DIAGNOSING CHILDHOOD MUSCULAR DYSTROPHIES

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1 DIAGNOSING CHILDHOOD MUSCULAR DYSTROPHIES Extracts from a review article by KN North and KJ Jones: Recent advances in diagnosis of the childhood muscular dystrophies Journal of Paediatrics and Child Health (1997) 33, Recent advances in molecular genetics research have revolutionised our understanding of the childhood muscular dystrophies. The first breakthrough came in 1987, with the identification of the gene for dystrophin, the protein that is abnormal in X-linked Duchenne muscular dystrophy. Dystrophin is bound to a complex of proteins in the muscle membrane, and primary abnormalities of these proteins have now been identified as the cause of some autosomally inherited forms of muscular dystrophy. A group of transmembrane proteins known as α- (adhalin), β-, γ- and δ-sarcoglycan are deficient in autosomal recessive limbgirdle muscular dystrophy, and the extracellular matrix protein merosin (α2-laminin), is deficient in a subset of patients with congenital muscular dystrophy. Identification of primary deficiencies in these dystrophin associated proteins will result in improved diagnostic accuracy, more accurate genetic counselling and, in some cases, the availability of prenatal diagnosis. Definition and Classification The myopathies are primary disorders of muscle, characterised by lower motor neurone weakness, and subdivided on the basis of the presence or absence of specific ultrastructural changes on muscle biopsy. Muscular dystrophies are genetically determined myopathies, not associated with specific ultrastructural abnormalities, but with dystrophic changes on muscle biopsy, that is an increase in connective tissue, degenerating and regenerating fibres and marked variation in fibre size. Classification of the muscular dystrophies has been based historically on a combination of clinical features including age of onset, clinical severity and rate of progression, distribution of muscle weakness and pattern of inheritance. In 1868, Duchenne described the rapidly progressive, pseudohypertrophic type of muscular dystrophy in boys that now bears his name. Weakness predominantly affects the limb girdle muscles. Becker muscular dystrophy is a related disorder (see below), with milder and more heterogeneous clinical manifestations. Other disorders distinguished by their characteristic pattern of weakness include facioscapulohumeral dystrophy, an autosomal dominant disorder affecting predominantly the facial and shoulder girdle muscles; and Emery-Dreifuss muscular dystrophy, an X-linked disorder with slowly progressive humero-peroneal muscle weakness and cardiomyopathy. Batten, in 1903, was the first to describe a muscular dystrophy presenting at birth, which was later called dystrophica muscularis congenita. Congenital muscular dystrophy is the term now used to describe a group of disorders presenting at birth or within the first few months of life, with static or slowly progressive muscle weakness, often associated with hypotonia, and variable involvement of the respiratory and bulbar muscles. The congenital muscular dystrophies are subclassified on the basis of the presence or absence of structural involvement of the central nervous system. Fukuyama congenital muscular dystrophy, Walker-Warburg syndrome and Muscle-eye-brain disease are associated with structural abnormalities of the brain, severe mental retardation and seizures; the latter two disorders with ocular malformations. Pure or classical congenital muscular dystrophy has

2 traditionally been defined as having no clinical involvement of the central nervous system and normal intellect. Although the brain is structurally normal, a subset of patients has been identified with white matter changes on CT or MRI. Muscular dystrophies that do not fall into these clinically defined groups have been classified historically as the limb-girdle muscular dystrophies, a heterogeneous group of disorders with marked clinical variability. The existence of limb-girdle muscular dystrophy as a separate entity has been challenged, and several disorders previously included in this group, such as Becker muscular dystrophy, are now distinguishable on a genetic basis. 1 In this article we will summarise recent advances in the understanding of the molecular pathogenesis of the muscular dystrophies, which have further clarified the classification of the limb-girdle muscular dystrophies and confirmed their existence as a separate entity. These advances will result in improved diagnostic accuracy, more accurate genetic counselling, and in some cases the availability of prenatal diagnosis.

3 Approach to the Diagnosis of the Childhood Muscular Dystrophies From p.197 Recent Advances in Diagnosis of the Childhood Muscular Dystrophies Muscle Weakness CK NORMAL ELEVATED Muscular dystrophy unlikely: consider other diagnoses MALE FEMALE DNA Analysis for dystrophin gene abnormalities Deletion detected No deletion detected Deletion not informative for severity Muscle Biopsy Routine histopathology dystrophin analysis (immunocytochemistry and Western Blot) Deletion informative for severity Positive family history ABNORMAL DYSTROPHIN NORMAL DYSTROPHIN DIAGNOSIS OF DYSTROPHINOPATHY (DMD or BMD) Merosin and adhalin analysis ie. Duchenne muscular dystrophy or Becker muscular dystrophy

4 As described above, the cloning of the dystrophin gene in 1987 led to a sequence of advances that have revolutionised the classification and diagnosis of the muscular dystrophies. Current classification of the limb-girdle muscular dystrophies (in terms of the mode of inheritance and gene localisation) is outlined in Table 1. When a patient presents with weakness in a pattern suggestive of a primary muscle disorder, ie predominantly proximal muscle involvement with wasting and diminished power, hypotonia, hyporeflexia +/- calf hypertrophy or contractures, we would suggest an approach to diagnostic testing as outlined in Figure 3. Serum creatine kinase (CK) is a useful screening test, as elevation above 1000 U/L is suggestive of a muscular dystrophy. Note that CK can be normal in some patients with a dystrophic biopsy. CK is also elevated in two thirds of female carriers of a dystrophin gene abnormality. 1 EMG is not useful in the differentiation of the muscular dystrophies as the findings are non specific, showing a myopathic pattern of low amplitude, polyphasic potentials of short duration. Muscle ultrasound and muscle MRI may be helpful in differentiating between a myopathy and a neuropathy, and in determining the extent of muscle involvement. If there is a high index of suspicion and the CK is elevated, EMG and imaging studies may be bypassed to proceed directly to muscle biopsy or DNA testing (see below). A dystrophic picture on muscle biopsy is characterised by marked variation in fibre size, increased connective tissue and the presence of degenerating and regenerating fibres (Figure 4). Biopsy is useful to exclude congenital myopathies with specific ultrastructural changes, such as nemaline rods or central cores, or a primary neuropathic disorder. Immunocytochemical studies may be performed on frozen sections from muscle biopsy using antibodies that react specifically with individual muscle proteins. Using antibodies to dystrophin, the protein may be shown to be completely deficient (Duchenne muscular dystrophy), or decreased in quantity (Becker muscular dystrophy) (Figure 5). Further quantitation of specific proteins and identification of proteins with abnormal molecular weight may be performed using Western blotting techniques. A similar process can be performed for the dystrophin associated proteins, and in addition to dystrophin, there are currently commercially available antibodies to adhalin (α-sarcoglycan) and merosin (α2 laminin) (Figure 6). If adhalin is abnormal on immunocytochemistry this may be due to a primary deficiency of adhalin or it may be the result of an abnormality of one of the other sarcoglycans. Primary and secondary sarcoglycan abnormalities can only be distinguished by the identification of a gene mutation. DNA diagnosis is now widely available for the X-linked dystrophinopathies and analysis of DNA from peripheral blood by PCR (polymerase chain reaction) results in detection of a dystrophin gene mutation in approximately 60% of patients with Duchenne or Becker muscular dystrophy. In some cases it is possible to predict clinical severity based on the nature of the mutation. This may obviate the need for muscle biopsy, however quantification of dystrophin by immunocytochemistry and Western blotting is more reliable in terms of predicting prognosis. DNA testing may ultimately replace muscle biopsy in the diagnosis of the muscular dystrophies, however, at present, mutation analysis for proteins other than dystrophin is available only on a research basis. Other investigations may be useful in determining the complete clinical phenotype associated with a particular muscular dystrophy. Cranial MRI may show leukodystrophic changes in merosin negative congenital muscular dystrophy and structural brain abnormalities in the non-classical congenital muscular dystrophies (eg. Fukuyama). ECG

5 and echocardiogram may show evidence of cardiac involvement in the dystrophinopathies, sarcoglycanopathies and Emery-Dreifuss muscular dystrophy. Improved diagnostic accuracy has resulted in more accurate genetic counselling, and in some cases prenatal diagnosis. Prenatal diagnosis is available for the dystrophinopathies (on chorionic villous biopsy or amniocentesis) by direct genetic testing for dystrophin gene abnormalities or by linkage studies in cases where the gene mutation is not known. Immunocytochemical analysis can be performed on chorionic villous biopsy for merosin abnormalities, as this protein is expressed in placental tissue. The technique of fetal muscle biopsy is currently being developed, and may enable direct study of protein expression in the fetal muscle while in utero. In addition, mutational analysis for the other protein abnormalities currently only in performed in research laboratories, will become more widely available, offering further prenatal testing options.