Association of nonsense mutation of dystrophin gene with disruption of sarcoglycan complex in X-linked dilated cardiomyopathy

Summary

Background

In a systematic analysis of inherited forms of cardiomyopathy, we previously identified a family with X-linked dilated cardiomyopathy characterised by a mutation in the rod region of dystrophin. We have now attempted to eludicate the genetic mechanism involved in this disease, as well as the role of dystrophin-associated glycoproteins.

Methods

The affected dystrophin epitope, which lacks binding to the dys-1 antibody, was analysed by single-strand conformation polymorphism analysis, reverse-transcription PCR, and DMA sequencing. Effects on dystrophin-associated glycoproteins were studied by immunohistochemistry and western blotting.

Findings

A translation-termination mutation (C4148T) in exon 29 of the dystrophin gene was found in all affected family members. Alternative splicing rescued the reading frame and led to the expression of a dystrophin molecule lacking 50 aminoacids both in cardiac and skeletal muscle. Immunohistochemical analysis of the dystrophin-associated proteins revealed a reduction of (3-sarcoglycan and 8-sarcoglycan in the sarcolemma of cardiac muscle but not skeletal muscle tissue. However, western blotting revealed similar amounts of sarcoglycan subunits in both tissues.

Interpretation

The molecular mechanism of this subtype of X-linked cardiomyopathy may be explained by a conformational change in exon-29-deleted dystrophin, resulting in disruption of the sarcoglycan assembly in heart muscle but not skeletal muscle.

Introduction

Idiopathic dilated cardiomyopathies are a group of heart-muscle diseases characterised by ventricular dilatation resulting in progressive heart failure or sudden cardiac death. Up to 35% of cases are inherited.1, 2 Genetic analyses have linked the X chromosome to familial dilated cardiomyopathy and also chromosomes 1, 2, 6, 9, 10, and 15.³ However, few mutations were characterised in these cases. Hereditary dilated cardiomyopathy was associated with mutations in a domain of the actin gene (15ql4) necessary for binding to dystrophin.⁴ Mutations in the dystrophin gene (Xp21) itself cause a group of dystrophinopathies which may present as classic Duchenne and Becker muscular dystrophy, X-linked dilated cardiomyopathy, X-linked mental retardation, or subclinical cases with raised concentrations of serum creatine kinase subtype MM.⁵ In the absence of skeletal-muscle weakness, dysfunction of other organs such as heart or brain can become the predominant clinical feature. Cardiac involvement is associated with dilated cardiomyopathy in more than 80% of cases; however, only 10% of affected boys die of heart failure.⁶ In rare instances of Becker muscular dystrophy, dilated cardiomyopathy has been the predominant symptom.⁷ Studies have also shown that a significant proportion of X-linked cases of dilated cardiomyopathy with subclinical skeletal muscle involvement are caused by dystrophin-gene defects.8, 9, 10, 11, 12, 13, 14

Dystrophin, a subsarcolemmal rod-shaped protein, is believed to stabilise the sarcomere by attaching the actin cytoskeleton via the dystrophin-associated glycoprotein complex to the extracellular matrix. Components of this complex are the dystroglycan and sarcoglycan subcomplexes. The two dystroglycans link dystrophin to Iaminin-α2.¹⁵ The sarcoglycan subcomplex is composed of four equimolar transmembrane glycoproteins with unknown function. Evidence that the sarcoglycans may be associated with cardiomyopathy derives from a report on a 13-year-old patient with dilated cardiomyopathy with a loss of staining of α-sarcoglycan and γ-sarcoglycan in cardiac and skeletal muscle.¹⁶ A primary defect in the γ-sarcoglycan gene results in autosomal recessive limb-girdle muscular dystrophy 2F, which is characterised by a severe Duchenne-like phenotype.¹⁷ An animal model for autosomal recessive cardiomyopathy and muscular dystrophy is the BIO 14·6 hamster, in which a deletion of the γ-sarcoglycan gene leads to a complete loss of all sarcoglycan subunits in heart and skeletal muscle.¹⁸

Previously, we have reported on a family with a severe form of X-linked dilated cardiomyopathy.⁹ Two brothers, Two brothers, the elder requiring heart transplantation, and a maternal cousin had raised creatine kinase concentrations. All complained of occasional cramping myalgia on exertion, but none had muscle weakness or an abnormal electromyogram. Molecular-genetic analyses with polymorphic markers linked the disease locus to the 5′ region of the dystrophin gene. Immunofluorescence analyses with N-terminal and C-terminal antibodies against the dystrophin protein showed a faint staining of the sarcolemmal membrane in cardiac and skeletal muscle. Western blot analyses revealed a reduction of dystrophin by 80% in both tissues. No reaction was found using the dys-1 antibody directed against the midrod region of the dystrophin protein. The goal of this study was the identification of the genetic defect of dystrophin and its effect on the dystrophin-associated glycoprotein complex.