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Myocardial fibrosis in arrhythmogenic cardiomyopathy: a genotype–phenotype correlation study

Myocardial fibrosis in arrhythmogenic cardiomyopathy: a genotype–phenotype correlation study Abstract Aims Arrhythmogenic right ventricular cardiomyopathy/dysplasia (ARVC/D) is a life-threatening entity with a highly heterogeneous genetic background. Cardiac magnetic resonance (CMR) imaging can identify fibrofatty scar by late gadolinium enhancement (LGE). Our aim is to investigate genotype–phenotype correlation in ARVC/D mutation carriers, focusing on CMR-LGE and myocardial fibrosis patterns. Methods and results A cohort of 44 genotyped patients, 33 with definite and 11 with borderline ARVC/D diagnosis, was characterized using CMR and divided into groups according to their genetic condition (desmosomal, non-desmosomal mutation, or negative). We collected information on cardiac volumes and function, as well as LGE pattern and extension. In addition, available ventricular myocardium samples from patients with pathogenic gene mutations were histopathologically analysed. Half of the patients were women, with a mean age of 41.6 ± 17.5 years. Next-generation sequencing identified a potential pathogenic mutation in 71.4% of the probands. The phenotype varied according to genetic status, with non-desmosomal male patients showing lower left ventricular (LV) systolic function. LV fibrosis was similar between groups, but distribution in non-desmosomal patients was frequently located at the posterolateral LV wall; a characteristic LV subepicardial circumferential LGE pattern was significantly associated with ARVC/D caused by desmin mutation. Histological analysis showed increased fibrillar connective tissue and intercellular space in all the samples. Conclusion Desmosomal and non-desmosomal mutation carriers showed different morphofunctional features but similar LV LGE presence. DES mutation carriers can be identified by a specific and extensive LV subepicardial circumferential LGE pattern. Further studies should investigate the specificity of LGE in ARVC/D. arrhythmogenic cardiomyopathy, cardiac magnetic resonance, desmin, late gadolinium enhancement, histology, myocardial fibrosis Introduction Arrhythmogenic right ventricular cardiomyopathy/dysplasia (ARVC/D) is a life-threatening inherited disease characterized by progressive fibrofatty replacement of the functional myocardium.1 This arrhythmogenic substrate leads to increased ventricular volumes, ventricular arrhythmias (VAs), and sudden cardiac death (SCD), especially among young people and athletes.2 The classic pathological features of ARVC/D consist of near-exclusive right ventricular (RV) wall involvement with extensive aneurysms with a ‘paper-thin’ appearance. However, disease expression is variable and may involve either or both ventricles (left-dominant pattern or biventricular pattern, respectively).3,4 Clinical diagnosis is based on demonstrating characteristic electrical, structural, and/or histological abnormalities. In addition, a positive family history for a pathogenic genetic mutation also contributes to the diagnosis.5 To date, 11 disease genes have been linked to the ARVC/D phenotype, highlighting its genetic heterogeneity. Approximately 50% of patients diagnosed with ARVC/D carry a pathogenic mutation in desmosomal genes, including desmoplakin (DSP), plakophilin-2 (PKP2), desmoglein-2 (DSG2), desmocollin-2 (DSC2), and plakoglobin (JUP).6 However, several non-desmosomal genes are increasingly recognized in ARVC/D pathogenesis, including desmin (DES),7 phospholamban (PLN), transforming growth factor beta-3 (TGFβ-3), transmembrane protein 43 (TMEM43), lamin A/C (LMNA),8 and the recently described filamin C (FLNC).9 Most of them are involved in structural and signalling functions. Over the last few decades, cardiac magnetic resonance (CMR) imaging has emerged as a powerful diagnostic tool in patients with suspected or diagnosed ARVC/D, providing accurate information even on the concealed form of ARVC/D. Recently, late gadolinium enhancement (LGE) has been recognized as an early and reliable means of assessing myocardial fibrofatty replacement10,11 and has been associated with an increased risk of SCD in non-ischaemic cardiomyopathy.12 Therefore, as genetic testing becomes more widespread, links between genetics and various patterns of ARVC/D are emerging. However, there is a lack of genotype–phenotype information and common dilemmas in differential diagnosis remain unclear, since current diagnostic criteria do not take account of the left-dominant form and new non-desmosomal genes are increasingly being recognized. The aim of this study is to investigate genotype–phenotype correlation in non-desmosomal and desmosomal ARVC/D mutation carriers in order to describe clinical features for diagnosis, focusing on CMR and fibrosis patterns. Methods Study population and clinical assessment From a cohort of patients referred to our tertiary medical centre (Virgen de las Nieves University Hospital, Granada, Spain) for evaluation of possible ARVC/D, we retrospectively included 44 individuals with a definite or borderline diagnosis of ARVC/D, based on 2010 diagnostic criteria, and in whom CMR was performed.5 Patients provided informed consent and the study was approved by the Institutional Review Board. Clinical evaluation All subjects underwent comprehensive clinical evaluation to determine ARVC/D diagnosis status according to International Task Force criteria.5 Clinical assessment included family history of ARVC/D or SCD, exhaustive medical history, 12-lead electrocardiogram (ECG), 24-h Holter monitoring, echocardiography, CMR, and genetic testing. A positive family history was considered if the patient had a first-degree relative in whom fibrofatty replacement had been histopathologically demonstrated in necropsy or a living relative with a definitive clinical diagnosis of ARVC/D. An abnormal ECG was defined as T-wave inversion beyond V3, lateral, or inferolateral leads, low QRS voltages, pathological Q waves, fragmented QRS, or non-sinus rhythm. Non-sustained ventricular tachycardia was defined as at least three consecutive ventricular beats at >100 bpm. The assessment of biventricular morphological and functional parameters was performed according to current recommendations.13 Cardiac magnetic resonance imaging All the patients gave their informed consent for the administration of gadolinium and underwent a CMR, which was performed on a 1.5-T MR system (General Electric, Signa EXCITE®, Milwaukee, WI, USA). The images were evaluated by four heart imaging experts (two cardiologists and two radiologists blinded to the genetic background and the aim of this study). The standard protocol included scout images (axial, sagittal, and coronal), double inversion recovery pulse sequence (dark blood images), structure and function module with balanced steady-state free precession (SSFP) sequence (short axis, four-chamber, three-chamber, two-chamber, and RV outflow tract). Finally, 0.1–0.2 mmol/kg of chelated gadolinium (Gadovist®) were administered, obtaining the LGE pictures using an inversion recovery sequence 7–10 min post-injection. Some additional sequences were acquired where clinically indicated. The post-processing analysis was performed using semi-automatic software (Reportcard®). The systolic function was assessed by volume measures, indexed to body surface area. Likewise, the ejection fraction was calculated by Simpson’s method. The volumes and ejection fraction were analysed by comparing them to the references values.14,15 Finally, the LGE images were visually analysed by describing their presence, location and distribution using an anatomical 17-segment left ventricular (LV) model and a 5-segment RV model. Genetic test Peripheral blood samples for the genetic analysis were obtained from the probands or the deceased index case, as applicable. We used a next-generation sequencing (NGS) gene panel containing 21 genes which had previously been reported as being associated with or are regarded as candidates for the development of arrhythmogenic cardiomyopathy (Supplementary data online, Methods). The pathogenicity of the identified variants was classified according to the current guidelines of the American College of Medical Genetics and Genomics (ACMG).16 After a potential disease-causing variant was identified in the index patient, genetic and clinical cascade screening was performed in all available family members. Histological analyses Tissue samples for histology were obtained from two deceased subjects (TMEM43 and FLNC carriers) and one explanted heart (DES carrier) under informed consent. Cardiac tissue samples were fixed in buffered 4% formaldehyde, dehydrated and embedded in paraffin following a conventional procedure.7,17 Sections of 5 µm thickness were prepared and stained with haematoxylin & eosin (HE) for morphological evaluation while the myofibril organization was evaluated with Heidenhain’s iron haematoxylin (HD) staining. Fibrillar collagens were stained using Masson’s trichrome (MS) and Picrosirius (PS, at light and polarized microscopy),17 whereas reticular collagens were stained with the Gomori silver stain (RET). Furthermore, in this study, a cardiac tissue sample from a deceased subject, without any cardiac disease, was used as a control (CTR). In addition, the percentage of fibrofatty infiltration was determined in sections stained with MS the ImageJ software (Bethesda, MD, USA) following a previously described methodology.18 Study design and statistical analysis The patients were divided into three groups: desmosomal mutation carriers, non-desmosomal mutation carriers, and patients with a negative genotype. After the data collection was finished, we reviewed all the case records and obtained further information from follow-up visits at 6 months and 1 year. Statistical analysis was performed using SPSS Statistics version 20.0 (IBM®, Armonk, NY, USA). The data were analysed for normality with the Shapiro–Wilk test. Clinical characteristics were compared using a χ2 or Fisher’s exact test for categorical variables and analysis of variance or Kruskal–Wallis for continuous variables. All continuous variables were described as mean value ± standard deviation for each measurement and categorical data were reported as frequencies and percentages. A P-value of <0.05 was considered statistically significant. Results Genetic and clinical evaluation Forty-four patients who had a borderline or definite ARVC/D diagnosis and in whom CMR was performed, were included in the study. Of these 44 patients, 22 were women (50%), and the mean age at diagnosis was 41.6 ± 17.5 years. Thirty-three (75%) fulfilled the current international Task Force Criteria for ARVC/D. The baseline characteristics are summarized in Table 1. Table 1 Baseline characteristics Overall (44) Non- desmosomal (25) Desmosomal (13) Negative (6) P-value Gender (female), n (%) 22 (50) 14 (56) 6 (46.2) 2 (33.3) 0.57 Age (years), mean ± SD 41.6 ± 17.5 37.04 ± 17.3 40.2 ± 16.8 56.7 ± 12.7 0.044 Fulfil TFC, n (%) 33 (75) 19 (76) 9 (69.2) 5 (83.3) 0.792 Asymptomatic, n (%) 27 (61.4) 19 (76) 7 (53.8) 1 (16.7) 0.022 VT/VF, n (%) 10 (22.7) 2 (8) 4 (30.8) 4 (66.7) 0.006 Family history SCD, n (%) 35 (79.5) 25 (100) 9 (69.2) 3 (50) 0.001 Phenotype,an (%)  RV 5 (11.4) 3 (12) 1 (7.7) 1 (16.7) 0.224  LV 10 (22.7) 9 (36) 0 (0) 1 (16.7)  BV 25 (56.8) 11 (44) 10 (76.9) 4 (66.7)  None 4 (9.1) 2 (8) 2 (15.4) 0 (0) Overall (44) Non- desmosomal (25) Desmosomal (13) Negative (6) P-value Gender (female), n (%) 22 (50) 14 (56) 6 (46.2) 2 (33.3) 0.57 Age (years), mean ± SD 41.6 ± 17.5 37.04 ± 17.3 40.2 ± 16.8 56.7 ± 12.7 0.044 Fulfil TFC, n (%) 33 (75) 19 (76) 9 (69.2) 5 (83.3) 0.792 Asymptomatic, n (%) 27 (61.4) 19 (76) 7 (53.8) 1 (16.7) 0.022 VT/VF, n (%) 10 (22.7) 2 (8) 4 (30.8) 4 (66.7) 0.006 Family history SCD, n (%) 35 (79.5) 25 (100) 9 (69.2) 3 (50) 0.001 Phenotype,an (%)  RV 5 (11.4) 3 (12) 1 (7.7) 1 (16.7) 0.224  LV 10 (22.7) 9 (36) 0 (0) 1 (16.7)  BV 25 (56.8) 11 (44) 10 (76.9) 4 (66.7)  None 4 (9.1) 2 (8) 2 (15.4) 0 (0) BV, biventricular; LV, left ventricle; RV, right ventricle; SCD, sudden cardiac death; TFC, Task Force Criteria; VT/VF, ventricular tachycardia/ventricular fibrillation. a Phenotype is defined by the presence of regional wall motion abnormalities and/or late gadolinium enhancement. Open in new tab Table 1 Baseline characteristics Overall (44) Non- desmosomal (25) Desmosomal (13) Negative (6) P-value Gender (female), n (%) 22 (50) 14 (56) 6 (46.2) 2 (33.3) 0.57 Age (years), mean ± SD 41.6 ± 17.5 37.04 ± 17.3 40.2 ± 16.8 56.7 ± 12.7 0.044 Fulfil TFC, n (%) 33 (75) 19 (76) 9 (69.2) 5 (83.3) 0.792 Asymptomatic, n (%) 27 (61.4) 19 (76) 7 (53.8) 1 (16.7) 0.022 VT/VF, n (%) 10 (22.7) 2 (8) 4 (30.8) 4 (66.7) 0.006 Family history SCD, n (%) 35 (79.5) 25 (100) 9 (69.2) 3 (50) 0.001 Phenotype,an (%)  RV 5 (11.4) 3 (12) 1 (7.7) 1 (16.7) 0.224  LV 10 (22.7) 9 (36) 0 (0) 1 (16.7)  BV 25 (56.8) 11 (44) 10 (76.9) 4 (66.7)  None 4 (9.1) 2 (8) 2 (15.4) 0 (0) Overall (44) Non- desmosomal (25) Desmosomal (13) Negative (6) P-value Gender (female), n (%) 22 (50) 14 (56) 6 (46.2) 2 (33.3) 0.57 Age (years), mean ± SD 41.6 ± 17.5 37.04 ± 17.3 40.2 ± 16.8 56.7 ± 12.7 0.044 Fulfil TFC, n (%) 33 (75) 19 (76) 9 (69.2) 5 (83.3) 0.792 Asymptomatic, n (%) 27 (61.4) 19 (76) 7 (53.8) 1 (16.7) 0.022 VT/VF, n (%) 10 (22.7) 2 (8) 4 (30.8) 4 (66.7) 0.006 Family history SCD, n (%) 35 (79.5) 25 (100) 9 (69.2) 3 (50) 0.001 Phenotype,an (%)  RV 5 (11.4) 3 (12) 1 (7.7) 1 (16.7) 0.224  LV 10 (22.7) 9 (36) 0 (0) 1 (16.7)  BV 25 (56.8) 11 (44) 10 (76.9) 4 (66.7)  None 4 (9.1) 2 (8) 2 (15.4) 0 (0) BV, biventricular; LV, left ventricle; RV, right ventricle; SCD, sudden cardiac death; TFC, Task Force Criteria; VT/VF, ventricular tachycardia/ventricular fibrillation. a Phenotype is defined by the presence of regional wall motion abnormalities and/or late gadolinium enhancement. Open in new tab The great majority of the patients were identified by family screening due to a positive family history of SCD or after a definitive family diagnosis of ARVC. Molecular-genetic screening identified a potential pathogenic mutation in 10 (71.4%) of the 14 index cases. Family cascade screening identified a positive genotype in 28 patients (Table 2). A mutation in the classic desmosomal genes (DSP, JUP, PKP2, DSG2, DSC2) was found in 13 patients (29.5%), being DSP the most prevalent gene affected. On the other hand, mutations in non-desmosomal genes were identified in 25 individuals (56.8%), being DES the most frequent gene (Figure 1). Figure 1 Open in new tabDownload slide Gene distribution. Figure 1 Open in new tabDownload slide Gene distribution. Table 2 Patients’ phenotypes by gene mutation and CMR pattern Gene Protein Nucleotide change Protein change Carriers LV RWMA RV RWMA LGE phenotype LGE LV extension DES Desmin c.1203G>C Missense 16 11 8 RV 1 LV 6 BV 9 Annular 13 Inferolateral 2 Inferior 1 DSP Desmoplakin c.3133C>T Nonsense 7 2 6 LV 1 BV 6 Annular 3 Septum 1 Inferolateral 2 Lateral 1 c.7697_7698insG Frameshift FLNC Filamin C c.4288 + 2T>G Nonsense 7 3 2 LV 5 RV 1 None 1 Inferolateral 3 Lateral 2 c.581_599delTGGTGG ACAACTGCGCCCC Nonsense DSG-2 Desmoglein2 c.875G>A Missense 5 3 4 BV 3 None 0 Annular 1 Lateral 2 c.535delA Nonsense PKP-2 Plakophilin2 c.1643delG Nonsense 1 None 1 1 LMNA Lamin A/C c.1541G>A Missense 1 None None None TMEM43 Luma c.1073C>T Missense 1 None 1 RV 1 Negative 6 2 5 LV 3 BV 2 None 1 Gene Protein Nucleotide change Protein change Carriers LV RWMA RV RWMA LGE phenotype LGE LV extension DES Desmin c.1203G>C Missense 16 11 8 RV 1 LV 6 BV 9 Annular 13 Inferolateral 2 Inferior 1 DSP Desmoplakin c.3133C>T Nonsense 7 2 6 LV 1 BV 6 Annular 3 Septum 1 Inferolateral 2 Lateral 1 c.7697_7698insG Frameshift FLNC Filamin C c.4288 + 2T>G Nonsense 7 3 2 LV 5 RV 1 None 1 Inferolateral 3 Lateral 2 c.581_599delTGGTGG ACAACTGCGCCCC Nonsense DSG-2 Desmoglein2 c.875G>A Missense 5 3 4 BV 3 None 0 Annular 1 Lateral 2 c.535delA Nonsense PKP-2 Plakophilin2 c.1643delG Nonsense 1 None 1 1 LMNA Lamin A/C c.1541G>A Missense 1 None None None TMEM43 Luma c.1073C>T Missense 1 None 1 RV 1 Negative 6 2 5 LV 3 BV 2 None 1 BV, biventricular; LGE, late gadolinium enhancement; LV, left ventricle; RV, right ventricle; RWMA, regional wall motion abnormality; SCD, sudden cardiac death. Open in new tab Table 2 Patients’ phenotypes by gene mutation and CMR pattern Gene Protein Nucleotide change Protein change Carriers LV RWMA RV RWMA LGE phenotype LGE LV extension DES Desmin c.1203G>C Missense 16 11 8 RV 1 LV 6 BV 9 Annular 13 Inferolateral 2 Inferior 1 DSP Desmoplakin c.3133C>T Nonsense 7 2 6 LV 1 BV 6 Annular 3 Septum 1 Inferolateral 2 Lateral 1 c.7697_7698insG Frameshift FLNC Filamin C c.4288 + 2T>G Nonsense 7 3 2 LV 5 RV 1 None 1 Inferolateral 3 Lateral 2 c.581_599delTGGTGG ACAACTGCGCCCC Nonsense DSG-2 Desmoglein2 c.875G>A Missense 5 3 4 BV 3 None 0 Annular 1 Lateral 2 c.535delA Nonsense PKP-2 Plakophilin2 c.1643delG Nonsense 1 None 1 1 LMNA Lamin A/C c.1541G>A Missense 1 None None None TMEM43 Luma c.1073C>T Missense 1 None 1 RV 1 Negative 6 2 5 LV 3 BV 2 None 1 Gene Protein Nucleotide change Protein change Carriers LV RWMA RV RWMA LGE phenotype LGE LV extension DES Desmin c.1203G>C Missense 16 11 8 RV 1 LV 6 BV 9 Annular 13 Inferolateral 2 Inferior 1 DSP Desmoplakin c.3133C>T Nonsense 7 2 6 LV 1 BV 6 Annular 3 Septum 1 Inferolateral 2 Lateral 1 c.7697_7698insG Frameshift FLNC Filamin C c.4288 + 2T>G Nonsense 7 3 2 LV 5 RV 1 None 1 Inferolateral 3 Lateral 2 c.581_599delTGGTGG ACAACTGCGCCCC Nonsense DSG-2 Desmoglein2 c.875G>A Missense 5 3 4 BV 3 None 0 Annular 1 Lateral 2 c.535delA Nonsense PKP-2 Plakophilin2 c.1643delG Nonsense 1 None 1 1 LMNA Lamin A/C c.1541G>A Missense 1 None None None TMEM43 Luma c.1073C>T Missense 1 None 1 RV 1 Negative 6 2 5 LV 3 BV 2 None 1 BV, biventricular; LGE, late gadolinium enhancement; LV, left ventricle; RV, right ventricle; RWMA, regional wall motion abnormality; SCD, sudden cardiac death. Open in new tab Cardiac imaging CMR morphofunctional analysis Among the 44 patients evaluated, 79.5% had LV involvement (35 patients), based on LGE and regional wall motion abnormalities (RWMAs). The distribution of ventricular affection patterns showed 5 patients (11.4%) with isolated RV disease (classic pattern), 10 (22.7%) with left-dominant affection, and 25 (56.8%) with biventricular involvement. Only four patients with positive genotypes had no structural heart involvement distinguishable by non-invasive imaging techniques; these were classified as silent carriers. Volume analysis is shown in Supplementary data online, Table S1. Overall, LV ejection fraction (LVEF) was mildly impaired (mean 48.9 ± 10%) or normal (47.7% had LVEF >50%). Men with non-desmosomal mutations showed slightly higher indexed LV end-diastolic volumes (iLVED) in comparison to men affected by desmosomal mutations and negative-genotype subjects (106.3 ± 25.6 mL/m2 vs. 80.7 ± 16.4 mL/m2 and 80.1 ± 17.3 mL/m2, respectively; P = 0.05). This was not observed in women. Men also tended to show lower LVEF values (44.6 ± 11.1% vs. 51.8 ± 6.6% and 56.4 ± 9.2%, respectively; P = 0.09). Female desmosomal mutation carriers showed a significantly lower RV ejection fraction (RVEF) in comparison to non-desmosomal carriers and negative-genotype patients (43.5 ± 9.8% vs. 52.6 ± 7.4% and 63.2 ± 5.3%, respectively; P = 0.02). Male desmosomal mutation carriers also showed lower RVEF values but the difference did not reach statistical significance. LV regional wall motion abnormalities (LV-RWMAs) were present in 21 patients (47.7%), distributed among 14 (56%) non-desmosomal mutation carriers, 5 (38.5%) desmosomal mutational carriers, and 2 (33.3%) negative-genotype subjects. Within LV-RWMAs, LV inferolateral wall involvement was frequent (15 subjects; 34.1%). With regard to the assessment of RV involvement, subjects with a desmosomal variant had a higher prevalence of RV-RWMAs than non-desmosomal mutation carriers (84.6% vs. 44%; P = 0.025). Late gadolinium enhancement A summary of LGE distribution is shown in Table 3. Fibrofatty replacement assessment with gadolinium was evaluated in the whole sample. Positive myocardial LV-LGE was present in 38 patients (79.5%). Biventricular distribution of LGE was present in 18 patients (45.4%). Exclusive LV presence of LGE was observed in 15 cases (34.1%). LGE location showed a predominance of subepicardial layer involvement (33; 86.8%). Mesocardial involvement was observed in two patients (one desmosomal mutation carrier and one negative genotype case). Table 3 Late gadolinium enhancement findings Non-desmosomal (25) Desmosomal (13) Negative (6) P-value LV-LGE positive, n (%) 20 (80) 10 (76.9) 5 (83.3) 0.94  Distribution LV-LGE, n (%) Subepicardial 20 (100) Subepicardial 9 (90) Mesocardial 1 (10) Subepicardial 4 (80) Mesocardial 1 (20) 0.17  Extension LV-LGE annular, n (%) 14 (56) 4 (30.8) 0 (0) 0.02 RV-LGE, n (%) 10 (40) 10 (76.9) 2 (33.3) 0.06 LGE pattern, n (%)  Isolated LV 11 (44) 1 (7.7) 3 (50) 0.27  Isolated RV 3 (12) 1 (7.7) 0 (0)  Biventricular 9 (36) 9 (69.2) 2 (33.3)  No 2 (8) 2 (15.4) 1 (16.7) Non-desmosomal (25) Desmosomal (13) Negative (6) P-value LV-LGE positive, n (%) 20 (80) 10 (76.9) 5 (83.3) 0.94  Distribution LV-LGE, n (%) Subepicardial 20 (100) Subepicardial 9 (90) Mesocardial 1 (10) Subepicardial 4 (80) Mesocardial 1 (20) 0.17  Extension LV-LGE annular, n (%) 14 (56) 4 (30.8) 0 (0) 0.02 RV-LGE, n (%) 10 (40) 10 (76.9) 2 (33.3) 0.06 LGE pattern, n (%)  Isolated LV 11 (44) 1 (7.7) 3 (50) 0.27  Isolated RV 3 (12) 1 (7.7) 0 (0)  Biventricular 9 (36) 9 (69.2) 2 (33.3)  No 2 (8) 2 (15.4) 1 (16.7) LGE, late gadolinium enhancement; LV, left ventricle; n, number; RV, right ventricle; RWMA, regional wall motion abnormality. Open in new tab Table 3 Late gadolinium enhancement findings Non-desmosomal (25) Desmosomal (13) Negative (6) P-value LV-LGE positive, n (%) 20 (80) 10 (76.9) 5 (83.3) 0.94  Distribution LV-LGE, n (%) Subepicardial 20 (100) Subepicardial 9 (90) Mesocardial 1 (10) Subepicardial 4 (80) Mesocardial 1 (20) 0.17  Extension LV-LGE annular, n (%) 14 (56) 4 (30.8) 0 (0) 0.02 RV-LGE, n (%) 10 (40) 10 (76.9) 2 (33.3) 0.06 LGE pattern, n (%)  Isolated LV 11 (44) 1 (7.7) 3 (50) 0.27  Isolated RV 3 (12) 1 (7.7) 0 (0)  Biventricular 9 (36) 9 (69.2) 2 (33.3)  No 2 (8) 2 (15.4) 1 (16.7) Non-desmosomal (25) Desmosomal (13) Negative (6) P-value LV-LGE positive, n (%) 20 (80) 10 (76.9) 5 (83.3) 0.94  Distribution LV-LGE, n (%) Subepicardial 20 (100) Subepicardial 9 (90) Mesocardial 1 (10) Subepicardial 4 (80) Mesocardial 1 (20) 0.17  Extension LV-LGE annular, n (%) 14 (56) 4 (30.8) 0 (0) 0.02 RV-LGE, n (%) 10 (40) 10 (76.9) 2 (33.3) 0.06 LGE pattern, n (%)  Isolated LV 11 (44) 1 (7.7) 3 (50) 0.27  Isolated RV 3 (12) 1 (7.7) 0 (0)  Biventricular 9 (36) 9 (69.2) 2 (33.3)  No 2 (8) 2 (15.4) 1 (16.7) LGE, late gadolinium enhancement; LV, left ventricle; n, number; RV, right ventricle; RWMA, regional wall motion abnormality. Open in new tab Different LGE distribution patterns were identified according to genetic background (Figure 2). There were no statistically significant differences in the presence of LV-LGE between desmosomal mutation carriers, non-desmosomal mutation carriers and negative-genotype patients (83.3% vs. 84% vs. 83.3%, respectively). The predominant LV-LGE distribution pattern in each group was subepicardial enhancement. Regarding the extent of LV-LGE, it was observed that individuals with non-desmosomal mutations, predominantly DES carriers, showed a characteristic annular or circumferential subepicardial LV-LGE pattern (Take home figure). This particular LV-LGE pattern was more frequently observed in subjects carrying a non-desmosomal mutation in comparison with desmosomal mutation carriers and negative-genotype subjects (76.5%, 23.5%, and 0%, respectively; P = 0.02). Among these individuals with non-desmosomal mutations, 13 were DES carriers (P = 0.006) and one had a truncating mutation in FLNC. This specific pattern was also observed in three DSP carriers and one DSG-2 carrier. On the other hand, a predominantly inferolateral distribution in the left ventricle was observed in most of the FLNC patients (Figure 2). Figure 2 Open in new tabDownload slide Representative late gadolinium enhancement images from non-desmosomal mutations (upper panels) and desmosomal mutations (lower panels). The white arrows show the enhancement zones. (A) DES mutation with circumferential LV enhancement. (B) FLNC mutation with lateral LV enhancement. (C) TMEM43 mutation patient with RV enhancement. (D) PKP-2 mutation with RV enhancement. (E) DSG-2 mutation with biventricular enhancement. (F) DSP mutation with biventricular enhancement. Figure 2 Open in new tabDownload slide Representative late gadolinium enhancement images from non-desmosomal mutations (upper panels) and desmosomal mutations (lower panels). The white arrows show the enhancement zones. (A) DES mutation with circumferential LV enhancement. (B) FLNC mutation with lateral LV enhancement. (C) TMEM43 mutation patient with RV enhancement. (D) PKP-2 mutation with RV enhancement. (E) DSG-2 mutation with biventricular enhancement. (F) DSP mutation with biventricular enhancement. Figure 3 Open in new tabDownload slide Histological images of human cardiac tissues affected by various mutations. Representative histological images from healthy (CTR) and genetically affected cardiac tissue stained using H&E (A) and HD (B) methods. Arrows indicate the increase in the fibrillar extracellular matrix components. Scale bar = 100 µm. Figure 3 Open in new tabDownload slide Histological images of human cardiac tissues affected by various mutations. Representative histological images from healthy (CTR) and genetically affected cardiac tissue stained using H&E (A) and HD (B) methods. Arrows indicate the increase in the fibrillar extracellular matrix components. Scale bar = 100 µm. Patients with desmosomal mutations notably tended to show a higher prevalence of RV-LGE in comparison to the non-desmosomal and negative-genotype groups (76.9% vs. 40% and 33.3%, respectively; P = 0.06). A summary of the genotype–phenotype correlation study, focusing on CMR findings, is shown in Table 3. Histological findings The histological analysis performed with HE staining showed an increase in the fibrillar connective tissue and intercellular space in all cardiac tissue from patients with genetic mutations (DES, FLNC, and TMEM43) as compared to CTR (Figure 3). The increase in the extracellular matrix was more evident in cardiac tissue from subjects affected by FLNC truncation with 51.42% and TMEM43 mutation with 40.35% of fibrofatty presence. Furthermore, certain degree of cardiomyocyte hypertrophy was observed in both mutations (Figure 3). In the case of DES mutation, HD staining was weaker than in other mutations and CTR, being suggestive of myofibrillar involvement. In addition, the presence of fibrofatty in the subject affected by DES mutation was 39.45% (Figure 3). In the case of the CTR the extracellular matrix (ECM) represented around 21.23%. Figure 4 Open in new tabDownload slide Histochemistry of fibrillar extracellular matrix components of human cardiac tissue affected by various mutations. Arrows indicate the increase in collagens, which were stained in green with MS (A) and red by PS (B) staining. PS polarized light microscopy (C) shows that the zones with collagens acquired a high level of arrangement and density. Gomori silver staining (D) shows the increase in reticular fibres as a black fibrillar precipitate. Scale bar = 100 µm. Figure 4 Open in new tabDownload slide Histochemistry of fibrillar extracellular matrix components of human cardiac tissue affected by various mutations. Arrows indicate the increase in collagens, which were stained in green with MS (A) and red by PS (B) staining. PS polarized light microscopy (C) shows that the zones with collagens acquired a high level of arrangement and density. Gomori silver staining (D) shows the increase in reticular fibres as a black fibrillar precipitate. Scale bar = 100 µm. The evaluation of the fibrillar ECM components with MS, PS, and RET stainings confirmed a clear increase in the intercellular space, which proved to be composed of fibrillar collagens (MS and PS) and reticular fibres (RET) in all affected cardiac tissues, differing with respect to the extracellular matrix of the CTR (Figure 4). Polarized-light microscopy analysis, performed with PS staining, revealed that in cardiac tissues affected by FLNC and TMEM43 mutations the increase in collagen fibres was accompanied by an increase in collagen birefringence, resulting from a complex parallel arrangement of these extracellular components. However, no birefringence for collagens was observed in cardiac tissue affected by DES mutation, where a pericellular increase in reticular fibres was, surprisingly, more evident (Figure 4). Take home figure Open in new tabDownload slide Characteristic circumferential LGE in a female patient affected by DES mutation. (A and C) Short-axis and four-chamber view SSFP cine sequences. (B and D) LGE images in short-axis and four-chamber view (white arrows) are shown. Take home figure Open in new tabDownload slide Characteristic circumferential LGE in a female patient affected by DES mutation. (A and C) Short-axis and four-chamber view SSFP cine sequences. (B and D) LGE images in short-axis and four-chamber view (white arrows) are shown. Discussion Since the first descriptions of ARVC/D cases appeared in the literature, the diagnosis of this entity has classically been based on gross and histological evidence of right ventricle fibrofatty transmural myocardial replacement.1 Advances in diagnosis have been made, especially in genetic testing and cardiovascular imaging. In the last decade, the disease spectrum has evolved considerably. NGS has expanded the disease beyond the classic desmosomal genes to include cytoskeletal7 or nuclear membrane genes8 with important roles in the mechanotransduction machinery.19 Our study describes the genotype–phenotype CMR and histological findings from a cohort of predominantly non-desmosomal ARVC/D patients, providing detailed information about different structural patterns of expression and fibrosis involvement. Specific LGE patterns have been identified in DES and FLNC mutation carriers, all of them at the subepicardial level. Characteristic fibrofatty replacement in ARVC/D can be non-invasively assessed using LGE-CMR, and this could be helpful in risk stratification and diagnosis of ‘concealed’ cases.20 However, LGE-CMR is not included in the revised current diagnostic criteria.5 In addition, it has been suggested that the non-desmosomal ARVC/D phenotype overlaps with the dilated phenotype and is considered a phenocopy with an increased risk of VAs and poor prognosis, recognizing the need for appropriate risk stratification, taking genetic background and LGE deposition into account.21 In the last few years, several genotype–phenotype correlations in ARVC/D have been proposed, mainly related to desmosomal mutations,22 but there is a lack of studies focusing on non-desmosomal mutations. Interestingly, our population was composed mainly of non-desmosomal mutation carriers. There is a lack of information in the literature on CMR findings in this population. CMR analysis showed abnormalities in most of the positive genotype subjects, the majority of whom were asymptomatic, highlighting its potential for identifying patients with an early or concealed stage of the disease. Myocardial fibrosis is a hallmark feature seen histologically in most cardiomyopathies. Fibrosis causes relaxation impairment and diastolic and systolic impairment and contributes to re-entrant mechanisms, increasing the risk of malignant VAs. Our data show that RV-LGE was more frequently observed in patients carrying a desmosomal mutation in comparison with the non-desmosomal and genotype-negative groups, although it did not reach statistical significance. Since the function of desmosomes is to link tissues mechanically, mutations in these proteins may lead to instability of the intercalated disk, making the cardiomyocyte more vulnerable to wall stress, particularly in a wall as thin as that of the right ventricle.23 Regarding LV-LGE involvement, it was localized at the subepicardial layer with progression to the mesocardium, characteristically in the posterolateral and septal LV wall, with no statistically significant differences between groups. Recently, these findings have been histologically correlated in desmosomal mutations carriers and PLN mutation carriers, being more pronounced in the latter.24 Our histological data suggest a similar cellular phenotype between groups with an increase in fibrillar connective tissue and intercellular space, as well as collagen network and arrangement. These similarities between the three samples suggest a common final pathway in non-desmosomal ARVC/D patients with impaired filament formation, cell membrane disruption and high incidence of fibrosis predisposing to VAs, as described by our group in a recent study.7 A novel and specific finding of our work is the LV-LGE subepicardial circumferential pattern observed in almost all patients carrying the recently described p.Glu401Asp variant in DES. Desmin is the main intermediate filament in cardiac muscle. With its multiple binding partners, it forms a three-dimensional scaffold that links various structures (the contractile apparatus, Z-discs, the nucleus, intercalated disks, sarcolemma, mitochondria, and other organelles). Desmin’s function is to integrate all these components, facilitating mechanochemical signalling and transport processes and crosstalk between different cellular organelles.25 Therefore, a dysfunctional desmin network may lead to severe cardiomyocyte dysregulation and cell death. To date, this pattern has not been described in association with any causal genotype of arrhythmogenic cardiomyopathy, even though it has been observed in some patients with biventricular involvement and no linked genotype.26 In addition, three DSP truncating-variant carriers showed this LGE distribution. Interestingly, truncating mutations in desmoplakin, which usually show a high risk of arrhythmia, seem to consistently cause extensive LV fibrosis, with globally distributed fibrosis being described in some patients.27 Desmoplakin is the most abundant component of the desmosome. It resides in the cytoplasmic surface of the desmosome and interacts with intermediate filaments, desmin in the cardiomyocyte, for desmosome assembly and cytoskeletal linkage.28 This similarity suggests that dysfunctional cytoskeletal-desmosome linkage and subsequent mechanochemical failure lead to the presence of a great amount of fibrosis with a specific circumferential subepicardial distribution and could therefore be easily identified using LGE images. Although the current data indicate that the subepicardial posterolateral pattern is suggestive of arrhythmogenic cardiomyopathy, this distribution has also been observed in other entities such as myocarditis, dilated cardiomyopathy, and sarcoidosis.29 LGE pattern, together with a reliable clinical scenario, family history of ARVC/D, the genetic background and propensity for VAs exceeding the degree of LV dilation could be helpful to distinguish ARVC/D from these entities.4,9 Therefore, up to now a specific non-invasive marker of the disease has been lacking. Here, we suggest that the distinctive circumferential subepicardial LGE pattern should be considered as suggestive of desminopathy or potentially as a failure of the major cardiomyocyte mechanochemical components. This genotype–phenotype correlation may improve the classification of arrhythmogenic cardiomyopathy and enhance understanding of the disease’s pathophysiology. Finally, most of the cases showed traces of LV involvement (including isolated or biventricular patterns). Classically, LV involvement has been described with minor increases in end-diastolic volumes and without any marked decrease in LVEF.30 LV affection was similar between the two groups, although men from the non-desmosomal group tended to show higher LV volumes and there was a significant decrease in RVEF in women in the group of desmosomal mutation carriers. Moreover, RWMAs are an important element of the current diagnostic criteria and CMR has great sensitivity for this purpose. However, LGE presence is an independent marker of VAs and SCD31 and a good marker of the presence of fibrosis and is not included in the current diagnostic criteria. In our cohort, RV-RWMAs were significantly frequent in the desmosomal mutations group. Interestingly, LV-RWMAs were similar in desmosomal and non-desmosomal mutation carriers. Nevertheless, LGE sequences demonstrated a higher presence of LGE than LV-RWMAs, indicating a high prevalence of LV fibrosis and constituting a more sensitive diagnostic tool. These findings reinforce the conclusion that diagnosis should not be based solely on the presence of RWMAs. Tissue characterization is particularly important in the LV phenotype, since we observed a high number of patients with a positive LGE presence who did not have RWMAs.22 Therefore, the absence of RWMAs in patients with suspected LV phenotypes should not rule out the disease. Further studies should investigate the specificity of LGE in a consecutive cohort of patients evaluated for ARVC/D. Limitations The small sample size enabled only preliminary conclusions to be drawn in terms of genotype–phenotype correlations. Clinical data exposed of the single pGlu401Asp variant may not be representative of the wide spectrum of DES mutation. It is possible that some potentially pathogenic genes have been missed in the negative mutations group. Finally, access to histological samples from patients affected by these mutations is limited so it not possible to perform statistical assumptions in terms of histopathological-LGE correlation. Conclusions Patients with ARVC/D carrying desmosomal and non-desmosomal mutations show different morphofunctional features in CMR, despite having similar LV posterolateral wall LGE distributions. Subjects with p.Glu401Asp in DES more frequently express a characteristic phenotype consisting of an extensive LV-subepicardial circumferential LGE pattern suggesting a different pathomechanism. Identifying phenotypic patterns suggestive of a genetic mutation may make it possibly to achieve early identification of forms of ARVC/D, which could enable clinical and therapeutic management and risk stratification to be individualized. In many cases, the presence of LGE is not associated with concomitant RWMAs, which highlights the role of tissue characterization with CMR in all patients with ARVC/D. Acknowledgements We thank Dr Cinta Moro, of the Instituto Nacional de Toxicología y Ciencias Forenses, for her kindness in providing the myocardial tissue samples. We are grateful for the technical assistance of Ms Fabiola Bermejo Casares, of the Department of Histology and Tissue Engineering Group, University of Granada, Spain. Conflict of interest: none declared. Funding Histological analyses were supported by the Tissue Engineering Group CTS-115 of the University of Granada, Spain (to V.C., D.D.-H., M.A., and A.C.). References 1 Thiene G , Nava A , Corrado D , Rossi L , Pennelli N. Right ventricular cardiomyopathy and sudden death in young people . N Engl J Med 1988 ; 318 : 129 – 33 . Google Scholar Crossref Search ADS PubMed WorldCat 2 Corrado D , Link MS , Calkins H. Arrhythmogenic right ventricular cardiomyopathy . N Engl J Med 2017 ; 376 : 61 – 72 . Google Scholar Crossref Search ADS PubMed WorldCat 3 Sen-Chowdhry S , Syrris P , Ward D , Asimaki A , Sevdalis E , McKenna WJ. Clinical and genetic characterization of families with arrhythmogenic right ventricular dysplasia/cardiomyopathy provides novel insights into patterns of disease expression . Circulation 2007 ; 115 : 1710 – 20 . Google Scholar Crossref Search ADS PubMed WorldCat 4 Sen-Chowdhry S , Syrris P , Prasad SK , Hughes SE , Merrifield R et al. Left-dominant arrhythmogenic cardiomyopathy: an under-recognized clinical entity . J Am Coll Cardiol 2008 ; 52 : 2175 – 87 . Google Scholar Crossref Search ADS PubMed WorldCat 5 Marcus FI , McKenna WJ , Sherrill D , Basso C , Bauce B et al. Diagnosis of arrhythmogenic right ventricular cardiomyopathy/dysplasia: proposed modification of the Task Force Criteria . Eur Heart J 2010 ; 31 : 806 – 14 . 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Google Scholar Crossref Search ADS PubMed WorldCat 19 Mestroni L , Sbaizero O. Arrhythmogenic cardiomyopathy: mechanotransduction Going Wrong . Circulation 2018 ; 137 : 1611 – 3 . Google Scholar Crossref Search ADS PubMed WorldCat 20 Tandri H , Saranathan M , Rodriguez ER , Martinez C , Bomma C et al. Noninvasive detection of myocardial fibrosis in arrhythmogenic right ventricular cardiomyopathy using delayed-enhancement magnetic resonance imaging . J Am Coll Cardiol 2005 ; 45 : 98 – 103 . Google Scholar Crossref Search ADS PubMed WorldCat 21 Spezzacatene A , Sinagra G , Merlo M , Barbati G , Graw SL et al. ; Familial Cardiomyopathy Registry . Arrhythmogenic phenotype in dilated cardiomyopathy: natural history and predictors of life-threatening arrhythmias . J Am Heart Assoc 2015 ; 4 : e002149 . Google Scholar Crossref Search ADS PubMed WorldCat 22 Rastegar N , Zimmerman SL , Te Riele A , James C , Burt JR et al. Spectrum of biventricular involvement on CMR among carriers of ARVD/C-associated mutations . JACC Cardiovasc Imaging 2015 ; 8 : 863 – 4 . Google Scholar Crossref Search ADS PubMed WorldCat 23 Padron-Barthe L , Dominguez F , Garcia-Pavia P , Lara-Pezzi E. Animal models of arrhythmogenic right ventricular cardiomyopathy: what have we learned and where do we go? Insight for therapeutics . Basic Res Cardiol 2017 ; 112 : 50. Google Scholar Crossref Search ADS PubMed WorldCat 24 Sepehrkhouy S , Gho J , van Es R , Harakalova M , de Jonge N et al. Distinct fibrosis pattern in desmosomal and phospholamban mutation carriers in hereditary cardiomyopathies . Heart Rhythm 2017 ; 14 : 1024 – 32 . Google Scholar Crossref Search ADS PubMed WorldCat 25 Capetanaki Y , Papathanasiou S , Diokmetzidou A , Vatsellas G , Tsikitis M. Desmin related disease: a matter of cell survival failure . Cur Opin Cell Biol 2015 ; 32 : 113 – 20 . Google Scholar Crossref Search ADS WorldCat 26 Sultan FAT , Ahmed MA , Miller J , Selvanayagam JB. Arrhythmogenic right ventricular cardiomyopathy with biventricular involvement and heart failure in a 9-year old girl . J Saudi Heart Assoc 2017 ; 29 : 139 – 42 . Google Scholar Crossref Search ADS PubMed WorldCat 27 Lopez-Ayala JM , Gomez-Milanes I , Sanchez Munoz JJ , Ruiz-Espejo F , Ortiz M et al. Desmoplakin truncations and arrhythmogenic left ventricular cardiomyopathy: characterizing a phenotype . Europace 2014 ; 16 : 1838 – 46 . Google Scholar Crossref Search ADS PubMed WorldCat 28 Garrod D , Chidgey M. Desmosome structure, composition and function . Biochim Biophys Acta 2008 ; 1778 : 572 – 87 . Google Scholar Crossref Search ADS PubMed WorldCat 29 Te Riele AS , Tandri H , Bluemke DA. Arrhythmogenic right ventricular cardiomyopathy (ARVC): cardiovascular magnetic resonance update . J Cardiovasc Magn Reson 2014 ; 16 : 50. Google Scholar Crossref Search ADS PubMed WorldCat 30 Igual B , Zorio E , Maceira A , Estornell J , Lopez-Lereu MP et al. Arrhythmogenic cardiomyopathy. Patterns of ventricular involvement using cardiac magnetic resonance . Rev Esp Cardiol 2011 ; 64 : 1114 – 22 . Google Scholar Crossref Search ADS PubMed WorldCat 31 Rodrigues P , Joshi A , Williams H , Westwood M , Petersen SE et al. Diagnosis and prognosis in sudden cardiac arrest survivors without coronary artery disease: utility of a clinical approach using cardiac magnetic resonance imaging . Circ Cardiovasc Imaging 2017 ; 10 : e006709. Google Scholar Crossref Search ADS PubMed WorldCat Author notes The first two Diego Segura-Rodríguez and Francisco José Bermúdez-Jiménez authors contributed equally to this study. Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2019. For permissions, please email: journals.permissions@oup.com. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png European Heart Journal - Cardiovascular Imaging Oxford University Press

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Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2019. For permissions, please email: journals.permissions@oup.com.
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2047-2404
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2047-2412
DOI
10.1093/ehjci/jez277
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Abstract

Abstract Aims Arrhythmogenic right ventricular cardiomyopathy/dysplasia (ARVC/D) is a life-threatening entity with a highly heterogeneous genetic background. Cardiac magnetic resonance (CMR) imaging can identify fibrofatty scar by late gadolinium enhancement (LGE). Our aim is to investigate genotype–phenotype correlation in ARVC/D mutation carriers, focusing on CMR-LGE and myocardial fibrosis patterns. Methods and results A cohort of 44 genotyped patients, 33 with definite and 11 with borderline ARVC/D diagnosis, was characterized using CMR and divided into groups according to their genetic condition (desmosomal, non-desmosomal mutation, or negative). We collected information on cardiac volumes and function, as well as LGE pattern and extension. In addition, available ventricular myocardium samples from patients with pathogenic gene mutations were histopathologically analysed. Half of the patients were women, with a mean age of 41.6 ± 17.5 years. Next-generation sequencing identified a potential pathogenic mutation in 71.4% of the probands. The phenotype varied according to genetic status, with non-desmosomal male patients showing lower left ventricular (LV) systolic function. LV fibrosis was similar between groups, but distribution in non-desmosomal patients was frequently located at the posterolateral LV wall; a characteristic LV subepicardial circumferential LGE pattern was significantly associated with ARVC/D caused by desmin mutation. Histological analysis showed increased fibrillar connective tissue and intercellular space in all the samples. Conclusion Desmosomal and non-desmosomal mutation carriers showed different morphofunctional features but similar LV LGE presence. DES mutation carriers can be identified by a specific and extensive LV subepicardial circumferential LGE pattern. Further studies should investigate the specificity of LGE in ARVC/D. arrhythmogenic cardiomyopathy, cardiac magnetic resonance, desmin, late gadolinium enhancement, histology, myocardial fibrosis Introduction Arrhythmogenic right ventricular cardiomyopathy/dysplasia (ARVC/D) is a life-threatening inherited disease characterized by progressive fibrofatty replacement of the functional myocardium.1 This arrhythmogenic substrate leads to increased ventricular volumes, ventricular arrhythmias (VAs), and sudden cardiac death (SCD), especially among young people and athletes.2 The classic pathological features of ARVC/D consist of near-exclusive right ventricular (RV) wall involvement with extensive aneurysms with a ‘paper-thin’ appearance. However, disease expression is variable and may involve either or both ventricles (left-dominant pattern or biventricular pattern, respectively).3,4 Clinical diagnosis is based on demonstrating characteristic electrical, structural, and/or histological abnormalities. In addition, a positive family history for a pathogenic genetic mutation also contributes to the diagnosis.5 To date, 11 disease genes have been linked to the ARVC/D phenotype, highlighting its genetic heterogeneity. Approximately 50% of patients diagnosed with ARVC/D carry a pathogenic mutation in desmosomal genes, including desmoplakin (DSP), plakophilin-2 (PKP2), desmoglein-2 (DSG2), desmocollin-2 (DSC2), and plakoglobin (JUP).6 However, several non-desmosomal genes are increasingly recognized in ARVC/D pathogenesis, including desmin (DES),7 phospholamban (PLN), transforming growth factor beta-3 (TGFβ-3), transmembrane protein 43 (TMEM43), lamin A/C (LMNA),8 and the recently described filamin C (FLNC).9 Most of them are involved in structural and signalling functions. Over the last few decades, cardiac magnetic resonance (CMR) imaging has emerged as a powerful diagnostic tool in patients with suspected or diagnosed ARVC/D, providing accurate information even on the concealed form of ARVC/D. Recently, late gadolinium enhancement (LGE) has been recognized as an early and reliable means of assessing myocardial fibrofatty replacement10,11 and has been associated with an increased risk of SCD in non-ischaemic cardiomyopathy.12 Therefore, as genetic testing becomes more widespread, links between genetics and various patterns of ARVC/D are emerging. However, there is a lack of genotype–phenotype information and common dilemmas in differential diagnosis remain unclear, since current diagnostic criteria do not take account of the left-dominant form and new non-desmosomal genes are increasingly being recognized. The aim of this study is to investigate genotype–phenotype correlation in non-desmosomal and desmosomal ARVC/D mutation carriers in order to describe clinical features for diagnosis, focusing on CMR and fibrosis patterns. Methods Study population and clinical assessment From a cohort of patients referred to our tertiary medical centre (Virgen de las Nieves University Hospital, Granada, Spain) for evaluation of possible ARVC/D, we retrospectively included 44 individuals with a definite or borderline diagnosis of ARVC/D, based on 2010 diagnostic criteria, and in whom CMR was performed.5 Patients provided informed consent and the study was approved by the Institutional Review Board. Clinical evaluation All subjects underwent comprehensive clinical evaluation to determine ARVC/D diagnosis status according to International Task Force criteria.5 Clinical assessment included family history of ARVC/D or SCD, exhaustive medical history, 12-lead electrocardiogram (ECG), 24-h Holter monitoring, echocardiography, CMR, and genetic testing. A positive family history was considered if the patient had a first-degree relative in whom fibrofatty replacement had been histopathologically demonstrated in necropsy or a living relative with a definitive clinical diagnosis of ARVC/D. An abnormal ECG was defined as T-wave inversion beyond V3, lateral, or inferolateral leads, low QRS voltages, pathological Q waves, fragmented QRS, or non-sinus rhythm. Non-sustained ventricular tachycardia was defined as at least three consecutive ventricular beats at >100 bpm. The assessment of biventricular morphological and functional parameters was performed according to current recommendations.13 Cardiac magnetic resonance imaging All the patients gave their informed consent for the administration of gadolinium and underwent a CMR, which was performed on a 1.5-T MR system (General Electric, Signa EXCITE®, Milwaukee, WI, USA). The images were evaluated by four heart imaging experts (two cardiologists and two radiologists blinded to the genetic background and the aim of this study). The standard protocol included scout images (axial, sagittal, and coronal), double inversion recovery pulse sequence (dark blood images), structure and function module with balanced steady-state free precession (SSFP) sequence (short axis, four-chamber, three-chamber, two-chamber, and RV outflow tract). Finally, 0.1–0.2 mmol/kg of chelated gadolinium (Gadovist®) were administered, obtaining the LGE pictures using an inversion recovery sequence 7–10 min post-injection. Some additional sequences were acquired where clinically indicated. The post-processing analysis was performed using semi-automatic software (Reportcard®). The systolic function was assessed by volume measures, indexed to body surface area. Likewise, the ejection fraction was calculated by Simpson’s method. The volumes and ejection fraction were analysed by comparing them to the references values.14,15 Finally, the LGE images were visually analysed by describing their presence, location and distribution using an anatomical 17-segment left ventricular (LV) model and a 5-segment RV model. Genetic test Peripheral blood samples for the genetic analysis were obtained from the probands or the deceased index case, as applicable. We used a next-generation sequencing (NGS) gene panel containing 21 genes which had previously been reported as being associated with or are regarded as candidates for the development of arrhythmogenic cardiomyopathy (Supplementary data online, Methods). The pathogenicity of the identified variants was classified according to the current guidelines of the American College of Medical Genetics and Genomics (ACMG).16 After a potential disease-causing variant was identified in the index patient, genetic and clinical cascade screening was performed in all available family members. Histological analyses Tissue samples for histology were obtained from two deceased subjects (TMEM43 and FLNC carriers) and one explanted heart (DES carrier) under informed consent. Cardiac tissue samples were fixed in buffered 4% formaldehyde, dehydrated and embedded in paraffin following a conventional procedure.7,17 Sections of 5 µm thickness were prepared and stained with haematoxylin & eosin (HE) for morphological evaluation while the myofibril organization was evaluated with Heidenhain’s iron haematoxylin (HD) staining. Fibrillar collagens were stained using Masson’s trichrome (MS) and Picrosirius (PS, at light and polarized microscopy),17 whereas reticular collagens were stained with the Gomori silver stain (RET). Furthermore, in this study, a cardiac tissue sample from a deceased subject, without any cardiac disease, was used as a control (CTR). In addition, the percentage of fibrofatty infiltration was determined in sections stained with MS the ImageJ software (Bethesda, MD, USA) following a previously described methodology.18 Study design and statistical analysis The patients were divided into three groups: desmosomal mutation carriers, non-desmosomal mutation carriers, and patients with a negative genotype. After the data collection was finished, we reviewed all the case records and obtained further information from follow-up visits at 6 months and 1 year. Statistical analysis was performed using SPSS Statistics version 20.0 (IBM®, Armonk, NY, USA). The data were analysed for normality with the Shapiro–Wilk test. Clinical characteristics were compared using a χ2 or Fisher’s exact test for categorical variables and analysis of variance or Kruskal–Wallis for continuous variables. All continuous variables were described as mean value ± standard deviation for each measurement and categorical data were reported as frequencies and percentages. A P-value of <0.05 was considered statistically significant. Results Genetic and clinical evaluation Forty-four patients who had a borderline or definite ARVC/D diagnosis and in whom CMR was performed, were included in the study. Of these 44 patients, 22 were women (50%), and the mean age at diagnosis was 41.6 ± 17.5 years. Thirty-three (75%) fulfilled the current international Task Force Criteria for ARVC/D. The baseline characteristics are summarized in Table 1. Table 1 Baseline characteristics Overall (44) Non- desmosomal (25) Desmosomal (13) Negative (6) P-value Gender (female), n (%) 22 (50) 14 (56) 6 (46.2) 2 (33.3) 0.57 Age (years), mean ± SD 41.6 ± 17.5 37.04 ± 17.3 40.2 ± 16.8 56.7 ± 12.7 0.044 Fulfil TFC, n (%) 33 (75) 19 (76) 9 (69.2) 5 (83.3) 0.792 Asymptomatic, n (%) 27 (61.4) 19 (76) 7 (53.8) 1 (16.7) 0.022 VT/VF, n (%) 10 (22.7) 2 (8) 4 (30.8) 4 (66.7) 0.006 Family history SCD, n (%) 35 (79.5) 25 (100) 9 (69.2) 3 (50) 0.001 Phenotype,an (%)  RV 5 (11.4) 3 (12) 1 (7.7) 1 (16.7) 0.224  LV 10 (22.7) 9 (36) 0 (0) 1 (16.7)  BV 25 (56.8) 11 (44) 10 (76.9) 4 (66.7)  None 4 (9.1) 2 (8) 2 (15.4) 0 (0) Overall (44) Non- desmosomal (25) Desmosomal (13) Negative (6) P-value Gender (female), n (%) 22 (50) 14 (56) 6 (46.2) 2 (33.3) 0.57 Age (years), mean ± SD 41.6 ± 17.5 37.04 ± 17.3 40.2 ± 16.8 56.7 ± 12.7 0.044 Fulfil TFC, n (%) 33 (75) 19 (76) 9 (69.2) 5 (83.3) 0.792 Asymptomatic, n (%) 27 (61.4) 19 (76) 7 (53.8) 1 (16.7) 0.022 VT/VF, n (%) 10 (22.7) 2 (8) 4 (30.8) 4 (66.7) 0.006 Family history SCD, n (%) 35 (79.5) 25 (100) 9 (69.2) 3 (50) 0.001 Phenotype,an (%)  RV 5 (11.4) 3 (12) 1 (7.7) 1 (16.7) 0.224  LV 10 (22.7) 9 (36) 0 (0) 1 (16.7)  BV 25 (56.8) 11 (44) 10 (76.9) 4 (66.7)  None 4 (9.1) 2 (8) 2 (15.4) 0 (0) BV, biventricular; LV, left ventricle; RV, right ventricle; SCD, sudden cardiac death; TFC, Task Force Criteria; VT/VF, ventricular tachycardia/ventricular fibrillation. a Phenotype is defined by the presence of regional wall motion abnormalities and/or late gadolinium enhancement. Open in new tab Table 1 Baseline characteristics Overall (44) Non- desmosomal (25) Desmosomal (13) Negative (6) P-value Gender (female), n (%) 22 (50) 14 (56) 6 (46.2) 2 (33.3) 0.57 Age (years), mean ± SD 41.6 ± 17.5 37.04 ± 17.3 40.2 ± 16.8 56.7 ± 12.7 0.044 Fulfil TFC, n (%) 33 (75) 19 (76) 9 (69.2) 5 (83.3) 0.792 Asymptomatic, n (%) 27 (61.4) 19 (76) 7 (53.8) 1 (16.7) 0.022 VT/VF, n (%) 10 (22.7) 2 (8) 4 (30.8) 4 (66.7) 0.006 Family history SCD, n (%) 35 (79.5) 25 (100) 9 (69.2) 3 (50) 0.001 Phenotype,an (%)  RV 5 (11.4) 3 (12) 1 (7.7) 1 (16.7) 0.224  LV 10 (22.7) 9 (36) 0 (0) 1 (16.7)  BV 25 (56.8) 11 (44) 10 (76.9) 4 (66.7)  None 4 (9.1) 2 (8) 2 (15.4) 0 (0) Overall (44) Non- desmosomal (25) Desmosomal (13) Negative (6) P-value Gender (female), n (%) 22 (50) 14 (56) 6 (46.2) 2 (33.3) 0.57 Age (years), mean ± SD 41.6 ± 17.5 37.04 ± 17.3 40.2 ± 16.8 56.7 ± 12.7 0.044 Fulfil TFC, n (%) 33 (75) 19 (76) 9 (69.2) 5 (83.3) 0.792 Asymptomatic, n (%) 27 (61.4) 19 (76) 7 (53.8) 1 (16.7) 0.022 VT/VF, n (%) 10 (22.7) 2 (8) 4 (30.8) 4 (66.7) 0.006 Family history SCD, n (%) 35 (79.5) 25 (100) 9 (69.2) 3 (50) 0.001 Phenotype,an (%)  RV 5 (11.4) 3 (12) 1 (7.7) 1 (16.7) 0.224  LV 10 (22.7) 9 (36) 0 (0) 1 (16.7)  BV 25 (56.8) 11 (44) 10 (76.9) 4 (66.7)  None 4 (9.1) 2 (8) 2 (15.4) 0 (0) BV, biventricular; LV, left ventricle; RV, right ventricle; SCD, sudden cardiac death; TFC, Task Force Criteria; VT/VF, ventricular tachycardia/ventricular fibrillation. a Phenotype is defined by the presence of regional wall motion abnormalities and/or late gadolinium enhancement. Open in new tab The great majority of the patients were identified by family screening due to a positive family history of SCD or after a definitive family diagnosis of ARVC. Molecular-genetic screening identified a potential pathogenic mutation in 10 (71.4%) of the 14 index cases. Family cascade screening identified a positive genotype in 28 patients (Table 2). A mutation in the classic desmosomal genes (DSP, JUP, PKP2, DSG2, DSC2) was found in 13 patients (29.5%), being DSP the most prevalent gene affected. On the other hand, mutations in non-desmosomal genes were identified in 25 individuals (56.8%), being DES the most frequent gene (Figure 1). Figure 1 Open in new tabDownload slide Gene distribution. Figure 1 Open in new tabDownload slide Gene distribution. Table 2 Patients’ phenotypes by gene mutation and CMR pattern Gene Protein Nucleotide change Protein change Carriers LV RWMA RV RWMA LGE phenotype LGE LV extension DES Desmin c.1203G>C Missense 16 11 8 RV 1 LV 6 BV 9 Annular 13 Inferolateral 2 Inferior 1 DSP Desmoplakin c.3133C>T Nonsense 7 2 6 LV 1 BV 6 Annular 3 Septum 1 Inferolateral 2 Lateral 1 c.7697_7698insG Frameshift FLNC Filamin C c.4288 + 2T>G Nonsense 7 3 2 LV 5 RV 1 None 1 Inferolateral 3 Lateral 2 c.581_599delTGGTGG ACAACTGCGCCCC Nonsense DSG-2 Desmoglein2 c.875G>A Missense 5 3 4 BV 3 None 0 Annular 1 Lateral 2 c.535delA Nonsense PKP-2 Plakophilin2 c.1643delG Nonsense 1 None 1 1 LMNA Lamin A/C c.1541G>A Missense 1 None None None TMEM43 Luma c.1073C>T Missense 1 None 1 RV 1 Negative 6 2 5 LV 3 BV 2 None 1 Gene Protein Nucleotide change Protein change Carriers LV RWMA RV RWMA LGE phenotype LGE LV extension DES Desmin c.1203G>C Missense 16 11 8 RV 1 LV 6 BV 9 Annular 13 Inferolateral 2 Inferior 1 DSP Desmoplakin c.3133C>T Nonsense 7 2 6 LV 1 BV 6 Annular 3 Septum 1 Inferolateral 2 Lateral 1 c.7697_7698insG Frameshift FLNC Filamin C c.4288 + 2T>G Nonsense 7 3 2 LV 5 RV 1 None 1 Inferolateral 3 Lateral 2 c.581_599delTGGTGG ACAACTGCGCCCC Nonsense DSG-2 Desmoglein2 c.875G>A Missense 5 3 4 BV 3 None 0 Annular 1 Lateral 2 c.535delA Nonsense PKP-2 Plakophilin2 c.1643delG Nonsense 1 None 1 1 LMNA Lamin A/C c.1541G>A Missense 1 None None None TMEM43 Luma c.1073C>T Missense 1 None 1 RV 1 Negative 6 2 5 LV 3 BV 2 None 1 BV, biventricular; LGE, late gadolinium enhancement; LV, left ventricle; RV, right ventricle; RWMA, regional wall motion abnormality; SCD, sudden cardiac death. Open in new tab Table 2 Patients’ phenotypes by gene mutation and CMR pattern Gene Protein Nucleotide change Protein change Carriers LV RWMA RV RWMA LGE phenotype LGE LV extension DES Desmin c.1203G>C Missense 16 11 8 RV 1 LV 6 BV 9 Annular 13 Inferolateral 2 Inferior 1 DSP Desmoplakin c.3133C>T Nonsense 7 2 6 LV 1 BV 6 Annular 3 Septum 1 Inferolateral 2 Lateral 1 c.7697_7698insG Frameshift FLNC Filamin C c.4288 + 2T>G Nonsense 7 3 2 LV 5 RV 1 None 1 Inferolateral 3 Lateral 2 c.581_599delTGGTGG ACAACTGCGCCCC Nonsense DSG-2 Desmoglein2 c.875G>A Missense 5 3 4 BV 3 None 0 Annular 1 Lateral 2 c.535delA Nonsense PKP-2 Plakophilin2 c.1643delG Nonsense 1 None 1 1 LMNA Lamin A/C c.1541G>A Missense 1 None None None TMEM43 Luma c.1073C>T Missense 1 None 1 RV 1 Negative 6 2 5 LV 3 BV 2 None 1 Gene Protein Nucleotide change Protein change Carriers LV RWMA RV RWMA LGE phenotype LGE LV extension DES Desmin c.1203G>C Missense 16 11 8 RV 1 LV 6 BV 9 Annular 13 Inferolateral 2 Inferior 1 DSP Desmoplakin c.3133C>T Nonsense 7 2 6 LV 1 BV 6 Annular 3 Septum 1 Inferolateral 2 Lateral 1 c.7697_7698insG Frameshift FLNC Filamin C c.4288 + 2T>G Nonsense 7 3 2 LV 5 RV 1 None 1 Inferolateral 3 Lateral 2 c.581_599delTGGTGG ACAACTGCGCCCC Nonsense DSG-2 Desmoglein2 c.875G>A Missense 5 3 4 BV 3 None 0 Annular 1 Lateral 2 c.535delA Nonsense PKP-2 Plakophilin2 c.1643delG Nonsense 1 None 1 1 LMNA Lamin A/C c.1541G>A Missense 1 None None None TMEM43 Luma c.1073C>T Missense 1 None 1 RV 1 Negative 6 2 5 LV 3 BV 2 None 1 BV, biventricular; LGE, late gadolinium enhancement; LV, left ventricle; RV, right ventricle; RWMA, regional wall motion abnormality; SCD, sudden cardiac death. Open in new tab Cardiac imaging CMR morphofunctional analysis Among the 44 patients evaluated, 79.5% had LV involvement (35 patients), based on LGE and regional wall motion abnormalities (RWMAs). The distribution of ventricular affection patterns showed 5 patients (11.4%) with isolated RV disease (classic pattern), 10 (22.7%) with left-dominant affection, and 25 (56.8%) with biventricular involvement. Only four patients with positive genotypes had no structural heart involvement distinguishable by non-invasive imaging techniques; these were classified as silent carriers. Volume analysis is shown in Supplementary data online, Table S1. Overall, LV ejection fraction (LVEF) was mildly impaired (mean 48.9 ± 10%) or normal (47.7% had LVEF >50%). Men with non-desmosomal mutations showed slightly higher indexed LV end-diastolic volumes (iLVED) in comparison to men affected by desmosomal mutations and negative-genotype subjects (106.3 ± 25.6 mL/m2 vs. 80.7 ± 16.4 mL/m2 and 80.1 ± 17.3 mL/m2, respectively; P = 0.05). This was not observed in women. Men also tended to show lower LVEF values (44.6 ± 11.1% vs. 51.8 ± 6.6% and 56.4 ± 9.2%, respectively; P = 0.09). Female desmosomal mutation carriers showed a significantly lower RV ejection fraction (RVEF) in comparison to non-desmosomal carriers and negative-genotype patients (43.5 ± 9.8% vs. 52.6 ± 7.4% and 63.2 ± 5.3%, respectively; P = 0.02). Male desmosomal mutation carriers also showed lower RVEF values but the difference did not reach statistical significance. LV regional wall motion abnormalities (LV-RWMAs) were present in 21 patients (47.7%), distributed among 14 (56%) non-desmosomal mutation carriers, 5 (38.5%) desmosomal mutational carriers, and 2 (33.3%) negative-genotype subjects. Within LV-RWMAs, LV inferolateral wall involvement was frequent (15 subjects; 34.1%). With regard to the assessment of RV involvement, subjects with a desmosomal variant had a higher prevalence of RV-RWMAs than non-desmosomal mutation carriers (84.6% vs. 44%; P = 0.025). Late gadolinium enhancement A summary of LGE distribution is shown in Table 3. Fibrofatty replacement assessment with gadolinium was evaluated in the whole sample. Positive myocardial LV-LGE was present in 38 patients (79.5%). Biventricular distribution of LGE was present in 18 patients (45.4%). Exclusive LV presence of LGE was observed in 15 cases (34.1%). LGE location showed a predominance of subepicardial layer involvement (33; 86.8%). Mesocardial involvement was observed in two patients (one desmosomal mutation carrier and one negative genotype case). Table 3 Late gadolinium enhancement findings Non-desmosomal (25) Desmosomal (13) Negative (6) P-value LV-LGE positive, n (%) 20 (80) 10 (76.9) 5 (83.3) 0.94  Distribution LV-LGE, n (%) Subepicardial 20 (100) Subepicardial 9 (90) Mesocardial 1 (10) Subepicardial 4 (80) Mesocardial 1 (20) 0.17  Extension LV-LGE annular, n (%) 14 (56) 4 (30.8) 0 (0) 0.02 RV-LGE, n (%) 10 (40) 10 (76.9) 2 (33.3) 0.06 LGE pattern, n (%)  Isolated LV 11 (44) 1 (7.7) 3 (50) 0.27  Isolated RV 3 (12) 1 (7.7) 0 (0)  Biventricular 9 (36) 9 (69.2) 2 (33.3)  No 2 (8) 2 (15.4) 1 (16.7) Non-desmosomal (25) Desmosomal (13) Negative (6) P-value LV-LGE positive, n (%) 20 (80) 10 (76.9) 5 (83.3) 0.94  Distribution LV-LGE, n (%) Subepicardial 20 (100) Subepicardial 9 (90) Mesocardial 1 (10) Subepicardial 4 (80) Mesocardial 1 (20) 0.17  Extension LV-LGE annular, n (%) 14 (56) 4 (30.8) 0 (0) 0.02 RV-LGE, n (%) 10 (40) 10 (76.9) 2 (33.3) 0.06 LGE pattern, n (%)  Isolated LV 11 (44) 1 (7.7) 3 (50) 0.27  Isolated RV 3 (12) 1 (7.7) 0 (0)  Biventricular 9 (36) 9 (69.2) 2 (33.3)  No 2 (8) 2 (15.4) 1 (16.7) LGE, late gadolinium enhancement; LV, left ventricle; n, number; RV, right ventricle; RWMA, regional wall motion abnormality. Open in new tab Table 3 Late gadolinium enhancement findings Non-desmosomal (25) Desmosomal (13) Negative (6) P-value LV-LGE positive, n (%) 20 (80) 10 (76.9) 5 (83.3) 0.94  Distribution LV-LGE, n (%) Subepicardial 20 (100) Subepicardial 9 (90) Mesocardial 1 (10) Subepicardial 4 (80) Mesocardial 1 (20) 0.17  Extension LV-LGE annular, n (%) 14 (56) 4 (30.8) 0 (0) 0.02 RV-LGE, n (%) 10 (40) 10 (76.9) 2 (33.3) 0.06 LGE pattern, n (%)  Isolated LV 11 (44) 1 (7.7) 3 (50) 0.27  Isolated RV 3 (12) 1 (7.7) 0 (0)  Biventricular 9 (36) 9 (69.2) 2 (33.3)  No 2 (8) 2 (15.4) 1 (16.7) Non-desmosomal (25) Desmosomal (13) Negative (6) P-value LV-LGE positive, n (%) 20 (80) 10 (76.9) 5 (83.3) 0.94  Distribution LV-LGE, n (%) Subepicardial 20 (100) Subepicardial 9 (90) Mesocardial 1 (10) Subepicardial 4 (80) Mesocardial 1 (20) 0.17  Extension LV-LGE annular, n (%) 14 (56) 4 (30.8) 0 (0) 0.02 RV-LGE, n (%) 10 (40) 10 (76.9) 2 (33.3) 0.06 LGE pattern, n (%)  Isolated LV 11 (44) 1 (7.7) 3 (50) 0.27  Isolated RV 3 (12) 1 (7.7) 0 (0)  Biventricular 9 (36) 9 (69.2) 2 (33.3)  No 2 (8) 2 (15.4) 1 (16.7) LGE, late gadolinium enhancement; LV, left ventricle; n, number; RV, right ventricle; RWMA, regional wall motion abnormality. Open in new tab Different LGE distribution patterns were identified according to genetic background (Figure 2). There were no statistically significant differences in the presence of LV-LGE between desmosomal mutation carriers, non-desmosomal mutation carriers and negative-genotype patients (83.3% vs. 84% vs. 83.3%, respectively). The predominant LV-LGE distribution pattern in each group was subepicardial enhancement. Regarding the extent of LV-LGE, it was observed that individuals with non-desmosomal mutations, predominantly DES carriers, showed a characteristic annular or circumferential subepicardial LV-LGE pattern (Take home figure). This particular LV-LGE pattern was more frequently observed in subjects carrying a non-desmosomal mutation in comparison with desmosomal mutation carriers and negative-genotype subjects (76.5%, 23.5%, and 0%, respectively; P = 0.02). Among these individuals with non-desmosomal mutations, 13 were DES carriers (P = 0.006) and one had a truncating mutation in FLNC. This specific pattern was also observed in three DSP carriers and one DSG-2 carrier. On the other hand, a predominantly inferolateral distribution in the left ventricle was observed in most of the FLNC patients (Figure 2). Figure 2 Open in new tabDownload slide Representative late gadolinium enhancement images from non-desmosomal mutations (upper panels) and desmosomal mutations (lower panels). The white arrows show the enhancement zones. (A) DES mutation with circumferential LV enhancement. (B) FLNC mutation with lateral LV enhancement. (C) TMEM43 mutation patient with RV enhancement. (D) PKP-2 mutation with RV enhancement. (E) DSG-2 mutation with biventricular enhancement. (F) DSP mutation with biventricular enhancement. Figure 2 Open in new tabDownload slide Representative late gadolinium enhancement images from non-desmosomal mutations (upper panels) and desmosomal mutations (lower panels). The white arrows show the enhancement zones. (A) DES mutation with circumferential LV enhancement. (B) FLNC mutation with lateral LV enhancement. (C) TMEM43 mutation patient with RV enhancement. (D) PKP-2 mutation with RV enhancement. (E) DSG-2 mutation with biventricular enhancement. (F) DSP mutation with biventricular enhancement. Figure 3 Open in new tabDownload slide Histological images of human cardiac tissues affected by various mutations. Representative histological images from healthy (CTR) and genetically affected cardiac tissue stained using H&E (A) and HD (B) methods. Arrows indicate the increase in the fibrillar extracellular matrix components. Scale bar = 100 µm. Figure 3 Open in new tabDownload slide Histological images of human cardiac tissues affected by various mutations. Representative histological images from healthy (CTR) and genetically affected cardiac tissue stained using H&E (A) and HD (B) methods. Arrows indicate the increase in the fibrillar extracellular matrix components. Scale bar = 100 µm. Patients with desmosomal mutations notably tended to show a higher prevalence of RV-LGE in comparison to the non-desmosomal and negative-genotype groups (76.9% vs. 40% and 33.3%, respectively; P = 0.06). A summary of the genotype–phenotype correlation study, focusing on CMR findings, is shown in Table 3. Histological findings The histological analysis performed with HE staining showed an increase in the fibrillar connective tissue and intercellular space in all cardiac tissue from patients with genetic mutations (DES, FLNC, and TMEM43) as compared to CTR (Figure 3). The increase in the extracellular matrix was more evident in cardiac tissue from subjects affected by FLNC truncation with 51.42% and TMEM43 mutation with 40.35% of fibrofatty presence. Furthermore, certain degree of cardiomyocyte hypertrophy was observed in both mutations (Figure 3). In the case of DES mutation, HD staining was weaker than in other mutations and CTR, being suggestive of myofibrillar involvement. In addition, the presence of fibrofatty in the subject affected by DES mutation was 39.45% (Figure 3). In the case of the CTR the extracellular matrix (ECM) represented around 21.23%. Figure 4 Open in new tabDownload slide Histochemistry of fibrillar extracellular matrix components of human cardiac tissue affected by various mutations. Arrows indicate the increase in collagens, which were stained in green with MS (A) and red by PS (B) staining. PS polarized light microscopy (C) shows that the zones with collagens acquired a high level of arrangement and density. Gomori silver staining (D) shows the increase in reticular fibres as a black fibrillar precipitate. Scale bar = 100 µm. Figure 4 Open in new tabDownload slide Histochemistry of fibrillar extracellular matrix components of human cardiac tissue affected by various mutations. Arrows indicate the increase in collagens, which were stained in green with MS (A) and red by PS (B) staining. PS polarized light microscopy (C) shows that the zones with collagens acquired a high level of arrangement and density. Gomori silver staining (D) shows the increase in reticular fibres as a black fibrillar precipitate. Scale bar = 100 µm. The evaluation of the fibrillar ECM components with MS, PS, and RET stainings confirmed a clear increase in the intercellular space, which proved to be composed of fibrillar collagens (MS and PS) and reticular fibres (RET) in all affected cardiac tissues, differing with respect to the extracellular matrix of the CTR (Figure 4). Polarized-light microscopy analysis, performed with PS staining, revealed that in cardiac tissues affected by FLNC and TMEM43 mutations the increase in collagen fibres was accompanied by an increase in collagen birefringence, resulting from a complex parallel arrangement of these extracellular components. However, no birefringence for collagens was observed in cardiac tissue affected by DES mutation, where a pericellular increase in reticular fibres was, surprisingly, more evident (Figure 4). Take home figure Open in new tabDownload slide Characteristic circumferential LGE in a female patient affected by DES mutation. (A and C) Short-axis and four-chamber view SSFP cine sequences. (B and D) LGE images in short-axis and four-chamber view (white arrows) are shown. Take home figure Open in new tabDownload slide Characteristic circumferential LGE in a female patient affected by DES mutation. (A and C) Short-axis and four-chamber view SSFP cine sequences. (B and D) LGE images in short-axis and four-chamber view (white arrows) are shown. Discussion Since the first descriptions of ARVC/D cases appeared in the literature, the diagnosis of this entity has classically been based on gross and histological evidence of right ventricle fibrofatty transmural myocardial replacement.1 Advances in diagnosis have been made, especially in genetic testing and cardiovascular imaging. In the last decade, the disease spectrum has evolved considerably. NGS has expanded the disease beyond the classic desmosomal genes to include cytoskeletal7 or nuclear membrane genes8 with important roles in the mechanotransduction machinery.19 Our study describes the genotype–phenotype CMR and histological findings from a cohort of predominantly non-desmosomal ARVC/D patients, providing detailed information about different structural patterns of expression and fibrosis involvement. Specific LGE patterns have been identified in DES and FLNC mutation carriers, all of them at the subepicardial level. Characteristic fibrofatty replacement in ARVC/D can be non-invasively assessed using LGE-CMR, and this could be helpful in risk stratification and diagnosis of ‘concealed’ cases.20 However, LGE-CMR is not included in the revised current diagnostic criteria.5 In addition, it has been suggested that the non-desmosomal ARVC/D phenotype overlaps with the dilated phenotype and is considered a phenocopy with an increased risk of VAs and poor prognosis, recognizing the need for appropriate risk stratification, taking genetic background and LGE deposition into account.21 In the last few years, several genotype–phenotype correlations in ARVC/D have been proposed, mainly related to desmosomal mutations,22 but there is a lack of studies focusing on non-desmosomal mutations. Interestingly, our population was composed mainly of non-desmosomal mutation carriers. There is a lack of information in the literature on CMR findings in this population. CMR analysis showed abnormalities in most of the positive genotype subjects, the majority of whom were asymptomatic, highlighting its potential for identifying patients with an early or concealed stage of the disease. Myocardial fibrosis is a hallmark feature seen histologically in most cardiomyopathies. Fibrosis causes relaxation impairment and diastolic and systolic impairment and contributes to re-entrant mechanisms, increasing the risk of malignant VAs. Our data show that RV-LGE was more frequently observed in patients carrying a desmosomal mutation in comparison with the non-desmosomal and genotype-negative groups, although it did not reach statistical significance. Since the function of desmosomes is to link tissues mechanically, mutations in these proteins may lead to instability of the intercalated disk, making the cardiomyocyte more vulnerable to wall stress, particularly in a wall as thin as that of the right ventricle.23 Regarding LV-LGE involvement, it was localized at the subepicardial layer with progression to the mesocardium, characteristically in the posterolateral and septal LV wall, with no statistically significant differences between groups. Recently, these findings have been histologically correlated in desmosomal mutations carriers and PLN mutation carriers, being more pronounced in the latter.24 Our histological data suggest a similar cellular phenotype between groups with an increase in fibrillar connective tissue and intercellular space, as well as collagen network and arrangement. These similarities between the three samples suggest a common final pathway in non-desmosomal ARVC/D patients with impaired filament formation, cell membrane disruption and high incidence of fibrosis predisposing to VAs, as described by our group in a recent study.7 A novel and specific finding of our work is the LV-LGE subepicardial circumferential pattern observed in almost all patients carrying the recently described p.Glu401Asp variant in DES. Desmin is the main intermediate filament in cardiac muscle. With its multiple binding partners, it forms a three-dimensional scaffold that links various structures (the contractile apparatus, Z-discs, the nucleus, intercalated disks, sarcolemma, mitochondria, and other organelles). Desmin’s function is to integrate all these components, facilitating mechanochemical signalling and transport processes and crosstalk between different cellular organelles.25 Therefore, a dysfunctional desmin network may lead to severe cardiomyocyte dysregulation and cell death. To date, this pattern has not been described in association with any causal genotype of arrhythmogenic cardiomyopathy, even though it has been observed in some patients with biventricular involvement and no linked genotype.26 In addition, three DSP truncating-variant carriers showed this LGE distribution. Interestingly, truncating mutations in desmoplakin, which usually show a high risk of arrhythmia, seem to consistently cause extensive LV fibrosis, with globally distributed fibrosis being described in some patients.27 Desmoplakin is the most abundant component of the desmosome. It resides in the cytoplasmic surface of the desmosome and interacts with intermediate filaments, desmin in the cardiomyocyte, for desmosome assembly and cytoskeletal linkage.28 This similarity suggests that dysfunctional cytoskeletal-desmosome linkage and subsequent mechanochemical failure lead to the presence of a great amount of fibrosis with a specific circumferential subepicardial distribution and could therefore be easily identified using LGE images. Although the current data indicate that the subepicardial posterolateral pattern is suggestive of arrhythmogenic cardiomyopathy, this distribution has also been observed in other entities such as myocarditis, dilated cardiomyopathy, and sarcoidosis.29 LGE pattern, together with a reliable clinical scenario, family history of ARVC/D, the genetic background and propensity for VAs exceeding the degree of LV dilation could be helpful to distinguish ARVC/D from these entities.4,9 Therefore, up to now a specific non-invasive marker of the disease has been lacking. Here, we suggest that the distinctive circumferential subepicardial LGE pattern should be considered as suggestive of desminopathy or potentially as a failure of the major cardiomyocyte mechanochemical components. This genotype–phenotype correlation may improve the classification of arrhythmogenic cardiomyopathy and enhance understanding of the disease’s pathophysiology. Finally, most of the cases showed traces of LV involvement (including isolated or biventricular patterns). Classically, LV involvement has been described with minor increases in end-diastolic volumes and without any marked decrease in LVEF.30 LV affection was similar between the two groups, although men from the non-desmosomal group tended to show higher LV volumes and there was a significant decrease in RVEF in women in the group of desmosomal mutation carriers. Moreover, RWMAs are an important element of the current diagnostic criteria and CMR has great sensitivity for this purpose. However, LGE presence is an independent marker of VAs and SCD31 and a good marker of the presence of fibrosis and is not included in the current diagnostic criteria. In our cohort, RV-RWMAs were significantly frequent in the desmosomal mutations group. Interestingly, LV-RWMAs were similar in desmosomal and non-desmosomal mutation carriers. Nevertheless, LGE sequences demonstrated a higher presence of LGE than LV-RWMAs, indicating a high prevalence of LV fibrosis and constituting a more sensitive diagnostic tool. These findings reinforce the conclusion that diagnosis should not be based solely on the presence of RWMAs. Tissue characterization is particularly important in the LV phenotype, since we observed a high number of patients with a positive LGE presence who did not have RWMAs.22 Therefore, the absence of RWMAs in patients with suspected LV phenotypes should not rule out the disease. Further studies should investigate the specificity of LGE in a consecutive cohort of patients evaluated for ARVC/D. Limitations The small sample size enabled only preliminary conclusions to be drawn in terms of genotype–phenotype correlations. Clinical data exposed of the single pGlu401Asp variant may not be representative of the wide spectrum of DES mutation. It is possible that some potentially pathogenic genes have been missed in the negative mutations group. Finally, access to histological samples from patients affected by these mutations is limited so it not possible to perform statistical assumptions in terms of histopathological-LGE correlation. Conclusions Patients with ARVC/D carrying desmosomal and non-desmosomal mutations show different morphofunctional features in CMR, despite having similar LV posterolateral wall LGE distributions. Subjects with p.Glu401Asp in DES more frequently express a characteristic phenotype consisting of an extensive LV-subepicardial circumferential LGE pattern suggesting a different pathomechanism. Identifying phenotypic patterns suggestive of a genetic mutation may make it possibly to achieve early identification of forms of ARVC/D, which could enable clinical and therapeutic management and risk stratification to be individualized. In many cases, the presence of LGE is not associated with concomitant RWMAs, which highlights the role of tissue characterization with CMR in all patients with ARVC/D. Acknowledgements We thank Dr Cinta Moro, of the Instituto Nacional de Toxicología y Ciencias Forenses, for her kindness in providing the myocardial tissue samples. We are grateful for the technical assistance of Ms Fabiola Bermejo Casares, of the Department of Histology and Tissue Engineering Group, University of Granada, Spain. Conflict of interest: none declared. Funding Histological analyses were supported by the Tissue Engineering Group CTS-115 of the University of Granada, Spain (to V.C., D.D.-H., M.A., and A.C.). References 1 Thiene G , Nava A , Corrado D , Rossi L , Pennelli N. Right ventricular cardiomyopathy and sudden death in young people . N Engl J Med 1988 ; 318 : 129 – 33 . Google Scholar Crossref Search ADS PubMed WorldCat 2 Corrado D , Link MS , Calkins H. 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Journal

European Heart Journal - Cardiovascular ImagingOxford University Press

Published: Apr 1, 2020

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