The limb-girdle muscular dystrophies are a genetically heterogeneous group of inherited progressive muscle disorders that affect mainly the proximal musculature, with evidence for at least three autosomal dominant and eight autosomal recessive loci. The latter mostly involve mutations in genes encoding components of the dystrophin-associated complex; another form is caused by mutations in the gene for the muscle-specific protease calpain 3. Using a positional cloning approach, we have identified the gene for a form of limb-girdle muscular dystrophy that we previously mapped to chromosome 2p13 (LGMD2B). This gene shows no homology to any known mammalian gene, but its predicted product is related to the C. elegans spermatogenesis factor fer-1. We have identified two homozygous frameshift mutations in this gene, resulting in muscular dystrophy of either proximal or distal onset in nine families. The proposed name 'dysferlin' combines the role of the gene in producing muscular dystrophy with its C. elegans homology.

Recently, a single gene, DYSF, has been identified which is mutated in patients with limb-girdle muscular dystrophy type 2B (LGMD2B) and with Miyoshi myopathy (MM). This is of interest because these diseases have been considered as two distinct clinical conditions since different muscle groups are the initial targets. Dysferlin, the protein product of the gene, is a novel molecule without homology to any known mammalian protein. We have now raised a monoclonal antibody to dysferlin and report on the expression of this new protein: immunolabelling with the antibody (designated NCL-hamlet) demonstrated a polypeptide of approximately 230 kDa on western blots of skeletal muscle, with localization to the muscle fibre membrane by microscopy at both the light and electron microscopic level. A specific loss of dysferlin labelling was observed in patients with mutations in the LGMD2B/MM gene. Furthermore, patients with two different frameshifting mutations demonstrated very low levels of immunoreactive protein in a manner reminiscent of the dystrophin expressed in many Duchenne patients. Analysis of human fetal tissue showed that dysferlin was expressed at the earliest stages of development examined, at Carnegie stage 15 or 16 (embryonic age 5-6 weeks). Dysferlin is present, therefore, at a time when the limbs start to show regional differentiation. Lack of dysferlin at this critical time may contribute to the pattern of muscle involvement that develops later, with the onset of a muscular dystrophy primarily affecting proximal or distal muscles.

Fatty liver has been linked to low aerobic fitness, but the mechanisms are unknown. We previously reported a novel model in which rats were artificially selected to be high capacity runners (HCR) and low capacity runners (LCR) that in a sedentary condition have robustly different intrinsic aerobic capacities. We utilized sedentary HCR/LCR rats (generation 17; max running distance equalled 1514 ± 91 vs. 200 ± 12 m for HCR and LCR, respectively) to investigate if low aerobic capacity is associated with reduced hepatic mitochondrial oxidative capacity and increased susceptibility to hepatic steatosis. At 25 weeks of age, LCR livers displayed reduced mitochondrial content (reduced citrate synthase activity and cytochrome c protein) and reduced oxidative capacity (complete palmitate oxidation in hepatic mitochondria (1.15 ± 0.13 vs. 2.48 ± 1.1 nm g −1 h, P < 0.0001) and increased peroxisomal activity (acyl CoA oxidase and catalase activity) compared to the HCR. The LCR livers also displayed a lipogenic phenotype with higher protein content of both sterol regulatory element binding protein-1c and acetyl CoA carboxylase. These differences were associated with hepatic steatosis in the LCR including higher liver triglycerides (6.00 ± 0.71 vs. 4.20 ± 0.39 nmol g −1 , P = 0.020 value), >2-fold higher percentage of hepatocytes associated with lipid droplets (54.0 ± 9.2 vs. 22.0 ± 3.5%, P = 0.006), and increased hepatic lipid peroxidation compared to the HCR. Additionally, in rats aged to natural death, LCR livers had significantly greater hepatic injury (fibrosis and apoptosis). We provide novel evidence that selection for low intrinsic aerobic capacity causes reduced hepatic mitochondrial oxidative capacity that increases susceptibility to both hepatic steatosis and liver injury.

Limb girdle muscular dystrophy type 2B (LGMD2B) and Miyoshi myopathy (MM), a distal muscular dystrophy, are both caused by mutations in the recently cloned gene dysferlin, gene symbol DYSF. Two large pedigrees have been described which have both types of patient in the same families. Moreover, in both pedigrees LGMD2B and MM patients are homozygous for haplotypes of the critical region. This suggested that the same mutation in the same gene would lead to both LGMD2B or MM in these families and that additional factors were needed to explain the development of the different clinical phenotypes. In the present paper we show that in one of these families Pro791 of dysferlin is changed to an Arg residue. Both the LGMD2B and MM patients in this kindred are homozygous for this mutation, as are four additional patients from two previously unpublished families. Haplotype analyses suggest a common origin of the mutation in all the patients. On western blots of muscle, LGMD2B and MM patients show a similar abundance in dysferlin staining of 15 and 11%, respectively. Normal tissue sections show that dysferlin localizes to the sarcolemma while tissue sections from MM and LGMD patients show minimal staining which is indistinguishable between the two types. These findings emphasize the role for the dysferlin gene as being responsible for both LGMD2B and MM, but that the distinction between these two clinical phenotypes requires the identification of additional factor(s), such as modifier gene(s).

This study confirms that the dysferlin gene is mutated in MM and LGMD2B and extends understanding of the timing of onset of the disease. Knowledge of the genomic organization of the gene will facilitate mutation detection and investigations of the molecular biologic properties of the dysferlin gene.