Human Molecular Genetics Advance Access originally published online on June 15, 2004
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Human Molecular Genetics, 2004, Vol. 13, No. 16 1727-1743
DOI: 10.1093/hmg/ddh185
Human Molecular Genetics, Vol. 13, No. 16 © Oxford University Press 2004; all rights reserved
Evidence for a dominant-negative effect in ACTA1 nemaline myopathy caused by abnormal folding, aggregation and altered polymerization of mutant actin isoforms
1The Institute for Neuromuscular Research, The Children's Hospital at Westmead, 2Discipline of Paediatrics and Child Health, Faculty of Medicine, University of Sydney, Australia, 3Centre for Neuromuscular and Neurological Disorders, University of Western Australia (UWA); Australian Neuromuscular Research Institute and Centre for Medical Research, UWA; West Australian Institute for Medical Research, Queen Elizabeth II Medical Centre, Nedlands, Western Australia, 4MRC Functional Genetics Unit, Department of Human Anatomy and Genetics, University of Oxford, UK and 5Department of Pathology, University of Geneva, Geneva, Switzerland
Received March 31, 2004; Accepted June 3, 2004
We have studied a cohort of nemaline myopathy (NM) patients with mutations in the muscle 
-skeletal actin gene (ACTA1). Immunoblot analysis of patient muscle demonstrates increased 
-filamin, myotilin, desmin and -actinin in many NM patients, consistent with accumulation of Z line-derived nemaline bodies. We demonstrate that nebulin can appear abnormal secondary to a primary defect in actin, and show by isoelectric focusing that mutant actin isoforms are present within insoluble actin filaments isolated from muscle from two ACTA1 NM patients. Transfection of C2C12 myoblasts with mutant actinEGFP constructs resulted in abnormal cytoplasmic and intranuclear actin aggregates. Intranuclear aggregates were observed with V163L–, V163M– and R183G–actinEGFP constructs, and modeling shows these residues to be adjacent to the nuclear export signal of actin. V163L and V163M actin mutants are known to cause intranuclear rod myopathy, however, intranuclear bodies were not reported in patient R183G. Transfection studies in C2C12 myoblasts showed significant alterations in the ability of V136L and R183G actin mutants to polymerize and contribute to insoluble actin filaments. Thus, we provide direct evidence for a dominant-negative effect of mutant actin in NM. In vitro studies suggest that abnormal folding, altered polymerization and aggregation of mutant actin isoforms are common properties of NM ACTA1 mutants. Some of these effects are mutation-specific, and likely result in variations in the severity of muscle weakness seen in individual patients. A combination of these effects contributes to the common pathological hallmarks of NM, namely intranuclear and cytoplasmic rod formation, accumulation of thin filaments and myofibrillar disorganization.
* To whom correspondence should be addressed at: Institute for Neuromuscular Research, The Children's Hospital at Westmead, Locked Bag 4001, Westmead, NSW 2145, Australia. Tel: +61 298451220; Fax: +61 298453078; Email: sandrac3{at}chw.edu.au
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