Limb-girdle Muscular Dystrophies

Pathophysiology

Dystrophin-associated proteins

Dystrophin, a large molecule located under the sarcolemma of myocytes, is mutated in DMD and BMD as described in Chapter 30 . Its N-terminus and rod domain interact with the cytoskeleton while its C-terminus interacts with several intracellular and transmembrane proteins, known as the dystrophin-associated proteins (DAPs) ( Figure 34.1 ).

Intracellular DAPs

The intracellular DAPs include the dystrobrevins and the syntrophins. Dystrobrevins share a similar structure to the C-terminal domain of dystrophin, with α-helical coiled-coils mediating their association. In addition, dystrobrevins interact with the sarcoglycan complex and neuronal nitric oxide synthase (nNOS). Both dystrophins and dystrobrevins bind syntrophins. To date, mutations in dystrobrevins or syntrophins have not been associated with muscle disease in humans. However, α-dystrobrevin deficient mice show evidence of a muscular dystrophy.

Transmembrane DAPs: Dystroglycan complex

The dystroglycan complex includes two proteins: β-dystroglycan, a transmembrane protein, binds the C-terminal region of dystrophin intracellularly and interacts with α-dystroglycan extracellularly. α-Dystroglycan, a heavily glycosylated protein, connects the dystrophin-β-dystroglycan axis of proteins to the G domain laminin α2, the heavy chain of laminin 211 (merosin). Laminin α2 ( LAMA2 ) mutations are one of the most common causes of congenital muscular dystrophy (see Chapter 29 ). In essence, the dystroglycan complex structurally links the intracellular dystrophin-cytoskeleton complex to the extracellular matrix. Mutations in dystroglycan itself are extremely rare, as they are usually incompatible with development. However, abnormal glycosylation of α-dystroglycan caused by mutations in a series of proven and putative glycosyltransferases, as well as cooperating components, can cause congenital muscular dystrophy or autosomal recessive LGMD, specifically LGMD2I (see the following).

Transmembrane DAPs: Sarcoglycan complex

The sarcoglycan complex in skeletal muscle includes four proteins: α-, β- , γ-, and δ-sarcoglycan. α-Sarcoglycan (50kDa) and γ-sarcoglycan (35kDa) are encoded on chromosome 17q21 and 13q12, respectively, and are almost exclusively expressed in skeletal muscle, though γ- and δ-sarcoglycan are expressed in smooth muscle as well. β-Sarcoglycan (43kDa) and δ-sarcoglycan (35kDa) are encoded on chromosome 4q12 and 5q33, respectively, and have more widespread expression.

Two additional widely expressed sarcoglycan-like molecules, ε- and ζ-sarcoglycan, have also been identified. While no human diseases are linked to ζ-sarcoglycan mutations, some patients with an autosomal dominant myoclonus-dystonia syndrome carry heterozygous ε-sarcoglycan mutations.

The exact nature of the association of the sarcoglycan complex with dystrophin is not known; however, associations with dystroglycans, synaptobrevin, dystrobrevin, and nNOS have been shown. γ-Filamin/filamin C, an actin binding protein, is an intracellular molecule that is known to interact with the sarcoglycan complex, an association that theoretically provides an additional point of interaction with the cytoskeleton. Biglycan provides an extracellular link between the sarcoglycan complex and dystroglycan. Recently, muscle specific aquaporin-4, a water channel, was shown to interact with the sarcoglycan complex via α1-syntrophin. Sarcospan, which is severely reduced in sarcoglycanopathies, is another protein that is intimately associated with the sarcoglycan complex. Sarcospan mutations are not linked to muscle disease, however, and its function remains elusive. These complex associations likely stabilize the DAPs and provide a structural and functional link to intracellular signals through different pathways, but the exact relevance of these interactions to muscle disease is yet to be identified.

Genetics and Mutations

Mutations in all four major sarcoglycan genes cause four genetically separate forms of autosomal recessive LGMD with almost indistinguishable clinical phenotypes. Although all four disorders appear to have a worldwide distribution, there are certain regional differences owing primarily to founder mutations (e.g. a high proportion of γ-sarcoglycanopathy in North Africa and the Mediterranean because of a founder mutation). Of the sarcoglycan mutations, α-sarcoglycanopathies are likely the most common type in Europe, North America, and Brazil, accounting for more than 50% of genetically proven sarcoglycanopathies. These are followed in varying proportion by β- and γ-sarcoglycan mutations, with δ-sarcoglycan as the least commonly mutated sarcoglycan in most series.

In α- and β-sarcoglycanopathy, in particular, there are high proportions of missense mutations, the majority of which are located in the respective extracellular domains. Approximately one-third of the α-sarcoglycan mutations map to the cadherin-like domains, suggesting a role for calcium-mediated protein interactions in the pathogenesis process. Among them, the single mutation Arg77Cys is the most frequent. Many other mutations are also identified, however, and genotype/phenotype correlations are difficult to evaluate because mutations often occur in compound heterozygous states. Even with homozygous mutations, there can be considerable phenotypic variability. Truncating or nonsense mutations likely give rise to a more severe phenotype, whereas missense mutations are much more variable.

Four β-sarcoglycan missense and nonsense mutations are known to occur. In the Indiana Amish, there is a founder mutation, Thr151Arg , associated with a variable phenotype. There is a relative clustering of mutations in the immediately extracellular domain, encoded on exon 3 of the gene. Similar to α-sarcoglycan, nonsense or truncating mutations lead to a more severe phenotype, but a higher proportion of missense mutations in β-sarcoglycan cause a phenotype at least as severe, particularly when the mutations are predicted to disrupt the secondary structure in the domain immediately following the transmembrane stretch. This may reflect the central position of β-sarcoglycan within the complex.

The most common mutation in γ-sarcoglycan is the frame-shifting deletion of a single thymidine, del521T , which originated in North Africa as a founder mutation and is readily found in Mediterranean countries or migrants from that area. As a result, γ-sarcoglycanopathy is the most common form of sarcoglycanopathy in these countries. Phenotypes may vary significantly even within the same families. Another founder mutation, Cys283Tyr , commonly found in the Romany (Gypsy) population in Europe, results in a consistently severe phenotype despite being a missense mutation.

Mutations in δ-sarcoglycan are the least common overall but are found commonly in Brazil, where the disorder was originally described. A single nucleotide deletion, nonsense mutations, and missense mutations have all been reported with rather severe phenotypes. Dilated cardiomyopathy has been seen in some families with heterozygous mutations in the δ-sarcoglycan gene, with or without skeletal muscle weakness. It has been hypothesized that these mutations may act in a dominant-negative way on the sarcoglycan complex as a whole, interfering with its function in cardiomyocytes.

Most milder sarcoglycanopathies are related to α-sarcoglycan mutations, including almost asymptomatic patients with high CK levels and lordosis only. β- and γ-sarcoglycan mutations have a higher proportion of severe early childhood cases though significant intrafamilial variability can be seen. δ-Sarcoglycan mutations are generally more severe.

Clinical Features

Clinical features of the four genetically distinct sarcoglycanopathies overlap significantly, with little difference among the different types. They predominantly affect young children with a median age of onset around 6 to 8 years with a broad range from even younger onset to adult onset disease or almost asymptomatic mutation carriers. Early motor milestones are usually normal, but there may be mild delay or toe walking in some children. Some children are slower than their peers in physical activities. (See Case Example 34.1 .)

Time course and distribution of motor symptoms

The first symptoms generally relate to pelvic muscle weakness, evidenced by a waddling gait, which limits activities such as running, getting up from the floor, or climbing stairs. Primary toe walking may be present in some children. Muscle cramps, pain, and exercise intolerance with or without myoglobinuria can also occur, the so-called “pseudometabolic presentation.” The distribution of weakness is reminiscent of dystrophinopathies: the glutei and adductors are more involved than the quadriceps, sartorius, and gracilis. However, unlike dystrophinopathies, anterior and posterior compartments of the thigh may be equally affected. Shoulder girdle weakness follows. The deltoid, infraspinatus, and biceps muscles are involved early in the disease. Scapular weakness tends to be more pronounced compared to dystrophinopathies ( Figure 34.2 ). Facial and extraocular muscles are spared. Late in the disease, distal muscles may be involved, starting in the anterior tibial compartment. Very mild facial involvement with a “transverse smile” may also be seen. In later stages—and similar to DMD—neck flexor weakness may occur. The weakness is quite rapidly progressive, with loss of strength towards the end of the first decade similar to DMD in the early onset cases, although there is more clinical variability compared to DMD. Loss of independent ambulation may occur around 12 to 16 years of age, although there again is considerable variability. CK levels are elevated 10- to 100-fold early in the course of the disease, but tend to decrease as weakness progresses.

Diagnosis

Sarcoglycanopathies can be suspected based on clinical grounds, in particular in a young patient with muscular dystrophy of Duchenne-like severity. The probability of an autosomal recessive LGMD in boys with a Duchenne-like phenotype is 6% to 8%. However, this probability obviously increases if the patient is a girl, or a boy with negative genetic studies of the dystrophin gene.

Definitive histological diagnosis requires examination of the muscle biopsy specimen, including immunohistochemical stains with antibodies against sarcoglycans and dystrophin. Histology usually shows marked degeneration and regeneration and severely dystrophic muscle. Dystrophin immunoreactivity is expected to be normal, though it can be reduced similarly to BMD or female carriers of dystrophin mutation. Western blot analysis of muscle usually shows dystrophin with a normal molecular weight and quantities within 10% of normal.

The pattern of in situ sarcoglycan immunoreactivity in sarcoglycanopathies is rather complex. The entire sarcoglycan complex tends to be affected as a unit, but in some cases the pattern of immunoreactivity of some components may appear more normal. For example, in α- and γ-sarcoglycanopathy, immunoreactivity of mutated proteins is reduced while all others may be preserved or only mildly affected. In contrast, in β- and δ-sarcoglycanopathies, the entire complex is severely reduced or completely absent. Thus, these patterns can be diagnostically helpful ( Figure 34.3 ).

Residual expression of α-sarcoglycan in some cases of missense mutations may correlate with milder clinical phenotypes, but this is not always true. For example, even complete absence of the protein has been reported with mild phenotypes in γ-sarcoglycanopathy.

Nevertheless, immunohistochemical stains can guide molecular genetic testing for the absent proteins in question in a stepwise manner.

Treatment

No specific therapies are available for sarcoglycanopathies, but unlike dystrophinopathies, sarcoglycanopathies are more amenable to gene therapy owing to the fact that cDNA for sarcoglycans are small enough to be suitable for package in viral vectors and delivery. Feasibility of local delivery of adeno-associated virus containing the α-sarcoglycan gene, with long-lasting expression of the wild-type gene without adverse effects, has been shown. Vascular delivery of the vector with systemic delivery remains an area of active investigation. Similarly, a phase I trial of adeno-associated virus γ-sarcoglycan gene therapy was recently conducted with promising proof of concept, though its widespread clinical use awaits larger studies and longer follow-up.

Anecdotal reports advocate the use of steroids in sarcoglycanopathies similar to that in DMD. However, no controlled studies have examined these effects systematically to date. Nifedipine was used in an animal model of δ-sarcoglycan deficient mice, and showed a positive effect on the course of cardiomyopathy attributed to coronary smooth muscle pathology, but the use of such agents in humans is not yet reported. Principles of respiratory and orthopedic management are similar to those for DMD (see Chapter 31, Chapter 44, Chapter 52, Chapter 53 ).

Pathophysiology and Genetics

This form, designated as LGMD 2I, was first identified and mapped in Tunisia to chromosome 19q13.3. FKRP was later identified based on its homology to fukutin, the protein deficient in Fukuyama congenital muscular dystrophy. It is thought to be involved in glycosylation of α-dystroglycan, in particular of its LARGE glycol-epitope, which is variably reduced in muscle by immunohistochemistry and Western blot in patients with LGMD 2I. These findings are also replicated in the mouse model of this disease.

The mutations identified so far include missense as well as null mutations. Most mutations are contained in exon 4; as a result, mutation analysis is not complicated. A missense mutation, Leu276Ile , is seen in almost all LGMD patients and results in a relatively milder phenotype in the homozygous state, compared to the compound heterozygous state with other, more severe mutations.

Clinical Features

The LGMD phenotype of FKRP mutations can present as a DMD phenocopy, with early loss of ambulation (before 10 years of age in some cases). The pattern of muscle weakness is very similar to DMD, showing a pronounced predilection for axial muscles, neck flexors, and proximal limb muscles with a prominent lordosis. In some patients, shoulder girdle muscles may be weaker than those of the pelvic girdle, with atrophy of the deltoid and pectoralis muscles. There can be tongue, calf, and brachioradialis hypertrophy. The hamstrings are generally more involved than the quadriceps. Mild facial weakness can also be seen. Scapular winging is uncommon. There is significant variability; patients homozygous for Leu276Ile may have a milder phenotype more similar to BMD, and there are adult-onset forms of the disorder. CK levels are high, 10 to 30 times the upper limit of normal.

Clinically significant dilated cardiomyopathy can occur even before loss of ambulation, while limb girdle weakness may still be relatively mild, and in occasional patients can be rapidly progressive. Respiratory failure necessitating nocturnal ventilatory support, on the other hand, typically manifests after loss of ambulation and should be evaluated and treated appropriately.

Diagnosis

LGMD 2I may be suspected on clinical grounds, based on features such as the peculiar hypertrophy of brachioradialis and tongue in some patients. Muscle biopsy immunohistochemical analysis usually shows prominently reduced α-dystroglycans and a mild reduction of laminin α2. While these are not specific, they can be helpful in prompting genetic analysis of FKRP gene in the right clinical setting.

Treatment

No specific treatments are available at this time. Similar to other muscular dystrophies causing cardiomyopathy and respiratory failure, the recognition, evaluation, and treatment of dilated cardiomyopathy and potential need for nocturnal ventilatory support is of utmost importance.

Background and Epidemiology

Calpainopathy, or LGMD 2A, one of the most common LGMDs, was the first autosomal recessive LGMD localized by linkage analysis. The first clinical description of this juvenile onset muscular dystrophy can be attributed to Wilhelm Erb in 1880s. Genetic linkage analysis in families of the Reunion Island off the coast of Africa identified the locus on chromosome 15q15. LGMD 2A is likely the most common juvenile onset form of LGMD and may account for 40% to 50% of all cases of LGMD.

Pathophysiology, Genetics, and Mutations

CAPN3 , the gene mutated in LGMD 2A, encodes calpain-3, a calcium-activated protease that localizes to the cytoplasm and nuclei of the cells. It has a nuclear localization signal where it is thought to help in processing of transcription factors relevant to muscle. It has been thought to stabilize NF-kB, which activates anti-apoptotic genes, and inhibit its degradation. Presence of apoptotic cell death in muscle cells of patients with calpainopathy further supports this mechanism. In addition, calpain-3 interacts with titin, a large sarcomeric protein. Additional studies propose a role for calpain-3 in membrane repair via its interactions with the dysferlin complex.

A large number of mutations in calpain-3 have been identified. These include (more commonly) missense mutations and (less commonly) large deletions and truncations. In general, truncating mutations tend to be associated with more severe phenotypes, though there are no strong genotype/phenotype correlations as a whole. There are no specific hotspots for mutations in the gene.

Clinical Features

Calpainopathy has a characteristic clinical phenotype, although atypical presentations do occur. The age of onset is slightly later than sarcoglycanopathies, between 8 and 15 years of age (range 2 to 40 years). Early milestones are generally normal, but some children are reported to be weaker than peers in physical activities. Primary toe walking and delayed walking are also reported in some individuals. Exercise-induced myalgias or muscle stiffness may predate muscle weakness. See Case Example 34.2 .