The Childhood Muscular Dystrophies: Diseases Sharing a Common Pathogenesis of Membrane Instability

Transcription

1 Carrell-Krusen Symposium Invited Lecture The Childhood Muscular Dystrophies: Diseases Sharing a Common Pathogenesis of Membrane Instability Jerry R. Mendell, MD; Zarife Sahenk, MD; Thomas W. Prior, PhD ABSTRACT New observations demonstrate that several childhood forms of muscular dystrophy share a common pathogenesis. In muscle, dystrophin occurs as part of a membrane complex (dystrophin-glycoprotein) linking the cytoskeleton to the basal lamina. In Duchenne muscular dystrophy, dystrophin deficiency disrupts the linkage of the integral glycoproteins of the sarcolemma and leads to muscle fiber necrosis. In severe childhood autosomal recessive muscular dystrophy, a selective deficiency of adhalin (50-kd glycoprotein) also causes dysfunction of the dystrophin-glycoprotein complex. Most recently, a form of congenital muscular dystrophy demonstrates deficiency of laminin M (merosin) further demonstrating that sarcolemmal instability results from defects in structural proteins of the basal lamina. Animal models have been identified also demonstrating defects in specific proteins linking the subsarcolemmal cytoskeleton to the extracellular matrix. The mdx mouse has a defect in the gene encoding dystrophin. The cardiomyopathic hamster shows a specific deficiency of adhalin in skeletal muscle. The dy/dy mouse has been found deficient in merosin. These animal models will help researchers to understand their human counterparts and provide a system for testing therapeutic strategies. (J Child Neurol 1995;10: ). As far back as 1975, Mokri and Engell proposed that a defect in the plasma membrane of the muscle fiber represented the earliest pathologic event leading to muscle fiber degeneration in Duchenne muscular dystrophy. Support for this hypothesis was substantiated when the causative molecular genetic abnormality was found to affect dystrophin, 2,3 a large subsarcolemmal protein. Since then, new observations demonstrate that other childhood forms of muscular dystrophy are related to a system of integrated proteins linking the subsarcolemmal cytoskeleton to the extracellular matrix.4-6 Animal models of childhood muscular dystrophies have also been identified, with molecular defects in these same transmembrane proteins, enabling scientists to better understand the human counterparts and providing a system for further exploration of therapeutic strategies. SARCOLEMMAL PROTEINS Dystrophin The characterization of dystrophin and its identification as the missing or deficient protein in Duchenne muscular Received Dec 14, Accepted for publication Dec 14, From the Departments of Neurology and Pathology, Ohio State University College of Medicine, Columbus, OH. Address correspondence to Dr Jerry R. Mendell, Department of Neurology, 1654 Upham Dr, Columbus, OH dystrophy was a landmark discovery in the understanding of genetic disease. 7,8 Although originally thought to represent only a small component of total skeletal muscle protein, dystrophin has now been shown to constitute 2% of sarcolemmal and 5% of sarcolemmal cytoskeletal proteins in muscle.9 Dystrophin is predicted to be a rod-shaped cytoskeletal protein composed of four domains.3, 10 The structure of dystrophin and its location emphasize a role in providing stability for the sarcolemma especially during the stress of muscle contraction. The amino-terminal end represents an actin-binding domain linking dystrophin to actin (the polymeric or filamentous form [F-actin]). The second domain is composed of repeating units (each consisting of 109 amino acids) bearing a striking similarity to spectrin (with repeats of 106 amino acids); in the erythrocyte membrane, spectrin confers structural integrity, enabling the membrane to withstand distortions necessary to migrate through capillaries. The third domain, a cysteine-rich region, and the fourth domain, the carboxyl terminal, share the important function of linking dystrophin to other sarcolemmal proteins. Dystrophin-Associated Proteins Dystrophin and the large oligomeric complex of proteins serve to link the subsarcolemmal cytoskeleton to the extracellular matrix. This collective group of proteins is referred

2 151 to as the dystrophin-glycoprotein complex.ll In addition to dystrophin, there are six proteins, classified on the basis of molecular weight and glycosylation as dystrophin-associated proteins and glycoproteins. The 59-kd protein, a member of the syntrophin family, binds to the carboxy terminus of dystrophin.12,13 Four of the dystrophin-associated glycoproteins span the membrane including: (1) a 43-kd glycoprotein, also called (3-dystroglycan, having a cytoplasmic tail that binds dystrophin; (2) a 50-kd glycoprotein, now called adha,lin; (3) a 35-kd glycoprotein; and (4) a 25-kd glycoprotein.14-l6 The fmal component, a 156-kd glycoprotein commonly referred to as a-dystroglycan, is extracellular and binds the basal lamina protein, laminin. A single complementary DNA encodes a- and #-dystroglycan. 17 Laminin The basal lamina surrounds each muscle fiber and is closely adherent to the surface of the sarcolemma. Constituents of the basal lamina include type IV collagen, the heparan-sulfate proteoglycan perlecan, and the glycoproteins laminin and entactin. Laminin is a large flexible complex of long polypeptide chains held together by disulfide bonds. Laminin M, also known as merosin, is a major isoform of laminin heavy chains in the basal lamina of postnatal muscle and nerve. a-dystroglycan is a highly specific laminin receptor, not binding to other extracellular matrix components. 17 A schematic model illustrating the dystrophin-glycoprotein complex is depicted in Figure 1. It is not surprising that defects in this transsarcolemmal support system result in clinical phenotypes comprising childhood forms of muscular dystrophy. DYSTROPHINOPATHIES Clinical Features The features of Duchenne and Becker muscular dystrophies have been well delineated. Clinical symptoms of Duchenne dystrophy are unusual in the neonatal period. Occasional patients, especially those with mental retardation, may exhibit delayed motor milestones. In most cases, the disease becomes clinically apparent between ages 2 and 3 years. The condition shows relentless progression, with weakness and wasting more profoundly affecting the proximal lower extremity muscles. As the disease progresses, contractures develop that limit function, especially at the ankles and hips. Scoliosis is common after wheelchair confinement, which typically occurs about age 12 years. Most patients die of complications of respiratory insufficiency at about age 20 years. Cardiac involvement is a consistent part of Duchenne dystrophy. The heart demonstrates fibrosis in the posterobasal portion of the left ventricular wall. The right ventricular septum and the right ventricular and atrial myocardium Figure 1. Schematic representation of the dystrophin-glycoprotein complex. Dystrophin occurs as part of a membrane complex of six proteins and glycoproteins. The amino terminus of dystrophin binds to actin. The carboxy terminus of dystrophin binds to the 59-kd protein (59DAP), a member of the syntrophin family. Dystrophin is tightly bound to the sarcolemma by (3-dystroglycan (43DAG) in close relationship to adhalin (50DAG) and other membrane glycoproteins (35DAG and 25DAG). a- Dystroglycan (156DAG) binds the basal lamina protein laminin M (merosin). (Modified from Matsumura K, Campbell KP: Dystrophin-glycoprotein complex: Its role in the molecular pathogenesis of muscular dystrophies. Muscle Nerve 1994;17:2-15 and Fallon JR, Hall ZW: Building synapses: Agrin and dystroglycan stick together. Trends Neurosci 1994;17: )

3 152 have much less involvement. Degenerative changes affecting the conduction system are infrequent. Despite known cardiac disease, most patients with Duchenne dystrophy remain surprisingly free of cardiovascular symptoms. Congestive heart failure and cardiac arrhythmias usually occur only in the late stages and especially during times of stress from intercurrent infections. Rarely, however, patients with Duchenne muscular dystrophy have overt signs of congestive heart failure and, in fact, may die of cardiac failure with relative sparing of respiratory muscle function. Clinical and pathologic involvement of smooth muscle of the gastrointestinal tract, although frequently overlooked, can be an important manifestation. A syndrome of acute gastric dilation, also referred to as intestinal pseudo-obstruction, consists of sudden episodes of vomiting associated with abdominal pain and distention, and may lead to death if not treated appropriately. 18,19 Patients dying of this syndrome show degeneration of the outer longitudinal smooth muscle layer of the stomach; other regions of the gastrointestinal tract can be affected, causing symptoms such as severe constipation. Dystrophin deficiency leads to degeneration of smooth muscle. The average IQ falls approximately one standard deviation below the mean.2 The impairment of intellectual function appears to be nonprogressive and affects the verbal ability more than performance. The neuropathologic correlate for mental retardation in Duchenne muscular dystrophy has not been established. In Becker muscular dystrophy, the pattern of muscle wasting closely resembles that seen in Duchenne dystrophy. The natural history of the illness permits distinction between Duchenne and Becker dystrophies. The majority of patients with Becker dystrophy initially experience difficulties between ages 5 and 15 years, although an onset in the 3rd or 4th decade, or even later, can occur. By definition, patients with Becker dystrophy ambulate beyond age 15 years, allowing clinical distinction from patients with Duchenne dystrophy. Patients with Becker dystrophy have a reduced life expectancy, but the majority of patients survive at least into the 4th or 5th decade. The preceding discussion implies a clear distinction between patients with Duchenne and Becker dystrophies, but a great heterogeneity of clinical presentation and course of illness can be recognized. A well-recognized subgroup of patients with an intermediate course between those typical of Duchenne and Becker dystrophies are referred to as outliers.21 These patients can be recognized usually by age 3 years by relative preservation of strength of neck flexion (antigravity neck flexor muscles), whereas patients with Duchenne dystrophy lack this ability throughout their entire life. Outliers also retain the ability to climb stairs and walk (after age 12 years but not beyond age 15 years) longer than patients with typical Duchenne muscular dystrophy. Other phenotypes of dystrophin deficiency have also been recognized, adding to the heterogeneity of the clinical dystrophinopathies. A condition with myalgias and myoglobinuria without persistent weakness has been described.22 Even a cardiomyopathy with few or no skeletal muscle signs can occur. Perhaps the future will hold observations indicating a selective deficiency of the brain dystrophin isoforms, accounting for some types of mental retardation. Molecular Genetics Duchenne muscular dystrophy is the most common X- linked recessive lethal disease, with an incidence of approximately one in 3500 newborns; it has been estimated that one third of the cases are the result of new mutations. The dystrophin gene, which causes Duchenne muscular dystrophy and its milder variants, is very large, spans more than 2000 kb of genomic DNA, and is composed of 79 exons that encode a 14-kb transcript. 2,7 Perhaps the enormous gene, which presents a large target for mutations, accounts for the high frequency of spontaneous mutations. Dystrophin is found in skeletal, smooth, and cardiac muscle, as well as in brain. 23 Immunohistochemistry demonstrates the localization of dystrophin to the cytoplasmic face of the sarcolemma and at postsynaptic membrane specializations on neurons. As already indicated, dystrophin binds strongly to the glycoprotein complex and provides muscle membrane stability.&dquo; In general, patients with Duchenne muscular dystrophy have little or no detectable dystrophin (Figure 2), whereas those with Becker muscular dystrophy have dystrophin of altered size or quantity.g S Approximately 65% of Duchenne and Becker dystrophy cases demonstrate large-scale deletions (of several kilobases to greater than one million base pairs) in the dystrophin gene. 24,25 Duplications are found in approximately 5% of cases. 26 The large gene size, particularly the introns, which average 35 kb, may account for part of the high deletion rate. The deletions are nonrandomly distributed and occur primarily in the center (80%) and less frequently near the 5 end (20%) of the gene. The larger deletions usually begin at the 5 end of the gene. Deletions disrupting the open reading frame result in the more severe Duchenne phenotype in the majority of cases,27 whereas in the milder Becker dystrophy, the deletion maintains the translational reading frame, and a semifunctional, truncated protein is produced. The readingframe hypothesis explains the phenotypic differences observed in about 92% of the Duchenne and Becker dystrophy cases. A major exception to the reading-frame rule has been the identification of Becker patients with out-offrame deletions affecting exons 3 through 7.28 To account for the milder phenotype observed with exon 3 through 7 deletions, it has been proposed that an alternate splicing mechanism or new cryptic translational start sites account for the production of dystrophin.29 There are now several reports of small mutations (point mutations and small deletions and duplications) detected in the dystrophin gene in Duchenne muscular dystrophy patients The majority of these point mutations have resulted in dystrophin truncation, consistent with the reading-frame hypothesis. However, unlike the deletion hot

4 153 Figure 2. Immune staining of the sarcolemma using antibody to the carboxy terminus of dystrophin. A, The sarcolemma is well defined by dystrophin localization to the membrane of normal muscle. (Original magnification, x 190.) B, Dystrophin staining is absent from muscle of Duchenne muscular dystrophy patient except for a single revertant fiber. (Original magnification, x 190.) spots, the small mutations are randomly distributed throughout the gene and unique to individual patients. With the ability to perform direct DNA diagnostics on the deletion and duplication cases, the accuracy of carrier detection has significantly improved. Nevertheless, the carrier state of the mother of an isolated case must be interpreted very cautiously from DNA testing. Even when the mother has no detectable mutation of the dystrophin gene, the risk of carrier status still has not been excluded, owing to the possibility of germline mosaicism In mothers not harboring mutations of the dystrophin gene in peripheral blood leukocytes, a mutation can be present in a percentage of the oocytes. Such examples of germline mosaicism have important counseling implications. The sisters of Duchenne patients should be investigated independently of the outcome of DNA testing of the mother. Furthermore, negative mutation results in the mother do not rule out a recurrence risk for future pregnancies. The exact recurrence risk in germline carriers is unknown because there is no method to estimate the size of the mutant clone in the mosaic mother. Recurrence risk for Duchenne muscular dystrophy in the mother of a sporadic case has been estimated to be as high as 14%. 42 In Duchenne muscular dystrophy families with undefined mutations, carrier detection and prenatal diagnosis will depend on linkage using restriction fragment length polymorphisms This method relies on the co-inheritance of the disease gene with DNA sequence variations known to be located close to the disease gene. Thus, even when the responsible gene mutation remains unknown, the restriction fragment length polymorphism technique allows one to trace the mutation through an affected family and make predictions about the inheritance of the disorder.46,47 The indirect restriction fragment length polymorphism approach can provide valuable information but has limitations. The intragenic recombination rate over the entire length of the dystrophin gene is estimated to be as high as 12%.48 The high recombinational error rate can be partially overcome by using polymorphisms at both ends of the gene. Restriction fragment length polymorphism results should be used cautiously for extended family members of sporadic cases because the proband may represent a new mutation carried only on his X chromosome. Unavailability of DNA from a deceased male or other key family members often makes restriction fragment length polymorphism studies inconclusive. Microsatellite sequences that correspond to short tandem repeats (dinucleotides, trinucleotides, or tetranucleotides), tend to be highly polymorphic, have been found in several locations in the dystrophin gene, and have significantly improved linkage analysis The variability of allele lengths at these loci makes them ideal for carrier detection and prenatal diagnosis, often providing information when the previous restriction fragment length polymorphism test was uninformative. In addition, these loci are of great diagnostic utility because they are easily assayed by polymerase chain reaction, thus reducing the time of analysis and cost. Molecular Pathogenesis of the Dystrophinopathies In Duchenne muscular dystrophy, the absence of dystrophin leads to a drastic reduction in all of the dystrophin-associated proteins.52 (Figure 3). This is a direct consequence of dystrophin deficiency and not a secondary effect of muscle degeneration. In dystrophin deficiency of skeletal muscle, components of the glycoprotein complex are normally synthesized, but either their assembly or integration into the sarcolemma is affected or there is accelerated degradation. Based on these observations, it is proposed that disruption of the dystrophin-glycoprotein complex plays a key role in the cascade of events leading to muscle cell necrosis in Duchenne muscular dystrophy.52 The absence of dystrophin causes a disruption of the linkage between the subsarcolemmal cytoskeleton and the extracellular matrix, leading to sarcolemmal instability, membrane tears, and eventual muscle cell necrosis.

5 154 Support for the delicate relationship within the components of the dystrophin-glycoprotein complex is further provided by the study of patients lacking the carboxyl-terminal domains of dystrophin. In some patients, immunohistochemistry reveals a drastic reduction in the components of glycoprotein complex even though dystrophin is properly localized to the sarcolemmal region.53 These results support the model indicating that the missing dystroglycan binding site at the carboxyl terminus of dystrophin is essential for membrane stability. The clinical pathological correlation with dystrophin-associated protein and glycoprotein localization also extends to patients with Becker muscular dystrophy. In patients with in-frame deletions in the amino terminal and rod domains of dystrophin, the reductions in the dystrophin-associated proteins and glycoproteins is usually milder than in patients with typical Duchenne dystrophy. This indicates that the rod domain may not be essential for the interactions with the glycoprotein complex. Nevertheless, mutations at the amino-terminal domain of dystrophin may affect anchorage of the dystrophin-glycoprotein complex to the subsarcolemmal actin cytoskeleton, resulting in an increased susceptibility for muscle fibers to undergo degeneration. 54 Rarely, a severe phenotype results from in-frame mutations at the amino terminal end of dystrophin. For example, we recently identified a single amino acid substitution in the actin binding domain resulting in a Duchenne phenotype.4 The severity may have been the result of an effect on protein conformation or membrane instability. In any case, the findings reinforce the functional importance of an intact actin-binding domain. SEVERE CHILDHOOD AUTOSOMAL RECESSIVE MUSCULAR DYSTROPHY A form of severe childhood muscular dystrophy, phenotypically very similar to Duchenne and Becker muscular dystrophies but inherited as an autosomal recessive trait has been referred to as severe childhood autosomal recessive muscular dystrophy. The condition affects boys and girls with equal penetrance. Deficiency of adhalin has been implicated in the pathogenesis of this condition. 4 Clinical Features Severe childhood autosomal recessive muscular dystrophy has clinical features overlapping with Duchenne and Becker muscular dystrophies.4,55-5s The age of onset varies Figure 3. Immune staining using specific antibodies to components of the dystrophinglycoprotein complex in a muscle biopsy from a patient with Duchenne muscular dystrophy. There is marked reduction of all components including (A) dystrophin, (B) a-dystroglycan, (C) 59-kd dystrophin-associated protein (syntrophin), (D) adhalin, (E) (3-dystroglycan, and (F) 35-kd dystrophin-associated glycoprotein. (Reprinted with permission from Matsumura K, Campbell KP: Dystrophin-glycoprotein complex: Its role in the molecular pathogenesis of muscular dystrophies. Muscle Nerve 1994;17:2-15.)

6 155 from 3 to 12 years. Truncal and proximal limb weakness is prominent, with sparing of cranial nerve musculature. The disorder is slowly progressive, with loss of ambulation between 10 and 25 years of age. Calf hypertrophy is common. Cardiac muscle can also be involved, leading to a clinically significant cardiomyopathy. The clinical phenotype has varied from a severe Duchenne dystrophy-like disorder to milder phenotypes typical of outliers or patients with Becker muscular dystrophy.4 Laboratory features are also consistent with the dystrophinopathies. Serum creatine kinase elevation is 20 to 50 times the normal upper limit. Muscle biopsy features are similar to those in Duchenne muscular dystrophy. There is marked variability in muscle fiber size, scattered necrotic and regenerating fibers, hypercontracted fibers, and connective tissue proliferation (Figure 4). Molecular Genetics A deficiency of adhalin has been demonstrated in patients with severe childhood autosomal recessive muscular dystrophy in the Arab countries of North Africa4 and in non- Arab populations including European,55 South Asian,57 and Brazilian.58 In addition, we recently identified a South African girl with severe childhood autosomal recessive muscular dystrophy and adhalin deficiency who had no familial links to North Africa. Confusion arises because the severe childhood autosomal recessive muscular dystrophy phenotype in North African families shows linkage to chromosome 13ql2,~ ~ whereas the adhalin gene has been mapped to chromosome 17q s1,s2 Recently, missense mutations in the adhalin gene were demonstrated in a large French family with severe childhood autosomal recessive muscular dystrophy, lending support for a specific mutation of the adhalin gene as causative for some cases. 62 However, the 13q-linked gene responsible for the disease observed in the Arab countries of North Africa remains to be elucidated. Figure 4. Photomicrograph of quadriceps muscle biopsy from patient with adhalin deficiency. Notice the striking variability in fiber size, increased number of internal nuclei, connective tissue proliferation, and hypercontracted fibers (arrows). (Original magnification, x 175.) Molecular Pathogenesis Adhalin deficiency can be demonstrated in both Duchenne muscular dystrophy and severe childhood autosomal recessive muscular dystrophy.4 In Duchenne dystrophy, the absence of dystrophin causes a secondary reduction in all dystrophin-associated proteins (Figure 3). In severe childhood autosomal recessive muscular dystrophy, adhalin is preferentially lost early in the disease (Figure 5), but other components of the dystrophin-glycoprotein complex are also lost in the later stages. These findings emphasize a common pathogenesis of muscle necrosis in both Duchenne and severe childhood autosomal recessive muscular dystrophies related to disruption of the dystrophin-glycoprotein complex. CONGENITAL MUSCULAR DYSTROPHY WITH MEROSIN DEFICIENCY Recent findings of a form of congenital muscular dystrophy caused by deficiency of laminin M (or merosin)5 demonstrate that sarcolemmal instability also results from defects in structural proteins of the basal lamina. Clinical Features As a group, congenital muscular dystrophies present at birth or in the first few months of life. Patients can be divided into those with only skeletal muscle disease and those with both central nervous system and skeletal muscle involvement.63 The muscle manifestations include hypotonia, proximal limb weakness, and joint contractures affecting elbows, hips, knees, and ankles. The contractures of congenital muscular dystrophy, referred to as arthrogryposis when present at birth, set this disease apart from other childhood dystrophies. Congenital hip dislocation may be present in occasional patients. Weakness of facial muscles may occur, but other cranial nerve musculature is spared. The finding of full extraocular motility differentiates congenital muscular dystrophy from centronuclear myopathy. The severity of congenital muscular dystrophy varies greatly. Approximately one half remain severely disabled, never achieving the ability to stand independently. Rarely, patients die of respiratory insufficiency during the first few years of life. Some patients exhibit delayed motor milestones but learn to walk, although difficulty in running and stair climbing persist. The best-characterized form of congenital muscular dystrophy with cerebral involvement is Fukuyama congenital muscular dystrophy. 64,65 This well-defined form of congenital muscular dystrophy is predominantly found in Japan, occurring with a frequency of seven to 12 per 100,000. Functional disability is severe in these patients; usually the maximum level of motor function achieved is crawling, and most patients with Fukuyama congenital muscular dystrophy never learn to walk. Patients usually become bedridden before 10 years of age, and most die by 20 years of age. Severe mental retardation is observed in all cases. IQ scores in most patients with Fukuyama congenital muscular dystrophy lie between 30 and 50. Seizures are common. The most characteristic change in

7 156 Figure 5. Immune staining using specific antibodies to components of the dystrophin-glycoprotein complex in a patient with adhalin deficiency. Immune staining is shown for (A) dystrophin, (B) a-dystroglycan, (C) 59-kd dystrophin-associated protein, and (D) adhalin. The adhalin is drastically reduced. (Reprinted with permission from Matsumura K, Campbell KP: Dystrophin-glycoprotein complex: Its role in the molecular pathogenesis of muscular dystrophies. Muscle Nerve 1994;17:2-15.) the central nervous system is micropolygyria of the cerebrum and cerebellum due to defects in migration of neurons. Hydrocephalus, focal interhemispheric fusion, and hypoplasia of the corticospinal tracts are also observed. Congenital muscular dystrophy in association with cerebro-ocular dysplasia represents another variant. 66,67 This disorder combines the features of Fukuyama congenital muscular dystrophy and cerebro-ocular dysplasia of Walker 61 (also called Warburg s syndrome 69). The clinical neuromuscular features parallel Fukuyama congenital muscular dystrophy, but patients with cerebro-ocular dysplasia-muscular dystrophy also have corneal abnormalities, cataracts, immature anterior chamber angle, ciliary body abnormalities, retinal dysplasia with or without retinal detachment, abnormal retinal pigment epithelium, aberrant retinal vascularization, and hypoplasia of the optic nerve. Hydrocephalus and macrocephaly are more common in cerebro-ocular dysplasia-muscular dystrophy than in Fukuyama congenital muscular dystrophy. The final variant with central nervous system involvement, congenital muscular dystrophy with hypomyelination, shows the neuromuscular features of other congenital muscular dystrophies combined with varying degrees of hypomyelination of the cerebral white matter demonstrable by computed tomography or magnetic resonance imaging.7 The low-density areas may be diffuse and symmetric or focal; the hypodensity may diminish or disappear with time, suggesting a delay in myelination. In contrast to Fukuyama congenital muscular dystrophy and cerebro-ocular dysplasia-muscular dystrophy, patients with congenital muscular dystrophy with hypomyelination have no apparent clinical signs of central nervous system disease except for delayed verbal development. Neither the ocular manifestations of cerebro-ocular dysplasiamuscular dystrophy nor the severe mental retardation of Fukuyama congenital muscular dystrophy is present. The congenital muscular dystrophies with and without cerebral involvement share similar serum creatine kinase, electromyographic, and muscle biopsy findings. The serum creatine kinase level ranges from normal up to elevations of 4 to 5 times, although we have observed cases with levels increased 30-fold. The electromyogram shows a myopathic pattern. Nonspecific muscle biopsy features include increased variability in muscle fiber size with greater numbers of central nuclei, loss of muscle fibers, scattered muscle fibers undergoing degeneration and regeneration, and prominent fat and connective tissue infiltration (Figure 6). Molecular Genetics Recent studies indicate that non-japanese congenital muscular dystrophy associated with central hypomyelination has a specific absence of the laminin M chain (merosin). 5 In normal human skeletal muscle, immunohistochemical studies show uniform labeling of merosin around each muscle fiber (Figure 6). In contrast, patients with merosin deficiency can be identified by western blot analysis and immunohistochemistry of muscle biopsy material (Figure 6). The specific gene defect causing merosin deficiency has not been identified, although the merosin gene localizes to chromosome 6q and will obviously be considered a candidate gene for causing this disease.

8 157 Merosin deficiency provides a further example of a childhood muscular dystrophy caused by disrupting the link between the subsarcolemmal cytoskeleton and the extracellular matrix resulting in muscle fiber changes. Additional studies are required to elucidate the relationship of the central hypomyelination to merosin deficiency. In Fukuyama congenital muscular dystrophy, a partial deficiency of merosin has also been demonstrated by immunohistochemistry; abnormalities, however, are not exclusive to merosin and involve other laminin subunits, including laminin Bl and B2.73 Of interest, Matsumura et al demonstrated reduced immunohistochemical staining of 43-kd dystrophin-associated glycoprotein in Fukuyama congenital muscular dystrophy74; localization of 43-kd dystrophin-associated glycoprotein to chromosome 3p21 also excludes this protein as a candidate for Fukuyama congenital muscular dystrophy. ANIMAL MODELS FOR CHILDHOOD MUSCULAR DYSTROPHIES Figure 6. Picture of quadriceps muscle biopsy from patient with congenital muscular dystrophy and merosin deficiency. A, There is very severe muscle fiber loss and marked connective tissue proliferation. Only islands of small muscle fibers have survived. (Hematoxylin and eosin stain; original magnification, x 230.) 8, Immune staining of muscle biopsy from congenital muscular dystrophy patient demonstrating absence of merosin. (Original magnification, x 400.) C, Immune staining with antibody to merosin in muscle biopsy from adult patient showing merosin localized to membrane of muscle fibers. (Original magnification, x 190.) Fukuyama congenital muscular dystrophy is inherited as an autosomal recessive trait. Recent linkage studies demonstrate localization of the gene for Fukuyama congenital muscular dystrophy to chromosome 9q The specific gene causing Fukuyama congenital muscular dystrophy has not been identified, but markers have been identified that are useful for presymptomatic, prenatal, and carrier diagnosis of family members. The chromosome 9 linkage excludes merosin as a candidate for causing this disease. Cerebro-ocular dysplasia-muscular dystrophy also appears to be autosomal recessive but no chromosomal linkage has been established. Molecular Pathogenesis The finding of merosin deficiency appears to be specific for congenital muscular dystrophy with hypomyelination. Three animal models have been identified demonstrating defects in specific proteins linking the subsarcolemmal cytoskeleton to the extracellular matrix. In the mdx mouse, the defect closely parallels the dystrophinopathies. The defect in the gene encoding dystrophin is well characterized.75 In addition to absence of dystrophin on the sarcolemma, the other components of the dystrophin-glycoprotein complex are markedly reduced, similar to what is observed in Duchenne dystrophy.76 The mdx mouse provides a useful system for testing therapeutic strategies, particularly for myoblast transfer and gene therapy. In the autosomal recessive cardiomyopathic hamster (BI014-6), a specific deficiency of the 50-kd dystrophinassociated glycoprotein occurs in skeletal muscles. 75 In the cardiac muscle, the 50-kd dystrophin-associated glycoprotein deficiency is accompanied by a decreased abundance of all dystrophin-associated proteins. The similarity between the 50-kd dystrophin-associated glycoprotein deficiency in the skeletal muscle of the cardiomyopathic hamster and in severe childhood autosomal recessive muscular dystrophy suggests that these two conditions may share the same pathogenesis and a defect within the same gene. Patients with severe childhood autosomal recessive muscular dystrophy also develop a cardiomyopathy.54 Work is underway to establish whether the gene encoding 50-kd dystrophin-associated glycoprotein is abnormal in the hamster and patients with severe childhood autosomal recessive muscular dystrophy. Potentially, the cardiomyopathic hamster will be important as a symptomatic animal model for testing gene therapies for severe childhood autosomal recessive muscular dystrophy and, perhaps, human cardiomyopathies. Finally, merosin has been found deficient in the dyldy mouse,78 a fatal murine muscular dystrophy inherited as an autosomal recessive trait. The dyldy mouse shows morphologic similarities to congenital muscular

9 158 dystrophy and has dysmyelination in the proximal part of the sciatic nerve and ventral and dorsal spinal roots. The dysmyelination of nerve has significance in light of the hypomyelination found in the non-japanese congenital muscular dystrophy patients. Further work needs to be done to clearly identify the gene defect in the dy/dy mouse to establish it as a definite model for a form of congenital muscular dystrophy. Acknowledgment Supported by a grant-in-aid from the Muscular Dystrophy Association. References 1. Mokri B, Engel AG: Duchenne dystrophy: Electron microscopic findings in pointing to a basic or early abnormality in the plasma membrane of the muscle fiber. Neurology 1975;25: Koenig M, Hoffman EP, Bertelson CJ, et al: Complete cloning of the Duchenne muscular dystrophy (DMD) cdna and preliminary genomic organization of the DMD gene in normal and affected individuals. Cell 1987;50: Koenig M, Monaco AP, Kunkel LM: The complete sequence of dystrophin predicts a rod-shaped cytoskeletal protein. Cell 1988;53: Matsumura K, Tome FMS, Collin H, et al: Deficiency of the 50K dystrophin-associated glycoprotein in severe childhood autosomal recessive muscular dystrophy. Nature 1992;359: Tome FMS, Evangelista T, Leclerc A, et al: Congenital muscular dystrophy with merosin deficiency. C R Acad Sci III 1994;317: Matsumura K, Campbell KP: Dystrophin-glycoprotein complex: Its role in the molecular pathogenesis of muscular dystrophies. Muscle Nerve 1994;17: Hoffman EP, Brown RH, Kunkel LM: Dystrophin: The protein product of the Duchenne muscular dystrophy locus. Cell 1987;51: Hoffman EP, Fischbeck K, Brown RH, et al: Dystrophin characterization in muscle biopsies from Duchenne and Becker muscular dystrophy patients. N Engl J Med 1988;318: Ohlendieck K, Campbell KP: Dystrophin constitutes 5% of membrane cytoskeleton in skeletal muscle. 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