A mutation update for the FLNC gene in myopathies and cardiomyopathies

Filamin C (FLNC) variants are associated with cardiac and muscular phenotypes. Originally, FLNC variants were described in myofibrillar myopathy (MFM) patients. Later, high‐throughput screening in cardiomyopathy cohorts determined a prominent role for FLNC in isolated hypertrophic and dilated cardiomyopathies (HCM and DCM). FLNC variants are now among the more prevalent causes of genetic DCM. FLNC‐associated DCM is associated with a malignant clinical course and a high risk of sudden cardiac death. The clinical spectrum of FLNC suggests different pathomechanisms related to variant types and their location in the gene. The appropriate functioning of FLNC is crucial for structural integrity and cell signaling of the sarcomere. The secondary protein structure of FLNC is critical to ensure this function. Truncating variants with subsequent haploinsufficiency are associated with DCM and cardiac arrhythmias. Interference with the dimerization and folding of the protein leads to aggregate formation detrimental for muscle function, as found in HCM and MFM. Variants associated with HCM are predominantly missense variants, which cluster in the ROD2 domain. This domain is important for binding to the sarcomere and to ensure appropriate cell signaling. We here review FLNC genotype–phenotype correlations based on available evidence.     

Prevalence of pathogenic and likely pathogenic variants in the RASopathy genes in patients who have had panel testing for cardiomyopathy

RASopathies are a group of developmental disorders caused by pathogenic variants in the RAS‐MAPK pathway. Cardiomyopathy is a major feature of this group of disorders, specifically hypertrophic cardiomyopathy (HCM). HCM can be the first presenting feature in individuals with RASopathies. We conducted a retrospective study of all individuals who have had a cardiomyopathy gene panel ordered through our institution to determine the prevalence of pathogenic or likely pathogenic variants in RAS pathway genes in individuals with cardiomyopathy. We evaluated variants in the following genes: BRAF, CBL, HRAS, KRAS, MAP2K1, MAP2K2, NF1, NRAS, PTPN11, RAF1, SHOC2, and SOS1. We reviewed 74 cases with cardiomyopathy, including 32 with HCM, 24 with dilated cardiomyopathy (DCM), nine with both left ventricular noncompaction (LVNC) and DCM, four with LVNC only, two with arrhythmogenic right ventricular cardiomyopathy (ARVC) and three with unspecified cardiomyopathy. We identified four patients (5.41%) with pathogenic or likely pathogenic variants in HRAS, PTPN11 and RAF1 (two individuals). Indication for testing for all four individuals was HCM. The prevalence of pathogenic or likely pathogenic variants in RASopathy genes in our HCM patient cohort is 12.5% (4/32). We conclude that the RASopathy genes should be included on multi‐gene panels for cardiomyopathy to increase diagnostic yield for individuals with HCM.     

High prevalence of genetic variants previously associated with LQT syndrome in new exome data

To date, hundreds of variants in 13 genes have been associated with long QT syndrome (LQTS). The prevalence of LQTS is estimated to be between 1:2000 and 1:5000. The knowledge of genetic variation in the general population has until recently been limited, but newly published data from NHLBI GO Exome Sequencing Project (ESP) has provided important knowledge on this topic. We aimed to investigate the prevalence of previously LQTS-associated variants in ESP (5400 individuals), in order to identify possible false-positive LQTS variants. With this aim, we performed a search for previously published LQTS-associated variants in ESP. In addition, a PolyPhen-2 prediction was conducted, and the four most prevalent LQTS-associated variants with significant functional effects present in ESP were genotyped in a second control population. We identified 33 missense variants previously associated with LQTS in ESP. These 33 variants affected 173 alleles and this corresponded to a LQTS prevalence of 1:31 in the ESP population. PolyPhen-2 predicted 30% of the 33 variants present in ESP to be benign compared with 13% among LQTS-associated variants not present in ESP (P=0.019). Genotyping of the four variants KCNH2 P347S; SCN5A: S216L, V1951L; and CAV3 T78M in the control population (n=704) revealed prevalences comparable to those of ESP. Thus, we identified a much higher prevalence of previously LQTS-associated variants than expected in exome data from population studies. Great caution regarding the possible disease causation of some of these variants has to be taken, especially when used for risk stratification in family members.     

Inherited cardiomyopathies mimicking arrhythmogenic right ventricular cardiomyopathy

Arrhythmogenic right ventricular cardiomyopathy (ARVC) represents an inherited cardiomyopathy that manifests clinically with malignant ventricular arrhythmias, sudden cardiac death, and less commonly heart failure. The condition is characterized by replacement of the myocardium, primarily of the right ventricle, with fibrofatty tissue. Extensive fibrofatty replacement of the myocardium has been previously thought to be pathognomonic of ARVC; however, this report details two other forms of inherited cardiomyopathy, namely hypertrophic cardiomyopathy (HCM) and the PRKAG2 cardiac syndrome, that were found to have significant fibrofatty myocardial replacement at pathologic examination. This report represents the first documentation of inherited cardiomyopathies mimicking ARVC and highlights the concept that other cardiac conditions can be associated with fibrofatty replacement of the myocardium.     

Phenotype‐driven molecular autopsy for sudden cardiac death

A phenotype‐driven approach to molecular autopsy based in a multidisciplinary team comprising clinical and laboratory genetics, forensic medicine and cardiology is described. Over a 13 year period, molecular autopsy was undertaken in 96 sudden cardiac death cases. A total of 46 cases aged 1–40 years had normal hearts and suspected arrhythmic death. Seven (15%) had likely pathogenic variants in ion channelopathy genes [KCNQ1 (1), KCNH2 (4), SCN5A (1), RyR2(1)]. Fifty cases aged between 2 and 67 had a cardiomyopathy. Twenty‐five had arrhythmogenic right ventricular cardiomyopathy (ARVC), 10 dilated cardiomyopathy (DCM) and 15 hypertrophic cardiomyopathy (HCM). Likely pathogenic variants were found in three ARVC cases (12%) in PKP2, DSC2 or DSP, two DCM cases (20%) in MYH7, and four HCM cases (27%) in MYBPC3 (3) or MYH7 (1). Uptake of cascade screening in relatives was higher when a molecular diagnosis was made at autopsy. In three families, variants previously published as pathogenic were detected, but clinical investigation revealed no abnormalities in carrier relatives. With a conservative approach to defining pathogenicity of sequence variants incorporating family phenotype information and population genomic data, a molecular diagnosis was made in 15% of sudden arrhythmic deaths and 18% of cardiomyopathy deaths.     

FLNC pathogenic variants in patients with cardiomyopathies: Prevalence and genotype-phenotype correlations

Pathogenic variants in FLNC encoding filamin C have been firstly reported to cause myopathies, and were recently linked to isolated cardiac phenotypes. Our aim was to estimate the prevalence of FLNC pathogenic variants in subtypes of cardiomyopathies and to study the relations between phenotype and genotype. DNAs from a cohort of 1150 unrelated index-patients with isolated cardiomyopathy (700 hypertrophic, 300 dilated, 50 restrictive cardiomyopathies, and 100 left ventricle non-compactions) have been sequenced on a custom panel of 51 cardiomyopathy disease-causing genes. An FLNC pathogenic variant was identified in 28 patients corresponding to a prevalence ranging from 1% to 8% depending on the cardiomyopathy subtype. Truncating variants were always identified in patients with dilated cardiomyopathy, while missense or in-frame indel variants were found in other phenotypes. A personal or family history of sudden cardiac death (SCD) was significantly higher in patients with truncating variants than in patients carrying missense variants (P = .01). This work reported the first observation of a left ventricular non-compaction associated with a unique probably causal variant in FLNC which highlights the role of FLNC in cardiomyopathies. A correlation between the nature of the variant and the cardiomyopathy subtype was observed as well as with SCD risk.     

NGS testing for cardiomyopathy: Utility of adding RASopathy‐associated genes

RASopathies include a group of syndromes caused by pathogenic germline variants in RAS‐MAPK pathway genes and typically present with facial dysmorphology, cardiovascular disease, and musculoskeletal anomalies. Recently, variants in RASopathy‐associated genes have been reported in individuals with apparently nonsyndromic cardiomyopathy, suggesting that subtle features may be overlooked. To determine the utility and burden of adding RASopathy‐associated genes to cardiomyopathy panels, we tested 11 RASopathy‐associated genes by next‐generation sequencing (NGS), including NGS‐based copy number variant assessment, in 1,111 individuals referred for genetic testing for hypertrophic cardiomyopathy (HCM) or dilated cardiomyopathy (DCM). Disease‐causing variants were identified in 0.6% (four of 692) of individuals with HCM, including three missense variants in the PTPN11, SOS1, and BRAF genes. Overall, 36 variants of uncertain significance (VUSs) were identified, averaging ～3VUSs/100 cases. This study demonstrates that adding a subset of the RASopathy‐associated genes to cardiomyopathy panels will increase clinical diagnoses without significantly increasing the number of VUSs/case.     

Truncating mutations on myofibrillar myopathies causing genes as prevalent molecular explanations on patients with dilated cardiomyopathy

Dilated cardiomyopathy (DCM) is one of the leading causes of heart failure with high morbidity and mortality. More than 40 genes have been reported to cause DCM. To provide new insights into the pathophysiology of dilated cardiomyopathy, a next‐generation sequencing (NGS) workflow based on a panel of 48 cardiomyopathies‐causing genes was used to analyze a cohort of 222 DCM patients. Truncating variants were detected on 63 unrelated DCM cases (28.4%). Most of them were identified, as expected, on TTN (29 DCM probands), but truncating variants were also identified on myofibrillar myopathies causing genes in 17 DCM patients (7.7% of the DCM cohort): 10 variations on FLNC and 7 variations on BAG3 . This study confirms that truncating variants on myofibrillar myopathies causing genes are frequently associated with dilated cardiomyopathies and also suggest that FLNC mutations could be considered as a common cause of dilated cardiomyopathy. Molecular approaches that would allow to detect systematically truncating variants in FLNC and BAG3 into genetic testing should significantly increase test sensitivity, thereby allowing earlier diagnosis and therapeutic intervention for many patients with dilated cardiomyopathy.     

Second report of SHMT2 related neurodevelopmental disorder with cardiomyopathy,spasticity, and brain abnormalities

Neurodevelopmental disorder with cardiomyopathy, spasticity, and brain abnormalities (NEDCASB; MIM# 619121) is a recently described metabolic disorder with characteristic features of mild dysmorphism, intellectual disability, spasticity, peripheral neuropathy, cardiomyopathy, and thin corpus callosum. Biallelic variants in SHMT2 (MIM 138450), encoding mitochondrial serine hydroxymethyltransferase enzyme, have been recently linked to this disorder. Till now, a total of seven variants including six missense and one deletion-insertion has been reported in SHMT2. We hereby report an additional individual with novel homozygous missense variant c.1133A > G in SHMT2 (NM_005412.6) identified by exome sequencing and review the phenotype and genotype of the previously reported individuals with NEDCASB.     

Molecular basis for clinical heterogeneity in inherited cardiomyopathies due to myopalladin mutations

Abnormalities in Z-disc proteins cause hypertrophic (HCM), dilated (DCM) and/or restrictive cardiomyopathy (RCM), but disease-causing mechanisms are not fully understood. Myopalladin (MYPN) is a Z-disc protein expressed in striated muscle and functions as a structural, signaling and gene expression regulating molecule in response to muscle stress. MYPN was genetically screened in 900 patients with HCM, DCM and RCM, and disease-causing mechanisms were investigated using comparative immunohistochemical analysis of the patient myocardium and neonatal rat cardiomyocytes expressing mutant MYPN. Cardiac-restricted transgenic (Tg) mice were generated and protein-protein interactions were evaluated. Two nonsense and 13 missense MYPN variants were identified in subjects with DCM, HCM and RCM with the average cardiomyopathy prevalence of 1.66%. Functional studies were performed on two variants (Q529X and Y20C) associated with variable clinical phenotypes. Humans carrying the Y20C-MYPN variant developed HCM or DCM, whereas Q529X-MYPN was found in familial RCM. Disturbed myofibrillogenesis with disruption of α-actinin2, desmin and cardiac ankyrin repeat protein (CARP) was evident in rat cardiomyocytes expressing MYPN(Q529X). Cardiac-restricted MYPN(Y20C) Tg mice developed HCM and disrupted intercalated discs, with disturbed expression of desmin, desmoplakin, connexin43 and vinculin being evident. Failed nuclear translocation and reduced binding of Y20C-MYPN to CARP were demonstrated using in vitro and in vivo systems. MYPN mutations cause various forms of cardiomyopathy via different protein-protein interactions. Q529X-MYPN causes RCM via disturbed myofibrillogenesis, whereas Y20C-MYPN perturbs MYPN nuclear shuttling and leads to abnormal assembly of terminal Z-disc within the cardiac transitional junction and intercalated disc.     

Systematic large-scale assessment of the genetic architecture of left ventricular noncompaction reveals diverse etiologies

PurposeTo characterize the genetic architecture of left ventricular noncompaction (LVNC) and investigate the extent to which it may represent a distinct pathology or a secondary phenotype associated with other cardiac diseases.MethodsWe performed rare variant association analysis with 840 LVNC cases and 125,748 gnomAD population controls, and compared results to similar analyses on dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM).ResultsWe observed substantial genetic overlap indicating that LVNC often represents a phenotypic variation of DCM or HCM. In contrast, truncating variants in MYH7, ACTN2, and PRDM16 were uniquely associated with LVNC and may reflect a distinct LVNC etiology. In particular, MYH7 truncating variants (MYH7tv), generally considered nonpathogenic for cardiomyopathies, were 20-fold enriched in LVNC cases over controls. MYH7tv heterozygotes identified in the UK Biobank and healthy volunteer cohorts also displayed significantly greater noncompaction compared with matched controls. RYR2 exon deletions and HCN4 transmembrane variants were also enriched in LVNC, supporting prior reports of association with arrhythmogenic LVNC phenotypes.ConclusionLVNC is characterized by substantial genetic overlap with DCM/HCM but is also associated with distinct noncompaction and arrhythmia etiologies. These results will enable enhanced application of LVNC genetic testing and help to distinguish pathological from physiological noncompaction.     

Investigation of de novo variation in pediatric cardiomyopathy

Pediatric cardiomyopathies can be caused by variants in genes encoding the sarcomere and cytoskeleton in cardiomyocytes. Variants are typically inherited in an autosomal dominant manner with variable expressivity. De novo variants have been reported, however their overall frequency is largely unknown. We sought to determine the rate of de novo, pathogenic and likely pathogenic (P/LP) variants in children with a diagnosis of hypertrophic, dilated, or restrictive cardiomyopathy (HCM, DCM, or RCM), and to compare disease outcomes between individuals with and without a de novo variant. A retrospective record review identified 126 individuals with HCM (55%), DCM (37%), or RCM (8%) ≤18 years of age who had genetic testing. Overall, 50 (40%) had positive genetic testing and 18% of P/LP variants occurred de novo. The rate of de novo variation in those with RCM (80%) was higher than in those with HCM (9%) or DCM (20%). There was evidence of germline mosaicism in one family with RCM. Individuals with de novo variants were more likely than those without to have a history of arrhythmia (p = .049), sudden cardiac arrest (p = .024), hospitalization (p = .041), and cardiac transplantation (p = .030). The likelihood of de novo variation and impact on family risk and screening should be integrated into genetic counseling.     

Reevaluation of genetic variants previously associated with arrhythmogenic right ventricular cardiomyopathy integrating population-based cohorts and proteomics data

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is one of the most common causes of sudden cardiac death in young people. Patients diagnosed with ARVC may experience increased likelihood of development of anxiety and depression, emphasizing the need for accurate diagnosis. To assist future genetic diagnosis and avoidance of misdiagnosis, we evaluated the reported monogenic disease-causing variants in ARVD/C Genetic Variants Database, Human Gene Mutation Database, and ClinVar. Within the aforementioned databases, 630 monogenic disease-causing variants from 18 genes were identified. In the genome Aggregation Database, 226 of these were identified; 68 of which were found at greater than expected prevalence. Furthermore, 37/226 genetic variants were identified amongst the 409 000 UK biobank participants, 23 were not associated with ARVC. Among the 14 remaining variants, 13 were previously found with greater than expected prevalence for a monogenic variant. Nevertheless, they were associated with serious cardiac phenotypes, suggesting that these 13 variants may be disease-modifiers of ARVC, rather than monogenic disease-causing. In summary, more than 10% of variants previously reported to cause ARVC were found unlikely to be associated with highly penetrant monogenic forms of ARVC. Notably, all variants in OBSCN and MYBPC3 were found, making these unlikely to be monogenic causes of ARVC.     

Multiplex targeted high‐throughput sequencing for Mendelian cardiac disorders

Mendelian cardiomyopathies and arrhythmias are characterized by an important genetic heterogeneity, rendering Sanger sequencing very laborious and expensive. As a proof of concept, we explored multiplex targeted high‐throughput sequencing (HTS) as a fast and cost‐efficient diagnostic method for individuals suffering from Mendelian cardiac disorders. We designed a DNA capture assay including all exons from 130 genes involved in cardiovascular Mendelian disorders and analysed simultaneously four samples by multiplexing. Two patients had familial hypertrophic cardiomyopathy (HCM) and two patients suffered from long QT syndrome (LQTS). In patient 1 with HCM, we identified two known pathogenic missense variants in the two most frequently mutated sarcomeric genes MYH7 and MYBPC. In patient 2 with HCM, a known acceptor splice site variant in MYBPC3 was found. In patient 3 with LQTS, two missense variants in the genes SCN5A and KCNQ were identified. Finally, in patient 4 with LQTS a known missense variant was found in MYBPC3, which is usually mutated in patients with cardiomyopathy. Our results showed that multiplex targeted HTS works as an efficient and cost‐effective tool for molecular diagnosis of heterogeneous disorders in clinical practice and offers new insights in the pathogenesis of these complex diseases.     

Cardiac troponin T mutation in familial cardiomyopathy with variable remodeling and restrictive physiology

We identified a unique family with autosomal dominant heart disease variably expressed as restrictive cardiomyopathy (RCM), hypertrophic cardiomyopathy (HCM), and dilated cardiomyopathy (DCM), and sought to identify the molecular defect that triggered divergent remodeling pathways. Polymorphic DNA markers for nine sarcomeric genes for DCM and/or HCM were tested for segregation with disease. Linkage to eight genes was excluded, but a cardiac troponin T (TNNT2) marker cosegregated with the disease phenotype. Sequencing of TNNT2 identified a heterozygous missense mutation resulting in an I79N substitution, inherited by all nine affected family members but by none of the six unaffected relatives. Mutation carriers were diagnosed with RCM (n = 2), non-obstructive HCM (n = 3), DCM (n = 2), mixed cardiomyopathy (n = 1), and mild concentric left ventricular hypertrophy (n = 1). Endomyocardial biopsy in the proband revealed non-specific fibrosis, myocyte hypertrophy, and no myofibrillar disarray. Restrictive Doppler filling patterns, atrial enlargement, and pulmonary hypertension were observed among family members regardless of cardiomyopathy subtype. Mutation of a sarcomeric protein gene can cause RCM, HCM, and DCM within the same family, underscoring the necessity of comprehensive morphological and physiological cardiac assessment in familial cardiomyopathy screening.     

Electronic health record phenotype in subjects with genetic variants associated with arrhythmogenic right ventricular cardiomyopathy: a study of 30,716 subjects with exome sequencing

PurposeArrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited heart disease. Clinical follow-up of incidental findings in ARVC-associated genes is recommended. We aimed to determine the prevalence of disease thus ascertained.MethodsIndividuals (n = 30,716) underwent exome sequencing. Variants in PKP2,DSG2,DSC2,DSP,JUP,TMEM43, or TGFβ3 that were database-listed as pathogenic or likely pathogenic were identified and evidence-reviewed. For subjects with putative loss-of-function (pLOF) variants or variants of uncertain significance (VUS), electronic health records (EHR) were reviewed for ARVC diagnosis, diagnostic criteria, and International Classification of Diseases (ICD-9) codes.ResultsEighteen subjects had pLOF variants; none of these had an EHR diagnosis of ARVC. Of 14 patients with an electrocardiogram, one had a minor diagnostic criterion; the rest were normal. A total of 184 subjects had VUS, none of whom had an ARVC diagnosis. The proportion of subjects with VUS with major (4%) or minor (13%) electrocardiogram diagnostic criteria did not differ from that of variant-negative controls. ICD-9 codes showed no difference in defibrillator use, electrophysiologic abnormalities or nonischemic cardiomyopathies in patients with pLOF or VUSs compared with controls.ConclusionpLOF variants in an unselected cohort were not associated with ARVC phenotypes based on EHR review. The negative predictive value of EHR review remains uncertain.     

Cardiomyopathies due to defective energy metabolism: morphological and functional features

Cardiomyopathies are defined as diseases of the myocardium associated with cardiac dysfunction and are classified by morphological characteristics as hypertrophic (HCM), dilated (DCM) arrhithmogenic right ventricular (ARVC) and restrictive cardiomyopathy. These were once considered as specific diagnoses but there is now considerable evidence that many different gene mutations can cause these pathologies. In recent years, big emphasis has been given to the possibility that deregulation of cardiac metabolism may play a role in the mechanisms that lead to cardiac maladaptive remodelling. Cardiac energy metabolism is tightly controlled in mammalian organisms during development and in response to diverse dietary, physiologic, and pathologic conditions. The cardiac phenotype of many genetic diseases caused by mutations in proteins involved in mitochondrial energy production and/or homeostasis, underscores the importance of energetic pathway on cardiac function. For example, inborn errors in nuclear-encoded mitochondrial fatty acid oxidation (FAO) pathway enzymes and defects in fatty acid uptake are an important cause of childhood HCM and sudden death. Abnormalities in mitochondrial respiratory chain function, particularly those caused by mitochondrial DNA (mtDNA) mutations, are responsible for a heterogeneous group of clinical disorders, including isolated HCM. Mitochondrial cardiomyopathies (MCM) are characterized by an adverse clinical course with biventricular dilation and failure, even at a young age. Mutations in genes encoding the gamma2 subunit of AMP-activated protein kinase (PRKAG2), alpha-galactosidase A (GLA) and lysosome-associated membrane proteine-2 (LAMP2) can cause profound myocardial hypertrophy in association with electrophysiological defects. Unlike HCM due to sarcomere gene mutations, which is characterized by myofiber disarray and fibrosis, large cytosolic vacuoles characterize cardiomyopathy due to defect in energy metabolism. Ultrastructural analysis revealed massive mitochondrial proliferation in MCM and glycogen in complexes with protein and/or lipids in cardiomyopathy due to PRKAG2, GLA and LAMP2 mutations.     

Identification of a metavinculin missense mutation, R975W, associated with both hypertrophic and dilated cardiomyopathy

We tested the hypothesis that perturbations in metavinculin may provide a pathogenic substrate for hypertrophic cardiomyopathy (HCM). HCM and dilated cardiomyopathy (DCM) are partially allelic disorders whereby identical genes have been implicated in the pathogenesis of both diseases. Mutations in metavinculin, a muscle-specific isoform of vinculin, were identified previously in DCM and shown to alter in vitro organization of actin filaments. Using denaturing high performance liquid chromatography and direct DNA sequencing, mutational analysis of the metavinculin-specific exon of vinculin (VCL, exon 19) was performed in a cohort of 389 unrelated patients with clinical HCM, previously genotyped for the 8 most common HCM-associated myofilament-encoding genes. Overall, 3 non-synonymous single nucleotide polymorphisms (A934V, P943A, and R975W) were detected in 4 patients. One patient with severely obstructive, mid-ventricular and apical hypertrophy harbored the previously published DCM-associated mutation, R975W. R975 is a highly conserved residue and R975W was absent in over 1400 reference alleles. Immunohistochemical analysis of the proband's myectomy specimen revealed a paucity of vinculin/metavinculin in the intercalated discs. Metavinculin mutations are pathogenic substrates for both HCM and DCM, further highlighting the allelic nature of these cardiomyopathies. Mutations in functionally distinct regions of certain cardiomyopathy-associated genes may have a dominant effect in determining a remodeling pathway of either maladaptive hypertrophy or dilation. However, this study demonstrates that the same fundamental mutation in humans can yield either cardiomyopathic phenotype, underscoring a critical role for modifier genes and/or environmental stressors in cardiac remodeling.     

Alpha-tropomyosin mutations in inherited cardiomyopathies

The inherited cardiac diseases hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) can both be caused by missense mutations in the TPM1 gene which encodes the thin filament regulatory protein α-tropomyosin. Different mutations are responsible for either HCM or DCM, suggesting that distinct changes in tropomyosin structure and function can lead to the different diseases. Various biophysical and physiological approaches have been used to investigate the structure–function effects of the mutations, and animal models developed. The reported effects of the mutations include changes to the secondary structure of tropomyosin, its binding to actin and its position on the thin filament, and alterations to actin–myosin interactions and myofilament Ca²⁺ sensitivity. The latter changes have been found to be particularly consistent, with HCM mutations increasing Ca²⁺ sensitivity and DCM mutations in general decreasing this parameter and uncoupling the effect of troponin phosphorylation upon Ca²⁺ responsiveness. As well as impacting on contractility, these changes are likely to alter intracellular Ca²⁺ handling and signaling, and a combination of these alterations may provide the trigger for disease remodeling.