A Comprehensive Analysis of Non-Desmosomal Rare Genetic Variants in Arrhythmogenic Cardiomyopathy: Integrating in Padua Cohort Literature-Derived Data

Arrhythmogenic cardiomyopathy (ACM) is an inherited myocardial disease at risk of sudden death. Genetic testing impacts greatly in ACM diagnosis, but gene-disease associations have yet to be determined for the increasing number of genes included in clinical panels. Genetic variants evaluation was undertaken for the most relevant non-desmosomal disease genes. We retrospectively studied 320 unrelated Italian ACM patients, including 243 cases with predominant right-ventricular (ARVC) and 77 cases with predominant left-ventricular (ALVC) involvement, who did not carry pathogenic/likely pathogenic (P/LP) variants in desmosome-coding genes. The aim was to assess rare genetic variants in transmembrane protein 43 (TMEM43), desmin (DES), phospholamban (PLN), filamin c (FLNC), cadherin 2 (CDH2), and tight junction protein 1 (TJP1), based on current adjudication guidelines and reappraisal on reported literature data. Thirty-five rare genetic variants, including 23 (64%) P/LP, were identified in 39 patients (16/243 ARVC; 23/77 ALVC): 22 FLNC, 9 DES, 2 TMEM43, and 2 CDH2. No P/LP variants were found in PLN and TJP1 genes. Gene-based burden analysis, including P/LP variants reported in literature, showed significant enrichment for TMEM43 (3.79-fold), DES (10.31-fold), PLN (117.8-fold) and FLNC (107-fold). A non-desmosomal rare genetic variant is found in a minority of ARVC patients but in about one third of ALVC patients; as such, clinical decision-making should be driven by genes with robust evidence. More than two thirds of non-desmosomal P/LP variants occur in FLNC.


Arrhythmogenic cardiomyopathy (ACM [MIM: 107970]
) is an autosomal dominant inherited disorder of the myocardium with a heterogeneous clinical presentation including life-threatening arrhythmias at risk of sudden cardiac death (SCD), especially in the young and athletes [1,2].
Herein, we report the frequency of rare genetic variants in ACM-related non-desmosomal genes in a well characterized ACM cohort in order to understand their relevance in disease diagnosis and clinical management.
Herein, we report the frequency of rare genetic variants in ACM-related nondesmosomal genes in a well characterized ACM cohort in order to understand their relevance in disease diagnosis and clinical management.

Results
Our cohort comprised 320 ACM unrelated Italian index patients without desmosome-related P/LP variants: 243 (76%) with ARVC and 77 (24%) with ALVC.Overall, 35 rare genetic variants in non-desmosomal disease-related genes were identified in 39 index cases: 3 classified as P, 20 as LP, and 12 as variants of uncertain significance (VUS) (Table 1).Clinically, only 16 index cases had an ARVC phenotype, whereas 23 had a ALVC phenotype, counting 6.6% among the ARVC cohort (16/243) and 29.9% of ALVC (23/77), respectively (Table 1).No PLN and TJP1 rare variants were identified in our cohort, but literature-reported variants were re-appraised (Figure 1, Table 2).Twenty-three of the 165 studies obtained from the initial literature search satisfied eligible criteria for qualitative and quantitative analysis in ACM carriers of TMEM43, DES, PLN, FLNC, CDH2, and TJP1 rare variants.In this setting, data collected from 104 index ACM cases highlighted 44 rare genetic variants in non-desmosomal disease-related genes (Table 2) of which 14 were classified as P, 19 as LP, 9 as VUSs, and 2 as benign.

TMEM43 Rare Variants and Cascade Genetic Screening
Transmembrane protein 43 was linked to ACM in 2008 [7], with the founder c.1073C>T (Ser358Leu) variant as the only P variant reported in 22 patients/families out of 553 investigated index cases since then (Table 2).The median age to develop ACM related to Ser358Leu is 32 years (95% CI: 28-35) for males and 44 years (95% CI: 39-48) for females.The disease is fully penetrant in males by the age of 63 and in females by the age of 76 years.
More recently, other TMEM43 rare variants were reported [28,34] but without sufficient evidence to determine their pathogenicity.In this setting, we identified the VUS missense c.349A>G (exon 4, Arg117Gly) variant in a 22-year-old patient with definite TFC but without family history of ACM or SCD (Figure S1-family D, #1, II-1, male, see Supplementary Results).

DES Rare Variants and Cascade Genetic Screening
Desmin was linked to ACM in 2009 [8] with the founder variant c.38C>T (Ser13Phe) in index cases exhibiting variable phenotypes characterized by high-grade AV block, rightventricle (RV) involvement and neurologic symptoms mainly affecting the lower limbs.The median age at presentation was 37 years for males (95% CI: 21-42) and 40.5 years for females (95% CI: 27-54).The disease is fully penetrant in males by the age of 49 and in females by the age of 51.5 years.To date, 14 rare variants have been reported in the literature in 21 ACM patients (Table 2) of which 6 classified as P, 6 as LP, 1 as VUS and 1 as benign.
In our cohort, genetic screening in DES revealed 9 rare genetic variants in 9 different probands (6 males, median age 35; 95% CI: 17-65), 5 of which were located at exon 1 encoding for the head ROD 1A protein domain (Table 1, Figure 1).Altogether, the sequence variant spectrum of DES includes 89% missense variants (8 out of 9; exemplified case in Figure 2).
Overall, 6 of 9 rare DES variants identified in our cohort were classified as LP due to variant properties, specifically mutational hotspots according to critical and well-stablished protein domains ("Head" and "IF rod") [35], literature evidence, and co-segregation analysis when available (Supplementary Results).Of note, the ALVC phenotype, both pathologically and clinically, is predominant in DES carriers, with a total of 6 index cases and 2 family members counting 66.7% of DES carriers.In detail, 55.6% of variants localize in the ROD 1A domain and comprise 3 cases with fatal event (one heart transplantation and two SCD).

FLNC Rare Variants and Cascade Genetic Screening
Filamin c was associated with ACM in 2016 [10], reporting 6 FLNC truncating variants in 7 patients (median age 38; 95% CI: 17-55) displaying infero-lateral negative T waves, mild to moderate LV systolic dysfunction, and regional dyskinesia.To date, 21 variants have been reported in the literature in approximately 53 ACM carriers, both index and family members (Table 2).

FLNC Rare Variants and Cascade Genetic Screening
Filamin c was associated with ACM in 2016 [10], reporting 6 FLNC truncating variants in 7 patients (median age 38; 95% CI: 17-55) displaying infero-lateral negative T waves, mild to moderate LV systolic dysfunction, and regional dyskinesia.To date, 21 variants have been reported in the literature in approximately 53 ACM carriers, both index and family members (Table 2).

CDH2 Rare Variants and Cascade Genetic Screening
In 2017, 2 P/LP CDH2 genetic variants in 3 ACM patients were reported simultaneously by two independent studies [11,12] engaging a total cohort of 78 patients.A more recent multicentric effort on 500 unrelated gene-elusive ACM patients, added 6 more rare CDH2 variants of which 4 were classified as P/LP (Table 2) [33].Overall, 8 ACM index patients harboring 7 P/LP CDH2 variants were described in the literature (4 males, median age 23, 95% CI: 14-59).However, disease penetrance and phenotypic characterization is still pending.
Among our 320 unrelated ACM patients only 2 CDH2 carried variants of unknown significance (Table 1 and Figure S4).

Burden of Rare Genetic Variants in Non-Desmosomal Genes
Taken together data obtained from our cohort and the literature, a 3.79-fold enrichment of rare non-synonymous TMEM43 variants was observed in ACM patients compared to the general population, with a frequency ranging from 1.9% (6/320) to 3.21% (28/873).Likewise, DES missense variants were enriched 10.31 times in ACM population and the frequency of this gene ranges from 2.8% (9/320) to 6.2% (30/486).FLNC rare non-synonymous variants frequency was calculated about 6.9-5.1% (22/320-42/818) but significant enrichment (107-fold) in the ACM population was evident only when considering radical variants (31/818).
Only 2 CDH2 VUS were found in our cohort (0.62%) summed with the scant literature evidence (10/898, 1.1%), leading to CDH2 genetic variant frequency estimation as 1%.This remark was further supported by no significant differences at the burden testing between ACM and control population for CDH2 rare non-synonymous variants.
As no rare PLN or TJP1 variants were found in our cohort, frequency calculation was based only upon literature data.Approximately 4.6% (31/670) of the affected ACM population may carry rare genetic variants in PLN, greatly influenced by the founder effect of the unique radical variant in the Dutch population.In TJP1, frequency was estimated about 0.55% (2/360) and no enrichment of rare genetic variants was evident probably due to the small cohort size evaluated.On the contrary, PLN was enriched over 100 times in ACM population due to its characteristic founder effect (Table 3).

Missense Variants Occur at Sites under Negative Selection
Evolutionary conserved sequences within proteins among species are crucial to maintain protein function and structure.In evolution, selection is a powerful force which works by constantly sorting through the variation that is produced by mutations to select the fit and remove the unfit, while ignoring neutral changes.As such, protein sequences of target genes were analyzed by studying the conservation of amino acids among 10 species calculating the Ka/Ks ratio, meaning the rate of non-synonymous substitutions to the rate of synonymous substitutions (Figure 1).TMEM43, DES, PLN and FLNC, are highly conserved protein sequences, with Ka/Ks ratio near 0, indicating negative selection pressure which tends to remove harmful variants.Instead, CDH2 presents a positive selection pressure particularly in 3 amino acids (Ka/Ks ratio > 1), indicating that missense variants in CDH2 tend to be enriched improving the fitness and consequently have less impact in protein structure.Although TJP1 is also under negative pressure, it is possible to observe how most amino acids have a ratio over 0.8, especially in the last 700 amino acids, indicating a less conservative region.
However, all missense variants examined in this study, obtained both from our cohort and the literature, are located in residues under negative selection pressure with Ka/Ks ratio < 0.3 as such were further assessed (Figure 1).

Discussion
About half of ACM patients fulfilling classical TFC harbor a causative rare variant in genes encoding cardiac desmosomal proteins [4].Many non-desmosomal genes have been recently linked to ACM pathogenesis displaying low disease penetrance and anecdotal frequencies in disease cohorts [36].In the clinical setting, current variant adjudication guidance is important for translating genetic data into information relevant to clinical diagnosis and family care.However, clinical genetic testing panels feature many disputed or limited-evidence genes.The present study provides a rigorous evaluation of rare variants in TMEM43, DES, PLN, FLNC, CDH2, and TJP1 genes and their incidence in ACM [7][8][9][10][11][12][13], integrating recent efforts from international scientific and clinical panels [6,37] to define the genetic architecture of monogenic ACM and its implication in the clinical management.Indeed, PLN and TMEM43 contribute to disease pathogenesis, particularly in geographic regions with well-characterized founder variants [7,9].DES was moderately associated with ACM by the ClinGen framework [6], whereas CDH2 and TJP1 belong to the category of genes recently associated with the disease with limited evidence of one or few rare P/LP variants detected in small families with ACM [6].For these genes, we replicated the screening in large cohort of gene elusive ACM cases in order to upgrade or not, the level of evidence.

Disputed Genes
The genes TJP1 and CDH2 exhibited a weak gene-disease relationship: Tight Junction Protein 1 gene, which encodes the multifunctional protein ZO-1 interacting with different proteins of cell-cell junctions in the so-called area composita, was linked to the disease phenotype in 2018 [38].Specifically, two rare variants were annotated in a mixed cohort of 40 Italian-Dutch-German ACM patients [13].Diseases associated with TJP1 include neovascular glaucoma and brain oedema and TJP1 related pathways include G-Beta gamma signalling and E-cadherin signalling in the nascent adherens junction.However, the absence of rare variants in the TJP1 gene in our local cohort of 320 ACM patients is well matched to the non-significant burden test obtained from literature data; as to support that this exceptional finding needs larger disease cohorts and more experimental evidence in order to assess its role in the disease pathogenesis.
The Cadherin-2 gene encodes a Ca 2+ -dependent cell adhesion protein, previously known also as N-cadherin, with a vital role in the intercalated disc [38].Diseases associated with CDH2 include agenesis of corpus callosum, cardiac, ocular and genital syndrome.In 2017, two independent studies demonstrated the implication of CDH2 missense variants to ACM pathogenesis [11,12].In our cohort we found two additional rare CDH2 variants both classified as VUS.Overall, gene prevalence was estimated less than 1% and enrichment analysis of rare genetic variants failed to show any difference of their burden in ACM with respect the control population.This observation along with the positive selection pressure in CDH2 reflect a disputed role of this gene in ACM pathogenesis.

Endemic Genes
The genes PLN and TMEM43 exhibited strong gene-disease association in limited geographic regions based on the accumulation of convincing genetic and experimental evidences.
Phospholamban is a Ca 2+ -ATPase, which triggered the hypothesis that Ca 2+ homeostasis might play an important role in the pathogenesis of arrhythmogenic phenotype in p.Arg14del carriers with ACM or dilated cardiomyopathy (DCM) [9].Multiple studies [9,28,34,39,40] support the conclusion that this variant is a founder mutation diffuse in the Dutch population.Even though the burden test for PLN rare genetic variants in the ACM population exhibited a 100-fold enrichment, no P variants were found in our cohort of 320 Italian index cases.The absence of PLN genetic variants in our cohort might be due to the effect of geographic limits underlying the importance of cohort location and ethnicity in order to avoid misinterpretation of genetic variants.However, this does not explain the spectrum of clinical features in patients with this variant adequately to suggest the possible involvement of other players in the disease development [41].
Transmembrane Protein 43 encodes for the formerly called LUMA protein, a fourtransmembrane domain protein which interacts with lamins and emerins and is involved in the structural organization of the nuclear membrane [42,43].Diseases associated with TMEM43 include Emery-Dreifuss muscular dystrophy type 7, whereas other studies have linked TMEM43 p.Ser358Leu variant to ACM as the founder pathogenic variant in limited regional cohorts [7,[19][20][21].Although this Ser358Leu is the only TMEM43 variant with pathogenic evidence till now, in our cohort TMEM43 frequency is about 1.9%.This frequency is similar to the one reported for some desmosomal genes, i.e., DSC2 and JUP [4] and a 3.79-fold enrichment was annotated for this gene in ACM patients.In particular, 5 patients, 3 with ARVC and 2 with ALVC, were carriers of the already reported p.Ser358Leu while the no-previously described p.Arg117Gly was identified in the sixth proband.However, the four small pedigrees available exhibited a disease penetrance of less than 100%, often with no signs or symptoms in elderly family carriers, suggesting the need for additional genetic or epigenetic factors to develop ACM instead of DCM.Larger multicentric cohort studies should be undertaken to confirm its univocal role in ACM.

Definitive Gene-Disease Association
Desmin is a structural intermediate filament protein present in the cytoskeleton of the leiomyocytes, rhabdomyocytes, and cardiomyocytes, and it is associated with different cellular structures.Its function is related to the maintenance of the structural integrity of the cardiomyocyte [44].DES-related myofibrillar myopathy is caused by missense rare genetic variants located in the central 2B domain of the protein, leading to skeletal muscle disease, which typically precedes cardiac involvement.However, P DES variants have also been described in DCM patients without skeletal muscle disease [45] and, in 2009, were linked to ACM typically affecting the RV or causing severe biventricular involvement [8,22].However, an ALVC pattern was highlighted in our cohort, counted in 66.7% of DES carriers.Of note, fatal events (heart transplantation and SCD) at a young age were noticed in 33.3% of our index cases, in keeping with a predominant LV involvement and high risk of ventricular arrhythmias, reported recently by a multicentric study [46].In contrast to this last [46], DES prevalence, combining the Padua and literature cohorts' data, is more frequent (6.2%, 30/486) with an enrichment of 10.31 times in the overall ACM cohort.This finding supports a definitive association of DES to the disease.Although the exact mechanism by which a single amino acid change could lead to disease development needs to be better elucidated, it is evident the involvement of DES variants in ACM with ARVC or ALVC or DCM or left-ventricular non-compaction [47].
Filamin C encodes gamma filamin, which is a muscle-specific protein playing a central role in muscle cells, participates in the anchoring of membrane proteins to the actin cytoskeleton in response to signalling events and displays structural functions at the Z lines in muscle cells [48].The frequency obtained from both Padua and the combined cohorts was in the range of 5.1%, confirming previously reported data on FLNC-related carriers (ranging from 3% to 7.5%) affected either by ACM or DCM [30,32].However, this gene has not yet been adjudicated by recent efforts of the ClinGen evidence-based framework [6].In this setting, burden testing of rare FLNC variants showed an astonishing 107-fold enrichment of radical variants in the ACM cohort, whereas this approach failed to show enrichment in the ACM population when radical and missense variants were summed together.Radical FLNC variants, which are expected to invoke haploinsufficiency via non-mediated decay, were found throughout the whole gene strongly associated to ALVC.This fact potentially could lead to Z-disc disarray and weakened cell-cell adhesion with subsequent impaired mechanotransduction.These structural alterations may promote fibrogenesis, contributing to the arrhythmogenesis, but functional data are still needed to determine the role of these variants in the ACM pathogenesis.Further, it is interesting to note that FLNC carriers demonstrated a later disease onset of the disease compared to desmosomal carriers, and muscular disabilities were not detectable or searched for.Specifically, only 5 of 34 FLNC carriers identified in our cohort fulfilled 2010 TFC showing a huge gap of these criteria in identifying ALVC which dropped disease incidence to 16.7%.Instead, 2020 Padua Criteria for ACM [49,50] were able to distinguish 8 ARVC, and 14 ALVC patients among FLNC carriers, estimating the disease penetrance about 71%.

Study Limitations
The small number of rare genetic variants in the most relevant non-desmosomal disease genes is well matched with the frequency reported in literature but does not exclude potential study bias.Co-segregation studies were limited due to the small number of relatives available for screening.The Padua cohort, and likely many cohorts derived from the literature, are gene-elusive ACM cohorts; as such the burden test analysis is referred to gene elusive ACM population.Public databases such as gnomAD provide some ancestry information; the exact ancestry of the individuals is not available and hence cannot be readily and directly compared to the ancestry of the case subjects.Potential bias derived from the use of gnomAD control database as control population cannot be excluded.These studies were filtered independently by three reviewers (M.C., M.B.M. and R.C.) and occasional disagreements that were unable to be resolved by the reviewers were settled by two additional authors (K.P. and C.B.).Only the first study published or the one containing more clinical details was included when the same cohort was reported by multiple studies fulfilling the inclusion criteria.
For each study, the following data were recorded: the first author, publication year, number of probands and family members tested, type of genetic analysis and genetic/aminoacidic variants.

Padua Cohort
The study cohort comprised 320 unrelated Italian index patients (233 males; median age: 45, 95% CI: 43-47) referred to the Referential Clinical Center for Inherited Cardiomyopathies of Padua.Specifically, 282 patients had a clinical diagnosis of ACM based on the 2010 Task Force Criteria (TFC) [3] or 2020 Padua criteria [49,50], in order to collect both dominant right-ventricular ACM with or without left-ventricular involvement (ARVC) and left-dominant ACM (ALVC); 38 index cases had a post-mortem diagnosis of ACM.
Prior genetic screening for single nucleotide variants or copy number variations in desmosome-encoding genes (PKP2, DSP, DSG2, DSC2 and JUP) showed no P or LP variants based on current American College of Medical Genetics and Genomics (ACMG) recommendations [51,52].
Clinical evaluation consisted of a detailed personal and family history, physical ex- These studies were filtered independently by three reviewers (M.C., M.B.M. and R.C.) and occasional disagreements that were unable to be resolved by the reviewers were settled by two additional authors (K.P. and C.B.).Only the first study published or the one containing more clinical details was included when the same cohort was reported by multiple studies fulfilling the inclusion criteria.
For each study, the following data were recorded: the first author, publication year, number of probands and family members tested, type of genetic analysis and genetic/ aminoacidic variants.

Padua Cohort
The study cohort comprised 320 unrelated Italian index patients (233 males; median age: 45, 95% CI: 43-47) referred to the Referential Clinical Center for Inherited Cardiomyopathies of Padua.Specifically, 282 patients had a clinical diagnosis of ACM based on the 2010 Task Force Criteria (TFC) [3] or 2020 Padua criteria [49,50], in order to collect both dominant right-ventricular ACM with or without left-ventricular involvement (ARVC) and left-dominant ACM (ALVC); 38 index cases had a post-mortem diagnosis of ACM.
Prior genetic screening for single nucleotide variants or copy number variations in desmosome-encoding genes (PKP2, DSP, DSG2, DSC2 and JUP) showed no P or LP variants based on current American College of Medical Genetics and Genomics (ACMG) recommendations [51,52].
Clinical evaluation consisted of a detailed personal and family history, physical examination, 12-lead ECG, 2-dimensional echocardiogram, signal-averaged ECG, and stress test ECG.CE-CMR was performed in selected cases with a previously detailed protocol [53].
The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Ethics Committee of Azienda Ospedale-Università Padova (protocol code 4501/AO/18 and date of approval 26 April 2018).Written informed consent was obtained from all subjects involved in the study.

Statistical Analysis
Statistical analysis was conducted with GraphPad Prism version 7.0 for Windows (GraphPad Software, La Jolla, CA, USA, www.graphpad.com).Age was expressed in terms of median ± 95%CI.Differences in variant distribution were assessed by Fisher's exact test for contingency analysis.Burden test in terms of differences in rare variant weight between ACM and gnomAD control population (considering as controls) were calculated by Fisher's exact test as previously reported [54].ACM allele count for each gene was determined taking together gene-elusive ACM population from the selected literature and Padua cohort whereas control alleles were extrapolated from gnomAD control database.Enrichment range of rare variants for each gene was calculated by Woolf logit interval for odd ratio as "relative risk" of having a causative rare variant in a determine gene when belonging to ACM cohort compared to the risk when belonging to control population.For a Detailed Description see Supplementary Materials.

Conclusions
Clinical diagnosis of ACM is still challenging, and genetic testing is often considered decisive, particularly when dealing with early ARVC and ALVC.Our study demonstrates that non-desmosomal genes often lack of evidence of gene-disease association and should not drive clinical decision-making.Additionally, some genes should be taken into account only in specific cohort location and ethnicity to reduce VUS, but penetrance calculation should be systematically reappraised.High frequency of missense DES and radical FLNC variants is observed in patients with a ALVC phenotype and high disease penetrance, supporting their strong pathogenic role.Indeed, only 6.6% of ARVC patients carry a rare genetic variant in non-desmosomal genes, at difference from 29.9% of ALVC patients.Functional studies are mandatory to clarify the role of non-desmosomal rare genetic variants in ACM pathogenesis and multicenter studies will profit in clarifying population divergences underlying phenotypic variability.Informed Consent Statement: Written informed consent was obtained from all subjects involved in the study.

Figure 1 .
Figure 1.Schematic representation of protein structure and aminoacidic variants localization.(A) Transmembrane protein 43 (TMEM43).(B) Desmin (DES).(C) Filamin C (FLNC).(D) Cadherin 2 (CDH2).Variants found in our cohort are highlighted in red.(E) Plot with amino acid sequences running on the x-axis and the selective pressure represented by Ka/Ks ratio on the y-axis.Red dots represent amino acidic changes found in our cohort or reported in literature.

Figure 1 .
Figure 1.Schematic representation of protein structure and aminoacidic variants localization.(A) Transmembrane protein 43 (TMEM43).(B) Desmin (DES).(C) Filamin C (FLNC).(D) Cadherin 2 (CDH2).Variants found in our cohort are highlighted in red.(E) Plot with amino acid sequences running on the x-axis and the selective pressure represented by Ka/Ks ratio on the y-axis.Red dots represent amino acidic changes found in our cohort or reported in literature.

Figure 2 .
Figure 2. Clinical setting of 17-year-old DES carrier affected by left-dominant ACM.Clinical-pathological correlation in 17-year-old male (#9) affected by ACM with left-dominant pattern who underwent cardiac transplantation for refractory heart failure (missense variant c.346A>G carrier).(A) 12leads ECG showing the typical epsilon waves in right precordial leads and T wave inversion on V1-V2.(B) Transthoracic echocardiogram with evidence of enlargement of right ventricular outflow tract (white arrow).(C) Cine-CMR confirming the dilatation (white asterisk).(D,E) Short-axis CE-CMR with LGE detectable on the entire RV wall (white arrow) and on the right side of the interventricular septum (white arrowhead).LV involvement in the inferior wall is also detectable (black arrow).(F) RV EMB demonstrating areas of replacement-type fibrosis and endocardial thickening (Heidenhain trichrome stain, panoramic view, scale bar 200 µm).(G) Gross view of the explanted heart with biventricular scars corresponding at histology (H) to transmural fibrosis of the RV free wall, subepicardial fibrosis in the LV and septal involvement on the RV-side (Heidenhaim trichrome stain, panoramic view, scale bar 5 mm).

Figure 2 .
Figure 2. Clinical setting of 17-year-old DES carrier affected by left-dominant ACM.Clinicalpathological correlation in 17-year-old male (#9) affected by ACM with left-dominant pattern who underwent cardiac transplantation for refractory heart failure (missense variant c.346A>G carrier).(A) 12-leads ECG showing the typical epsilon waves in right precordial leads and T wave inversion on V1-V2.(B) Transthoracic echocardiogram with evidence of enlargement of right ventricular outflow tract (white arrow).(C) Cine-CMR confirming the dilatation (white asterisk).(D,E) Short-axis CE-CMR with LGE detectable on the entire RV wall (white arrow) and on the right side of the interventricular septum (white arrowhead).LV involvement in the inferior wall is also detectable (black arrow).(F) RV EMB demonstrating areas of replacement-type fibrosis and endocardial thickening (Heidenhain trichrome stain, panoramic view, scale bar 200 µm).(G) Gross view of the explanted heart with biventricular scars corresponding at histology (H) to transmural fibrosis of the RV free wall, subepicardial fibrosis in the LV and septal involvement on the RV-side (Heidenhaim trichrome stain, panoramic view, scale bar 5 mm).

Figure 3 .
Figure 3. Flow diagram summarizing the literature review and inclusion/exclusion process.

Figure 3 .
Figure 3. Flow diagram summarizing the literature review and inclusion/exclusion process.
and B.B. are members of the European Reference Network for Rare and Low Prevalence Complex Diseases of the Heart (ERN GUARD-Heart).Institutional Review Board Statement:The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Ethics Committee of Azienda Ospedale-Università Padova (protocol code 4501/AO/18 and date of approval 26 April 2018).

Table 1 .
Clinical data of 39 Caucasian carriers of non-desmosomal gene variants.

Table 3 .
Burden test of non-synonymous rare genetic variants in ACM population.

Table 3 .
Cont.Significant differences were annotated between FLNC missense and radical variants.ACM allele count is calculated by combining cases in the literature and the Padua cohort.GnomAD database was considered as control population.Fischer's exact test and odd ratio calculation were performed for contingency analysis.Ctrls: control cases from gnomAD population; OR: Odd ratio. *