Variants in ACTC1 underlie distal arthrogryposis accompanied by congenital heart defects

Abstract

Contraction of the human sarcomere is the result of interactions between myosin cross-bridges and actin filaments. Pathogenic variants in genes such as MYH7, TPM1, and TNNI3 that encode parts of the cardiac sarcomere cause muscle diseases that affect the heart, such as dilated cardiomyopathy and hypertrophic cardiomyopathy. In contrast, pathogenic variants in homologous genes such as MYH2, TPM2, and TNNI2 that encode parts of the skeletal muscle sarcomere cause muscle diseases affecting skeletal muscle, such as distal arthrogryposis (DA) syndromes and skeletal myopathies. To date, there have been few reports of genes (e.g., MYH7) encoding sarcomeric proteins in which the same pathogenic variant affects skeletal and cardiac muscle. Moreover, none of the known genes underlying DA have been found to contain pathogenic variants that also cause cardiac abnormalities. We report five families with DA because of heterozygous missense variants in the gene actin, alpha, cardiac muscle 1 (ACTC1). ACTC1 encodes a highly conserved actin that binds to myosin in cardiac and skeletal muscle. Pathogenic variants in ACTC1 have been found previously to underlie atrial septal defect, dilated cardiomyopathy, hypertrophic cardiomyopathy, and left ventricular noncompaction. Our discovery delineates a new DA condition because of variants in ACTC1 and suggests that some functions of ACTC1 are shared in cardiac and skeletal muscle.

Keywords: Mendelian disease; Mendelian disorder; cardiac defect; cardiomyopathy; congenital contractures; distal arthrogryposis; exome sequencing; gene discovery; molecular dynamics simulations.

Figure 1

Phenotypic characteristics of individuals with DA because of heterozygous variants in ACTC1 (A–D) The characteristics shown include webbed neck, bilateral clubfoot, camptodactyly of the fingers, and hypoplastic flexion creases in family A (A; II-1 and III-1); camptodactyly, webbed neck, bilateral clubfoot, camptodactyly of the fingers and toes, and hypoplastic flexion creases in family B (B; II-2 and III-1); webbed neck, bilateral clubfoot, webbed neck, bilateral clubfoot, and camptodactyly of the fingers and toes in family C (C; II-2); and ptosis, webbed neck, camptodactyly of the fingers, and scoliosis in family D (D; II-1). Table 1 contains a detailed description of the clinical findings of each affected individual, and Figure S1 provides a pedigree for each family.

Figure 2

Genomic model of ACTC1 and ACTA1 Illustrated are each of the variants found in ACTC1 that underlie DA and the homologous sites in ACTA1 that result in severe nemaline myopathy when mutated. ACTC1 and ACTA1 are each composed of 7 exons and consist of protein-coding (blue) and non-coding (orange) sequences. The proteins are nearly identical except for four residues (represented by single-letter amino acid codes immediately above and below the green line). The approximate location of each pathogenic variant (red text) is indicated by an arrow.

Figure 3

Molecular structures of the globular and filamentous forms of human cardiac actin (A) Globular actin (g-actin) monomers are comprised of four subdomains (subdomains 1–4 [SD1–SD4]) arranged around the nucleotide binding pocket. The g-actin monomer simulated in this study contains ATP in the binding pocket. The atoms of four residues corresponding to variant sites examined in this study are shown as spheres: T68 (orange), R185 (magenta), G199 (purple), and R374 (blue). (B) Actin monomers polymerize into protofibrils, which then associate with one another to form filamentous actin (F-actin). In this F-actin pentamer, chains A, C, and E form one protofibril, and chains B and D form the other. The pentamer simulated here has ADP molecules in the nucleotide binding pockets. The location of residue T68 is denoted on chain C.

Figure 4

p.Thr68Asn alters the structure and dynamics of g-actin SD2 (A and B) Comparison of representative MD-derived snapshots of WT (A) and p.Thr68Asn (B) g-actin highlights the structural and dynamic changes induced by p.Thr68Asn (red ribbon). Side-chain atoms of relevant residues are shown and annotated. (C) Bar heights correspond to the fraction of time that select residue pairs spent in contact with one another, averaged over triplicate simulations (error bars correspond to SD). p.Thr68Asn (orange bars) led to shifts in several amino acid interactions relative to the WT (black bars). Statistically significant differences between the WT and p.Thr68Asn contact frequencies are denoted (ns, not significant; ∗p ≤ 0.05, ∗∗p ≤ 0.01). (D) p.Thr68Asn (orange) increased the α helix secondary structure content of residues 40–50 within the D-loop relative to the WT (black). (E) The p.Thr68Asn variant (orange) led to a decrease in the SASA of D-loop residues (41–56) relative to the WT (black). The histogram shows the SASA probability density of all replicate simulations combined.

Figure 5

DA-associated variants lead to structural changes within SD2 (A–D) The percent simulation time for which residue-residue contacts endured were compared between the four mutant simulations (p.Thr68Asn [A; orange], p.Arg185Trp [B; magenta], p.Gly199Ser [C; purple], and p.Arg374Ser [D; blue]), and the WT simulations (black). For each mutant-WT comparison, residue-residue contacts that were present for statistically different percentages of the simulations were mapped onto the reference crystal structure of g-actin. Contacts that were present more frequently in the WT simulations are denoted by black pipes, and contacts present more frequently in the mutant simulations are colored orange, magenta, purple, or blue. The thickness of the pipes corresponds to the difference in percent simulation time that the contact was present between the WT and mutant simulations (larger pipes indicate that a contact was observed more frequently). Although the variants were distributed throughout the structure, they all led to statistically significant (see Table S2 for test statistics) changes in the structure of SD2 (orange ribbons).

Figure 6

p.Thr68Asn alters inter-chain interactions made by SD2 in F-actin Residue-residue interactions formed between SD2 of chain C and chains A and B were analyzed in the GaMD simulations of the WT and p.Thr68Asn F-actin. (A) p.Thr68Asn led to statistically significant differences in residue-residue contacts formed by SD2 of chain C (denoted by pipes as in Figure 5). Differences were found in contacts formed between SD2 of chain C and SD1 of chain A as well as in contacts formed between SD2 of chain C and the SD3-SD4 linker of chain B. (B) The total number of atom-atom interactions formed between SD2 of chain C and all atoms in chains A and B were monitored in the WT and p.Thr68Asn GaMD simulations. Relative to the WT simulation (black), the p.Thr68Asn simulation (orange) had fewer inter-chain contacts involving chain CSD2. (C–E) In the reference cryo-EM structure and WT simulation, the D-loop of SD2 in chain C fits into a pocket formed by SD1 and SD3 of chain A. The p.Thr68Asn simulations instead sampled non-native conformations in which the D-loop exited this binding pocket. In the reference cryo-EM structure and the WT simulation (D), the D-loop of chain C is stabilized via a network of hydrophobic interactions formed with Y171 of chain A as well as a hydrogen bond network involving Arg 41 (chain C), Thr 68 (chain C), and Glu 272 (chain B). These interactions were disrupted in the p.Thr68Asn simulation (E).