MDA Special Research Update
#77a

LIMB-GIRDLE MUSCULAR DYSTROPH LIMB-GIRDLE MUSCULAR DYSTROPHIES (LGMDs) and otherY MDA-supported researchers are planning for clinical trials for the disorders.

The results of recent meetings of MDA-supported researchers and officials of the Food and Drug Administration (FDA) provided a clear indication that LGMD clinical trials will be underway shortly, particularly for those forms of LGMD associated with loss of the sarcoglycans known to cause specific forms of the disease.

The following information is presented to show the number of presently known forms of LGMD. (See chart at end of this report -- more are expected to be found.) It needs to be appreciated that LGMD is a very complex group of disorders, some for which the gene at fault is known and others for which the gene is yet to be determined. This complexity and variability is what researchers and the clinicians must contend with in the differential diagnosis of the different forms of LGMD.

As a result of a late-summer meeting of gene therapy experts at which the FDA was represented, plans are being formulated for a multicenter effort to begin to identify those whose LGMD is caused by a specific gene defect. This may require both a muscle biopsy and blood sample. This information will then be stored in a central database, to be retrieved when LGMD clinical trials begin. Those with that particular type of LGMD may be selected to participate in the clinical trials. Other factors relating to the disorder may be important in the selection process. Because of the small size of the sarcoglycan genes, permitting them to be easily inserted into a very efficient viral vector (adeno-associated vector), those with sarcoglycanopathy forms of LGMD will more than likely be the first selected for clinical trials.

It’s hoped that by presenting the most recently known classifications of LGMD identifying all the known forms, it may help to understand the complexity and variability of LGMDs and what may be necessary before a clinical trial can begin. Please visit the clinical trial section on the MDA Web site for continuos updates on clinical trials in LGMD.

LIMB-GIRDLE MUSCULAR DYSTROPHY / NEW FORM LIMB-GIRDLE MUSCULAR DYSTROPHIES (LGMDs) are characterized by progressive muscle weakness, muscle wasting and muscle-cellular changes as seen upon microscopic examination of biopsies. These microscopic changes include variation in muscle fiber size, degeneration of muscle myofibers, necrosis or destruction of myofibers, regeneration of muscle and fibrosis replacing necrotic tissue. Generally, it’s the shoulder and pelvic girdle muscle groups that are first affected. Some forms of LGMD are more progressive than others and, thus, may begin to affect other muscle groups. Facial muscles generally are spared. There is high variability between the different forms of LGMD in respect to age at onset, rate of progression, degree of muscle involvement, and clinical severity.

At least 12 distinct disease-causing chromosome locations (loci) for the different forms of LGMD have been reported by various research groups. In a study of a large Hutterite family, researchers found affected members with a mild form of autosomal recessive LGMD. The locus was mapped within the chromosomal region also linked to FUKUYAMA CONGENITAL MUSCULAR DYSTROPHY (FCMD), but was found to actually lie more distally along the chromosome, away from the FCMD locus. From these data, researchers strongly suggest yet another gene locus for LGMD, which the research team name LGMD2H. Researchers have mapped this form to chromosome 9q31-33.

Researchers report that more than 40 genes have been mapped to this locus, but none appears to be a convincing candidate. The gene Hexabrachion, which is centromeric to the region, has been detected in Duchenne muscular dystrophy and, since it may be implicated in this disorder, is still under consideration. Two different mouse phenotypes of muscular dystrophy map to the mouse chromosomal region corresponding to the region in human chromosomes (syntenic regions) for LGMD2H. (Weiler, T., et al. A gene for autosomal recessive limb-girdle muscular dystrophy in Manitoba Hutterites maps to chromosome region 9q31-q33: evidence for another limb-girdle muscular dystrophy locus. American Journal of Human Genetics. 63:140-147:1998

MIYOSHI MYOPATHY / LGMD2B / DISTAL MYOPATHY / GENE FOUND MIYOSHI MYOPATHY (MM) is an adult-onset, autosomally recessive type of DISTAL MUSCULAR DYSTROPHY. Researchers developed an artificial chromosome to span the segment of chromosome 2 that has been linked to both Miyoshi myopathy and LIMB-GIRDLE MUSCULAR DYSTROPHY TYPE 2B (LGMD2B). This development enabled the researchers to more definitively study the segment of DNA and they were able to find five new markers, which enabled the discovery of the gene causing MM, LGMD2B and a third muscular dystrophy disorder DISTAL MYOPATHY WITH ANTERIOR TIBIALIS MUSCLE ONSET. Researchers report this new gene as a cDNA of 6.9 kb in length and named the expressed protein DYSFERLIN. The designation for the human gene itself is DYSF. Mutations are described from nine families; five of the mutation types are predicted to prevent expression. What was very significant in this study, which helps explain the suspected allelism of Miyoshi myopathy and LGMD2B, was that the same mutation even within the same family could produce the different phenotypic disorders. In one family, one patient was reported with MM, while two sisters with the exact mutation showed LGMD2B phenotype. Another mutation produced MM in one family, while in another family it produced distal myopathy with anterior tibialis muscle onset. Many genetic studies have indicated the importance of genetic background and environmental factors on the expression of a given gene. This study strongly indicates this type of interaction. Further study of this gene and its effects should elucidate what some of the factors are that influence the expression of this mutated gene. Even though there is different muscle involvement seen in these different disorders, all of the patients show childhood or early adult onset and pronounced elevations of serum CK levels.

Genetic analysis of this gene shows it to have 6,243-bp that code for the protein dysferlin, which is believed to contain 2,080 amino acids. The protein shows some similarity to the protein fer-1 found in the well-studied flatworm, C. elegans. The study indicates that the protein may target for interaction with the sarcoplasmic reticulum, a membrane complex within the cell, but also may have some nuclear membrane targeting capacity as well. Further study is required in order to determine the protein’s normal function and what it’s doing when mutated to cause the disease.

Researchers point out that in muscular dystrophies other than myotonic, the disorders can be characterized in three basic categories: 1) those with loss of integrity of the muscle cell membrane as in Duchenne muscular dystrophy, 2) altered enzymatic function of calpains as in LGMD2A and 3) altered energy generation as in metabolic myopathies. However, researchers in this study have found the membrane proteins and energy generation to be normal in these patients with dysferlin gene mutations. Researchers suggest that these dysferlinopathies may represent a new, fourth category of muscular dystrophy. (Liu, J., et al. Dysferlin, a novel skeletal muscle gene, is mutated in Miyoshi myopathy and limb-girdle muscular dystrophy. Nature Genetics. 20:Sept. 1998.)

NEW ANIMAL MODEL LGMD 2C

In the study of human disease, animal models have been instrumental in the understanding of a particular disorder and the development of therapies to combat the disease process. Until only recently, researchers didn’t have the advantage of any animal model for LGMD. However, the cardiomyopathic hamster had the same genetic mutation as LGMD Type 2F in humans and was used as the animal model for this form. This animal model paved the way for the development of gene therapy for the sarcoglycanopathy forms of LGMD. Now researchers have announced a new mouse model for LGMD Type 2C that is very similar to the phenotype seen in the human disorder. Researchers have successfully developed a mouse without gamma-sarcoglycan gene expression, and the data from the study of this animal model suggest some interesting relationships that wouldn’t have been seen without it.

The sarcoglycans alpha-, beta- gamma-, delta- and epsilon- contribute, to one degree or another, to the stability of the muscle membrane for contraction by forming a complex with dystrophin, alpha- & beta-dystroglycans and laminin (referred to as the dystrophin-associated complex, DAC) to secure the muscle cell to the extracellular membrane. Recent studies have shown that mutations in any single sarcoglycan gene can result in variable secondary reduction of other sarcoglycans. Epsilon-sarcoglycan is expressed in non-muscle tissue as well as skeletal and cardiac muscle as are the other sarcoglycans.

Gamma-sarcoglycan is a transmembrane, dystrophin-associated protein expressed in skeletal and cardiac muscle. Researchers report that the mice they developed lacking gamma-sarcoglycan expression (gsg-/-) in muscle showed pronounced dystrophic muscle changes in early life. By 20 weeks of age (mouse life-span ~ 2 years), these mice develop cardiomyopathy and died prematurely. The loss of gamma-sarcoglycan expression also produced secondary reduction of beta-, and delta-sarcoglycans, while only partial reduction of alpha- and epsilon-sarcoglycan, suggesting that beta-, gamma- and delta-sarcoglycan function as a unit. Dystrophin, beta-dystroglycan and laminin expression and localization was essentially normal and apparently unaffected in these animals. The muscles in these mice developed membrane abnormalities, as seen in dystrophin-deficient muscle, and yet dystrophin was present. Researchers suggest from this evidence that it’s gamma-sarcoglycan that is the likely mediator of pathology in these disorders.

These mice develop pathology like human LGMD, showing muscle pseudohypertrophy, elevated serum CK, degeneration and regeneration of skeletal muscle. These mice (gsg-/-) also develop marked cardiomyopathy. The similar phenotype in these gamma-sarcoglycan-deficient mice and the cardiomyopathic BIO 14.6 hamster suggest that both gamma- and delta-sarcoglycan have critical and distinct roles in cardiac and skeletal muscle.

The gsg-/- mice differ from mdx mice (model for Duchenne muscular dystrophy) in that they show a strong cardiomyopathy and a more severe skeletal muscle phenotype, a much higher CK, extensive fibrosis and other features of dystrophic effects seen in human but not very well in the mdx mouse.

In the gsg-/- mice muscle, even though dystrophin is present, it isn’t capable of stabilizing the sarcoglycans and can’t prevent the dystrophic process. Researchers suggest that the absence of gamma-sarcoglycan may lead to abnormal interactions within the DAC and the development of muscular dystrophy. Whether restoration of the sarcoglycan complex is sufficient to prevent the dystrophic phenotype is unknown. Since sarcoglycan deficiency is the common feature in both DMD and LGMD, it’s a likely mediator of the dystrophic process in both these disorders. Researchers suggest that the development of new approaches to inhibiting cell death or the stabilization of the sarcoglycan subunits, may be reasonable therapeutic approaches to the treatment of Duchenne and similar dystrophies. (Hack, AA., Gamma-sarcoglycan deficiency leads to muscle membrane defects and apoptosis independent of dystrophin. Journal of Cell Biology. 142:(5):1-9:1998.)

The following is the most recent classification compiled by MDA research staff of the different forms of LGMD as reported in peer-reviewed journals (references indicated).

CLASSIFICATION OF LIMB-GIRDLE MUSCULAR DYSTROPHIES.

(Chromosome (Chr) location and affected gene if known.)

LGMD Type 1 are the autosomal dominant forms and are heterogenic*.

LGMD1A on Chr 5q22.3-31.3288
. (Bethlem myopathy is not considered to be an allelic** variation of LGMD1A but is also on Chr 5q and is also dominant.)
LGMD1B on Chr 1q11-21330
LGMD1C on Chr 3p25 - CAV-3 or M-Caveolin gene.355
LGMD1D?? - FDC-CDM346
-Familial dilated cardiomyopathy, conduction defect with LGMD. Chr 6q23346

LGMD Type 2 are the autosomal recessive forms and are also heterogenic*:

DAG(Duchenne-like)
2E on Chr 4q12--beta-sarcoglycan--A3b--43 kD DAG
2F on Chr 5q33-34-delta -sarcoglycan314-35 kD DAG
2G on Chr 17q11-12341. (gene unknown)
2H on Chr 9q31-33364. (gene unknown, syntenic to two mouse disorders)