RF10.83 – GENOTYPE-PHENOTYPE
CHARACTERIZATION OF THE PATHOGENETIC
EXPANSION OF THE C9ORF72 GENE
IN A LARGE SERIES OF ITALIAN FAMILIAL
AND SPORADIC ALS PATIENTS
Responsabile scientifico del progetto
ADRIANO CHIO
Università di Torino – AOU San Giovanni Battista
U.O.5 – Mauro Cozzolino
Fondazione Santa Lucia
Ministero della Salute – Ricerca Finalizzata
Art.12 e 12bis, D.L.vo 502/92 – Finanziamento 2010
Sezione III: Attività per progetti
CONTRIBUTORS
1 – AOU San Giovanni Battista, I Division of Neurology, Università di Torino:
Adriano Chio
2 – Fondazione Salvatore Maugeri IRCCS, Director ALS Center, Milano:
Gabriele Mora
3 – ASO OIRM Sant’Anna, Director Laboratory of Molecular Genetics, Torino:
Gabriella Restagno
4 – NEMO Center, Director of the Center, Fondazione Serena and Ospedale
Niguarda Ca’ Granda, Milano: Massimo Corbo
5 – Fondazione Santa Lucia IRCSS, Laboratory of Neurochemistry, Roma:
Mauro Cozzolino
BACKGROUND
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder
of cortical, bulbar and spinal motor neurons, clinically characterized by
progressive paralysis and ultimately death due to respiratory failure typically
within 3 years from symptom onset.
The clinical heterogeneity of ALS is increasingly recognized: its phenotypes
(classic, bulbar, flail arm, flair leg and upper motor neuron predominant) appear
to define clinical subtypes with marked demographic, clinical and outcome
differences; besides, about 30% of patients develop some degree of frontotemporal involvement. However, it is likely that the biological bases of these
heterogeneities are to be rooted in the underlying biological etiology of ALS.
Approximately 5 to 10% of cases are familial in nature (fALS), whereas the
majority of cases occur sporadically in the community (sALS). Among fALS,
a total of 25% of cases is due to pathogenic mutations of four main genes, i.e.
superoxide dismutase 1 (SOD1), TAR DNA binding protein (TAROBP), fused
in sarcoma (FUS), and optineurin (OPTN). These genes, however, account also
for 2% of apparently sporadic ALS patients. Recently, we have found that a
large hexanucleotide (GGGGCC) repeat expansion in the first intron of
C9ORF72 gene, located on chromosome 9p21, causes about 30% of fALS in
populations of European ancestry [Renton et al., 2011].
Of note, repeat expansion of this gene account also for about one third
of Finnish and 12% of American patients with frontotemporal dementia
(FTD) [Dejesus Hernandez et al., 2011]. The repeat expansion is dominantly
transmitted in the families until now reported [Renton et al., 2011; Dejesus
Hernandez et al., 2011], but sporadic cases carrying the expansion have been
described, at least in the Finnish population [Renton et al., 2011]. Since the
pathogenic effect of the C9ORF72 gene expansion in ALS has been identified
only recently, the understanding of the clinical and pathological characteristics
of patients carrying this expansion is still extremely poor. Moreover, accurate
measurements of the repeat expansions size have not yet been performed, since
they require Southern blot analysis. This measurement is necessary to establish:
a) the instability of the repeat expansion from generation-to-generation;
614
2013
RF10.83 – Genotype-phenotype characterization of the pathogenetic expansion...
b) the relationship between the size of the repeat with the age at onset and the
clinical course of the disease.
AIMS
Our aims are:
a) A full characterization of fALS and sALS carrying the pathogenetic
expansion of the C9ORF72 gene from the clinical, neuropsychological, neurophysiological, neuroradiological and biochemical/functional point of view.
b) Through the accurate analysis of the patients pedigrees, to determine the
penetrance of the C9ORF72 pathogenetic expansions and look for the presence
of an anticipation phenomenon related to the size of the hexanucleotide expansion.
c) To evaluate the effect of the size of the amplification, evaluated through
southern blot, on the age at onset and the progression rate of the disease.
d) To compare the characteristics of patient carrying the pathogenetic
expansion of the C9ORF72 gene to those carrying mutations of other ALSrelated genes (SOD1, TARDBP, FUS and OPTN) in order to establish the relative
frequency of the different genes and the specific phenotypes related to each
mutation.
e) To assess the subcellular distribution of RNA foci containing the repeats
in lymphocytes and the expression and localization of the C9ORF72 protein
in lymphocytes of patients with C9ORF72 repeat expansion compared with
patients with other ALS-related mutations.
MAIN EXPECTED RESULTS AND IMPACT
The phenotype of sporadic and familial ALS patients carrying C9ORF72
repeat expansions will be determined and compared with the phenotypes of
other mutations. The analysis of the penetrance and size of amplification with
respect to patients clinical phenotype will have an impact for genetic
counseling. The characterization of biochemical alterations in leukocytes of
patients with C9ORF72 repeat expansions will provide information on its
pathogenetic mechanisms.
Si allega il programma dettagliato dell’Unità Operativa 5 che fa capo alle
Fondazione Santa Lucia.
2013
615
Sezione III: Attività per progetti
U.O.5 - Fondazione Santa Lucia
Mauro Cozzolino
The role of the unit of Fondazione Santa Lucia is to identify the molecular
mechanism(s) whereby C9ORF72 repeat expansions induce motor neurons
degeneration in ALS. The working hypotheses are that either the abnormally
expanded RNA alleles sequester RNA-binding proteins, leading to misregulation of one or more steps of mRNA metabolism, or that they interfere
with the expression of the C9ORF72 protein, thus affecting its functions,
which are still unknown.
These two hypotheses will be initially addressed in mouse motoneuronal-like
NSC34 cells that will be engineered for the overexpression of non-pathological and
repeats of this hexanucleotide, as well as for the overexpression and downregulation of the C9ORF72 protein (both isoform 1 and 2). In these cells, we will:
1. Characterize the function of the C9ORF72 protein.
2. Determine the primary outcomes of GGGGCC RNA toxicity.
This will be achieved by:
a) Identifying the proteins bound by GGGGCC RNA repeats, by an in-vitro
biotinylated RNA pull-down assay followed by Mass Spec analysis. This will
permit to identify possible targets of the pathogenic process that will be validated
in both the motoneuronal cellular model as well as in lymphocytes from patients.
b) Looking in NSC-34 cells for molecular phenotypes commonly associated to
RNA mis-regulation, such as:
- Redistribution of RNA granules in the cell.
- SnRNAs abundance and snRNPs assembly.
- Changes in alternative splicing.
c) Monitoring cell viability, that will be assessed using various methods
(caspases activation and cytocrome c release, staining with Hoechst, AnnexinVFITC binding, MTT assay) already successfully used in the proponent’s lab.
d) Studying mitochondria functionality. Indeed, alterations in mitochondria
and accumulation of oxidative stress appear to be common to ALS. Thus, mitochondria will be analysed by monitoring different parameters, with techniques
already set up in the proponent’s lab: polarization of mitochondria, intracellular
ATP, activity of respiratory complexes, mitochondrial GSH/GSSG ratio. Similarly,
oxidative stress will be monitored with specific probes and methods commonly
used by the proponent.
e) Monitoring neurite outgrowth and maintenance in NSC34 cells. Indeed,
accumulation of RNA repeat expansions, might impair the ability of cells to
maintain viable neuronal processes, similarly to what has been observed in cells
overexpressing other ALS genes.
The phenotypes that will stem from these studies will be related to the
accumulation and subcellular distribution of RNA foci containing the repeats,
which will be monitored using RNA FISH analysis. Moreover, the severity of
616
2013
RF10.83 – Genotype-phenotype characterization of the pathogenetic expansion...
phenotypes will be related to the number of expressed repeats. Finally,
lymphocytes of patients with C9ORF72 expansion will be also assayed to validate
relevant phenotypes that will come out from cell culture model.
Results from this study will increase our current understanding of the
molecular mechanisms underlying the pathogenesis of familial ALS and possibly
offer a clue on the causes of selective motor neuron vulnerability in ALS.
2013
617