0-TESTO COMPLETO.pdf - Fondazione Santa Lucia

Sezione III: Attività per progetti
has been thus proposed that D4Z4 hypomethylation might be a hallmark of
chromatin modification at D4Z4 in FSHD-like patients.
To test this possibility, we selected 73 FSHD-like patients, who are clinically
identical to FSHD affected subjects but have an unaltered D4Z4 repeat.
To this purpose, we will examine two CpG methylation-sensitive restriction
sites (BsaAI and FseI) in the first (proximal) unit of D4Z4 repeat array on
chromosome 4q35.
Results of this analysis will provide additional tools for FSHD diagnosis
and will lay the basis for transcriptional studies.
H. Analysis of gene expression in FSHD-like patients – The clinical and molecular
screening described above will allow us to select a number of patients
that show clinical feature of FSHD without carrying the molecular hallmark
of the disease, the D4Z4 deletion.
To test the possibility that other molecular defects might affect expression of
4q35 genes we aim at evaluating 4q35 transcription levels in muscle biopsies
obtained from patients clinically affected by FSHD with no D4Z4 deletion.
Expression analysis will be performed on total RNA extracted from part of muscle
biopsy previously obtained for diagnostic purposes. Biopsies have been stored
in Neuromuscular bank of tissues and DNA samples (NMTB) (telethon grant:
GTF05003) directed by Angelini, Bank of DNA Cell Lines and Nerve-Muscle cardiac
tissues NMUNIT-UNIMIOM (telethon grant GTF02008) directed by Moggio.
The study will include biopsies obtained from sex and age matched controls
from healthy volunteers or normal muscle biopsies from subjects biopsied
for other reasons.
This analysis may bring an important result for FSHD diagnosis. Detection
of specific over-expression of one or more 4q35 genes will provide a
molecular marker to study FSHD-like cases. It is also important to note that
these findings will support further investigations to establish which mechanism,
besides D4Z4 deletion, causes transcriptional de-repression at 4q35.
Methods
1. To define the clinical features of a large Italian population of FSHD
and to study the genotype-phenotype correlation in FSHD
A. Clinical studies – The proposed study includes:
a) patients clinically affected with FSHD, confirmed by molecular diagnosis
(one p13E-11 EcoRI allele < 35 kb);
b) patients clinically affected with FSHD carrying one p13E-11 EcoRI
allele between 35 kb and 45 kb;
c) patients clinically affected with FSHD carrying one p13E-11 EcoRI
allele > 50 kb;
d) all family members, both symptomatic and asymptomatic, in which
molecular analysis has not been performed.
Asymptomatic family members are included the clinical investigation.
All subjects enrolled in the study will be interviewed using the clinical
data questionnaire which focuses on the patient’s clinical history. Each
778 2009