. 599 600 601 602 603 3.3 Immunohistochemical analysis of fixed myocardial tissue 604 605 606 Histological examination of eleven cardiac specimens from sudden death victims (numbered 607 608 1 to 11, Appendix Table A4) carrying FLNC mutations was performed at post mortem. Mean 609 610 weight was 471.3 ± 58.9 g. All but two cases had normal internal left ventricular measurements 611 612 (mean LV diameter 37.4 ± 11.0 mm). Wall thickness was within normal limits in all hearts (11- 613 614 13 mm). 615 616 617 Evidence of fibrosis was present in both ventricles in ten samples; fibrofatty replacement 618 619 was evident in three samples whilst four specimens showed signs of fibrosis and inflammation, 620 621 mainly in the left ventricle. Distribution of fibrofatty infiltration in the left ventricle was 622 623 circumferential in 7 (mesocardial in 4 and subepicardial in 3) and inferolateral subepicardial in 624 625 626 4 cases. Infiltration which was predominant in the inferolateral wall, extended from the basal 627 628 to the apical segments. Inflammatory infiltrates affecting the left ventricle were multifocal in 629 630 2 and extensive in another 2. There was only one specimen (case no 9) with remarkable 631 632 inflammatory infiltrates in the right ventricle, which also had extensive left involvement. The 633 634 same sample had no evidence of fibrosis at PM, however, fat infiltration was present. 635 636 Characteristic histology images are shown in Appendix Figure A2. 637 638 639 Cardiac specimens used for immunohistochemistry experiments originated from ACM 640 641 patients with FLNC variants who had suffered sudden cardiac death. Those included three 642 643 deletions and eight single nucleotide substitutions (two splice site, four termination and two 644 645 646 647 648 11 649 650 651 652 missense variants). Two unrelated cases carried the same nonsense mutation: c.5398G>T; 653 654 655 p.G1800X. A list of FLNC variants in fixed tissue samples is given in Appendix Table A4. 656 657 Immunoreactive signal for filamin C was strong and indistinguishable from controls in RV 658 659 samples from ten ACM cases but it was found to be reduced in LV specimens from all eleven 660 661 cases. The signal for plakoglobin was strong and indistinguishable from controls in nine cases 662 663 664 in both RV and LV samples. Junctional signal for Cx43 was reduced in two RV samples and 665 666 six LV samples. Moreover, signal for the desmosomal protein desmoplakin was found to be 667 668 reduced at cell-cell junctions in eight RV samples and five LV samples. In contrast, GSK3β 669 670 was present in the cytosol in all FLNC cases examined as in control myocardium samples. 671 672 Finally, immunoreactive signal for SAP97 appeared reduced in the majority of RV and LV 673 674 samples whilst in two cases this protein was only detected in the sarcomere. Characteristic 675 676 confocal microscopy images from case no 6 are displayed in Figure 3. Immunohistochemistry 677 678 data from RV and LV specimens from all eleven cases are summarised in Appendix Figure 679 680 681 A3. 682 683 4. Discussion 684 685 686 For the first time we performed clinical characterization of FLNC variants in an ACM 687 688 cohort; previous studies have focused on pure DCM or DCM with an arrhythmogenic 689 690 component [8-10]. We observed marked phenotypic differences in ACM associated with FLNC 691 692 null variants compared to classic ARVC caused by desmosomal gene mutations. 693 694 695 Repolarization (e.g. T wave inversion) and depolarization (such as prolonged terminal 696 697 activation duration and epsilon waves) abnormalities are considered typical diagnostic features 698 699 for classic forms of ARVC [6]. However, in our FLNC cohort only three patients (19%) had 700 701 Task Force diagnostic ECG repolarization and depolarization abnormalities. In contrast, the 702 703 704 majority of FLNC null variant carriers (69%) had ECG repolarization and depolarization 705 706 707 12 708 709 710 711 abnormalities, such as right bundle branch block (RBBB) and loss of inferior R waves, which, 712 713 714 in isolation, are not considered diagnostic criteria [6]. Similarly, standard echocardiographic 715 716 imaging has been shown to detect structural abnormalities in the majority of ARVC patients 717 718 [14] but 69% of our cases had no detectable echocardiographic disease features. Moreover, the 719 720 most striking observation was that MRI showed the presence of LGE with preserved ventricular 721 722 function in all index cases and the majority of gene positive family members, a clinical feature 723 724 which is not currently a diagnostic criterion for ARVC [6]. Consequently, in these cases, strict 725 726 adherence to the Task Force diagnostic criteria, can lead to individuals at risk being incorrectly 727 728 classified as either unaffected or being at low risk of complications. This highlights the 729 730 731 importance of genetic evaluation of asymptomatic family members and the need of more 732 733 detailed phenotyping targeting recognized features of particular subtypes of ACM. In this study 734 735 familial evaluation limited to ECG and echocardiography would not have identified the 736 737 majority of at-risk individuals whereas significant abnormalities were detected with ECG 738 739 monitoring and CMRI, for example individuals IV:1 (Family A) and II:1 and III:4 (Family C). 740 741 742 In summary, we present the clinical phenotype of FLNC families with index cases who fulfil 743 744 Task Force diagnostic criteria for ARVC. It is characterized by predominant LV involvement; 745 746 frequently non-diagnostic electrocardiography and echocardiography; frequent ventricular 747 748 ectopy or non-sustained VT on 24-hour Holter monitoring and fibrosis (late gadolinium 749 750 enhancement) on MRI. Notably there is a high incidence of adverse cardiovascular events, 751 752 highlighted in our cohort with a family history of multiple sudden cardiac death victims at a 753 754 755 young age. This clinical pattern appears similar to emerging experience of other genetically 756 757 determined arrhythmogenic cardiomyopathies caused by mutations in TMEM43, 758 759 phospholamban, desmin and lamin A/C [15-18]. All may present with life threatening 760 761 arrhythmia, myocardial structural abnormalities, usually predominantly of the left ventricle, 762 763 though patients have been reported who fulfil Task Force diagnostic criteria. This highlights 764 765 766 13 767 768 769 770 the need for evolution of the current classification with use of the term ‘arrhythmogenic 771 772 773 cardiomyopathy’ which incorporates ARVC as well as other inherited and acquired forms of 774 775 ACM. The recent Heart Rhythm Society guidelines for the diagnosis and management of 776 777 arrhythmogenic cardiomyopathy recognize this evolving scenario [1]. 778 779 780 Previous studies have highlighted the importance of immunohistochemistry of cardiac tissue 781 782 from mutation carriers in investigating the disease mechanisms related to ACM [19, 20]. In 783 784 this study, we sought to characterize the molecular profile of fixed RV and LV specimens from 785 786 ACM sudden death cases. It has been previously shown that filamin C displays a strong 787 788 localisation at the intercalated disc that decreases or is completely absent in patients with 789 790 restrictive and dilated cardiomyopathy carrying FLNC mutations [8, 21]. Here, staining for 791 792 FLNC showed a decreased immunoreactive signal intensity in the left ventricle in all eleven 793 794 ACM cases highlighting a predominant left ventricle disease pattern associated with mutations 795 796 797 in this gene. This is consistent with a recent study that reported reduced immunohistochemical 798 799 staining signal for filamin C in left ventricle samples from an arrhythmogenic DCM patient 800 801 who carried the p.G1891Vfs61X mutation [10]. 802 803 804 It is now well established that in ARVC plakoglobin translocates from the intercalated discs 805 806 to intracellular pools [19], an observation that is considered as a “hallmark” of disease 807 808 pathogenesis [22]. However, the signal for plakoglobin was strong and indistinguishable from 809 810 controls in nine cases (82%) examined in our FLNC cohort in both left and right ventricular 811 812 samples. Interestingly, signal for plakoglobin was decreased in both RV and LV specimens 813 814 carrying the two missense variants (p.K35N and p.T160K). However, at present, the 815 816 significance of this finding is unclear. Similar to plakoglobin mislocalization, the enzyme 817 818 GSK3β is re-distributed from the cytosol to the intercalated disc in classic ARVC [23]. 819 820 821 However, none of the eleven FLNC cases examined showed this re-distribution. 822 823 824 825 14 826 827 828 829 Immunoreactive signal for the major gap junction protein Cx43 is usually significantly 830 831 832 depressed at cardiac intercalated discs in patients with ARVC [23]. In our FLNC cohort, 833 834 junctional signal for Cx43 was reduced only in two (20%) RV and six LV (60%) cases. 835 836 Considering the advanced disease state and predominant LV involvement in all our FLNC 837 838 cases, it is unclear whether Cx43 remodelling played a primary role in ACM pathogenesis or 839 840 was a result of the histological changes in the myocardium. The signal for the desmosomal 841 842 protein desmoplakin was found to be reduced at cell-cell junctions mainly in RV samples 843 844 (80%) and when DSP staining intensity was reduced in the left ventricle, the corresponding 845 846 RV signal for the same case was also reduced. Altered desmoplakin localisation has been 847 848 849 reported in left dominant arrhythmogenic cardiomyopathy [24] and, as our FLNC cases had a 850 851 predominantly left dominant pattern of disease, the observed reduction of desmoplakin signal 852 853 is in line with this phenotype. 854 855 856 SAP97 is a membrane-associated guanylate kinase reported to show consistently decreased 857 858 immunohistochemistry staining intensity in both the sarcomeric and junctional pools in the 859 860 myocardium of desmosomal ACM patients independently of the specific causative mutation 861 862 [25]. In our cohort SAP97 was reduced in the majority of RV and LV specimens whilst in two 863 864 cases SAP97 signal was detectable in the sarcomeres but not at the intercalated discs. This 865 866 finding is consistent with previous reports on myocardial samples from patients with end-stage 867 868 ischemic, dilated and hypertrophic cardiomyopathy [25]. 869 870 871 Overall, we observed a specific localization pattern in our cohort for three proteins: FLNC 872 873 signal was reduced in all cases; plakoglobin signal was normal in the large majority of RV and 874 875 LV specimens and GSK3β signal was normal in all cases tested. All these findings point to a 876 877 disease pattern different to classic ARVC and are consistent with the hypothesis that ACM 878 879 880 associated with FLNC variants presents with a left dominant arrhythmogenic cardiomyopathy 881 882 phenotype which may manifest via a different mechanism to typical right ventricular 883 884 15 885 886 887 888 arrhythmogenic cardiomyopathy. Begay et al 2018 observed similar immunohistochemistry 889 890 891 results to our data for a patient with the p.G1891Vfs61X FLNC mutation, namely normal 892 893 plakoglobin immunostaining signal, typical GSK3β cytoplasmic distribution and reduced DSP 894 895 signal compared to control samples [10]. 896 897 898 Collectively, our analysis suggests that the clinical and molecular “signature’ of FLNC 899 900 cardiomyopathy is distinct to that of ARVC. These results indicate that these two clinical 901 902 entities reflect different molecular mechanisms of pathogenesis. 903 904 905 906 907 4.1 Limitations 908 909 910 This study is limited by the small number of recruited FLNC variant carriers which, in part, 911 912 is attributed to the low frequency of causative FLNC variants in ACM and the high genetic 913 914 heterogeneity that characterizes the disorder. As it is common in studies of cardiomyopathy 915 916 patients, small family sizes have restricted our ability to perform extensive segregation analysis 917 918 919 in all FLNC cases. 920 921 Immunohistochemical analysis relied on the availability of paraffin fixed tissue. The 922 923 challenges in collecting human heart samples are well known. Therefore, this study was also 924 925 926 hindered by limited quantity of tissue for each case and some samples were not immunostained 927 928 for a complete set of proteins. 929 930 5. Conclusion 931 932 933 ACM related to FLNC variants presents with a distinctive phenotype that may not be 934 935 recognized by current Task Force ARVC diagnostic criteria or by familial evaluation limited 936 937 to ECG or echocardiography. Physicians should be aware of “non-diagnostic” disease features 938 939 in asymptomatic gene positive individuals. 940 941 942 943 16 944 945 946 947 The molecular mechanism of pathogenesis of this form of ACM is markedly different to 948 949 950 classic ARVC and does not involve mislocalization of plakoglobin or GSK3β. 951 952 953 954 955 956 957 958 959 960 961 962 963 964 965 966 967 968 969 970 971 972 973 974 975 976 977 978 979 980 981 982 983 984 985 986 987 988 989 990 991 992 993 994 995 996 997 998 999 1000 1001 1002 17 1003 1004 1005 1006 References 1007 1008 1009 [1] Towbin JA, McKenna WJ, Abrams DJ, Ackerman MJ, Calkins H, Darrieux FCC, et al. 2019 HRS 1010 Expert Consensus Statement on Evaluation, Risk Stratification, and Management of Arrhythmogenic 1011 Cardiomyopathy. Heart Rhythm. (2019). 1012 [2] Sekiguchi M, Kinoshita O. [From arrhythmogenic right ventricular 1013 dysplasia/cardiomyopathy(ARVD/ARVC) to a broader concept of ABCDE syndrome]. Nihon Rinsho. 58 1014 (2000) 108-116. 1015 [3] Basso C, Corrado D, Thiene G. Arrhythmogenic right ventricular cardiomyopathy: what's in a 1016 name? From a congenital defect (dysplasia) to a genetically determined cardiomyopathy 1017 (dystrophy). Am J Cardiol. 106 (2010) 275-277. 1018 [4] Basso C, Bauce B, Corrado D, Thiene G. Pathophysiology of arrhythmogenic cardiomyopathy. Nat 1019 1020 Rev Cardiol. 9 (2011) 223-233. 1021 [5] Corrado D, Basso C, Judge DP. Arrhythmogenic Cardiomyopathy. Circ Res. 121 (2017) 784-802. 1022 [6] Marcus FI, McKenna WJ, Sherrill D, Basso C, Bauce B, Bluemke DA, et al. Diagnosis of 1023 arrhythmogenic right ventricular cardiomyopathy/dysplasia: proposed modification of the task force 1024 criteria. Circulation. 121 (2010) 1533-1541. 1025 [7] Marian AJ, van Rooij E, Roberts R. Genetics and Genomics of Single-Gene Cardiovascular 1026 Diseases: Common Hereditary Cardiomyopathies as Prototypes of Single-Gene Disorders. J Am Coll 1027 Cardiol. 68 (2016) 2831-2849. 1028 [8] Begay RL, Tharp CA, Martin A, Graw SL, Sinagra G, Miani D, et al. FLNC Gene Splice Mutations 1029 Cause Dilated Cardiomyopathy. JACC Basic Transl Sci. 1 (2016) 344-359. 1030 [9] Ortiz-Genga MF, Cuenca S, Dal Ferro M, Zorio E, Salgado-Aranda R, Climent V, et al. Truncating 1031 FLNC Mutations Are Associated With High-Risk Dilated and Arrhythmogenic Cardiomyopathies. J Am 1032 Coll Cardiol. 68 (2016) 2440-2451. 1033 [10] Begay RL, Graw SL, Sinagra G, Asimaki A, Rowland TJ, Slavov DB, et al. Filamin C Truncation 1034 Mutations Are Associated With Arrhythmogenic Dilated Cardiomyopathy and Changes in the Cell- 1035 Cell Adhesion Structures. JACC Clin Electrophysiol. 4 (2018) 504-514. 1036 [11] Lopes LR, Zekavati A, Syrris P, Hubank M, Giambartolomei C, Dalageorgou C, et al. Genetic 1037 complexity in hypertrophic cardiomyopathy revealed by high-throughput sequencing. J Med Genet. 1038 50 (2013) 228-239. 1039 [12] Asimaki A, Protonotarios A, James CA, Chelko SP, Tichnell C, Murray B, et al. Characterizing the 1040 Molecular Pathology of Arrhythmogenic Cardiomyopathy in Patient Buccal Mucosa Cells. Circ 1041 Arrhythm Electrophysiol. 9 (2016) e003688. 1042 [13] Richards S, Aziz N, Bale S, Bick D, Das S, Gastier-Foster J, et al. Standards and guidelines for the 1043 interpretation of sequence variants: a joint consensus recommendation of the American College of 1044 Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med. 17 (2015) 1045 405-424. 1046 [14] Yoerger DM, Marcus F, Sherrill D, Calkins H, Towbin JA, Zareba W, et al. Echocardiographic 1047 findings in patients meeting task force criteria for arrhythmogenic right ventricular dysplasia: new 1048 insights from the multidisciplinary study of right ventricular dysplasia. J Am Coll Cardiol. 45 (2005) 1049 860-865. 1050 1051 [15] Merner ND, Hodgkinson KA, Haywood AF, Connors S, French VM, Drenckhahn JD, et al. 1052 Arrhythmogenic right ventricular cardiomyopathy type 5 is a fully penetrant, lethal arrhythmic 1053 disorder caused by a missense mutation in the TMEM43 gene. Am J Hum Genet. 82 (2008) 809-821. 1054 [16] van der Zwaag PA, van Rijsingen IA, Asimaki A, Jongbloed JD, van Veldhuisen DJ, Wiesfeld AC, et 1055 al. Phospholamban R14del mutation in patients diagnosed with dilated cardiomyopathy or 1056 arrhythmogenic right ventricular cardiomyopathy: evidence supporting the concept of 1057 arrhythmogenic cardiomyopathy. Eur J Heart Fail. 14 (2012) 1199-1207. 1058 1059 1060 1061 18 1062 1063 1064 1065 [17] van Tintelen JP, Van Gelder IC, Asimaki A, Suurmeijer AJ, Wiesfeld AC, Jongbloed JD, et al. 1066 Severe cardiac phenotype with right ventricular predominance in a large cohort of patients with a 1067 1068 single missense mutation in the DES gene. Heart Rhythm. 6 (2009) 1574-1583. 1069 [18] Quarta G, Syrris P, Ashworth M, Jenkins S, Zuborne Alapi K, Morgan J, et al. Mutations in the 1070 Lamin A/C gene mimic arrhythmogenic right ventricular cardiomyopathy. Eur Heart J. 33 (2012) 1071 1128-1136. 1072 [19] Asimaki A, Tandri H, Huang H, Halushka MK, Gautam S, Basso C, et al. A new diagnostic test for 1073 arrhythmogenic right ventricular cardiomyopathy. N Engl J Med. 360 (2009) 1075-1084. 1074 [20] Fidler LM, Wilson GJ, Liu F, Cui X, Scherer SW, Taylor GP, et al. Abnormal connexin43 in 1075 arrhythmogenic right ventricular cardiomyopathy caused by plakophilin-2 mutations. J Cell Mol Med. 1076 13 (2009) 4219-4228. 1077 [21] Tucker NR, McLellan MA, Hu D, Ye J, Parsons VA, Mills RW, et al. Novel Mutation in FLNC 1078 (Filamin C) Causes Familial Restrictive Cardiomyopathy. Circ Cardiovasc Genet. 10 (2017). 1079 [22] Asimaki A, Kleber AG, Saffitz JE. Pathogenesis of Arrhythmogenic Cardiomyopathy. Can J Cardiol. 1080 31 (2015) 1313-1324. 1081 [23] Asimaki A, Saffitz JE. Remodeling of cell-cell junctions in arrhythmogenic cardiomyopathy. Cell 1082 Commun Adhes. 21 (2014) 13-23. 1083 [24] Kaplan SR, Gard JJ, Carvajal-Huerta L, Ruiz-Cabezas JC, Thiene G, Saffitz JE. Structural and 1084 molecular pathology of the heart in Carvajal syndrome. Cardiovasc Pathol. 13 (2004) 26-32. 1085 [25] Asimaki A, Kapoor S, Plovie E, Karin Arndt A, Adams E, Liu Z, et al. Identification of a new 1086 modulator of the intercalated disc in a zebrafish model of arrhythmogenic cardiomyopathy. Sci 1087 Transl Med. 6 (2014) 240ra274. 1088 1089 1090 1091 1092 1093 1094 1095 1096 1097 1098 1099 1100 1101 1102 1103 1104 1105 1106 1107 1108 1109 1110 1111 1112 1113 1114 1115 1116 1117 1118 1119 1120 19 1121 1122 1123 1124 Figure legends 1125 1126 1127 1128 Fig. 1. Pedigrees of ACM families with FLNC variants. 1129 1130 1131 Squares indicate males; circles, females; slashes, deceased individuals; black symbols, definite 1132 1133 diagnosis of ARVC based on current Task Force diagnostic criteria [6] or evidence of ARVC 1134 1135 at post mortem; grey symbols, borderline diagnosis of ARVC; hatched symbols, gene positive 1136 1137 individuals with possible diagnosis of ARVC; (+), positive genotype for FLNC variant; (-), 1138 1139 negative genotype for FLNC variant; Arrows indicate the index case in each family. ALVC, 1140 1141 arrhythmogenic left ventricular cardiomyopathy; PM, post mortem; SCD, sudden cardiac 1142 1143 death. Gene negative individuals with a possible diagnosis of ARVC based solely on family 1144 1145 1146 history are depicted as unaffected. 1147 1148 1149 1150 1151 Fig. 2. ECG and cardiac MR images from individual IV:1 (Family A) who was clinically 1152 1153 screened due to family history of ACM. 1154 1155 1156 A). Electrocardiogram showing inferior lead T-wave inversion (III and aVF). 1157 1158 1159 B). CMRI two-chamber view of the left ventricle (left image) and short axis view (right image) 1160 1161 showing basal lateral subepicardial late gadolinium enhancement (white arrows). 1162 1163 1164 1165 1166 Fig. 3. Immunohistochemistry staining of paraffin-embedded cardiac specimens from case no 1167 1168 6 carrying the p.Y705X FLNC variant. 1169 1170 1171 Top panel, control sample; middle panel, RV sample; bottom panel, LV sample. 1172 1173 Immunoreactive signal for plakoglobin and GSK3β at the intercalated discs appear normal 1174 1175 compared control samples. Signal for FLNC appears normal in the RV but reduced in the LV. 1176 1177 1178 1179 20 1180 1181 1182 1183 Signal for connexin 43, desmoplakin, and SAP97 is reduced in both RV and LV. N-cadherin 1184 1185 1186 is used as a marker of tissue quality and is normal in all specimens. 1187 1188 1189 1190 1191 1192 1193 1194 1195 1196 1197 1198 1199 1200 1201 1202 1203 1204 1205 1206 1207 1208 1209 1210 1211 1212 1213 1214 1215 1216 1217 1218 1219 1220 1221 1222 1223 1224 1225 1226 1227 1228 1229 1230 1231 1232 1233 1234 1235 1236 1237 1238 21 1239 1240 1241 1242 Table 1 1243 1244 1245 Clinical characteristics of FLNC null variant carriers in families A-D. 1246 1247 Family - FLNC NYHA class / Age Sex ECG Echocardiography Arrhythmia CMRI Task Force 1248 Individual Genotype Symptoms Diagnostic 1249 Criteria 1250 (m, M) / Diagnosis 1251 1252 Description LVEF 24 hr VE RV RVEF (%) LV LVEF LGE 1253 count / type EDV EDV (%) distribution (%) of arrhythmia (ml) (ml) 1254 1255 Family A p.Arg991X NYHA II 76 F Permanent AF, Borderline LV 50 19,248 146 49 122 41 Basal lateral 2m and 1M 1256 II:2 low QRS voltage dilatation with LGE Non- Definite 1257 in limb leads mild LV systolic (index dysfunction sustained VT 1258 case) Late potentials 1259 Family A p.Arg991X Asymptomatic 44 F Low QRS Normal 55-60 12,935 Normal Normal 172 52 Normal / No 1m and 1M 1260 III:2 voltage in limb biventricular LGE 1261 leads size and Borderline 1262 function 1263 1264 Family A p.Arg991X Palpitations 20 F T-wave Normal 55-60 69 212 54 206 59 Basal lateral 2m 1265 IV:1 inversion biventricular sub-epicardial inferior leads size and Sustained VT LGE Possible 1266 function presentation, 1267 RBBB 1268 morphology 1269 Family A p.Arg991X Asymptomatic 18 F Unremarkable Normal 55-60 1 168 58 184 56 Normal / No 1m 1270 IV:2 biventricular LGE 1271 size and Unaffected 1272 function 1273 1274 1275 1276 1277 1278 22 1279 1280 1281 1282 1283 Family B c.7252-1G>A Presyncope 64 M Incomplete Normal 55-60 535 2m 1284 II:1 RBBB biventricular 1285 size and Possible 1286 function 1287 1288 Family B c.7252-1G>A Asymptomatic 62 M Low QRS Normal 55-60 22 210 61 209 65 Sub-epicardial 1M* II:2 voltage biventricular LGE basal-mid 1289 size and anterolateral Possible 1290 (index function and 1291 case) inferolateral 1292 walls

1293 Family C p.L1573X Asymptomatic 76 M Loss of inferior Normal 59 0 147 67 156 64 Basal lateral None 1294 II:1 R waves biventricular epicardial LGE 1295 size and Unaffected 1296 function 1297 1298 Family C p.L1573X Syncope 54 M Low QRS Non-dilated LV 45-50 211 51 183 51 Extensive basal 1m and 1M III:1 voltage in limb with mild LV to mid sub- 1299 leads dysfunction endocardial LGE Borderline 1300 (index in the Sustained VT case) RV regional wall anterolateral 1301 of LBBB motion and 1302 morphology abnormality inferolateral 1303 with superior (dyskinetic walls 1304 RVOT and RV axis 1305 free wall) 1306 Family C p.L1573X Palpitations 50 F T wave Normal 60 166 Normal Normal 112 70 Equivocal basal 1m 1307 III:4 inversion V6 biventricular LGE 1308 size and Unaffected 1309 function 1310 1311 Family C p.L1573X Asymptomatic 19 M Unremarkable Normal 59 3 180 54 173 57 Normal / No None IV:1 biventricular LGE 1312 size and Unaffected 1313 function 1314 1315 1316 1317 1318 1319 23 1320 1321 1322 1323 1324 Family C p.L1573X Asymptomatic 26 F Poor R-wave Normal 60 0 141 64 161 60 Normal / No None 1325 IV:3 progression in biventricular LGE 1326 anterior leads size and Unaffected 1327 function 1328 1329 Family D p.Arg482X Asymptomatic 73 M Unremarkable Non-dilated LV 45 1,093 141 41 196 48 Circumferential 1m and 1M II:1 and mild LV basal LGE and 1330 dysfunction mid-inferior Borderline 1331 and 1332 inferolateral 1333 LGE

1334 Family D p.Arg482X NYHA II SCD M T-wave Dilated LV and 35 9,249 Normal normal 257 52 Extensive 2m and 1M 1335 II:4 71 inversion V6 severe LV inferior and 1336 dysfunction Non- inferolateral Definite sustained VT 1337 subepicardial basal LGE 1338 1339 Family D p.Arg482X Asymptomatic 46 F Unremarkable Normal 60-65 6 131 71 149 68 Normal / No 2m 1340 III:2 biventricular LGE Non- Possible 1341 size and function sustained VT 1342 1343 Family D p.Arg482X Asymptomatic 43 M Unremarkable Normal 60-65 3 161 57 146 68 Subtle streak of 1m 1344 III:3 biventricular non-ischaemic 1345 size and LGE in the basal Unaffected 1346 function inferolateral 1347 wall 1348 Family D p.Arg482X NYHA II 50 F Low QRS Borderline LV 40-45 5,197 227 62 187 62 Basal 1m and 1M 1349 III:6 voltage in dilatation with inferolateral Borderline 1350 precordial leads mild to and inferior (index moderate LV wall LGE 1351 case) systolic 1352 dysfunction 1353 1354 1355 AF, atrial fibrillation; CMRI, cardiac magnetic resonance imaging; SCD, sudden cardiac death; ECG, electrocardiogram; EDV, end diastolic 1356 1357 1358 volume; EF, ejection fraction; NYHA, New York Heart Association classification; LBBB, left bundle branch block; LGE, late gadolinium 1359 1360 24 1361 1362 1363 1364 1365 enhancement; LV, left ventricle; LVEF, left ventricular ejection fraction; LVIDD, end-diastolic internal dimension; m, minor Task Force ARVC 1366 1367 1368 diagnostic criterion; M, major Task Force ARVC diagnostic criterion; RBBB, right bundle branch block; RV, right ventricle; RVEF, right 1369 1370 ventricular ejection fraction; RVOT, right ventricle outflow tract; VE, ventricular ectopic; VT, ventricular tachycardia; * one major diagnostic 1371 1372 criterion due to family history. 1373 1374 1375 1376 1377 1378 1379 1380 1381 1382 1383 1384 1385 1386 1387 1388 1389 1390 1391 1392 1393 1394 1395 1396 1397 1398 1399 1400 1401 25 1402 1403

Author Agreement Form – International Journal of Cardiology

Manuscript Title: Filamin C variants are associated with a distinctive clinical and immunohistochemical arrhythmogenic cardiomyopathy phenotype

List of all Authors: Charlotte L. Hall, Mohammed M. Akhtar, Maria Sabater-Molina, Marta Futema, Angeliki Asimaki, Alexandros Protonotarios, Chrysoula Dalageorgou, Alan M. Pittman, Mari Paz Suarez, Beatriz Aguilera, Pilar Molina, Esther Zorio, Juan Pedro Hernández, Francisco Pastor, Juan R. Gimeno, Petros Syrris, William J. McKenna

Corresponding Author: Petros Syrris, PhD Centre for Heart Muscle Disease, Institute of Cardiovascular Science, University College London, London, WC1E 6DD, UK Tel: +44 207 679 6464; Fax: +44 207 679 6463. Email: [email protected]

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Filamin C variants are associated with a distinctive clinical and

immunohistochemical arrhythmogenic cardiomyopathy phenotype

Charlotte L. Hall, Mohammed M. Akhtar, Maria Sabater-Molina, Marta Futema, Angeliki

Asimaki, Alexandros Protonotarios, Chrysoula Dalageorgou, Alan M. Pittman, Mari Paz

Suarez, Beatriz Aguilera, Pilar Molina, Esther Zorio, Juan Pedro Hernández, Francisco Pastor,

Juan R. Gimeno, Petros Syrris, William J. McKenna

Appendix

Supplementary methods

Clinical testing

ECGs were recorded at rest using a standard protocol (10mm/mV in speed 25mm/s) in the

standard lead position. Incomplete RBBB was defined in this study as QRS width <120ms with an R wave peak time in V1 or V2>50ms. Low voltage was defined as QRS voltage less than 5mm in all limb leads and less than 10mm in all precordial leads. Poor R-wave progression was defined by R wave height ≤ 3mm in V3. Epsilon waves (reproducible low-amplitude signals between the end of QRS complex and the onset of T wave) and T-wave inversion were studied on all leads. T-wave inversion was defined as T-waves of negative amplitude ≥0.1mV.

QRS duration in leads V1–V6 and terminal activation duration of QRS complex (TAD) in leads

V1–V3 (from the nadir of S wave to the end of QRS, including R′) were measured. Signal averaged electrocardiograms were performed using time-domain analysis with a bandpass filter of 40Hz in individuals with QRS complex duration <110ms on standard ECG. 24h Holter monitoring was recorded on an outpatient basis. Ventricular extrasystoles and episodes of ventricular tachycardia (≥3 consecutive ventricular complexes at a rate of ≥100 beats/min) were

1 noted. No individual was receiving antiarrhythmic or other drugs known to affect the QRS complex at the time of acquisition of the ECG tracings.

All individuals underwent echocardiography included standard 2-dimensional measurements of left ventricular end-diastolic diameter (LVEDd) and left ventricular ejection fraction (LVEF) by the Simpson bi-plane method. Henry’s formula was used to correct dimensions for age and body surface area. Right ventricular (RV) outflow-tract end-diastolic diameter was measured on parasternal long-axis view (RVOT-PLAX). Wall motion abnormalities (hypokinesia, akinesia, dyskinesia, and aneurysm) of the right and left ventricles were documented.

CMRI was being performed on a 1.5 Tesla scanner (Magnetom Avanto, Siemens Medical

Solutions) using a cardiac 32-channel phased array coil. Balanced steady-state free precession cine imaging are used to acquire 10–12 short axis slices (8mm slice thickness, 2mm gap) with one slice per breath-hold. Four-chamber, two-chamber and LV inflow/outflow views and a short-axis stack from mitral annulus to apex were obtained. Sequence parameters are 1.5ms echo time, 3.1ms repetition time, and acquired voxel size usually were 1.9 × 1.9mm with a typical FOV of 350mm in the phase encoding direction. Late gadolinium enhancement (LGE) imaging was acquired with a standard segmented ‘fast low-angle shot’ two-dimensional inversion-recovery gradient echo sequence or a respiratory motion-corrected, free-breathing single shot SSFP averaged phase sensitive inversion recovery (PSIR) sequence at 15 minutes following the injection of 0.1 mmol/kg of a gadolinium based contrast medium. Volumetry and all tissue characterisation analyses were performed on a standard post-processing platform

(cvi42, version 5.6.5, Circle Cardiovascular imaging, Calgary, Canada). Manual epicardial and endocardial contours were drawn on the LV-SAX stack to measure LV volumes at end-diastole and end-systole. Papillary muscles and trabeculae were included within the LV cavity volume.

LGE was deemed present if viewed on SAX stack imaging with verification in one LAX view,

LGE will be recorded on 16 segments according to the American Heart Association 17 segment

2 model (segment 17 not assessed) along with the location (sub-endocardial, sup-epicardial, mid- wall or transmural) and pattern of fibrosis.

Bioinformatics analysis of WES data and variant filtering

Paired-end sequence reads were aligned with NovoAlign (Novocraft Technologies) against the reference human genome assembly GRCh37 (hg19). Duplicate reads removal, format conversion and indexing were performed with Picard (http://picard.sourceforge.net/). The

Genome Analysis Toolkit (GATK) (https://www.broadinstitute.org/gatk/) was used to recalibrate base quality scores and to perform local realignments around possible indels. The

HaplotypeCaller 3.1 package in GATK was used to call variants and to generate a multi-sample joint genotyping.

Variants were annotated using ANNOVAR software [1] and the Variant Effect Predictor

(VEP) [2] tool from Ensembl. Pathogenicity of the identified missense variants was predicted using multiple bioinformatics in silico tools, namely HumVar-trained PolyPhen-2 model [3],

SIFT [4] and MutationTaster [5].Variants were also annotated with frequencies as reported in large sequencing studies, with the Genome Aggregation Database (gnomAD, http://gnomad.broadinstitute.org/) [6] being the largest. Variants identified by WES with a minor allele frequency (MAF) higher or equal to 0.001 (0.1%) in the publically available gnomAD database were removed from further analysis. Variants outside of the coding and splice site regions were also filtered out. Remaining genetic changes were filtered by their predicted functional effect, which prioritized variants that are likely to result in a loss-of- function (stop gain, frameshift deletion or insertion), non-synonymous or altering splicing.

Copy number variants (CNVs) analysis

The analysis of large rearrangements in the coding regions of genes was performed using a read depth strategy designed to overcome biases associated with sequence capture and high-

3 throughput sequencing. This set of tools is implemented in the ExomeDepth software package

(freely available at the Comprehensive R Archive Network) [7].

Immunohistochemistry

Formalin-fixed, paraffin-embedded tissue sections (5μm) were deparaffinised, dehydrated, rehydrated and heated in citrate buffer (10mmol/l, pH 6.0) to enhance specific immunostaining.

After being cooled to room temperature, the tissue sections were simultaneously permeabilized and blocked by incubation in phosphate-buffered saline (PBS) containing 1% Triton X-100,

3% normal goat serum and 1% bovine serum albumin [8]. The sections were then incubated first with a primary antibody and then with indocarbocyanine-conjugated goat anti-mouse or anti-rabbit rabbit IgG. Primary antibodies included mouse monoclonal N-cadherin (Sigma, concentration 1:400), rabbit polyclonal Cx43 (Sigma, 1:400), mouse monoclonal plakoglobin

(Sigma, 1:1000), mouse monoclonal desmoplakin (Fitzgerald, 1:10), rabbit polyclonal SAP97

(Santa Cruz Biotechnology, 1:50), rabbit polyclonal anti-GSK3β (Cell Signaling Technology,

1:80) and rabbit monoclonal anti-FLNC (Abcam, 1:200).

4 References

[1] Wang K, Li M, Hakonarson H. ANNOVAR: functional annotation of genetic variants from high-throughput sequencing data. Nucleic Acids Res. 38 (2010) e164. [2] McLaren W, Pritchard B, Rios D, Chen Y, Flicek P, Cunningham F. Deriving the consequences of genomic variants with the Ensembl API and SNP Effect Predictor. Bioinformatics. 26 (2010) 2069-2070. [3] Adzhubei IA, Schmidt S, Peshkin L, Ramensky VE, Gerasimova A, Bork P, et al. A method and server for predicting damaging missense mutations. Nat Methods. 7 (2010) 248-249. [4] Ng PC, Henikoff S. SIFT: Predicting amino acid changes that affect protein function. Nucleic Acids Res. 31 (2003) 3812-3814. [5] Schwarz JM, Rodelsperger C, Schuelke M, Seelow D. MutationTaster evaluates disease- causing potential of sequence alterations. Nat Methods. 7 (2010) 575-576. [6] Lek M, Karczewski KJ, Minikel EV, Samocha KE, Banks E, Fennell T, et al. Analysis of protein- coding genetic variation in 60,706 humans. Nature. 536 (2016) 285-291. [7] Plagnol V, Curtis J, Epstein M, Mok KY, Stebbings E, Grigoriadou S, et al. A robust model for read count data in exome sequencing experiments and implications for copy number variant calling. Bioinformatics. 28 (2012) 2747-2754. [8] Asimaki A, Protonotarios A, James CA, Chelko SP, Tichnell C, Murray B, et al. Characterizing the Molecular Pathology of Arrhythmogenic Cardiomyopathy in Patient Buccal Mucosa Cells. Circ Arrhythm Electrophysiol. 9 (2016) e003688. [9] Marcus FI, McKenna WJ, Sherrill D, Basso C, Bauce B, Bluemke DA, et al. Diagnosis of arrhythmogenic right ventricular cardiomyopathy/dysplasia: proposed modification of the task force criteria. Circulation. 121 (2010) 1533-1541. [10] Richards S, Aziz N, Bale S, Bick D, Das S, Gastier-Foster J, et al. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med. 17 (2015) 405-424. [11] Li Q, Wang K. InterVar: Clinical Interpretation of Genetic Variants by the 2015 ACMG- AMP Guidelines. Am J Hum Genet. 100 (2017) 267-280. [12] Hebsgaard SM, Korning PG, Tolstrup N, Engelbrecht J, Rouze P, Brunak S. Splice site prediction in Arabidopsis thaliana pre-mRNA by combining local and global sequence information. Nucleic Acids Res. 24 (1996) 3439-3452. [13] Reese MG, Eeckman FH, Kulp D, Haussler D. Improved splice site detection in Genie. J Comput Biol. 4 (1997) 311-323.

5 Fig. A1. Pedigrees of ACM families with FLNC VUS variants.

Squares indicate males; circles, females; slashes, deceased individuals; black symbols, definite diagnosis of ARVC based on current Task Force diagnostic criteria [9] or evidence of ARVC at post mortem; grey symbols, borderline diagnosis of ARVC; hatched symbols, gene positive individuals with possible diagnosis of ARVC; (+), positive genotype for FLNC variant; (-), negative genotype for FLNC variant. Arrows indicate the index case in each family. PM, post mortem; SCD, sudden cardiac death. Gene negative individuals with a possible diagnosis of

ARVC based solely on family history are depicted as unaffected.

Fig. A2. Masson’s trichrome staining histology images of left ventricular myocardium obtained at post mortem from a carrier of the R1370X FLNC variant (case no 8) showing A).

Fatty (asterisks) and fibrous infiltration (arrow heads) (x250) and B). Additionally patchy degeneration of cardiomyocytes (arrows) (x400).

Fig. A3. Summary of immunohistochemistry staining data for RV and LV fixed tissue specimens from ACM cases with FLNC variants.

6 Table A1

FLNC variants identified by WES in cohort of ACM patients without pathogenic variants in known cardiomyopathy and arrhythmia genes.

FLNC cDNA FLNC amino acid gnomAD ACMG classification Sample ID change change frequency [6] [10, 11]

Family A II:2 c.2971C>T p.R991X Novel Pathogenic

Predicted abnormal

Family B II:2 c.7252-1G>A exon splicing* Novel Pathogenic

Family C III:1 c.4718T>A p.L1573X Novel Pathogenic

Family D III:6 c.1444C>T p.R482X Novel Pathogenic

Family E

II:3 c.174-185del p.59_62DLQRdel Novel VUS

Family F

II:2 c.6779A>G p.K2260R 4.554 x 10-5 VUS

Index case G c.6173A>G p.Q2058R Novel VUS

Index case H c.2141T>C p.I714T Novel VUS

Index case I c.5644A>G p.I1882V 0.001122 VUS

Variant classification according to ACMG guidelines was performed using the InterVar online tool [10, 11]. VUS, variant of unknown significance. *predicted effect on exon splicing by

NETGENE2 (http://www.cbs.dtu.dk/services/NetGene2/) [12] and Berkeley Drosophila

Genome project (BDGP; http://www.fruitfly.org/seq_tools/splice.html) [13].

7 Table A2

Incidents of sudden cardiac death in families with FLNC variants.

Documented SCD Age at death Family PM report Comment (n) (yrs)

20 ALVC Individual III:3

2 Not shown in A 59 N/A pedigree

Individual III:1

1 20 ACM FLNC c.7252-1G>A B carrier

N/A Not shown in 40 3 pedigree

N/A Not shown in C 60 pedigree

N/A Not shown in 70 pedigree

Individual III:5 27 Biventricular dilatation

3 Individual II:5 29 N/A

D Individual II:4

Definite diagnosis, 71 N/A FLNC R482X

carrier

1 Individual II:1 E 40 N/A

8 Individual III:1

1 36 ACM FLNC K2260R F carrier

26 ACM Pedigree not shown

G 2 60 N/A Pedigree not shown

H 1 42 N/A Pedigree not shown

I 1 47 N/A Pedigree not shown

ACM, arrhythmogenic cardiomyopathy; ALVC, arrhythmogenic left ventricular cardiomyopathy; PM, post mortem; SCD, sudden cardiac death.

9 Table A3

Clinical characteristics of FLNC VUS variant carriers in cases E-I.

Family - FLNC NYHA class- Age Sex ECG Echocardiography Arrhythmia CMRI Task Force Individual Genotype Symptoms Criteria (m, M) / Diagnosis Description LVEF 24 hr VE RV RVEF LV EDV LVEF LGE (%) count / EDV (%) (ml) (%) distribution Type of (ml) arrhythmia Family E p.59_62 palpitations 68 F Inferolateral T- Moderate to 35-40 AF, N/A N/A N/A N/A N/A 3m and 1M II:3 DLQRdel NYHA I wave inversion, severe LV Non- (index dysfunction sustained Definite case) late potentials VT RV regional wall abnormality (dyskinesia plus RV dilatation) Family E p.59_62 Asymptomatic 38 F Unremarkable Unremarkable 55 750, N/A N/A N/A N/A N/A 1m and 1M III:1 DLQRdel Non- sustained Borderline VT Family F p.K2260R Asymptomatic 74 F Low QRS Unremarkable 55 1 89 69 77 75 Sub- 1M II:2 voltage and epicardial (index poor R wave at basal Possible case) progression, mid to SAECG normal inferior wall Family F p.K2260R Asymptomatic 16 F Unremarkable Unremarkable 60 0 Normal Normal 141 63 No LGE 1M* IV:2 Possible Index p.Q2058R Palpitations 67 F Low QRS Unremarkable 55 paroxysmal Normal Normal 112 69 Basal 1M* case G voltages AF, 84 lateral non- Possible infarct fibrosis Index p.I714T NYHA II and 64 M T-wave Dilated RV, 50 138 290 44% 227 57 No LGE 1m and 2M case H Syncope inversion V1-V4 RV lateral wall akinesia Definite

10 Non- sustained VT Index p.I1882V NYHA I 78 M Unremarkable Moderate RV 55 N/A N/A N/A N/A N/A N/A 1m and 2M* case I ECG dilatation and RV aneurysm Definite Late potentials

AF, atrial fibrillation; CMRI, cardiac magnetic resonance imaging; ECG, electrocardiogram; EDV, end diastolic volume; EF, ejection fraction;

NYHA, New York Heart Association classification; LGE, late gadolinium enhancement; LV, left ventricle; m, minor Task Force ARVC diagnostic criterion; M, major Task Force ARVC diagnostic criterion; RV, right ventricle; SAECG, signal averaged electrocardiogram; VE, ventricular ectopics; VT, ventricular tachycardia; * one major diagnostic criterion due to family history (pedigree not shown for index case G).

11 Table A4

FLNC variants identified in cardiac tissue samples used in immunohistochemistry analysis.

ACMG FLNC genomic or cDNA FLNC amino gnomAD Case no classification change acid change frequency [6] [10, 11]

g.128470694_128498579del ^ 1 p.0? Novel Pathogenic (c.3_*2del)

2 c.105G>C p.K35N Novel VUS

3 c.249C>G p.Y83X Novel Pathogenic

4 c.479C>A p.T160K Novel VUS

5 c.1965_1966delTG p.A656PfsX8 Novel Pathogenic

6 c.2115_2120delTGCCCA p.Y705X Novel Pathogenic

Predicted

7 c.4288+2T>G abnormal exon Novel Pathogenic

splicing *

8 c.4108C>T p.R1370X Novel Pathogenic

9 c.5398G>T p.G1800X Novel Pathogenic

10 c.5398G>T p.G1800X Novel Pathogenic

Predicted

11 c.5298+21C>T abnormal exon Novel VUS

splicing *

Variant classification according to ACMG guidelines was performed using the InterVar online tool [10, 11]. VUS, variant of unknown significance. *predicted effect on exon splicing by NETGENE2 (http://www.cbs.dtu.dk/services/NetGene2/) [12] and Berkeley Drosophila Genome project (BDGP; http://www.fruitfly.org/seq_tools/splice.html) [13]. ^Genomic coordinates of the FLNC gene according to Ensembl human genome assembly GRCh37.p13.

12