Exploring the complex spectrum of dominance and recessiveness in genetic cardiomyopathies

Discrete categorization of Mendelian disease genes into dominant and recessive models often oversimplifies their underlying genetic architecture. Cardiomyopathies (CMs) are genetic diseases with complex etiologies for which an increasing number of recessive associations have recently been proposed. Here, we comprehensively analyze all published evidence pertaining to biallelic variation associated with CM phenotypes to identify high-confidence recessive genes and explore the spectrum of monoallelic and biallelic variant effects in established recessive and dominant disease genes. We classify 18 genes with robust recessive association with CMs, largely characterized by dilated phenotypes, early disease onset and severe outcomes. Several of these genes have monoallelic association with disease outcomes and cardiac traits in the UK Biobank, including LMOD2 and ALPK3 with dilated and hypertrophic CM, respectively. Our data provide insights into the complex spectrum of dominance and recessiveness in genetic heart disease and demonstrate how such approaches enable the discovery of unexplored genetic associations.


Gene summaries for recessive cardiomyopathy genes
Below are summaries of the evidence for the association of genes with recessive forms of cardiomyopathies.For full details of the studies, genetic variants and clinical and phenotypic details of the cardiomyopathy patients, see Supplementary Table 1.The genes here are divided into those replicated in multiple family pedigrees (and therefore considered validated) and those found in single pedigrees (and therefore considered as still being candidate recessive cardiomyopathy genes).

Validated recessive cardiomyopathy genes ALPK3
ALPK3, encoding for ɑ-protein kinase 3, has been associated with a recessive form of cardiomyopathy in 26 individuals from 17 families across 9 publications [1][2][3][4][5][6][7][8][9] .The phenotypes described are variable but are often associated with a diagnosis of DCM at birth or infancy which can progress to LV hypertrophy and HCM over a period of months.There is also widespread variability in severity and outcomes for these cases -while there have been some reports of early death and heart transplantation, most patients were alive at the time of reporting and there have been several reports of later onset disease.Additional extra-cardiac, particularly dysmorphic, features are often observed in cases but there does not seem to be a consistent syndromic pattern to these.In these early reports, most heterozygous relatives of patients with biallelic truncating ALPK3 variants were reported to be healthy, although a minority (5/37, 13.5% 6 ) were diagnosed with HCM in adulthood.Subsequently a large cohort study found an association for heterozygous ALPK3 truncating variants with adult-onset HCM, with such variants being detected in 1.6% of HCM cases across two cohorts and characterised by age-related penetrance in families (perhaps explaining the large proportion of apparently unaffected heterozygous relatives in earlier studies who are likely to be relatively young adult relatives of paediatric cases) 10 .While most of the earlier reported cases involved biallelic truncating variants, recent studies have also included missense variants (either as homozygotes or compound heterozygotes with truncating variants).As we now know that heterozygous truncating variants are associated with adult-onset HCM, the role of some missense variants (when compound heterozygous with truncating variants) may be questionable, especially for later onset disease.The ALPK3 transcript has recently been updated (from a 1907 amino acid coding transcript to a 1705 residue one) as the previous exon 1 is now believed to be non-coding (it contains many common truncating variants in gnomAD).Because of this, three compound heterozygous cases (from two families) described by Herkert et al. were not included in this analysis, as one of the reported variants affected the previous exon 1 6 .Note that there may also therefore be a discrepancy with the variant descriptions of earlier reports.ALPK3 was classified as having Strong evidence for association with "Infant-onset HCM/DCM" by the ClinGen HCM curation in 2018 (based on the initial reports) 11 and was upgraded to Definitive in February 2022.ALPK3 is also a strong candidate to be the causal gene at a locus that has been associated with HCM, DCM and several related LV traits in multiple GWAS [12][13][14] .ALPK3 demonstrates only marginal constraint of loss-of-function variants in gnomAD (LOEUF=0.91).Homozygous ALPK3 null mice generally recapitulate phenotypes of biallelic patients, displaying myocardial hypertrophy and disarray, as well as DCM-associated features like reduced cardiac output and ejection fraction 15 .
Heterozygous ALPK3 null mice had normal contractile function and survival but developed hypertrophy after one year 16 .In summary, there is an abundance of evidence for the association of ALPK3 truncating variants with cardiomyopathy for both recessive and dominant inheritance.

Function of ALPK3
A recent study found that ALPK3 is actually a pseudokinase, with its kinase domain sequence having diverged to the point of lacking any catalytic activity 16 .ALPK3 was shown to colocalise with myomesin proteins (MYOM1, MYOM2) at the nuclear envelope and sarcomere M-band.Loss-of-function ALPK3 variants caused these myomesin proteins to mislocalize and dysregulated several additional M-band proteins involved in sarcomere protein turnover.

BAG5
BAG5, encoding for Bcl-2-associated athanogene co-chaperone 5, has been associated with a recessive form of isolated DCM in 6 individuals from 5 families in two publications 17,18 .In the main study evaluating 4 Japanese families, BAG5 was initially highlighted through a transcriptomics study as an upregulated gene in failing hearts, with biallelic truncating variants in BAG5 then identified in 5/536 individuals with DCM (though this cohort apparently included both probands and affected relatives, so it is difficult to estimate the overall disease frequency) 17 .For one family, the pathogenicity of the BAG5 homozygous variant was verified through homozygosity mapping, exome sequencing and variant filtering in two affected and two unaffected individuals.The BAG5 patients had early onset disease (four between 11-14 years and one at 34 years) characterised by rapid progression, the development of ventricular tachycardia/fibrillation and the requirement for LVAD support.Although heterozygous relatives were unaffected, heterozygous BAG5 truncating variants were detected in 3/26 individuals with tachycardia-induced cardiomyopathy (a reversible cardiomyopathy phenotype), suggesting such variants may act as cardiomyopathy risk factors.Knock-in mice for the recurrent BAG5 DCM-associated variant p.Arg197* displayed ventricular dilatation, reduced systolic function and arrhythmogenicity which was ameliorated by administration of an AAV-BAG5 wild type vector.BAG5 may be particularly prevalent in Japan as the recurrent p.Arg197* variant has a MAF of 0.000168 for in the ToMMo database of 38,000 Japanese individuals.BAG5 is not constrained for loss-of-function variants in gnomAD (LOEUF=1.16).A subsequent genome sequencing study on a cohort of paediatric cardiomyopathy patients from Canada identify one case with the homozygous p.Gln125* variant 18 .The demonstration of multiple affected families (including through genome-wide analysis in one family) and strong supporting evidence in knock-in mice studies support a role for this gene in cardiomyopathy.

Function of BAG5
BAG5 acts as a co-chaperone for HSC70 (for which it acts as a nucleotide exchange factor) which is responsible for maintaining proteostasis (the correct folding of proteins and refolding of misfolded proteins).BAG5 is localised to junctional membrane complexes (JMC) which are critical for calcium handling and loss-of-function variants lead to reduced levels of JMC protein, disturbed T-tubule morphology and perturbed calcium handling 17 .

CAP2
CAP2 encodes adenylyl cyclase-associated protein 2, one of the two isoforms (the other being CAP1) of the ancestral protein CAP, which is important for actin organisation regulation in a wide-range of organisms.A total of 4 papers concerning the CAP2-cardiomyopathy association in humans have been published (two family-based, one cohort-based and a case report).Family-based and cohort-based analyses describing single patients concern a large consanguineous Bedouin family from Israel, a trio of unspecified ethnic background (both sequenced with WES) and a Pakistani cohort [19][20][21] .The four affected individuals across the different studies were homozygous carriers of ultra-rare proteintruncating variants and were all affected by severe forms of early-onset DCM (LVEFs 5-34%, ages of onset 0-12 years, data not reported for the patient from the Pakistani cohort) leading to congestive heart failure and/or death or enlistment for heart transplant.The Pakistani proband had a positive family history, with two siblings who had previously died of DCM.A fourth paper that has been excluded from our analysis reports on a family with two affected children with a homozygous variant in CAP2, but does not provide variant details 22 .Of note, the gnomAD-based LOEUF intolerance score of CAP2 is 0.407, with an observed/expected protein-truncating variant ratio of 0.19, indicating that truncating variants are constrained in the population.Functional validation experiments demonstrated the protein truncating effects of the variant segregating in the Bedouin family and supported a nonsense-mediated-decay disease-causing mechanism for the nonsense variant observed in the trio.Tissue analysis revealed the presence of nemaline rods in the skeletal muscles of the trio's proband.In terms of non-human evidence, four CAP2 mouse models were published [23][24][25][26] .The majority of homozygous knockout mice developed severe DCM and malignant ventricular arrhythmias and died suddenly.However, the phenotype observed in mutant mice was not always consistent with that observed in human variant carriers, as besides their cardiac phenotype knockout mice were observed to suffer also from microphtalmia, and females tended to survive close to expected levels and with live normal life spans 24 .Furthermore, heterozygous mice have also been observed to suffer from malignant arrhythmias 25 .The most recent of these papers focuses entirely on the skeletal muscle phenotype and does not describe the animals' cardiac phenotype 26 .In summary, there is convincing evidence for the association of CAP2 with recessive DCM with four independent families (albeit one without variant details) and equivalent data in mice.

Function of CAP2
CAP2 is a 477-residue multifunctional protein that localises to the centre of the sarcomere (M-line), in close proximity to thin filament pointed ends in the heart.CAP2 inhibits actin incorporation into thin filaments and promotes thin filament depolymerization in a tropomyosin-dependent manner by increasing the activity of cofilin by two orders of magnitude.Different from other pointed-end proteins, CAP2's function is not enhanced but inhibited by tropomyosin and it does not directly control thin filament lengths.In addition, CAP2 plays a key role in cardiomyocyte maturation by modulating pre-sarcomeric actin assembly and regulating alpha-actin composition in mature filaments.CAP2 interacts with filamentous actin via a helical-folded domain located in the N-terminal region, and with globular actin via a Wiskott-Aldrich-homology 2 (WH2) and a CAP-retinitis pigmentosa (CARP) domain, both located in the C-terminal region.

FBXO32
An association between FBXO32 (F-box only protein 32) and autosomal recessive DCM was found in two studies with six affected biallelic individuals across two families, one from Saudi Arabia and the other from Iran 27,28 .Both of these MENA region countries are associated with high consanguinity rates and indeed the parents in both families are first cousins.The associated biallelic variants were nontruncating: a missense variant, p.Gly243Arg, in the Saudi family and an inframe indel variant, p.Lys295del, in the Iranian family.All heterozygous individuals were unaffected.The LOD score in the Saudi family reached a significant value of 3.37.The variants were associated with adolescent to young adult onset DCM (median age of onset 20 years; range 14-26 years).There was significant variation in outcome -most of the individuals with DCM were relatively asymptomatic, whereas a 26 year old proband developed decompensated heart failure and underwent a heart transplant.Another affected individual was reported to experience shortness of breath only.Histology of an explanted diseased heart after heart transplantation from one of the probands showed hypertrophied cardiomyocytes, and immunohistochemistry revealed apparent reduced FBXO32 protein expression.FBXO32 is a putative causal gene at a locus associated with HCM using a multi-trait analysis of GWAS (MTAG) approach 29 .According to the GWAS Catalog, common variant loci mapped to FBXO32 have also been associated with atrial fibrillation [30][31][32][33] and ECG traits 34 in a number of GWAS analyses.While there are only two genetic studies for the FBXO32 association, replication in independent families including a significant LOD score of 3.37 in one has been demonstrated, in conjunction with additional supporting GWAS evidence.

Function of FBXO32
FBXO32 encodes the F-box only protein 32.This constitutes one of the four subunits of the SCF protein complex (Skp, Cullin, F-box containing complex); the other three subunits are Skp1, Cullin and Rbx1.The particular type of F-box protein in an SCF complex varies, and so confers substrate specificity.SCF acts as the E3 ubiquitin-ligase component in the ubiquitin-proteasome system.An E2 ubiquitinconjugating enzyme binds to the Rbx1 subunit of SCF, inducing the transfer of ubiquitin from E2 to the target protein bound by the F-box subunit of the SCF.This marks the target protein for degradation by the proteasome.In human heart samples from DCM patients homozygous for the p.Gly243Arg variant in FBXO32, Al-Yacoub et al. showed an upregulation of the CHOP transcription factor and dysregulation of endoplasmic-reticulum stress proteins, compared to control human hearts 35 .It was therefore suggested that the defective FBXO32 protein stimulates the unfolded protein response, a type of stress response caused by the presence of unfolded or misfolded proteins in the lumen of the endoplasmic reticulum.This response leads to increased CHOP expression which activates an apoptosis pathway, thereby putatively causing DCM and heart failure.

FLII
The Al-Hassnan et al. cohort study of 205 Saudi Arabian unrelated probands with paediatric cardiomyopathy reported two consanguineous families with a variant in FLII (FLII Actin Remodelling Protein), associating it with paediatric autosomal recessive dilated cardiomyopathy 36 .In 2023, Ruijmbeek et al. reported again on the same 2 families, providing more clinical details, and on a third (non-consanguineous) pedigree from the Netherlands 37 .All the families were sequenced with WES, which revealed homozygosity for the FLII p.Arg1240Cys and p.Leu674Val missense variants in the first two families, and compound heterozygosity of p.Arg1168Trp and the protein-truncating p.Gln454* variant in the Dutch pedigree.Parents were sequenced in all cases and were unaffected heterozygous carriers of a single FLII variant passed on to the proband.In gnomAD, p.Arg1240Cys and p.Arg1168Trp are present in heterozygous form with the rare frequencies of 0.06% in East Asians and 0.009% in Finns, respectively, while p.Leu674Val and p.Gln454* are absent.Probands (two girls and one boy) were characterized by an early onset (< 1 year) and were all reported alive at the ages of 2, 3 and 7 years.The phenotype appears consistent across these three cases, with disease presentation characterized by a markedly reduced LV function (LVEF in the 23-32% range), tricuspid and mitral regurgitation and the absence of heart rhythm abnormalities or extra-cardiac features.In all cases, the proband showed either stable disease or improved cardiac function at follow-up.Using CRISPR-Cas9, Ruijmbeek et al. created genome-edited zebrafish with genotypes corresponding to those observed in 2 of the 3 families (i.e.homozygosity for p.Arg1230Cys -corresponding to human p.Arg1240Cys -and compound heterozygosity for p.Arg1158Trp and p.Ser449*, mimicking the variants carried by the Dutch proband).The phenotype of both zebrafish models resembled the one observed in the families, with fish showing a normal heart rate accompanied by significantly reduced contractility and EF.Using confocal microscopy coupled with 3D rendering, authors also showed that fish homozygous for p.Arg1230Cys had less organized and more primitive cardiac trabeculae besides less densely packed and less organized myofibrils and intercalated discs compared with controls.A range of other functional validation experiments were performed by Ruijmbeek et al on KO fish from a previously published line with a premature stop codon in place of a leucine residue at position 111, characterized by embryonic lethality 38 .These experiments showed how KO animals manifested a very severe cardiac phenotype characterized by, for example, a markedly decreased ventricular complexity (i.e.low number of trabeculae), cell architectural changes (i.e.cardiomyocytes extrusions), irregular and poorly defined filament organization, and thinner and unbundled myofibrils.In addition, expression and immunohistochemical analyses showed how FLII dysfunction also results in the dysregulation of DCM-related signaling pathways (Notch and Hippo) during ventricular morphogenesis.Of note, embryonic lethality was also observed for KO mice 39 .

Function of FLII
FLII encodes the FLII actin remodelling protein, otherwise known as the flightless I actin binding protein.FLII contains a gelsolin actin binding domain and a leucine rich repeat protein interaction domain.Using the gelsolin domain, FLII binds to F-and G-actin and controls actin filament assembly and disassembly, for example FLII has the ability to sever actin filaments 40 .The FLII protein is involved in early embryogenesis and structural organisation, and published data overall strongly suggest a crucial role in striated muscle function.

JPH2
JPH2, which codes for the junctophilin-2 protein, has been found to be associated with recessive paediatric DCM in 7 individuals from 7 families across 6 publications 36,[41][42][43][44][45] .Four families were from the MENA region, known for higher consanguinity rates, and one of the families was Finnish-a known genetically isolated founder population.A mixture of biallelic truncating and non-truncating variants were associated using gene sequencing panels and WES.The recessive variants in JPH2 seem to be associated with early-onset paediatric DCM (median age of onset 3 years; range 0.67-11 years) with poor outcomes (66% deceased).JPH2 truncating variants are marginally constrained (LOEUF=0.78) in gnomAD.JPH2 has been classified with moderate evidence for association with DCM via a semidominant mode of inheritance by the ClinGen curation 46 ; semi-dominant due to one JPH2 DCM study that claimed dominant inheritance 47 .In the studies above, individuals heterozygous for the truncating and non-truncating variants were not affected.However, heterozygous missense variants in JPH2 are known to be associated with autosomal dominant adult-onset HCM, with the gene classified as having moderate evidence for the association by the ClinGen curation 11 .JPH2 homozygous knockout mice exhibited embryonic lethality 48 .In summary, there is substantial genetic evidence that JPH2 is associated with recessive DCM, although further data will be required to fully define the genotypephenotype relationships for DCM and HCM.

Function of JPH2
Junctophilin-2 is part of the junctophilin family of proteins, which are junctional complex proteins.Junctophilin-2 is the predominant paralog in cardiac tissue 48 .The protein stretches from its membrane occupation and recognition nexus (MORN) domain located at the t-tubular sarcolemma, to its hydrophobic C-terminus located at the sarcoplasmic reticulum (SR) membrane 49 .In doing so, junctophilin-2 connects L-type calcium channels in the t-tubule sarcolemma to the ryanodine receptors in the SR membrane, allowing for dyad formation and effective calcium-induced calcium release 49 .

KLHL24
KLHL24, encoding for the ubiquitin ligase substrate receptor Kelch-like (KLHL) protein 24, has been associated with a recessive form of isolated HCM in 9 individuals from 4 families across 3 publications 36,50,51 .Hedberg-Oldfors et al. identified two consanguineous families with biallelic loss-offunction variants in KLHL24 -a nonsense variant (p.Glu350*) in two siblings of Iraqi origin with HCM and a missense variant (p.Arg206His) in five affected individuals in a large pedigree of Iranian ancestry 50 .The latter was identified through linkage/homozygosity mapping and WES, yielding a LOD score of 3.6.The affected individuals in these pedigrees were diagnosed at relatively early ages (16-28 years), with three other sudden deaths of individuals in their 20s reported in these families (though with unknown genotype), suggesting KLHL24 variants are associated with a severe HCM phenotype.Additional cases identified through WES included a HCM patient diagnosed at 11 years reported in a large case series of childhood onset cardiomyopathy in Saudi Arabia (with the nonsense variant p.Trp387*) 36 and a patient of Middle Eastern origin who had a mixed cardiomyopathy phenotype of HCM, DCM and LVNC features (with the same p.Arg306His variant described in the large Iranian pedigree) 51 .Morpholino knockdown of the KLHL24a homologue in zebrafish resulted in similar heart defects observed with other HCM-causing variants which could not be rescued by KLHL24a mRNA carrying the equivalent variants observed in HCM patients (in contrast to wild type KLHL24a mRNA) 50 .Truncating variants in KLHL24 are rarely observed and somewhat constrained (LOEUF=0.65) in gnomAD, suggesting this gene is likely to be a very rare cause of HCM and may be largely restricted to populations of high consanguinity.The role of KLHL24 in cardiomyopathies is supported by the fact that 40% of cases with KLHL24 gain-of-function start lost variants causing epidermolysis bullosa simplex, a hereditary skin fragility disorder, were also diagnosed with DCM 52,53 .In summary, there is convincing evidence for association of KLHL24 with recessive HCM, established by strong genetic data in multiple families (including robust segregation evidence in one with a LOD score of 3.6) and other supporting evidence.

Function of KLHL24
Several KLHL proteins act as adaptors for the recruitment of substrates to Cul3-based E3 ubiquitin ligases and therefore contribute to the turnover of structural proteins in muscle cells.An upregulation of desmin was detected in the heart and skeletal muscle of the HCM patients described by Hedberg-Oldfors et al., suggesting it might be a substrate of KLHL24 in myocytes 50 .This was confirmed by a study that showed KLHL24 start-lost gain-of-function variants conversely led to a tenfold reduction in desmin protein levels (using engineered heart tissue generated from patients with such variants) 54 .

LDB3
LDB3, encoding for the PDZ-LIM domain-binding factor (also known as ZASP -Z-disc Alternatively Spliced Protein) has been associated with a recessive form of DCM in 5 individuals from 5 families in a single publication 55 .All cases were associated with a severe early onset phenotype, although one was terminated during pregnancy upon discovery of cardiac defects by ultrasound and biallelic LDB3 variants by prenatal testing.Four of the cases were from consanguineous families, suggesting this gene is likely to be more prevalent in populations of high consanguinity.The role of LDB3 in recessive DCM is supported by animal model data, with both knockout mice (global and cardiac-specific) and zebrafish morpholino knockdown leading to severe DCM phenotypes [56][57][58][59] .While heterozygous missense variants in LDB3 have been implicated in DCM in a number of studies, the gene was classified as having Limited evidence for association by the ClinGen curation, due to a lack of robust statistical genetics and functional evidence (note, this curation occurred prior to the publication of the study above) 46 .All relatives with heterozygous truncating variants in the recessive study were unaffected, although loss-of-function variants are somewhat constrained in gnomAD (LOEUF=0.68).LDB3 is a reported mapped gene for PR interval 60 and ECG morphology 34 according to the GWAS Catalog.Notably, there are both short and long isoforms of LDB3 expressed -variants affecting the short isoform have been implicated in skeletal myopathies.The one patient (aborted foetus) with biallelic variants affecting the short isoform displayed irregular Z-disc formation in skeletal muscle whereas the four cases with variants affecting the long isoform only had cardiac restricted phenotypes.Although only one study has thus far associated LDB3 with recessive DCM, the demonstration of multiple affected families and strong supporting evidence from animal models support a role for this gene in recessive cardiomyopathy.

Function of LDB3
LDB3/ZASP is involved in the formation, structural stability and function of the sarcomere Z-disc, through key interactions with proteins like actin, alpha-actinin 2, myotilin and protein kinase C.

LEMD2
An association between LEMD2 (LEM Domain Nuclear Envelope Protein 2) and autosomal recessive arrhythmic cardiomyopathy was found in one study of two families with 11 affected biallelic individuals overall 61 .These families were from the Hutterite isolated endogamous ethnoreligious population in Canada -all of the parents of the above mentioned individuals were consanguineous.The associated biallelic variant, found using WES, was a missense variant, p.Leu13Arg, in LEMD2.The homozygous phenotype was described as "arrhythmic" cardiomyopathy since the patients did not meet the diagnostic criteria for arrhythmogenic cardiomyopathy, but nonetheless presented with severe ventricular arrhythmias resulting in sudden cardiac death, often as the first symptom.Left ventricular fibrosis and mild impairment of LV systolic function was also characteristic of the reported cardiomyopathy.A DCM phenotype was present in one homozygous individual.There appears to be age-dependent penetrance, with onset of sudden cardiac arrest around the third and fourth decades of life.This adult onset cardiomyopathy had a median age of onset of 31 years (range 15-46 years).The outcomes were of moderate severity (death or heart transplant in 27% of the affected individuals at median age 28 years, range 27-42 years).Of the individuals genotyped as heterozygous, all showed no cardiac abnormalities on echo-and electrocardiography.The biallelic phenotype also included juvenile cataracts.A separate, earlier study from the United States in 2016 also linked the same homozygous LEMD2 variant to juvenile-onset cataracts by a recessive mode of inheritance in three Hutterite families-of note, six individuals with cataracts also presented with sudden cardiac death 62 .Homozygous LEMD2 knockout mice died in-utero, by E11.5 63 .The p.Leu13Arg variant from the human Hutterite studies was engineered into two independent knock-in mouse models -both studies reported a DCM-like phenotype with fibrosis and arrhythmia in homozygous mice 64,65 .Wild-type LEMD2 delivered using AAV9 rescued the phenotype in the Caravia et al. study 65 .Although only a single LEMD2 variant in a founder population has been associated with recessive cardiomyopathy, the strong segregation data in multiple pedigrees, combined with equivalent phenotypes in mouse knockin studies, support the pathogenicity of this gene.

Function of LEMD2
LEMD2 encodes the LEM domain nuclear envelope protein 2, which localises to the inner membrane of the nuclear envelope.It is thought to be involved in DNA replication, mitosis and nuclear structure organisation 66,67 .Other genes that also encode inner nuclear membrane proteins, such as LMNA, are a known cause of DCM.The 2019 Hutterite study noted earlier showed abnormal nuclear envelope morphology and condensed peripheral heterochromatin in TEM micrographs of patient cardiac tissue and fibroblasts 61 .Assays on these fibroblasts showed decreased proliferation, increased senescence and a prolonged G1 phase compared to controls.The p.Leu13Arg mouse knock-in cardiomyocytes displayed more nuclear membrane invaginations, nuclear envelope associated heterochromatin disorganisation, DNA damage and apoptosis.The interaction between mutant LEMD2 and BAF (barrier-to-autointegration factor) was found to be disrupted; BAF is required for the nuclear envelope repair process.Therefore, impaired nuclear structure organisation, possibly via disruption of nuclear envelope rupture repair, may underpin the pathophysiology of a rare recessive arrhythmic cardiomyopathy described in the Hutterite population.

LMOD2
LMOD2 encodes leiomodin 2 (LMOD2), an actin-binding protein.Published genetic evidence of biallelic variant pathogenicity in humans concerns a total of 5 families from 5 studies which describe 6 neonate patients affected by an extremely early-onset and severe form of DCM [68][69][70][71][72] .An additional case was not sequenced for LMOD2 although her phenotype and outcome was essentially identical to that of her variant-carrying sister and has been excluded by our analysis.Of the 6 neonates included in our work, one had a later-than-usual onset at 10 months of age (potentially triggered by a viral infection, followed by heart transplant 4 months later) but in all the others disease appeared hours/days after birth.Of these neonate patients, two received heart transplant before 1 year of age, while the other 3 died within hours/weeks.All the 6 neonates were carriers of biallelic ultra-rare LMOD2-truncating variants in homozygosity or compound heterozygosity, with the gene characterised by a gnomAD-derived LOEUF intolerance score of 0.86 and an observed/expected ratio for proteintruncating variants of 0.48, indicating that the gene is marginally constrained toward this type of variants.Of note, one of these alleles was carried by two distinct probands, and for all these variants no homozygous individuals are present in gnomAD.Where reported, LVEF of the described neonates was in the 14-35% range and in three cases the heart was described as almost non-contractile or immobile, though not always dilated.Four of the 6 cases also developed ventricular tachycardia.Tissue staining showed in three papers that carriers' cardiomyocytes possessed significantly shorter thin filaments vs controls 68,70,72 .Of note, 2 of the 6 reported patients were carriers of the same variant (p.Trp398*) and showed some differences in terms of age of onset (0 vs 4 months), histology (while significant vs controls in both cases, the shortening of thin filaments was not of comparable severity) and electrical activity (ventricular tachycardia and ectopy vs ventricular tachyarrhythmia).Functional validation results also demonstrated the total absence of LMOD2 protein from the heart of a homozygous nonsense variant carrier characterised by a 5-fold decrease in contraction force 68 , and the lack of full-length LMOD2 in a carrier of an essential splice site variant 70 .Non-human evidence comprises two mouse models.A LMOD2-knockout mouse was characterised by abnormally short thin filaments, severe contractile dysfunction and ventricular enlargement consistent with DCM and leading to death around 3 weeks of age 73 .A knock-in mouse carrying the homologous proteintruncating variant to that detected in one of the aforementioned neonates engineered to escape nonsense-mediated decay unexpectedly had a substantially less severe phenotype, with no correlation between expression level and severity (milder phenotype observed even with the lowest expression levels) 68 .In summary, consistent evidence across multiple independent studies and families, as well as mouse knockout data, support the association of LMOD2 with recessive DCM.

Function of LMOD2
LMOD2 functions as an important regulator of thin filament by promoting elongation of actin through polymerization at pointed ends.Leiomodin is a homolog of tropomodulin, a capping protein that has the opposite effects: while tropomodulin plays a role in capping of filament ends to terminate elongation, leiomodin acts as powerful nucleator of actin polymerization in vitro to lengthen thin filaments.LMOD2 competes for binding with Tmod1 and displaces it, allowing for actin polymerization/thin filament elongation 74 .

MYZAP
MYZAP, encoding for the myocardial zonula adherens protein, has been associated with a recessive form of isolated DCM in 8 individuals from 3 families (from Finland and Slovenia) in two publications 75,76 .Homozygous truncating variants were identified in each family through exome sequencing.Age of onset ranged from 14-41 years but the cases were associated with relatively severe outcomes, with 75% of the patients either dying or requiring heart transplantation.Heterozygous relatives of the patients were all unaffected and MYZAP is not constrained of loss-of-function variants in gnomAD (LOEUF=1.24).Knockdown of the MYZAP homologue in zebrafish caused severe cardiomyopathy 77 , while knockdown in mice led to heart failure and death after transverse aortic constriction 78 .A common missense variant in MYZAP, p.Gln254Pro, was associated with atrial fibrillation in a study using cases/controls from Iceland, UK Biobank, Norway and USA (OR=1.38)-the variant has a MAF of 0.011 in Iceland, 0.016 in Finland but only 0.0036 in UKBB/gnomAD-NFE 79 .Although only two studies have thus far implicated MYZAP variants in recessive cardiomyopathy, replication in multiple families with consistent phenotypes and supporting evidence from animal models and GWAS together provide substantial evidence for association.

Function of MYZAP
MYZAP is located at the intercalated disc structure of intercellular junctions between cardiomyocytes, where it interacts with desmoplakin and zonula adherens proteins.MYZAP is part of the broader GRINL1A complex transcription unit which comprises genes/transcripts for GCOM1, MYZAP and POLR2M.GTEx exon expression (PEXT metric) indicates that the cardiac tissue expression is restricted to the exons of MYZAP, suggesting it is the relevant cardiac gene/transcript at this locus.

NRAP
NRAP, encoding for nebulin-related anchoring protein, has been associated with a recessive form of isolated DCM in 24 individuals from 22 families across 6 publications 36,41,[80][81][82][83] .In the initial reports, biallelic truncating variants were identified by exome sequencing in small family pedigrees 41,81,83 and a large case series of childhood onset cardiomyopathy in Saudi Arabia 80 .Cases were largely early onset (though this may partially reflect publication bias towards childhood disease) with severe outcomes, although two reports of unaffected homozygous carriers in their mid-30s suggests incomplete penetrance.In the cohort of childhood onset cardiomyopathy in Saudi Arabia reported by Al-Hassnan et al., NRAP accounted for 14.3% of all DCM cases with positive results, indicating it is likely to be a prevalent disease gene in regions of high consanguinity (perhaps driven by recurrent variants present at low frequency in these populations) 36 .Koskenvuo et al. recently published an enrichment analysis for NRAP, with double variants (including at least one truncating variant) detected in 1.9% of 577 DCM cases compared to none in 25912 controls and 5150 non-DCM cardiac cases (p<0.0001,OR=1052) 82 .There are some caveats with this dataset however.Only 6/11 cases were confirmed biallelic / compound heterozygous (based on homozygosity or segregation analysis).Additionally, 6/11 cases had one truncating and one missense variant, although 4 of these carried the missense variant p.Gln24His which is strongly predicted to affect splicing.While ancestry data was not provided for these cases, this study was conducted by Blueprint Genetics in Finland -the recurrent variants described (p.Thr1458Glnfs36, p.Arg1502*, p.Tyr448*, p.Gln24His) are particularly enriched in Finnish individuals in gnomAD, indicating the relatively high prevalence of NRAP biallelic variants may be restricted to such populations.Koskenvuo et al. also highlighted an enrichment of single heterozygous NRAP truncating variants in DCM cases (1.9%) compared to controls (0.3%) and non-DCM cardiac cases (0.5%) (p<0.0001,OR=6.7), suggesting these variants may be risk factors of intermediate effect sizes in DCM patients 82 .There is no constraint of loss-of-function variants in gnomAD however (LOEUF=1.0).NRAP is a reported mapped gene for QT interval according to the GWAS Catalog 84 .Upregulation of NRAP has been observed in mouse models of DCM 85 and in human DCM patients 86 , while cardiacspecific experimental overexpression of NRAP led to right ventricular cardiomyopathy in mice 87 .In summary, there is abundant evidence that biallelic truncating variants in NRAP are associated with isolated DCM, they lead to severe and relatively early onset disease (though not always in infancy/childhood) and are likely to be particularly prevalent in regions with bottleneck populations or high consanguinity.

Function of NRAP
NRAP is involved in myofibrillogenesis during cardiomyocyte development in the foetal heart.In the adult heart, it anchors terminal actin filaments to the membrane and plays a role in tension transmission from the sarcomere to the extracellular matrix.NRAP comprises a LIM domain, which interacts with alpha-actinin and talin, and 46 nebulin repeats.The latter comprises a domain of 11 simple repeats, which interacts with actin, KLHL41 and muscle LIM protein (CSRP3), and a C-terminal section of 5 super repeats, which interacts with filamin C and vinculin.

PLEKHM2
PLEKHM2, which codes for the pleckstrin homology and RUN domain containing M2 protein, has been associated with recessive DCM with left ventricular non-compaction (LVNC) in 5 individuals, four males and one female, across two families in two studies.In a study on a Bedouin family from the Negev, Muhammad et al. described four DCM individuals homozygous for a truncating frameshift variant (p.Lys645Alafs*12) in PLEKHM2, identified using WES and homozygosity mapping; the 11 heterozygotes were unaffected 88 .Fibroblasts from the individuals displayed impaired autophagy flux, perinuclear distribution of lysosomes and abnormal distribution of endosomes.PLEKHM2 cDNA transfection rescued the lysosome distribution.A case study from the United States described a 21 year old female with DCM; panel testing uncovered compound heterozygosity for two PLEKHM2 variants-a paternally inherited frameshift variant (p.Gly919Alafs*34) and a maternally inherited synonymous splice site variant (p.Ser662Ser) 89 .An in-silico analysis of the synonymous splice region variant predicted loss of consensus splicing (SpliceAI donor loss score = 0.72).Her father displayed syncope with bradycardia at 42 before receiving a pacemaker; SCD in her paternal grandfather and great-grandfather was reported at 65 years and 40s respectively.Her mother and siblings were healthy.Immunostaining for PLEKHM2 showed reduced expression in the patient versus control.The recessive variants in PLEKHM2 seem to be associated with an approximately adolescent-onset DCM (median age of onset 16 years; range 7-21 years) with moderately poor outcomes (two deceased and one heart transplantation).PLEKHM2 truncating variants are somewhat constrained (LOEUF=0.54) in gnomAD.PLEKHM2 has been classified with limited evidence for association with DCM via an autosomal recessive mode of inheritance by the ClinGen curation 46 , due to a lack of studies -at the time of curation only the first study was published.PLEKHM2 is a putative causal gene for a genomewide significant locus associated with DCM-relevant LV cardiac traits in a UK Biobank study 14 .Although only two studies have thus far implicated biallelic PLEKHM2 variants with cardiomyopathy, their identification in independent pedigrees with consistent phenotypes supports this association.

Function of PLEKHM2
The pleckstrin homology and RUN domain containing M2 protein, encoded by PLEKHM2, is part of a complex which links kinesin-1, a motor protein that moves to the plus-end of microtubules, to lysosomes 90 .More specifically, the WD domains of the PLEKHM2 protein bind to the light chains of kinesin-1, and its N-terminal RUN domain binds to Arl8-GTP which itself is bound to the lysosome.The PLEKHM2 protein also contains a C-terminal PH domain which is free -it is speculated to have functions in motor or membrane traffic regulation.Lysosomes degrade unnecessary or dysfunctional cellular components and as such their motility is critical in facilitating effective autophagy.Therefore, impaired autophagy may be important in the pathogenesis of DCM in patients with PLEKHM2 truncating variants.

PPA2
PPA2 encodes the inorganic phosphatase 2, a ubiquitously expressed mitochondrial protein important for the phosphate metabolism of cells.Published research concerning the association of PPA2 variants with human cardiomyopathies comprises 7 papers reporting on 32 families (with 56 affected individuals carrying biallelic variants and 64 unaffected heterozygotes) of various ethnic backgrounds 41,44,[91][92][93][94][95] .The largest share of these families (N=23 of 32) are described in two papers published by the same group 92,94 .The vast majority (79%) of the reported biallelic variant carriers died suddenly at a very young age (mean age of death 3.6 years), either as a result of sudden arrhythmogenic events or of sudden heart failure.The rest either had an unknown clinical phenotype/outcome or were reported as alive (following transplant or ICD implantation and/or in some cases with recurrent arrhythmogenic events or severe cardiomyopathy).One was reported as affected by syndromic disease with no cardiac symptoms, and has been excluded from our analysis.In cases in which an autopsy was performed, a viral infection and/or alcohol ingestion seemed to have acted as a trigger for the sudden deterioration of cardiac function or arrhythmogenic death.Other features shared by many cases were emesis before death, necrotic/fibrotic areas of the myocardium and lactic acidosis.Of note, cardiomyopathy or the presence of dysmorphic ventricles were described for a minority of cases (23%).Reported extra-cardiac features ranged from none to severe multi-organ involvement.The vast majority of variants reported in affected individuals are rare missense alleles carried in homozygosity or in compound heterozygosity.All reported variants are rare, with the commonest being p.Glu172Lys, observed in 0.095% of Non-Finnish Europeans in gnomAD (but no homozygotes) and remarkably recurrent in the affected individuals reported in the literature, being carried by members of 17 different families.Of note, other recurrent variants were also observed in up to 5 distinct families.Functional validation experiments showed that specific variants (such as p.Glu172Lys) caused a substantial to near-complete inactivation of the phosphatase activity and their association with decreased levels of PPA2 protein in the myocardium 91 .Other experiments showed the loss of phosphatase in fibroblasts to be associated with a drastic reduction of mitochondrial respiratory chain Complex IV, and in silico modelling predicted specific variants to disrupt stabilising interactions and create new ones in the hydrophobic core of the protein 41 .No PPA2-related animal models are reported in the literature to date.In summary, there is an abundance of evidence that biallelic variants in PPA2 are associated with sudden cardiac death and an arrhythmogenic cardiomyopathy in young people, although considerable heterogeneity exists in the reported phenotypes.

Function of PPA2
The inorganic phosphatase 2 hydrolyses inorganic pyrophosphate, generated by many nucleotidedependent reactions.This activity is essential for the correct regulation of mitochondrial membrane potential, and mitochondrial organisation and function 92 .The mechanism through which inorganic pyrophosphatase deficiency can lead to heart or global multiorgan dysfunction is not well understood.A hypothesis is that the accumulation of pyrophosphate beyond a certain threshold has an impact on the regulation of mitochondrial inner membrane potential and thus leads to chronic ADP build-up, with the most disruptive consequences observed in energy-consuming organs like heart and brain 94 .

PPP1R13L
PPP1R13L codes for the protein phosphatase 1 regulatory subunit 13 like, also called inhibitor of apoptosis stimulating protein of p53 (iASPP).PPP1R13L is expressed mainly in skin, testes, heart and stomach.This gene has been associated with a recessive, severe, early-onset form of DCM/ACM in 6 studies 36,[96][97][98][99][100] .These describe a total of 11 families originally from Eastern Europe (5 pedigrees) and Middle Eastern countries or ancestries (6 pedigrees), all of which were investigated with WES/WGS.In most of the 15 reported variant carriers, the disease is not described specifically as DCM or ACM, but as a "cardio-cutaneous" syndrome with a certain variability in terms of extra-cardiac symptoms and their severity.While for 20% of the biallelic PPP1R13L variant carriers no extra-cardiac features are reported, in the majority hair and/or skin and nail anomalies were observed in addition to cardiomyopathies, with other features reported including teeth and/or head/neck anomalies, cleft lip/palate and neurological complications or cognitive delay.Two individuals from an Israeli family were not sequenced and have been excluded from our analysis, although the phenotype and clinical history were remarkably similar to that of ascertained variant carriers from the same pedigree.Most PPP1R13L variants detected in these homozygous or double heterozygous variant carriers are predicted to have a protein-truncating effect with the rest being missense variants.All variants are ultra-rare in the gnomAD database with no homozygous carriers.Heterozygous variant carriers (N=23) in the described families are reported as not having a related disease phenotype.Of note, PPP1R13L has a gnomAD-derived LOEUF intolerance score of 0.49 with an observed/expected ratio of predicted protein-truncating variants of 0.28, indicating that the gene is constrained for loss-of-function variants.The cardiac phenotype of the described biallelic variant carriers is overt DCM or (in 1 case only) biventricular arrhythmogenic CM progressing to reduced systolic function and heart failure.Onset was consistently during early childhood (by age 4), with the exception of a single case where disease was reported to have manifested at 8 years of age.LVEF, where provided, was in the range 9-23% and in the majority of cases outcomes were either death (in all cases before 5 years of age) or heart transplant (at 2-8 years of age).Three biallelic variant carriers (of whom the oldest was 6 at the time) were reported alive.Functional validation experiments demonstrated fibroblasts from some of the biallelic variant carriers to have lower mRNA compared with control cells and no iASPP protein (supporting nonsense-mediated decay as the disease mechanism), coupled with higher mRNA levels of specific cytokine-coding genes, suggesting a pro-inflammatory effect of the pathogenic variants.This was observed also in PPP1R13L-knockdown murine cardiomyocytes.As far as animal models are concerned, one of the aforementioned family-based studies 96 reports on mice with spontaneous recessive PPP1R13L mutations that had been previously studied 101,102 .These mice get born blind with open eyelids and develop rapidly progressing and fatal DCM.Furthermore, differentially expressed genes in these mice compared with wild-type animals were reported enriched for inflammationrelated pathways, and injection of a pro-inflammatory molecule in mutant mice caused death while having no deadly effect in variant-free mice.In summary, there is abundant genetic and animal model evidence that biallelic loss-of-function variants in PPP1R13L are associated with severe early-onset recessive cardiomyopathy with a cardio-cutaneous phenotype.

Function of PPP1R13L
iASPP is one of the most evolutionarily conserved inhibitors of the transcriptional activity of NF-kB and p53.The inhibition of p53 is possibly exerted by preventing the association between p53/TP53 and ASPP1 or ASPP2.According to the hypothesis tested by Falik-Zaccai et al., the absence of iASPP would unleash NF-kB to increase transcription of pro-inflammatory mediators and, as a consequence, would lower the heart's threshold to inflammatory response, induce prolonged inflammatory processes and eventually DCM 96 .

RPL3L
Four studies have found an association between RPL3L, which codes for ribosomal protein L3 like, and autosomal recessive infantile-onset DCM.In total, the association was found in 9 individuals, five male and four female, from 6 families in 4 studies 36,[103][104][105] .Affected individuals were compound heterozygous or homozygous for generally non-truncating variants, identified using WGS or WES.All were missense except for one family who were compound heterozygous for a missense and frameshift variant, though the latter was C-terminal variant predicted to escape NMD.Parents were consanguineous in two of the families (from Saudi Arabia and Colombia).All heterozygous individuals were unaffected.The recessive variants in RPL3L are associated with very early-onset infantile DCM (median age of onset 46 days; range 1-274 days) with very poor outcomes (death or heart transplant in all individuals at median age 91 days; range 15-486 days).Non-rare missense (p.Ala75Val) and splice donor (c.1167+1G>A) variants in RPL3L were associated with atrial fibrillation in a study using cases/controls from Iceland, UK Biobank, Norway and USA (OR=1.2 and 1.5 respectively) 79 .Two other missense variants (pArg4Gln, p.Arg242Trp) enriched in the Finnish population were associated with atrial fibrillation in a FinnGen GWAS 106 .RPL3L is also a reported mapped gene for p-wave duration according to the GWAS Catalog 107 .In summary, the identification of biallelic RPL3L variants in multiple independent families with consistent phenotypes provides robust evidence for association with recessive DCM.

Function of RPL3L
The human eukaryotic 80S ribosome, responsible for protein synthesis, is a ribonucleoprotein complex consisting of the 60S large subunit and 40S small subunit.The 60S subunit is composed of rRNA and 46 ribosomal proteins.RPL3 encodes one of these proteins, the ribosomal protein large 3. RPL3L, ribosomal protein large 3-like, is a paralog of RPL3 which has a tissue specific high level of expression in skeletal and cardiac muscle.An in-vitro myoblast cell line study showed a reduction in myotube growth when RPL3L was expressed via an inducible promoter gene expression system 108 .It follows that pathogenic variants in RPL3L may therefore cause DCM via some impairment of ribosomal function -in particular, dysregulated cardiac muscle growth may be involved.

SLC30A5
One study from Germany reported an association between SLC30A5 (Solute Carrier Family 30 Member 5) and very severe and early onset recessive cardiomyopathy 109 .Four children across two families were described, all of whom were severely affected; the parents in both families were unaffected and consanguineous.Death from cardiac symptoms occurred in all four children -of these, cardiomyopathy was detected in three.One died in-utero, the other three died in the first few days after birth.Other non-cardiac phenotypes were described, such as dysmorphic features, cystic hygromas, small ribs, dystrophic nails and intraventricular haemorrhage, though the cardiac phenotype seemed predominant.Exome sequencing revealed all three children in family one were homozygous for the p.Ile278Phefs*33 frameshift variant in SLC30A5; the parents were unaffected and heterozygous.The child in family two was homozygous for the p.His661Tyrfs*10 frameshift variant in SLC30A5, and the parents were heterozygous.These variants are not present in the gnomAD database.Loss-of-function variants in SLC30A5 are constrained in gnomAD (LOEUF=0.46).One SLC30A5 knockout mouse study showed sudden cardiac death in more than 60% of the knockout mice -the mice also displayed a non-cardiac, skeletal phenotype of reduced bone density, as well as poor growth 110 .Although there have been limited genetic studies published thus far for SLC30A5, the identification of biallelic truncating variants in two independent families, coupled with supporting mouse knockout data, suggests this gene is associated with a severe early-onset form of recessive cardiomyopathy.

Function of SLC30A5
SLC30A5 (Solute Carrier Family 30 Member 5), also known as ZnT-5 (Zinc Transporter 5), encodes one of a family of zinc transporters.These membrane transport proteins facilitate the efflux of cytoplasmic zinc ions out of the cell, or efflux from the cytoplasm into vesicles 111 .The paper by Lieberwirth et al. therefore speculates that impaired zinc homeostasis may be one rare cause of lethal perinatal autosomal recessive cardiomyopathy 109 .

TRIM63
Biallelic loss-of-function variants in TRIM63, which encodes the E3 ubiquitin protein ligase TRIM63 (also known as muscle-specific RING finger protein 1 (MuRF1)), were associated with HCM in a large case-control study 112 , detected in 15 (0.4%) of 4,867 HCM probands compared with 0% in 3,136 controls.The phenotype was largely adult-onset in the 15 probands and 4 affected relatives, ranging from teens to 60s at presentation.The patients were characterised by concentric LV hypertrophy and comparatively high rates of cardiac fibrosis, LV systolic dysfunction and arrhythmias.The TRIM63 variants were a mix of biallelic truncating (8 probands), biallelic missense (6) and mixed (1).While a previous study from 2014 proposed a modifier role for heterozygous missense variants in TRIM63 (and TRIM55) in patients with HCM 113 , all 32 heterozygous relatives in this study were reported to be healthy.TRIM63 is also not constrained of loss-of-function variants in gnomAD (LOEUF=1.11).We recently demonstrated that biallelic TRIM63 variants are highly enriched in HCM patients from Egypt compared to population-matched controls (8/374, 2.14% vs 0/400, 0%) -the five-fold enrichment in Egyptian vs the predominantly European ancestry cases in the prior study demonstrates that TRIM63 is likely to be a more prevalent HCM gene in populations of high consanguinity 114 .These reports built upon two earlier studies on individual patients with stable HCM and mild skeletal muscle phenotypes, both of whom has the p.Gln247* in homozygosity 115,116 .While the p.Gln247* variant has also been found in both cohort studies above, it is particularly common in Ashkenazi Jews (MAF=0.008),indicating that TRIM63 could also be a relatively common cause of HCM in individuals of this ancestry.TRIM63 was previously curated as having limited evidence for association with HCM by the ClinGen curation, although this was assessed before the publication of the two cohort studies described above 11 .TRIM63 is a reported mapped gene for myocardial longitudinal strain in UK Biobank data 29 , as well as for QRS complex/amplitude 117 and ECG morphology 34 according to the GWAS Catalog.Knockout mice data for TRIM63 has revealed complex phenotype correlations.TRIM63 knockouts alone are healthy with normal cardiac muscle.Double knockouts of TRIM63 and TRIM55 (MURF2) display severe cardiac hypertrophy 118 .In the single gene knockout models however, TRIM63 knockout mice developed TAC-induced LV hypertrophy whereas TRIM55 knockout mice did not, suggesting it is TRIM63/MURF1 that is responsible for the hypertrophy phenotype 119 .Although all reports of biallelic TRIM63 variants in HCM were based on panel sequencing studies rather than unbiased genome-wide approaches, the clear and significant enrichment of variants in two cohorts provides robust evidence for its role as a recessive HCM gene.

Function of TRIM63
TRIM63/MURF1 localises to the Z-disk and M-line of the sarcomere where it acts to regulate the degradation of sarcomeric proteins through ubiquitylation.

Gene summaries for recessive cardiomyopathy genes without replication
The following genes are associated with recessive cardiomyopathies in single reported family pedigrees only (or 2-3 probands in one cohort-based study).The evidence available, including supporting data from animal models, GWAS etc., is described below.Further studies are required however to establish their pathogenic role in cardiomyopathy.

AASDH
The Al-Hassnan et al. cohort study of 205 Saudi Arabian unrelated probands with paediatric cardiomyopathy reported one family with a variant in AASDH (Aminoadipate-Semialdehyde Dehydrogenase), associating it with paediatric autosomal recessive dilated cardiomyopathy 36 .WES revealed homozygosity for the AASDH p.Tyr1061Cysfs*3 frameshift variant (this variant occurs in the last exon of the gene); the parents were consanguineous, heterozygous and unaffected.The girl presented with DCM 5 months after birth and was reported still alive at 6 years of age.In gnomAD, the variant is very rare (maximum population frequency of 0.05% in Latinos/Admixed-Americans) with no homozygotes; the gene is only marginally constrained for loss-of-function variants (LOEUF = 0.83).

Function of AASDH
AASDH encodes the aminoadipate-semialdehyde dehydrogenase enzyme.This is a non-ribosomal peptide synthetase, these enzymes usually catalyse the biosynthesis of a particular member of the class of peptides known as non-ribosomal peptides, which are named as such since they do not require ribosomes for their synthesis.

ACACB
The Al-Hassnan et al. cohort study of 205 Saudi Arabian unrelated probands with paediatric cardiomyopathy reported one family with a variant in ACACB (Acetyl-CoA Carboxylase Beta), associating it with paediatric autosomal recessive left ventricular non-compaction cardiomyopathy 36 .WES revealed homozygosity for the ACACB p.Arg2102Gln missense variant; the parents were consanguineous, heterozygous and unaffected.The girl presented with LVNC 21 days after birth and died at 1 year of age.In gnomAD, the variant is very rare (maximum population frequency of 0.0063% in Africans/African-Americans) with no homozygotes.ACACB knockout mice had a normal lifespan with higher fatty acid oxidation rate and lower fat 120 .

Function of ACACB
ACACB encodes the acetyl-CoA carboxylase beta enzyme.This catalyses the carboxylation of acetyl-CoA into malonyl-CoA, which is a key substrate in the biosynthetic pathway for fatty acid production.

BICD2
One study presented evidence for an association between homozygosity for a BICD2 (bicaudal-D cargo adaptor protein 2) variant and dilated cardiomyopathy 121 .In the studied family from China, healthy consanguineous parents had seven children, of which, two sons and one daughter were affected with DCM.Echocardiography in the proband showed poor systolic function (LVEF 26.8%) and LV dilatation (LVESV 357ml).WES of the parents and two of the affected children identified novel homozygous variants in five gene candidates, with the BICD2 p.Arg810His missense variant chosen as most likely to be associated with a cardiac phenotype.BICD2 is highly expressed in the skin and oesophageal mucosa, less so in the heart (GTEx).The unaffected parents were heterozygous for the BICD2 variant, whereas two of the affected children were homozygous.The variant is associated with poor outcomes: one of the affected individuals died after a heart transplant, the other two are presumably alive.The p.Arg810His variant is rare, with a maximum population frequency of 0.014% in gnomAD and no homozygous carriers.Immunohistochemistry demonstrated BICD2 expression in the human heart.BICD2 knock-out zebrafish displayed a greater rate of embryonic lethality; echocardiography showed a reduction in cardiac output but no significant change in cardiac area or volume, unlike DCM.RNAseq of the zebrafish showed an altered transcriptome, relative to wild-type, though the findings were not particularly specific to DCM.BICD2 variants are also associated with autosomal dominant spinal muscular atrophy 122 .

Function of BICD2
BICD2 is one of the two paralogs of the gene encoding the bicaudal-D cargo adaptor protein.BICD2 binds to activated RAB6A, a GTPase located on the membrane surface of vesicles located on the trans side of the Golgi apparatus 123 .This allows recruitment of the dynein-dynactin complex to the Nterminal end of BICD2; dynein is a motor protein that travels towards the minus end of microtubules, and dynactin is an enhancer of dynein activity.This motor complex then transports its cargo in a retrograde manner from the Golgi apparatus to the endoplasmic reticulum 124 .The study by Luo et al.  is therefore suggestive of dysfunction in retrograde COPI-independent Golgi-ER transport as a potential rare cause of autosomal recessive DCM.

CASZ1
The Al-Hassnan et al. cohort study of 205 Saudi Arabian unrelated probands with paediatric cardiomyopathy reported one family with a variant in CASZ1 (Castor Zinc Finger 1), associating it with paediatric autosomal recessive left ventricular non-compaction and dilated cardiomyopathy 36 .WGS revealed homozygosity for the CASZ1 p.Ser237Cys missense variant; the parents were consanguineous, heterozygous and unaffected.The girl presented with LVNC and DCM 5 months after birth and was reported still alive at 6.5 years of age.The variant is absent from gnomAD.CASZ1 knockout mice display embryonic lethality, display abnormal heart development, heart shape and Z line formation 125 .CASZ1 is a reported mapped gene for atrial fibrillation and ECG traits according to the GWAS Catalog [31][32][33][34]126 .

Function of CASZ1
CASZ1 encodes the castor zinc finger 1 protein, which is a zinc finger transcription factor.It has been associated with the regulation of gene expression during cardiac development in Xenopus and mouse models 125,127 .

GATAD1
One study found an association between GATAD1, which codes for the GATA zinc finger domain containing 1 protein, and autosomal recessive DCM 128 .Three individuals were affected, two female and one male, in a Norwegian consanguineous family where the parents were first cousins.The two female patients had DCM whereas the male displayed idiopathic left ventricular enlargement.Linkage analysis and homozygosity mapping identified a critical region (7q21) with LOD score of 3.1; WES after variant filtration revealed the p.Ser102Pro missense variant in GATAD1 located within this critical region.The three affected individuals were homozygous for the variant and had late-onset DCM (median age of onset 53 years old; range 50-57 years) without poor outcomes-both females are still alive at median age 75 years old (range 74-76 years) and the male died at 73 years of age but from cancer (13 heterozygotes in the family were unaffected).Two other variants were found by WES in the affected individuals after filtering: MYLK3, which encodes a cardiac specific kinase, and SETD1A, which encodes a protein involved in epigenetic gene regulation.However, these genes were ruled out since an unaffected individual was homozygous for these variants.Immunohistochemistry using GATAD1 antibodies showed abnormal intracellular GATAD1 localisation compared to control healthy tissue and tissues from other DCM mutations.Histology showed globular, rather than the usual rodlike, cardiomyocyte morphology.There has also been an experimental study on GATAD1's association with DCM using adult zebrafish 129 .In-situ hybridization showed high GATAD1 RNA expression in the heart, but the highest in the brain.Transgenic expression of GATAD1-GFP showed subcellular localisation near the nucleus and actin filaments.GATAD1 knock-out zebrafish showed significant decreases in survival rates and compromised swimming capacity, however these fish were also treated with ethanol and a high-cholesterol diet, in addition there was no change in body weight adjusted ventricular area.The human GATAD1 gene with the S102P variant was introduced into zebrafishthere was a reduction in survival but this did not reach significance, there was no significant difference in swimming capacity and only one of the six transgenic fish displayed an enlarged heart.Based on the evidence described above, the ClinGen curation classified GATAD1 as having limited evidence for association with DCM via an autosomal recessive mode of inheritance 46 .

Function of GATAD1
GATAD1 encodes GATA zinc finger domain containing 1; this protein has been identified as a H3K4me3 histone code reader protein 130 .H3K4me3 (trimethylation of the fourth residue, a lysine, in histone 3) is a post-translational modification of the tail of a histone.GATAD1 is ubiquitously expressed across all tissues, as seen in the GTEx Portal.Patterns of H3K4me3 marks were found to be different between healthy human LV specimens and that of DCM patients with heart failure 131 .It is suggested then, that deleterious variants in GATAD1 may cause DCM via epigenetic dysregulation.

GET3/ASNA1
An association between biallelic GET3 (guided entry of tail-anchored proteins factor 3, ATPase) variants and dilated cardiomyopathy was found in one study from the Netherlands 132 .This gene was previously known as ASNA1 (arsenite-stimulated ATPase) and is referred to as such in that paper.Two females were affected in a small family with non-consanguineous parents.Dilatation of the heart and poor LV contractility were observed in both.WES and filtration to novel variants revealed that they were compound heterozygous for the GET3 variants p.Cys289Trp and p.Gln305* on the paternal copy, and p.Val163Ala on the maternal copy.A GET3 p.Val163Ala heterozygote was unaffected.The patients had very early onset DCM (9/14 days) with very poor outcomes (death at 12/49 days).GET3 is constrained for loss-of-function variants (LOEUF=0.35) in gnomAD.Histology of the explanted hearts showed reduced GET3 protein expression, disorganised intercalated discs and irregularly shaped nuclei, compared to controls.GET3 protein with the Val163Ala variant showed reduced insertion efficiency of tail-anchored proteins into the endoplasmic reticulum, measured by the presence of glycosylated tail-anchored proteins, compared to wild-type 132 .Homozygous GET3 knockout zebrafish died by 9 days post fertilisation.Injections of wild-type GET3 mRNA slowed the progression of cardiac failure whereas p.Val163Ala and p.Cys289Trp-p.Gln305* mutant GET3 mRNA did not.It is therefore suggested that disturbances in tail-anchored protein insertion could be a cause of DCM.

Function of GET3
The GET3 gene product is a chaperone that facilitates the insertion of tail-anchored proteins from the cytosol into the membrane of the endoplasmic reticulum.

KIF20A
One study from Belgium described an association between KIF20A (Kinesin Family Member 20A) and lethal paediatric restrictive cardiomyopathy 133 .Two, one male and one female, out of the three children of non-consanguineous parents developed restrictive cardiomyopathy prenatally; they developed heart failure & died at 93 and 71 days of age respectively.WES revealed the two affected children as compound heterozygous for the missense p.Arg182Trp and frameshift p.Ser635Thrfs*15 variants in KIF20A.The unaffected child did not possess any of these variants, the parents were each heterozygous for one of the variants.The highest frequency in gnomAD for these variants was 0.0065% for the p.Arg182Trp variant in South Asians; no homozygotes are listed in gnomAD for either variant.KIF20A has a LOEUF of 0.74 in gnomAD, which is somewhat constrained but not as much as other genes linked to recessive cardiomyopathy.Lower abundances of KIF20A transcripts and protein were measured in the fibroblasts of the RCM patients.Immunostaining of these cells revealed an abnormal subcellular localisation of KIF20A, and subsequent failure of the Aurora B protein (trafficked by KIF20A) to localise at the spindle midzone during mitosis.ATPase assays showed that the mutant KIF20A motor protein had an almost complete loss of function compared to wild-type.Zebrafish were subjected to KIF20A knockdown using Morpholinos; 90% of these fish developed cardiac oedema, pooling of red blood cells proximal to the atrium, tachycardia and increased fractional shortening by 6 days post fertilisation.Wild-type KIF20A cDNA partially rescued the phenotype, whereas mutant cDNA did not.Histology of the fish showed increased ventricular thickness in the mutants.In a different study, KIF20A homozygous knockout mice die by 3-4 weeks of age 134 .The authors speculate that impaired cell division may be one rare cause of lethal paediatric recessive restrictive cardiomyopathy 133 .

Function of KIF20A
KIF20A (Kinesin Family Member 20A) encodes a kinesin, a motor protein which transports cargo from the minus end of microtubules to the plus end (anterograde transport) and transports the Aurora B kinase protein 133 , as well as Golgi-associated vesicles.Aurora B is a protein involved in attachment of the mitotic spindle to the centromeres of chromosomes during mitosis, more specifically, it inhibits binding of the spindle microtubules to the kinetochore until there sufficient tension is generated.

PHACTR2
A single study, conducted on an Bedouin family from Israel, associated biallelic PHACTR2 variation with early-onset DCM with LVNC 135 .The variant associating with disease in the family is the rare, missense allele p.Arg511His and family in question is consanguineous and comprises two heterozygous parents, one affected homozygous male child and his 5 healthy siblings (3 of whom heterozygous for the variant, 2 homozygous for the reference allele).The proband had a dilated LV with decreased function (and normal morphology) at gestation week 32.His birth was normal, but symptoms of severe heart failure appeared at 10 weeks of age, with subsequent examinations revealing cardiomegaly, pulmonary edema, a severely dilated LV with reduced function (EF 37-40%) and LVNC.As outcome, he is reported alive in his teens (authors don't provide an age indication), under pharmacological treatment and suffering from constant fatigue and exercise intolerance (EF = 44%).Functional follow-up performed by the authors included several experiments.Using the crystal structure of PHACTR1 (given that that the variant site is conserved across all PHACTR proteins and down to zebrafish) they predicted how the variant would cause misfolding of the second RPEL repeat, which interacts with actin, and would disrupt the entire PHACTR-actin complex.While the variant had no effect on the subcellular localization of the protein, it had significant effects on actin dynamics as observed comparing the proband's fibroblasts to control cells.Among the significant changes observed in the patient's cells were a decreased globular/filamentous actin ratio, an increased accessibility of actin to depolymerizing agents and a decreased repolymerization rate.Furthermore, the proband's cells were dysmorphic (rounded) following actin depolymerization compared with control cells, and a wound healing assay showed control cells to migrate more quickly than the patient's.

Function of PHACTR2
PHACTR2 is a member of a family of four phosphatases and actin regulator (PHACTRs) proteins that exhibit the modulatory activity of protein phosphatase 1 (PP1) and an actin-binding activity 136 .Its expression is ubiquitous, but about twice as high in the heart compared with the skeletal muscle.

RHBDF1
The Al-Hassnan et al. cohort study of 205 Saudi Arabian unrelated probands with paediatric cardiomyopathy reported three independent families with a variant in RHBDF1 (Rhomboid 5 Homolog 1), associating it with paediatric autosomal recessive dilated cardiomyopathy 36 .WES and WGS revealed homozygosity for the p.Gly665Trp RHBDF1 missense variant in the proband of the first family, and the p.Phe405Serfs*16 frameshift variant in the second and third families.The parents in all three families were consanguineous, heterozygous and unaffected.The boy in the first family presented with DCM at 1 month and was still reported to be alive at 8 years.The boy in the second family presented at 2 months and died at 2.5 years.In the third family, the boy presented at 7 months and was still reported to be alive at 1.67 years.In gnomAD, the frameshift variant is absent, and the missense variant is present at a very rare frequency in heterozygous form (0.00093% in non-Finnish Europeans).The gene is only marginally constrained for loss-of-function variants (LOEUF = 0.94) in gnomAD.

Function of RHBDF1
RHBDF1 encodes the rhomboid 5 homolog 1 protein, otherwise known as the iRhom2 protein (i for inactive).Rhomboid proteases are intramembrane serine proteases-they possess a proteolytic active site which is embedded inside the lipid bilayer.The RHBDF1 protein does not possess the catalytic serine residue and so is an enzymatically inactive rhomboid protease.Despite this, they still appear to have functions, such as in regulation of the epidermal growth factor receptor signalling pathway 137 .

SLC6A6
SLC6A6, encoding for the taurine transporter, was associated with recessive cardiomyopathy in one family in a single study 138 .Two siblings from a consanguineous Pakistani family had mild hypokinetic cardiomyopathy with systolic dysfunction, along with cone-rod retinopathy and were homozygous for the p.Gly399Val variant (detected by WES and homozygosity mapping).Blood taurine levels were almost undetected and functional analysis revealed that transport capacity was only 15% of normal.Taurine supplementation therapy for 24 months completely corrected the cardiomyopathy.Supporting evidence for a role for SLC6A6 in cardiomyopathy comes from knockout mice studies which demonstrated cardiac dysfunction and fractional shortening in older mice 139 .It is also a putative causal gene at a significant GWAS locus in both HCM and DCM case-control studies 29,140 .Taurine deficiency in dogs and cats also leads to cardiomyopathy, with improvement in echocardiography parameters observed upon diet taurine supplementation 141 .

Function of SLC6A6
The SLC6A6-encoded taurine transporter is a multi-pass membrane protein and member of the family of sodium and chloride-ion dependent transporters whose function is to transport taurine and betaalanine.

SOD2
SOD2, encoding for superoxide dismutase 2 or manganese-superoxide dismutase, was associated with recessive DCM in one family in a single study 142 .The p.Gly181Val homozygous variant was identified in a new-born patient (born to distantly related parents) with severe biventricular DCM who died at 4 days.Functional studies confirmed dysfunction of the patient's superoxide dismutase enzyme.While there is only a single family with biallelic SOD2 variants currently reported, Sod2 knockout mice can also develop early onset DCM and death (although phenotypes differed depending on strain) 143,144 .SOD2 is also the putative causal gene at a myocardial fibrosis (interventricular septum and LV free wall) GWAS locus 145 .

Function of SOD2
SOD2 is one of three superoxide dismutase genes which convert superoxide anions into hydrogen peroxide and protect cells from damage by reactive oxygen species.

TAF1A
One study reported an association between TAF1A (TATA-Box Binding Protein Associated Factor, RNA Polymerase I Subunit A) and recessive DCM 146 .The two daughters of unaffected parents were diagnosed via echocardiography with DCM (LV enlargement; LVEF 20% and 27%), each at 2.5 years of age.The condition of each child deteriorated and they underwent heart transplants at 3 years.WES and subsequent variant filtration revealed compound heterozygosity in both children for the TAF1A p.Leu84Ser and p.Gly341Arg missense variants.The parents were heterozygous for one of each of these variants.The gnomAD database shows that these variants are very rare with no homozygotes.Histology of the explanted hearts showed interstitial fibrosis, cardiomyocyte hypertrophy and nucleolar segregation, the latter of which was not present in other DCM hearts-suggesting the involvement of TAF1A which is expressed in the nucleolus.Their zebrafish TAF1A knockout model showed a heart failure like phenotype; early lethality (all were dead by 11 days post fertilisation); pericardial oedema; reduction in ventricular fractional shortening; however, there was no mention of cardiac dilatation.An entry exists in ClinVar of a girl (0-9 years old) of African descent (Jamaican) with restrictive cardiomyopathy who is compound heterozygous for the TAF1A variants p.Gly341Arg and p.Thr261Pro.

Function of TAF1A
TAF1A encodes the TATA box-binding protein-associated factor RNA polymerase I subunit A protein.
The transcription factor SL1, selective factor 1, is composed of one TATA-binding protein (TBP) and three TBF-associated factor proteins (TAFs), one of which is TAF1A 147 .SL1 binds to the promoter of ribosomal DNA (rDNA) genes and forms part of the complex of transcription factors which recruit RNA polymerase I (Pol I) and initiate transcription.Pol I transcribes 45S rDNA in the nucleolus.The resulting 45S rRNA is eventually processed and combined with 5S rRNA to form the 80S human ribosome.

ULK1
The Al-Hassnan et al. cohort study of 205 Saudi Arabian unrelated probands with paediatric cardiomyopathy reported one family with a variant in ULK1 (Unc-51 Like Autophagy Activating Kinase 1), associating it with paediatric autosomal recessive DCM 36 .WES revealed homozygosity for the ULK1 p.Arg691Trp missense variant in the proband.The parents were consanguineous, heterozygous and unaffected.The boy presented at 1.5 months with DCM and died at 2 years of age.In gnomAD, the missense variant is present in heterozygous form but is rare (0.018% in non-Finnish Europeans).

Function of ULK1
ULK1 encodes the Unc-51 like autophagy activating kinase 1 protein.The Atg1/ULK1 protein-kinase complex is involved in the process of autophagy; specifically in the formation of autophagosomes, which are sealed double-membranes that engulf unwanted proteins and organelles 148 .In the cardiac context, ULK1 has been associated with protective mitophagy under stress conditions, and double knock-out ULK1/ULK2 perinatal mice develop cardiomyopathy [149][150][151] .