The genetics of hypertrophic cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is most commonly transmitted as an autosomal dominant trait, caused by mutations in genes encoding cardiac sarcomere proteins1–3. Other inheritable causes of the disease include mutations in genes coding for proteins important in calcium handling or that form part of the cytoskeleton4–6. At present, the primary clinical role of genetic testing in HCM is to facilitate familial screening to allow the identification of individuals at risk of developing the disease7,8. It is also used to diagnose genocopies, such as lysosomal9–11 and glycogen storage disease which have different treatment strategies, rates of disease progression and prognosis12–14. The role of genetic testing in predicting prognosis is limited at present, but emerging data suggest that knowledge of the genetic basis of disease will assume an important role in disease stratification15–17 and offer potential targets for disease-modifying therapy in the near future18.


GENETIC ARCHITECTURE OF HCM
Familial HCM is characterized by locus and allelic heterogeneity, with a high frequency of novel individual mutations 7,19 . The first genetic mutation to be identified was a single base substitution in the MYH7 gene encoding ß-myosin heavy chain, a key component of the cardiac sarcomere 20 . Since then, many different mutations in MYH7 and other genes of the cardiac sarcomere have been identified. In 5-10% of cases, HCM is caused by mutations in genes that cause metabolic disorders [21][22][23] , neuromuscular disease [24][25][26] or inherited genetic syndromes including Noonan syndrome [27][28][29] (Figure 1).

THE CARDIAC SARCOMERE
Every cardiomyocyte is composed of myofibrils that run longitudinally along the cell and are transversely subdivided into contractile units called sarcomeres 30,31 . The sarcomere constitutes the fundamental motor unit of the cardiomyocyte and is composed of two principal components -the thick filament composed of around 300 molecules of myosin, each made up of 2 protein units of βor α-myosin heavy chain and 4 myosin light chain molecules, and the thin filament composed of repeating actin molecules, closely associated with the regulatory troponin complex (troponin T (TnT), troponin I (TnI)  Reproduced from Ref. [31]. Abbreviations: TnI, Troponin I; TnC, Troponin C; TnT, Troponin T; Tm, Tropomyosin; c-MYBP-C, cardiac myosin binding protein-c; ELC, Essential Light Chain; RLC, Regulatory Chain; LMM, Light meromyosin. The domains of cMyBP-C are numbered from C0-C10; m is the regulatory motif between domains C1 and C2 and contains the PKA phosphorylation site; PA represents a proline / alanine-rich linker sequence between domains C0 and C1. and troponin C (TnC)) and α-tropomyosin [31][32][33] . An additional protein, cardiac myosinbinding protein C, contributes to the regulation of actin-myosin interaction and crossbridge kinetics 31 ( Figure 2).
Muscle contraction results from the interaction between myosin and actin in which the globular head of the myosin molecule bends towards and then binds to actin, contracts, releases actin, and then initiates a new cycle. The links between the myosin head and actin are called cross-bridges; the contraction of the myosin S1 region which requires the hydrolysis of ATP to release energy is called the power stroke 31,34 .
Electrical activation of the heart and cardiomyocyte contraction are coupled through the intracellular movement of calcium. Depolarisation of the cardiomyocyte cell membrane activates the L-type voltage-dependent calcium channels in the T tubule causing an influx of calcium into the cell that triggers opening of ryanodine-receptor channels in the adjacent sarcoplasmic reticulum with a rapid increase in cytosolic calcium 31 . Calcium binds to TnC, inducing an allosteric conformational change in TnI and TnT that is transmitted to tropomyosin and exposes the myosin-binding sites of actin allowing the cross-bridges to form 33 . The myosin heavy chain head, with ADP and inorganic phosphate bound to its nucleotide-binding pocket, then interacts with the exposed actin-binding sites followed by the release of ADP and inorganic phosphate, which occurs simultaneously with the power stroke. ATP then binds to the nucleotidebinding pocket of the myosin heavy chain head, which detaches from actin and myosin then hydrolyses ATP into ADP and inorganic phosphate restarting the contraction cycle 31 .

SARCOMERE MUTATIONS IN HCM
Mutations in ßMYH7 and MYBPC3 account for 60-70% of HCM patients with pathogenic variants 1,19,32,35 . Mutations in other sarcomere and associated protein genes are listed in Table 1. Mutations in MYH7 are predominantly missense, with single nucleotidebase substitutions resulting in a non-synonymous single amino acid substitution 20,36 . In contrast, the majority of mutations in MYBPC3 are nonsense mutations due to insertion/deletions, splice-site variants or frameshifts causing a premature stop codon that results in a truncated protein transcript 1,32,37 .
The majority of missense mutations are believed to have a dominant negative effect in which the mutant protein is incorporated into the sarcomere, but its interaction with the normal wild-type protein disrupts normal sarcomeric assembly and function (poison polypeptide hypothesis) 31,38 . Individual missense mutations may change an amino acid in a highly-conserved residue, alter important kinase domains (affecting ligand interaction) or change surface-exposed portion of a molecule altering protein-protein interaction. Missense mutations can also cause protein misfolding and accelerated degradation by ubiquitin-proteasomal surveillance pathways.
In truncating mutations, haploinsufficiency is thought to be the major disease mechanism 39,40 . The reported absence of detectable truncated myosin-binding protein C in western-blot analysis of myectomy specimens of patients with this mutation may be due to nonsense mediated mRNA decay of abnormal transcripts or ubiquitin-mediated proteasomal degradation of aberrant truncated protein [40][41][42][43] .
Allelic heterogeneity can be partly explained by the effect of different mutations on the structure and function of the complete peptide. ß-myosin heavy chain, for example, consists of a globular head, an α-helical rod and a hinge region. The globular head contains binding sites for ATPase and actin as well as interaction sites for regulatory and essential light chains in the head-rod region 31 . The majority of disease-causing ß-myosin heavy chain mutations are found in one of four locations: the actin binding site, the nucleotide binding pocket, the hinge region adjacent to the binding site for two reactive thiols and in the α-helix close to the essential light chain interaction site 44 . Thus, different effects on protein function might be expected depending on the position of the mutation.
It has been speculated that the HCM disease phenotype results from reduced contractile function caused by altered actin-myosin interactions, and consequent inappropriate compensatory hypertrophic remodelling 45,46 . However, some MYH7 mutations are associated with increased cardiomyocyte mechanical contractile forces in vitro and show an increase in calcium sensitivity, leading to increases in tension generation and ATPase activity. Animal and cell studies have also confirmed altered calcium homeostasis as a key contributor to the pathophysiological processes that lead to the development of LV hypertrophy 47,48 .
Troponin T mutations account for less than 5% of all cases of HCM but often show a particular phenotype. TNNT2 mouse models show varying degrees of myocyte disarray and fibrosis with minimal LVH, in common with TNNT2 disease expression in humans 49,50 . Troponin-mutated mice exhibit severely impaired myocardial relaxation, independent of the degree of fibrosis, and consistent with the finding of increased calcium sensitivity [51][52][53] . Some studies suggest that some TNNT2 mutations are associated with a high incidence of sudden death which may also relate to calcium loading in cardiomyocytes 54,55 .
Findings from murine models of HCM suggest that the increased contractility seen with some mutations is at the expense of inefficient ATP utilization. Furthermore, animal Table 1 List of genes in which pathogenic mutations are associated with hypertrophic cardiomyopathy. The chromosome location and the proportion of HCM cases attributed to mutations in these specific genes are included. and human studies suggest that HCM is associated with depleted energy stores and abnormal ATP/ADP ratios [56][57][58][59] . Inefficient utilization of ATP is also seen in metabolic disorders or mitochondrial cytopathies, which can produce a pattern of LV hypertrophy similar to that in sarcomeric HCM.
Recently, mutations in genes encoding z-disc proteins including myozenin (MYOZ2), telethonin (TCAP), alpha-actinin-2 (ACTN2), muscle LIM protein (CRP3) and nexilin (NEXN) have been implicated in HCM 31 . Some mutations in Z-disc proteins have a pleotropic effect, causing an HCM phenotype in certain individuals and a DCM phenotype in others within the same family 60 .

CHILDHOOD-ONSET HCM
The importance of sarcomeric protein gene mutations in childhood hypertrophic cardiomyopathy is unknown. The observation that the development of left ventricular hypertrophy in individuals with familial disease often occurs during the period of somatic growth in adolescence has led to the suggestion that sarcomeric protein disease in very young children is rare 61,62 . However, studies of children with HCM have shown that, as in adults, sarcomeric protein gene mutations account for approximately 50% of cases of idiopathic HCM, even in infants and young children 63,64 .

PHENOTYPIC VARIABILITY
There is a substantial variation in the expression of identical mutations indicating that other genetic and possibly environmental factors influence disease expression. The effect of age is perhaps the best characterized factor, most patients developing ECG and echocardiographic manifestations of the disease after puberty and before the age of thirty 65,66 . Sex also appears to influence disease expression in sarcomere protein disease 67,68 . Other potential modifying factors include renin-angiotensin-aldosterone system gene polymorphism [69][70][71] , and the occurrence of homozygosity and compound heterozygosity [72][73][74] .

GENETIC ADVANCEMENTS
The impact of more rapid, cost-effective gene sequencing methods, together with improved cellular models including induced pluripotent stem cells, gene-editing technology, larger sequenced control cohorts and deep clinical phenotyping in cases means that we are at an exciting crossroads in the genetics of HCM. This provides researchers with significant opportunity to identify novel candidate causative genes and to potentially allow clearer genotype-phenotype correlations to be made, thereby laying the foundation for more personalised patient care.