ReviewMuscle disease caused by mutations in the skeletal muscle alpha-actin gene (ACTA1)
Introduction
ACTA1 gene mutations have been shown to cause three different congenital myopathies [1].
(i) The first of these is ‘actin myopathy’ (AM), a term first applied by Goebel et al. [2] for a disease phenotype described previously [3], [4] in which patients’ biopsies reveal homogeneous filamentous inclusions containing actin, occupying areas devoid of sarcomeres, which would normally be part of the myofibrillar filament lattice (Fig. 1, Table 1).
(ii) The second is intranuclear rod myopathy (IRM) with characteristic intranuclear inclusions [2], [5] (Fig. 1, Table 1).
(iii) The third, but commonest, disease is nemaline myopathy (NEM) with characteristic sarcoplasmic nemaline bodies (rods) [6], [7] (Fig. 1, Table 1).
More than one phenotype may be caused by one ACTA1 mutation [1] (Table 2, Fig. 2). Further ACTA1 gene mutations are continually being identified in patients with the three conditions. Over 60 ACTA1 mutations are therefore now known (Table 2, Fig. 2). In addition to the nemaline and intranuclear rods and actin accumulations, ACTA1 mutations also effect changes in size and distribution of muscle fibre types.
AM and IRM are rare, but NEM is a more common and better known congenital myopathy [13]. NEM ranges in severity from a paucity of spontaneous movements at birth requiring immediate mechanical ventilation, to mild disease compatible with life to adulthood [14]. The European Neuromuscular Centre (ENMC) International Consortium on Nemaline Myopathy has divided NEM into six different subtypes: severe, intermediate, typical, mild, adult onset and other forms based on the severity of the disease, age of onset and additional features [15] (Table 1). In addition to ACTA1mutations [1], [9], [10], [12], NEM has been associated with mutations in four other genes coding for muscle thin filament proteins. These are: (1) slow α-tropomyosin (TPM3) [16], [17], [18]; (2) nebulin (NEB) [19]; (3) slow troponin T (TNNT1) [20]; and (4) β-tropomyosin (TPM2) [21]. In addition, mutations in the skeletal muscle ryanodine receptor gene (RYR1) can cause nemaline bodies as well as central cores in the mixed muscle disorder core–rod myopathy [22], [23]. Linkage analysis indicates that NEB mutations cause the majority of NEM cases. ACTA1 mutations appear to be the second most common cause of NEM [8] at around 20% of cases. The small size of the actin gene makes it easier to find the ACTA1 NEM mutations than those in the giant NEB gene, so more disease-causing mutations are known in ACTA1, with many NEB mutations remaining unidentified.
Mutations in human actin genes, including the cardiac actin gene, ACTC, which produce dilated [24] or hypertrophic [25], [26] cardiomyopathy are a relatively recent discovery. In Drosophila melanogaster, the fruitfly, mutants of the Act88F muscle actin gene, whose expression is largely restricted to flight muscles and which is not therefore required for organism viability, have been known for some time [27], [28]. Mutations in this animal model have clarified the molecular functions of actin and its dysfunction in muscle development and can aid our understanding of human actin mutations.
In this review we will concentrate on 69 characterised ACTA1 gene mutations (Table 2, Fig. 2) with a view to developing an understanding of genotype–phenotype correlations and suggesting future studies. Mutations are distributed through all six coding exons of ACTA1 (Fig. 2). There is as yet no overlap between the mutant ACTA1 residues that produce skeletal myopathies and those in ACTC that cause cardiomyopathies, despite the proteins being 99% identical [29].
Section snippets
The genetics of ACTA1 mutations
ACTA1 mutations cause both dominant and recessive disease, with all of the sporadic cases for whom parental DNA has been available having de novo dominant mutations not present in either parent. Sixty mutations are associated with NEM, seven with AM and eight with IRM. Some mutations are associated with more than one type of muscle pathology, e.g. Val163Leu with both AM and IRM (Table 2, Fig. 2). In two instances, Val163Leu and Met227Ile, two different DNA mutations cause the same amino acid
Actin
Actin is found in all cells and forms, as polymerised F-actin, a major part of the cytoskeleton. Within muscle cells, actin, myosin and their associated proteins form the specialised contractile structure known as the sarcomere. The roles of actin in the cytoskeleton and sarcomere require interactions with a great variety of proteins. These have likely restrained actin evolution, probably explaining why it is one of the most conserved proteins known [29].
The actin monomer is a globular protein
The effects of functional null alleles of the ACTA1 gene
A recessive ACTA1mutant producing a severe NEM phenotype is due to a nonsense mutation of codon 39 (R39X), a premature translation termination signal. A similar mutation, KM88 (or T79X) in the D. melanogaster Act88F actin gene causes a dominant flightless phenotype with severe sarcomeric abnormalities [32]. KM88 flight muscles show no evidence that the N-terminal peptide, if produced, accumulates. The messenger RNA appears to be degraded [32], an effect well known as nonsense-mediated mRNA
Do actin mutants causing skeletal muscle disease cluster within the actin structure?
If the dominance of most ACTA1 disease mutants is caused by disruption of specific actin interactions within the sarcomere, one might expect the mutant residues to cluster on the surface, or just below it, especially within or close to the binding sites for actin-binding proteins (ABPs). Important interactions would include actin–actin contacts in F-actin, contacts with other sarcomeric proteins, or with proteins involved in actin filament assembly, such as profilin, gelsolin, etc. ACTA1
Which actin functions are affected by the disease-causing mutations?
Our purpose in what follows is to use the knowledge of actin structure and function gained from model systems to attempt a prediction of the molecular consequences of specific ACTA1mutations. We hope to provide insights into how the disease phenotypes arise, or at least to detect patterns, propose testable models and perhaps indicate other candidate proteins for AM, IRM and NEM.
Molecular modelling of all the known ACTA1 mutations is beyond the scope of this review and would not often predict,
Intranuclear rod myopathy (IRM)
The actin mutants that cause IRM are His40Tyr, Ala138Pro, Asp154Asn, Val163Leu, Val163Met, Lys336Ile and Ile357Leu. These mutants do not cluster and clearly do not define a single binding site.
(i–iii) Val163Leu and Ala138Pro described above, along with Val163Met are likely to affect the hinge, with effects on cleft opening and nucleotide exchange.
(iv) Asp154Asn (see above) is part of a phosphate-binding loop and will affect nucleotide binding.
(v) Lys336 lies within the ‘hinge’ and forms part of
Nemaline rod myopathy (NEM)
NEM is the commonest ACTA1mutant phenotype. The NEM mutant residues are not clustered (Fig. 4), nor do they tend to occur on the surface where ABP-binding sites are likely. This makes it difficult to discern functional patterns among the mutant changes.
In the following section, we have grouped the mutant residues by their likely effects. It is very important to remember that these effects are functionally interrelated. Thus changes in G-actin conformation and actin–actin contacts will affect
Potential actin–ABP interactions affected by ACTA1 myopathies
Interaction sites of ABPs have been deduced by ‘docking’ binding partners and actin atomic structures, restricted by the ‘mass envelopes’ determined by image processing of high resolution EMs. This approach is not accurate enough to assign confidently particular residues to many of the contacts and largely ignores conformational adjustments involved in protein binding. However, through a combination of biochemical and genetic studies [64], [65], [66], residues have been assigned to binding
Actin-tropomyosin
Tropomyosin (Tpm), is a dimer, which by end-to-end association forms a continuous filament wound around F-actin. It has an important role in the control of striated muscle contraction (see above) but also probably stabilises F-actin in vivo. F-actin decorated with Tpm in the presence/absence of Ca2+ and the myosin S1 fragment showed structures [69] representing the three thin filament regulatory states proposed by McKillop et al. [70]. Gordon et al. [31] projected the Tpm in these
α-Actinin
α-Actinin is an F-actin cross-linker and a major Z-disc component. EM studies of F-actin decorated with an N-terminal α-actinin domain [71] showed binding to two neighbouring actin monomers, consistent with biochemical studies that localised binding to residues 86–117 and 350–375 [72], [73], [74], [75]. ACTA1NEM mutations possibly affecting α-actinin binding include Ala114Thr, Asn115Ser/Thr, Arg116His, Ile357Leu, Val370Phe, Arg372Ser and Lys373Gln. The resolution of the EM approach prevents
Nebulin
Nebulin is a giant filamentous protein, spanning the thin filament length. It may act as a template to control thin filament length during myofibrillogenesis [76]. EM reconstructions of F-actin decorated with a nebulin fragment [77] show unexpectedly that nebulin binding occurs at three separate locations, suggesting that nebulin lies on, or moves between, different positions on the F-actin helix. ACTA1 mutations in the nebulin contact sites include Met227Ile/Thr/Val, Arg256His/Leu, Glu259Val
Discussion
ACTA1 mutations cause three histological types of myopathy, AM, IRM and NEM. If these are distinct myopathies, then the mutant effects on specific actin functions might be restricted to one myopathy. We predict that all the AM mutations will affect nucleotide binding and/or hinge flexibility. Similarly most of the IRM mutations seem likely to affect nucleotide binding and hinge flexibility while the remainder can be included by considering evidence of communication between events at the
Acknowledgements
We thank A. Clark, J. Hertz, E. Honey, D. Hutchinson, H. Jungbluth, C. von Kaisenberg, J. McGaughran, G. Matthijs, F. Muntoni, Y. Nevo, H. Schmallbruch, J.M. Schroeder for unpublished information on their patients for whom ACTA1 mutations were found. J.C.S. is supported by the BBSRC (UK) and the British Heart Foundation; K.J.N. is a CJ Martin Fellow of the Australian National Health and Medical Research Council; N.G.L. is supported by The Australian National Health and Medical Research Council
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