0-TESTO COMPLETO.pdf - Fondazione Santa Lucia

Sezione III: Attività per progetti
2. To study the D4Z4 deleted alleles and the associated polymorphisms
in a large cohort of FSHD families and sporadic cases.
Patients. The study will focus on myopathic patients with D4Z4 fragment >
50 kb. Clinical examination will search for the inclusion criteria outlined
above. Morphological and immunohistochemical study will be performed on
muscle biopsy to exclude muscle diseases other than FSHD (limb-girdle muscular
dystrophy with defect of structural proteins, mitochondrial and other
metabolic myopathies, inflammatory myopathies). Genetic analysis of the
FRP gene will identify the patients with muscular dystrophy due to defect of
fukutin-related protein, which can present facial weakness. The analysis will
be conducted in the center directed by Angelini (P3).
The clinical features of all the FSHD-like patients will be discussed in
meetings including all the clinical centers, organized by the clinical coordinator;
in particular the neurological examination will be filmed so that the clinical
data can be shared among different clinicians; ideally all or most of the
centers should agree about the FSHD phenotype of the patient under discussion.
Thus we should be able to select patients with FSHD-like myopathy that
will be considered for further molecular studies.
Methylation studies. D4Z4 DNA methylation analysis will be performed on
Genomic DNA from FSHD and FSHD-like patients, both family members and
unrelated individuals.as previously described [van Overveld et al. 2003]. After
DNA digestion with the restriction enzymes EcoRI, BglII, BlnI, FseI and
BsaAI, the methylation level of the D4Z4 unit will be diagnosed by comparison
of the signal intensity of BglII-BglII fragment with BglII-BsaAI fragment
and the comparison of BglII-BglII fragment with BglII-FseI fragment. In
hypomethylation condition the BglII-BsaAI and BglII-FseI fragments have a
signal of greater intensity in comparison to BglII-BglII fragments, because
BsaAI and FseI cut the DNA only when specific CpGs in their restriction sites
are not methylated.In conclusion, in agreement with the hypothesis that
removal of silencing at 4q35 causes FSHD, we expect a hypomethylation pattern
in those patients who don’t carry the D4Z4 deletion, but have a clinical
FSHD phenotype.
Analysis of gene expression. Only in presence of ascertained clinical signs, a
selected group of patients will be subsequently subjected to muscular biopsy
and consequent analysis of 4q35 gene expression. To assess gene expression
level in relation to FSHD pathogenesis a precise quantitative analysis of 4q35
gene expression in affected muscle will be performed.
Muscle specimens from healthy age- and sex-matched individuals as normal
controls will guarantee a good validation of the study.
Gene expression will be analyzed by TaqMan assay. Total RNA will be isolated
from muscle samples and used for the synthesis of cDNA with random
examers and reverse transcriptase. Primers and probes has been designed
using the Beacon Designer software for RealTime PCR assay on the iCycler iQ
Detection System. Because of the high degree of homology among the FRG1
and FRG2 multi-copy gene family, we have performed a preliminary test by
780 2009