Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2019 Dec; 163(4):309-317. Genetic architecture of recent-onset dilated cardiomyopathy in Moravian region assessed by whole-exome sequencing and its clinical correlates Anna Chaloupkaa, Lenka Piherovab, Ilga Grochovaa, Jana Binovac, Jan Krejcia, Lenka Spinarovaa, Viktor Straneckyb, Stanislav Kmochb, Milos Kubanekc Aims. Recent-onset dilated cardiomyopathy (RODCM) is a disease of heterogeneous aetiology and clinical outcome. In this pilot study, we aimed to assess its genetic architecture and correlate genotype with left ventricular reverse remodelling (LVRR). Patients and Methods. In this multi-centre prospective observational study, we enrolled 83 Moravian patients with RODCM and a history of symptoms of less than 6 months, for whole-exome sequencing (WES). All patients underwent 12-month clinical and echocardiographic follow-up. LVRR was defined as an absolute increase in left ventricular ejec- tion fraction > 10% accompanied by a relative decrease of left ventricular end-diastolic diameter > 10% at 12 months. Results. WES identified at least one disease-related variant in 45 patients (54%). LVRR occurred in 28 patients (34%), most often in carriers of isolated titin truncated variants, followed by individuals with a negative, or inconclusive WES and carriers of other disease-related variants (56% vs. 42% vs. 19%, P=0.041). Conclusion. A substantial proportion of RODCM cases have a monogenic or oligogenic genetic background. Carriers of non-titin disease-related variants are less likely to reach LVRR at 12- months than other individuals. Genetic testing could contribute to better prognosis prediction and individualized treatment of RODCM. Key words: dilated cardiomyopathy, familial cardiomyopathy, next generation sequencing, genetic architecture, cardiac remodelling Received: July 31, 2018; Accepted: September 7, 2018; Available online: September 24, 2018 https://doi.org/10.5507/bp.2018.054 © 2019 The Authors. This is an open access article licensed under the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/). a1st Internal Clinic of Cardio-angiology, St. Anne’s University Hospital and Faculty of Medicine, Masaryk University, Brno, Czech Republic bResearch Unit for Rare Diseases, Department of Paediatrics and Adolescent Medicine, 1st Faculty of Medicine, Charles University, Prague, Czech Republic cDepartment of Cardiology, Institute for Clinical and Experimental Medicine, Prague, Czech Republic. Corresponding author: Anna Chaloupka, e-mail: [email protected] INTRODUCTION tial improvement of LV systolic function accompanied by reduction of LV volumes, has been identified as a marker Dilated cardiomyopathy (DCM) is a prevalent disease of favourable prognosis in RODCM (ref.8-10). LVRR may leading to chronic heart failure (CHF). It has a hetero- indicate either the beneficial effect of pharmacotherapy, geneous aetiology including genetic, inflammatory, toxic disappearance of myocardial toxic or inflammatory insult. and metabolic causes1,2. The clinical diagnosis of DCM Due to the permanent character of myocardial damage, is based on the presence of left ventricular (LV) systolic LVRR should be less prevalent in genetic forms of DCM dysfunction in the absence of abnormal loading condi- (ref.7,11). tions or significant coronary artery disease sufficient to Tremendous advances have been made in understand- cause global systolic impairment2,3. In routine practice, ing the genetic basis of DCM over the last 30 years. To the aetiology of DCM remains often unexplained due to date, hundreds of mutations in more than 40 genes en- incomplete assessment of family history or recognition of coding proteins with various cellular functions have been clinical red-flags, suggesting genetic etiology4. In addition, identified as causing DCM (ref.12-14). The predominating potentially reversible causes of DCM like myocarditis and molecular cause seem to be truncating titin variants, toxic injury may be undiagnosed due to insufficient ac- responsible for almost a quarter cases of familial DCM curacy of non-invasive diagnostic tools to confirm myo- (ref.14). Most familial forms of DCM are monogenic carditis, or concealed history of alcohol, or drug abuse5,6. disorders, most commonly with autosomal dominant in- An ultimate diagnostic and prognostic challenge are heritance15,16. Some genotypes like mutations of LMNA, patients with recent-onset DCM (RODCM) who may PLN, and RBM20 have been associated with severe forms have variable clinical outcomes ranging from complete re- of CHF and / or malignant ventricular arrhythmias17-19. covery of LV systolic dysfunction to rapid progression to Cardiomyopathy gene panels are usually used in clini- end-stage CHF or sudden cardiac death7. Importantly, left cal settings to screen the most common disease-causing ventricular reverse remodelling (LVRR), defined as a par- genes. Discovery of next-generation sequencing (NGS) en- 309 Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2019 Dec; 163(4):309-317. ables us to screen whole protein-coding genome (exome) a pilot study of the first WES results derived from a large in a timely and cost-effective way and thus substantially project of WES in RODCM. increase mutation detection in both sporadic and familial cases of DCM (ref.20). Recognition of the genetic com- ponent, particularly in patients with RODCM or their MATERIALS AND METHODS asymptomatic relatives, could improve risk stratification and thus diagnostic and therapeutic management. Patients and study design The above formed the motivation for deciding to Patients were recruited in the Department of 1st assess the genetic architecture of RODCM with whole- Internal Cardio-angiology, St. Anne’s University Hospital exome sequencing (WES), and to study the genotype- in Brno, Czech Republic. This multi-centre prospective phenotype correlates. Specifically, we wanted to analyse observational study was approved by the Institutional the relationship between the results of WES and left ven- Ethics Committee at all respective participating institu- tricular reverse remodelling. The analysis was designed as tions and all patients signed the written informed consent. Table 1. Patient characteristics. Variable Baseline 12-month follow-up (n=83) (n=83) Age [years] 48 (40-56) - Gender (males/females) 68 (82%) - 15 (18%) Arterial hypertension 33 (40%) - Diabetes mellitus 10 (12%) - Positive family history of DCM 14 (17%) - NYHA class I – 3 (4%) I – 22 (26%) II – 40 (48%) II – 44 (53%) III – 39 (47%) III – 17 (21%) IV – 1 (1%) IV – 0 (0%) Systolic BP [mmHg] 120 (115-130) 128 (115-145) Diastolic BP [mmHg] 80 (75-87) 87 (80-95) Heart rate [bpm] 80 (69-90) 70 (60-80) Sinus rhythm 82 (99%) 80 (96%) QRS duration [ms] 100 (80-120) 108 (100-129) LVEDD [mm] 65 (60-70) 61 (55-67) LVEDV [mL] 214 (179-260) 187 (153-237) LVEF [%] 25 (17-30) 35 (27-45) E/Em 13 (10-19) 9 (7-11) Left atrium (PLAX) [mm] 45 (42-49) 42 (37-46) RVEDD (PLAX) [mm] 34 (30-38) 32 (28-35) TAPSE [mm] 18 (16-21) 21 (18-24) RV Sm [cm/s] 10 (9-12) 13 (11-15) ACEi 63 (76%) 73 (88%) ARB 6 (7%) 10 (12%) Equivalent dose of ACEi or ARB ≥ 50% 25 (30%) 39 (47%) Beta-blockers 74 (89%) 83 (100%) Equivalent dose of betablockers ≥ 50% 14 (17%) 27 (32%) Aldosterone receptor blockers 58 (70%) 66 (79%) Furosemide 62 (75%) 70 (84%) Furosemide ≥ 40 mg daily 45 (54%) 46 (55%) Sodium [mmol/L] 140 (138-142) 141 (139-142) (n=68) Creatinine [μmol/L] 86 (76-98) 81 (75-91) (n=70) NT-proBNP [ng/mL] 1664 (751-3232) 446 (109-779) (n=69) Patient characteristics at baseline and 12-month follow-up. Continuous variables are shown as median and IQR, discrete variables as absolute and relative frequency. DCM – dilated cardiomyopathy, NYHA class – New York Heart Association class, BP – blood pressure, LVEDD – left ventricular end-diastolic diameter, LVEDV – left ventricular end-diastolic volume, LVEF – left ventricular ejection fraction, PLAX – parasternal long axis, RVEDD – right ventricular end-diastolic diameter, TAPSE – tricuspid annular plane systolic excursion, RV Sm – right ventricular Sm wave, ACEi – angiotensin converting enzyme inhibitor, ARB – angiotensin receptor blocker. 310 Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2019 Dec; 163(4):309-317. All clinical, genomic data and samples were independent- sequencing was performed on the captured barcoded ly anonymized and stored in secured local databases ac- DNA library using the Illumina Hiseq 2500 system at the cording to the provisions of the Czech data protection Genomic facility in Motol University Hospital in Prague. authority (www.uoou.cz) and in accordance with Act The resulting FASTQ files were aligned to the Human 373/2011 Sb (§28-29). We prospectively enrolled 83 con- Genome Reference (hg19) using Novoalign (3.02.10). secutive patients with recent-onset DCM, specified by a Following genome alignment, conversion of SAM for- history of heart failure of less than 6 months. DCM was mat to BAM and duplicate removal were performed us- defined in accordance with the ESC definition by the ing Picard Tools (1.129). The Genome Analysis Toolkit, presence of LV dilatation (LVEDD 33mm/m in men and GATK (3.3) (ref.24) was used for local realignment around 32 mm/m in women) and LV systolic dysfunction (LVEF indels, base recalibration and variant recalibration and < 45%) in the absence of abnormal loading conditions genotyping. (hypertension, valve disease) or coronary artery disease sufficient to cause global systolic impairment2. Other in- Variant annotation and classification clusion criteria were age ≥ 18 years and signed informed Variant annotation was performed with SnpEff (ref.25) consent. DCM was considered familial when at least 2 and GEMINI (ref.26). Only the sequence having a frequen- genetically related first-degree relatives were diagnosed cy lower than 0.0005 in the dbSNP, ExAC (http://exac. with DCM, or in cases of premature sudden cardiac death broadinstitute.org/), gnomAD (http://gnomad.broadinsti- or CHF in a first-degree relative. Exclusion criteria, except tute.org/) and our internal exome database were priori- for the already mentioned hypertension, valvular disease tized for further analysis.