N. De Luna

Dysferlin expression in monocytes: A source of mRNA for mutation analysis

Dysferlin protein is expressed in peripheral blood monocytes. The genomic analysis of the DYSF gene has proved to be time consuming because it has 55 exons. We designed a mutational screening strategy based on cDNA from monocytes to find out whether the mutational analysis could be performed in mRNA from a source less invasive than the muscle biopsy. We studied 34 patients from 23 families diagnosed with dysferlinopathy. The diagnosis was based on clinical findings and on the absence of protein expression using either immunohistochemistry or Western blot of skeletal muscle and/or monocytes. We identified 28 different mutations, 13 of which were novel. The DYSF mutations in both alleles were found in 30 patients and only in one allele in four. The results were confirmed using genomic DNA in 26/34 patients. This is the first report to furnish evidence of reliable mutational analysis using monocytes cDNA and constitutes a good alternative to genomic DNA analysis.

A new phenotype of dysferlinopathy with congenital onset

We report two patients with a new phenotype of dysferlinopathy presenting as congenital muscular disease. Both patients showed weakness in proximal lower limbs and neck flexor muscles at birth. The presence of normal CK levels during the first years should be noted. Initial MRI showed no abnormalities but short-time-inversion-recovery (STIR) sequences revealed a striking myoedema in gastrocnemius and hamstring muscles at the age of 5. Muscle biopsy showed mild dystrophic features and the absence of dysferlin. Dysferlin gene (DYSF) analysis revealed a p.Ala927LeufsX21 mutation in a homozygous state in both siblings. This new phenotype widens the clinical spectrum of dysferlin myopathies.

Antidisialosyl antibodies in chronic idiopathic ataxic neuropathy

Abstract. Antidisialosyl antibodies were found in two out of 13 patients with chronic idiopathic ataxic neuropathy (CIAN) and not in 32 patients with different sensory neuropathies of known cause. This finding confirms the association of antidisialosyl antibodies and CIAN regardless of the absence of the M band. These antibodies may have pathogenic relevance; however, larger series are needed to establish their clinical significance.

G.P.287

Dysferlinopathies are autosomal recessive inherited muscular dystrophies caused by mutations in the gene DYSF. Dysferlin is primarily expressed in skeletal muscle, cardiac muscle and peripheral blood monocytes. Dysferlin expression in skeletal muscle and monocytes strongly correlate in the healthy and disease states. We evaluated the efficiency of the monocyte assay to detect carriers and to determine the carrier frequency of dysferlinopathies in the general population. We enrolled 149 healthy volunteers and collected peripheral blood samples for protein analysis. While eighteen of these individuals with protein levels in the range of 40–64% were predicted to be carriers by the monocyte assay, subsequent DYSF sequencing analysis in 14 of these 18 volunteers detected missense variants in only four. Analysis of DNA methylation patterns at DYSF locus showed no changes in DNA methylation levels at CpG island and shores between samples. Our results suggest that: (1) dysferlin expression can also be regulated by factors outside of the dysferlin gene, but not related to DNA methylation; (2) carrier frequency and therefore the number of affected individuals could be higher than previously estimated; and (3) although reliable for evaluating dysferlinopathies, results of the monocyte assay can not be used when determining carrier status and molecular analysis of DYSF must be performed.

P.19.4 PDGF-BB is expressed by muscle fibers in myopathies and regulates growth and migration of satellite cells and pericytes

PDGF-AA and BB belong to the family of Platelet Derived Growth factors (PDGFs). They activate two different receptors: PDGF-RcfN and PDGF-RcfOfnrespectivelyf∣fnPDGF-AA has been recently involved in the process of fibrosis in muscle dystrophies. In contrast, PDGF-BB could be important in myoblast activation and differentiation. Its role in pathological conditions has not been investigated. Our objective is to study the expression and function of PDGF-BB in human skeletal muscle both in normal and pathological conditions. We studied PDGF-BB and PDGF-Rcβ expression on muscle biopsies from controls and patients with different muscle dystrophies and also in vitro in myoblasts, pericytes and myotubes using immunohistochemistry, WB and ELISA. To study the effect of PDGF-BB on myoblasts and pericytes we performed growth curves, differentiation studies and transwells assays. PDGF-BB is normally expressed in vessels but not in muscle fibers. Muscle fiber expression of PDGF paralleled the degree of muscle degeneration/regeneration in myopathies. In vitro experiments demonstrated that myoblasts and pericytes expressed PDGF-RcfO in the membrane, but did not produce PDGF-BB. In contrast, myotubes did express low levels of PDGF-BB, that increased after injury with ionomycine, SDS or a mechanical damage using glass beads. Moreover, we also observed that myotubes from dystrophinopathy patients expressed higher levels of PDGF-BB than myotubes from controls, facioscapulohumeral muscular dystrophy or dysferlinopathy patients. Finally, PDGF-BB promoted an increase in the growth ratio of myoblasts and pericytes and increased cell migration in the transwell-assays. Our results indicate that PDGF-BB is expressed in muscle fibers during degeneration/regeneration. Further, PDGF-BB may have an important role in growth, activation and migration of muscle stem cells with myogenic potential.