O-1. High frequency of hypomethylation in patients with fshd-like phenotype

We studied 10 sporadic patients presenting with typical FSHD clinical phenotype and absence of D4Z4 deletion on chromosome 4q35 tested by classical Southern blot technique. Clinical features included facial and shoulder girdle muscle weakness in association or not with pelvic, abdominal, and anterior forelegs muscle involvement. Muscle biopsies showed variable degree of muscular dystrophy, absence of abnormalities of citoskeletal proteins routinely analyzed by immunohistochemistry and Western Blot. We performed calpain 3 and valosin containing protein (VCP) mutation analysis by direct sequencing, extensive D4Z4 genotyping by PFGE analysis and DNA methylation studies of the D4Z4 repeat. We found 2 patients presenting with already known calpain 3 mutations, 1 patient carrying a deletion of the probe region used for FSHD diagnosis, and 5 patients presenting with D4Z4 hypomethylation. No patients were found carrying VCP gene mutations. For 2 out of 10 patients molecular diagnosis is still lacking. We conclude that 1) even in absence of calpain3 deficiency detected by Western blot, this diagnosis has to be considered in patients with symmetric limb girdle muscular dystrophy and facial involvement; 2) PFGE analysis has to be performed routinely in FSHD diagnostic workout to exclude deletion of the probe region (alternatively these deletions can be detected by re-hybridizations with probe D4Z4) and more complex genetic rearrangements related to FSHD, and 3) the percentage of patients presenting with hypomethylation of D4Z4 not associated with D4Z4 deletion is very high and further studies are needed to better define this condition. We will provide a clinical and histological description of these 5 cases and a short discussion on features that they have in common.