Sclerosi Laterale Amiotrofica:

Sclerosi Laterale Amiotrofica:
Clinica, Genetica,
Nuove Prospettive Terapeutiche
Nicola Ticozzi
U.O.Neurologia e Laboratorio Neuroscienze
Università degli Studi di Milano
IRCCS Istituto Auxologico Italiano
Sclerosi Laterale Amiotrofica
Charcot, J. M. & Joffory, A.
Deux cas d’atrophie musculaire progressive avec
lesions de la substance grise et des faisceaux anterolateraux de la moelle epiniere.
Arch. Physiol. Neurol. Pathol. 2, 744–754 (1869).
 Malattia neurodegenerativa
dei motoneuroni
 Paralisi progressiva della
muscolatura volontaria
 Età esordio: 55-65 anni
 Sopravvivnza: 3 anni
 Incidenza: 2x100.000/anno
 Prevalenza: 6-8/100.000
 Lifetime risk: 1:400 – 1:600
 Nessuna terapia efficace
Une leçon du Docteur Charcot à la Salpêtrière – André Brouillet, 1887
SLA e le altre malattie neurodegenerative
Bertram L and Tanzi RE, The Journal of Clinical Investigation 2005
SLA e Malattie
del Motoneurone
Una malattia da vulnerabilità
selettiva di un sistema
Motoneurone e vulnerabilità
Transporto Assonale
Mitocondrio
The majority of patients with
adult-onset motor neuron
disease will be found to have
IDIOPATHIC ALS
Eziopatogenesi
Focalità all’ esordio
Diffusione
Ravits et al., 2009
Diffusione
Ravits et al., 2007
Clinical syndromes of ALS
Syndrome
Main clinical features
Prognosis
Classic (“Charcot”) ALS
Limb onset (spinal);
bulbar involvement usual; UMN +
LMN signs;
M:F ratio 3:2.
60-70% of all cases at presentation;
median survival 3-4 yrs.
Progressive bulbar palsy
(PBP)
Onset with dysarthria, then
progressive speach and swallowing
difficulties;
limb involvement follows (months or
yrs);
M:F ratio: 1:1 (PBP > common in
older women).
20% of all case at presentation;
median survival 2-3 yrs.
Progressive muscular atrophy
(PMA)
Almost always limb onset;
> 50% develop UMN signs;
85% develop bulbar symptoms;
heterogeneous condition but
majority are ALS;
M:F ratio 3-4.
10% all cases at presentation;
overlap with “flail arm” and “flail leg”
syndromes;
median survival 5 yrs;
more long survivors (>10 yrs).
Primary Lateral Sclerosis
(PLS)
Clinically progressive pure UMN
syndrome;
after few yrs may convert to ALS.
10 yrs or more.
Clinical syndromes of ALS (cont.)
Syndrome
Main clinical features
Prognosis
“Flail arm syndrome”; man in a Syndrome of predominantly LMN
weakness of both arms;
barrel syndrome; VulpianUMN signs develop in 50-70%;
Bernhard syndrome
About 10% of all cases;
M:F ratio 9:1;
prognosis better than in ALS
syndrome more common in African
and Asian patients.
“Flail leg syndrome”;
“pseudopolyneuritic form” of
ALS; Patrikios syndrome
Syndrome of progressive leg
weakness, predominantly LMN.
Rare;
slow progression;
DD difficult.
Monomelic forms of ALS
Rare variants of ALS with slowly
progressive focal (upper > lower
limb UMN and LMN syndrome);
Distinct LMN form most common in
Asia (monomelic juvenile onset
amyotrophy; Hirayama’s syndrome);
DD with multifocal motor
neuropathy.
Juvenile onset form progressive
over months or several yrs and then
stabilises;
does not generalises;
pathology unknown.
ALS-dementia syndrome
(ALS-D)
Dementia of fronto-temporal type
present in 5% of all ALS cases;
20-40% ALS patients have subtle
cognitive changes of “frontal” type;
ALS-D may present first with
dementia or ALS progressing to
dementia, or with combination of
both; about 50% familial.
Usually 2 to 5 yrs.
often slow progression;
pathology is that of ALS.
(“Charcot” ALS)
Unusual initial signs and symptoms
• Hemiparetic form (Mills’ variant)
• Head drop (cervical extensor muscle weakness)
• Fasciculations
• Weight loss
• Respiratory failure
• Monomelic presentation
• Symmetrical onset
• Diffuse onset
Mitsumoto et al, 1998
La diagnosi di SLA
Steps in the diagnosis of ALS
suggested by WFN guidelines
Steps
Rationale
1. History, physical examination
Ascertain clinical findings that may
suggest level of certainty of diagnosis
2. EMG examination
Ascertain findings that confirm LMN
degeneration in clinically involved regions;
Identify LMN degeneration in clinically
uninvolved regions;
Exclude other disorders
3. Neuroimaging
Ascertain findings that may exclude other
disease processes
4. Clinical laboratory examinations
5. Neuropathologic examinations
6. Repetition of clinical and EMG
(6 months apart)
Ascertain possible ALS-related syndromes.
Ascertain findings confirming/excluding ALS
Ascertain evidence of progression
Mitsumoto et al, 1998, 2006
CRITERI CLINICI
Trofismo
Tono
Stenia
ROT
Segni patologici
REGIONS
Bulbar
Cervical
Thoracic
Abdominal
UMN
+
+
+/-
+
LMN
+
+
+
+
Clinically definite ALS
Lombar
Criteri di El Escorial
Neurofisiologia
EMG
ENG
TMS-MEPs
EMG
200msec
msec L
200
L 100uV
100uV
• A riposo
– Attività spontanea
patologica (fibrillazione, onde
lente positive, fascicolazioni,
scariche ad alta frequenza,
scariche miotoniche)
• Lieve contrazione
– Morfologia Potenziali di Unità
Motoria (PUM): alterazioni
quantitative e qualitative
• Massima Contrazione
– Reclutamento PUM
EMG
200 msec L 200 uV
• A riposo
– Attività spontanea
patologica (fibrillazione, onde
lente positive, fascicolazioni,
scariche ad alta frequenza,
scariche miotoniche)
• Lieve contrazione
– Morfologia Potenziali di Unità
Motoria (PUM): alterazioni
quantitative e qualitative
• Massima Contrazione
– Reclutamento PUM
EMG
200 msec L 1mV
• A riposo
– Attività spontanea
patologica (fibrillazione, onde
lente positive, fascicolazioni,
scariche ad alta frequenza,
scariche miotoniche)
• Lieve contrazione
– Morfologia Potenziali di Unità
Motoria (PUM): alterazioni
quantitative e qualitative
• Massima Contrazione
– Reclutamento PUM <
NUOVI CRITERI ELETTROMIOGRAFICI
DI EL ESCORIAL PER LA DIAGNOSI DI LMN
EMG
SEGNI DI DENERVAZIONE IN FASE ATTIVA
• Potenziali di fibrillazione.
• Onde positive appuntite o sharp waves.
NUOVI CRITERI ELETTROMIOGRAFICI
DI EL ESCORIAL PER LA DIAGNOSI DI LMN
EMG
PRESENZA DI FASCICOLAZIONI
• La presenza è utile nella diagnosi anche quando
sono registrabili in muscoli in cui non sono presenti
segni di denervazione sia attiva che cronica.
• L’assenza non preclude la diagnosi.
NUOVI CRITERI ELETTROMIOGRAFICI
DI EL ESCORIAL PER LA DIAGNOSI DI LMN
EMG
SEGNI DI DENERVAZIONE CRONICA
• Potenziali di Unità Motoria (PUM) di ampiezza e durata
incrementata.
• per la presenza di una sofferenza del UMN si ha una
riduzione del reclutamento sia spaziale che temporale e
quindi una riduzione della frequenza di scarica.
• Potenziali di Unità Motoria instabili.
NUOVI CRITERI ELETTROMIOGRAFICI
DI EL ESCORIAL PER LA DIAGNOSI DI LMN
ENG
Richiesta per la diagnosi per definire ed escudere
altre patologie del nervo periferico, della giunzione
neuromuscolare e dei muscoli che possano mimare
una SLA.
NUOVI CRITERI ELETTROMIOGRAFICI
DI EL ESCORIAL PER LA DIAGNOSI DI LMN
ENG
• I parametri di conduzione nervosa motoria sono all’inizio della
malattia generalmente normali o lievemente alterati. Importante la
ricerca di eventuali blocchi di conduzione (riduzione dell’ampiezza del
MAP >30% senza dispersione temporale) in sedi non usuali di
compressione.
• I parametri di conduzione nervosa sensitiva devono essere normali.
(sono alterati nella sindrome di Kennedy).
• Presenza di onda F di ampiezza aumentata e monomorfa.
VALUTAZIONE DELLE
ALTERAZIONI UMN
Potenziali Evocati Motori
PEM
•Nella SLA in fase iniziale
Ampiezza Motot Evoked Potential (MEP) corticale ridotta
T.C.M.C normale
T.C.M.P. normale o lievementa aumentato
•Nella SLA in fase avanzata
Ampiezza MEP corticale ridotta o MEP assente
T.C.M.C aumentato
T.C.M.P. aumentato o MEP radicolare assente
PEM: CONCLUSIONI
Le alterazioni del TCMC, della soglia di eccitabilità corticale, e del
rapporto MEP/MAP sono comunque variabili anche quando sono
presenti segni bulbari.
Se sono presenti alterazioni di questi parametri i PEM supportano la
diagnosi ma se sono assenti non la escludono
Le percentuali di alterazione del PEM nelle varie casistiche variano
dal 38% (Mills and Nithi, 1998) al 100% (Hugon et al, 1987)
Neuroimaging studies in the diagnosis of ALS
MRI
• Brain atrophy (parietal, insular, frontal temporal, corpus callosum).
• Spinal cord atrophy (rarely documented).
• CST hyperintensity in T2- and proton-density weighted MRI<
(usually bilateral and symmetrical, 17 to 100% in studies<).
• Neocortical hypointensity (in T2, bilateral, in pre- and post-central
gyrus, mean 52% reported).
Neuroimaging
Filippini et al., Neurology, 2010
Diagnosis flow for ALS patients
1) First consultation
• Hearing
• Neurological exam
3) ALS diagnosis
Diagnosis Standard of ＷＦＮ (El
Escorial)
Therapy (riluzole)
Therapeutical Plan (Rare Disease)
Follow-up visit in 1-2 months
•
•
•
•
5) Confirmation of Disease
2) Exclusion of other dubious diseases
(hospitalization)
• Blood Biochemistry
• Needle EMG (electromyogram)
• Nerve conduction study
• ＭR
• C.S.F.
• (Muscle biopsy)
•
•
After 3-6 months
Second opinion
6) Progression of Disease
•
•
•
ALSFRS-R
BMI
FVC/ Pulmonary functional test/ Blood
gas test
Diseases that can masquerade as ALS/MND
Anatomical abnormalities/compression syndromes:
Arnold-Chiari-1 and other hindbrain malformations
Cervical, foramen magnum or posterior fossa region tumors
Cervical disc herniation with osteochondrosis
Cervical meningeoma
Retropharyngeal tumour
Spinal epidural cyst
Spondylotic myelopathy and/or motor radiculopathy
Syringomyelia
Acquired enzyme defects
Adult GM2 gangliosidosis (hexosaminidase-A or B- deficiency)
Familial amyloid polyneuropathy (FAP)
Polyglucosan body disease
Autoimmune syndromes:
Monocloncal gammopathy with motor neuropathy
Multifocal motor neuropathy with/without conduction bloks (MMN)
Dysimmune LMN syndromes (with GM1, GD1b, and asialo-GM1 antibodies)
Other dys-immune LMN syndrome including CIDP
Multiple sclerosis
Myastenia gravis
Endocrine abnormalities
Diabetic “amyotrophy”
Insulinoma causing neuropathy
Hyperthyroidism with myopathy
Hyperparathyroidism
Hypokalemia (Conn’s syndrome)
Exogenous toxins
Lead (?), mercury (?), cadmium, aluminum, arsenic, thallium, manganese,
organic, pesticides, neurolathyrism, konzo
EFNS Task Force, 2005
Diseases that can masquerade as ALS/MND (cont.)
Infections:
Acute poliomyelits
Post-poliomyelitis progressive muscular atrophy
HIV-1 (with vacuolar myelopathy)
HTLV-1cassociated myelopathy (HAM, tropical spastic paraplegia)
Neuroborreliosis
Spinal encephalitis lethargica, varicells-zoster, brucellosis, cat-scratch disease,neuro-syphilis, prion disorders
Myopathies:
Cachectic myopathy
Carcinoid myopathy
Dystrophin-deficient myopathy
Inclusion body myositis (IBM)
Inflammatory myopathies
Polymyositis
Sarcoid Myositis
Neoplastic syndromes:
Chronic lymphocytic leukemia
Intramedullary glioma
Lymphoproliferative disorders with paraproteinemia and/or oligoclonal bands in the CSF
Pancoast tumor syndromes
Paraneoplastic Encephalomyelitis (PEM) with anterior horn cell involvement
Stiff-Person-Plus syndromes
Physical injury:
Electric shock neuronopathy
Radiation-induced radiculo-plexopathies and/myelopathy
Vascular Disorders:
Arterioveneous malformation
Dejerine anteriori bulbar artery syndrome
Stroke
Vasculitis
EFNS Task Force, 2005
Diseases that can masquerade as ALS/MND (cont.)
Other neurological conditions:
Wester pacific atypical forms of MND/ALS (Guam, New Guinea, Kii Peninsula Japan)
Carribean atypical forms of MND-dementia-PSP (Guadeloupe)
Madras-form of juvenile onset MND/ALS (South India)
Frontotemporal dementia with MND/ALS (FTD, including Pick’s disease with amyotrophy)
Multiple System Atrophy (MSA)
Olivo-ponto cerebellar atrophy (OPCA/SCA) syndromes
Primary lateral sclerosis (PLS; some subtypes not related to ALS)
Progressive supranuclear palsy (PSP)
Hereditary spastic paraplegia (HSP; many variants, some subtypes with distal amyotrophy)
Progressive spinal muscular atrophy (PMA; some subtypes not related to ALS)
Spinobulbar muscular atrophy with/without androgen receptor mutation (SBMA)
SMA I-IV
Brown-Vialetto-van Laere syndrome (early onset bulbar and spinal ALS with sensorineural deafness
Fazio-Londe syndrome (infantile PBP)
Harper-Young syndrome (laryngeal and distal SMA)
Monomelic sporadic spinal muscular atrophy (BFA, including Hirayama Syndrome)
Polyneuropathies with dominating motor symptoms (HMSN type 2)
Benign fasciculations
Myokymia
EFNS Task Force, 2005
The most important of the acquired
diseases of the spinal cord in
simulating ALS:
Spondylotic Myelopathy
Spondylotic Myelopathy
• May lead to spinal cord compression and ischemia with/without nerve
root compromise.
• Neck pain common but not invariable clinical feature.
• Some patients develop UMN signs in the legs and, with central grey
matter or nerve root involvement or both, they may have LMN signs in
the arms (simulating ALS).
• 5% of ALS patients have had cervical or lumbar laminectomy early in
their course.
• Unlike ALS, proprioceptive loss in the lower and upper extremities and
sphincter abnormalities.
• Cervical MR often discloses abnormal signal on FLAIR sequences
intrinsic to the spinal cord.
• EMG: active and cronic denervation in both arms and legs, bulbar and
thoracic EMG should be normal.
Spinobulbar Muscular Atrophy (SBMA)
DD: 1 in 35 patients initially diagnosed as having ALS may have SBMA
• X-linked SMA, CAG expansion (9-36 to 40-62), in men
• slowly progressive, at age 30-60 yrs
• muscle cramps/fasciculations, then bulbar and proximal limb
• atrophy/weakness, symmetrical, tendon reflexws <, no UMN signs
• whelchair in 2-3 decades
• rarely sensory symptoms at onset, then mild sensory < vibration
(feet) and < sensory nerve conduction potentials
• signs of mild androgen insensitivity (gynecomastia 50%, etc.)
• hand postural tremor early or late
• female carriers asymptomatic (minority with cramps or tremor)
Spinobulbar Muscular Atrophy (SBMA)
• CK >
• EMG:
chronic denervation and partial reinnervation
fibrillation potentials not prominent
some patients: decrement of low-frequency repetitive nerve stimulation studies
• SURAL BIOPSY: loss of large-diameter axons
• MUSCLE BIOPSY:
signs of chronic denervation with grouped atrophy of myofibers
fiber-type grouping
• GENETIC TESTING: CAG repeat in the AR gene (Xq11-q12) (9-36 CAG to 40-62)
anticipation is not a prominent feature of SBMA
• PATHOLOGY: dorsal root ganglion cell loss + MN loss
Spinal Muscular Atrophy (SMA)
Type I
Mild adult onset SMA
Focal Spinal Muscular Atrophy (SMA): Hirayama
• monomelic amyotrophy of the upper limb (oblique amyotrophy), rarely
bilateral, no UMN signs
• development in months, then stability
• lower limb rarer
• > male, in early adult life, no family history
• MR: cervical lesion in flexion
• DD: flail-arm syndrome, monomelic ALS, multifocal motor neurophaty
Dorsal interosseous muscles
Hirayama et al., 1987
Spinobulbar Muscular Atrophy: Brown-Vialetto-van Laere
• AD, AR, X-linked
• progressive weakness
• bilateral cranial nerves VII to XII
• bilateral sensoryneural deafness
• variable progression
• DD: Fazio-Londe (AR, rapidly progressive bulbar degeneration)
SLA e Disturbi Cognitivi
Jean-Martin Charcot, 1874
“ patients are not demented
and cognition is spared “
Aran – doctoral thesis, 1850
ALS patient as “perfectly conscious of his
condition, remember the most precise details of
his disease, and all in all have normal functions
except those of movement”
Annali di Neurologia, 25, 273-287, 1907
Rassegna di Studi Psichiatrici, 30, 705-722, 1941
• frontal impairment clearly mentioned
5 to 15%
25 to 50%
Strong
et al.,et2009
Strong
al., 2099
5% dei Pazienti FTD hanno segni clinici o subclinici di sofferenza LMN
Anomalie neuropsicologiche nella SLA
Phukan
Phukan et
et al.,
al., 2007
2007
2011
Terapia della SLA
Sintomatica
Nutrizione
Respirazione
Fisioterapia
Palliazione
NUTRIZIONE
Disfagia
II° MN
(V, VII, IX, XII)
+/perdita
innervazione I°
PERDITA DI PESO & MALNUTRIZIONE
DISTURBI PSICOLOGICI
RITARDATO SVUOTAMENTO GASTRICO
CONSTIPAZIONE
> DISPENDIO ENERGETICO GIORNALIERO
THE NUTRITIONAL STATUS IN ALS PATIENTS
SCIENTIFIC DATA
• Resting energy expenditure (REE) in 36 ALS patients on riluzole
(22.5 months)
• STATE OF HYPERMETABOLISM CONFIRMED (+ 16.9% + 14.5%
above the normal expected value)
• NO CORRELATION WITH THE VC
• COLLERATION WITH AGE, GENDER (>MEN), LEUCOCYTOSIS
INDEPENDENTLY
Desport et al., 2000
MALNUTRITION IN ALS
• 21% OF 47 ALS PATIENTS ARE MODERATELY OR SEVERELY MALNOURISHED
(tested using TSF, MAMC, DIETARY ANALYSIS)
• NO DIFFERENCES BETWEEN BULBAR- OR SPINAL-ONSET PATIENTS
• MEN MORE MALNOURISHED THAN WOMAN
CONCLUSION: MALNUTRITION MORE PREVALENT THAN APPRECIATED IN
ALS PATIENTS, INCLUDING THOSE WITH NO SWALLOWING DIFFICULTIES
Worwood and Leigh, 1998
NUTRITIONAL STATUS AS PROGNOSTIC FACTOR FOR
SURVIVAL
(Desport et al., 1999)
SURVIVAL (Kaplan-Meier) WORSE FOR MALNURISHED ALS
(p=<0.0001), with 7.7 fold increased risk of death
Only VC (p < 0,001) and MALNUTRITION (p < 0,01) have significant
independent prognostic value
HOW DETECT DYSPHAGIA?
• CAREFUL HISTORY
•
•
•
•
QUESTIONS REVEALING (MEAL DURATION, etc.)
PHYSICAL EXAMINATION
EVALUATE SWALLOW DURING A MEAL
ADMINISTER MODIFIED BARIUM - SWALLOW WITH VIDEOFLUOROSCOPY
BUT
• NO SINGLE TEST
• SWALLOWING STUDY INADEQUATE
NUTRIZIONE ENTERALE
NFT
55% prescribed EN, 90% failures
PEG
93% prescribed EN, no failure
PEJ
Alternative strategy
RIG/PRG
Better tolerated
Practice Parameter, AAN, 2009
Refeeding Syndrome
•
•
•
•
•
Ipofosfatemia
Ipomagnesemia
Ipopotassiemia
Deficit vitaminici (Tiamina)
Ritenzione di liquidi
•
Complessa sindrome con instabilità
cardiovascolare
•
Mortale nella SLA nel 1° mese,
particolarmente nelle PEG tardive
Respirazione
Indicazioni per una NIV



Paziente con insufficienza respiratoria
cronica clinicamente stabile o ad evoluzione
lentamente progressiva:
Significativa ritenzione diurna di CO2 (>50
mmHg) a pH compensato
Aumento moderato diurno o notturno di CO2
(45 o 50 mmHg) associato a sintomi
attribuibili ad ipoventilazione (cefalea diurna,
sonno agitato, incubi notturni, nicturia,
sonnolenza diurna….)
Ipoventilazione notturna significativa o
desaturazione ossiemoglobinica
Indicazioni per una NIV
Devono però esser rispettate le seguenti condizioni:
 La terapia farmacologica deve esser la più idonea
al caso
 Il paziente deve esser in grado di rimuovere
adeguatamente le secrezioni
 Devono esser trattate in modo congruo tutte le
patologie reversibili associate (OSAS,
ipotiroidismo, scompenso cardiaco, alterazioni
elettrolitiche…)
NIV: vantaggi



Rapidità e facilità d’applicazione
Eliminazione dei rischi legati all’aggressione della trachea
determinata dall’intubazione
L’alternarsi di periodi di ventilazione e di respirazione
spontanea (ritmo d’applicazione variabile)
Durante la ventilazione
 Diminuzione della CO2
 Diminuzione dell’attività elettromiografica dei muscoli
respiratori
All’arresto della ventilazione
 Mantenimento della diminuzione di CO2
 Diminuzione della dispnea
 Aumento della forza inspiratoria massima
NIV: svantaggi








Instabilità dell’interfaccia
Impossibilità di garantire una ventilazione continua di lunga
durata
La necessità di cooperazione da parte del paziente
(Pz. Bulbari!)
Lesioni cutanee a livello della radice del naso
Insufflazione gastrica
Perdite d’aria
Congiuntiviti
Pause respiratorie (in caso di Bilevel senza frequenza di
sicurezza) con vere e proprie apnee
What are the limits of NIV ?
1) When ventilator dependency is quite total (20-24 h / d)
Then the quite continuous use of NIV, although non absolutely
impossible (Bach), becomes difficult and more dangerous
2) When airways must be protected related to
swallowing disturbancies and repeated aspirations which are
usually associated with a high ventilator dependency and
generalized motor impairment (ALS)
Who needs a tracheostomy ?
Tracheostomy is still used
1. When NIV reachs its limit
2.Or, even, still as an elective method due to its more constant and
stable efficacy in term of ventilation
Symptomatic treatment
• Scialorrea
–
–
–
–
–
–
–
–
–
Amitriptiline 25-50 mg oral x 3 a day
Atropine drops (IV) 0.25-0.75 mg x 3 a day
Glycopyrrolate (nebulized or iv form)
Scopolamine (oral or dermal patch)
Scopolamine transdermal (1.5 mg every 5 days
(II)
Benztropine (I)
Botulinum toxin type A (IV)
No study in type B
Radiological intervention (IV): external irradiation
or low dosage palliative radiation of single fraction
of 7-8 Gy
Symptomatic treatment
• Pseudobulbar emotional lability
– Dextromethorphan and quinidine (IA)
– Fluvoxamine
– Amitriptyline
– Citalopram
– Dopamine
– Lithium
Symptomatic treatment
• Cramps
– Quinine sulphate 200 mg x 2 and vitamin E (I)
– Physiotherapy
– Carbamazepine
– Diazepam
– Phenytoin
– Verapamil
– Gabapentin
Symptomatic treatment
• Spasticity
–
–
–
–
–
–
–
–
–
–
–
Physical therapy (IIB)
Hydroterapy in heated pool (III)
Cryoterapy
Oral baclofen (up to 80 mg daily)
Intrathecal baclofen
Gabapentin (900-2400 mg daily)
Tizanide (6-24 mg daily)
Memantine (10-60 mg daily)
Dantrolene (25-100 mg daily)
Diazepam (10-30 mg daily)
Botulin toxin A
Symptomatic treatment
• Depression, anxiety, and insomnia
– Amitriptyline
– Sertraline
– Fluoxetine
– Paroxetine
– Zolpidem
– Diazepam
– Sub-lingual lorazepam
Symptomatic treatment
• Pain
– Paracetamol
– Weak opioids (tramadol)
– Strong opioids (morphine
or ketobemidon)
ALS: Nutritional and Respiratory Issues
Both have potentially profound effects on survival:
PEG (left, from Mazzini et al) and BiPAP (right, from Kleopa et al.)
Impatto dei Centri Terziari sulla
sopravvivenza dei pazienti SLA
Chiò et al., 2006
Terapia della SLA
“Dopo quanto vi ho detto finora sulla malattia, dovrei forse
trattenervi più a lungo riguardo al problema della terapia?
I tempi non sono ancora maturi perché questo argomento
possa essere trattato seriamente”
J.M. Charcot, Leçons du Mardi à la Salpêtrière, 1869
Eziologica
Farmacologica
Terapia genica?
Cellule staminali?
1994
Traynor et al., 2006
CL201 Part 2: Slope estimates for
ALSFRS-R total scores
slope 50 mg = -1.283
slope 300 mg = -1.021
imputes placebo slope = -1.337
Relative slope reduction = 20.4%
Imputed placebo decline
KNS-760704 (dexpramipexole)
KNS-760704: Survival estimates
Log rank test: p = 0.0708
Includes all study deaths to Week 28.
Terapia Genica?
= Completed
Determine Antisense oligo Distribution in
CNS Following ICV Administration
Rat
Human
Identification of
Rat SOD-1 ASOs
Identification
of Human
SOD-1 ASO Candidates
ASO Medicinal
Chemistry
Demonstrate SOD-1
Inhibition in Liver Following
Systemic Administration
Demonstrate SOD-1
Inhibition in CNS Following
ICV Administration
Examine Dose
Schedule Requirements,
PK & Histopathology
Test in Human Fibroblasts
from A4V Patients
SOD1 A4V
Test in Primary Hepatocytes
from Transgenic
(A4V/G93A) Mice & Rats
Test Lead ASOs for
SOD-1 Inhibition in
Transgenic Mice/Rats via
Systemic & ICV TX
Select Human Candidate
Primate PK & Toxicology
3, 145-1456
Stem cells
Cova and Silani
SLA: malattia extramotoneuronale?
Tg SOD1
Ilieva et al., 2009
2010
Human Clinical
Trials (2010)
Chen et al., 2007
Chew et al.,
2007
Mazzini et al., 20042008
Cashman et al., 2008
Appel et al.,
2008
Huang et al.,
2000
Deda et al.,
2009
Martinez et al.,
2009
Huang et al.,
2009
Blanquer et al., 2010
1 PD , 14 yrs after grafting
TH
VMAT2
DAT
No neuromelanin
GENETICA DELLA SLA
SLA familiare e SLA sporadica
Clinicamente e neuropatologicamente indistinguibili
UNICA MALATTIA
SALS 90%
Fattori Genetici
+
Fattori Ambientali
Malattia
multifattoriale con
eziopatogenesi ignota
h2 = 0.38 – 0.78
Altri
geni
FALS 10%
Geni sconosciuti
FUS
TDP43
Altri
SOD1
SOD1 SOD1
Malattia monogenica
mendeliana con
eziopatogenesi nota
Perché studiare la SLA familiare?
Invecchiamento
Fattori
genetici
Tossine
ambientali
?
?
?
SALS
Modello animale
(SOD1, TARDBP…)
MODELLO
PATOGENETICO
FALS
Mutazione in un
singolo gene
TERAPIA
DELLA SLA
Genetica della SLA Familiare
ALS-type
Onset
Inheritance
Locus
Gene
Protein
ALS1
Adult
AD (AR)
21q22.1
SOD1
Cu/Zn superoxide dismutase
ALS2
Juvenile
AR
2q33-35
ALS2
Alsin
ALS3
Adult
AD
18q21
Unknown
-
ALS4
Juvenile
AD
9q34
SETX
Senataxin
ALS5
Juvenile
AR
15q15-21
SPG11
Spatacsin
ALS6
Adult
AD2
16p11.2
FUS
Fused in sarcoma
ALS7
Adult
AD
20p13
Unknown
-
ALS8
Adult
AD
20q13.33
VAPB
VAMP-associated protein B
ALS9
Adult
AD
14q11
ANG
Angiogenin
ALS10
Adult
AD
1q36
TARDBP
TAR DNA-binding protein
ALS11
Adult
AD
6q21
FIG4
PI(3,5)P(2)5-phosphatase
ALS12
Adult
AR/AD
10p15-p14
OPTN
Optineurin
ALS-FTD1
Adult
AD
9q21-22
Unknown
-
ALS-FTD2
Juvenile
AD
9p13.2-21.3
Unknown
-
ALS
Adult
AD
12q24
DAO
D-amino acid oxidase
ALS
Adult
AD
7q21.3
PON
Paraoxonase
ALS
Adult
AD
9p12-13
VCP
Valosin Containing Protein
VCP
CHMP2B
MAPT
FTD
TARDBP
FUS
Pure
LMN
CMT
HMN
SBMA
PMA
ALS
PLS HSP/SPG
UMN
HSP 27
NF-L
Senataxin
SOD1
Dynactin (DCTN1)
VAPB
Glycyl tRNA synthetase
PON
HSP 22
SMN1
Seipin (BSCL2)
IGHMBP2
Androgen receptor
Pure
ALSIN
Spastin
OPTN
FIG4
Paraplegin
Atlastin
NIPA1
KIF5A
HSP 60
Spartin
Superossido Dismutasi 1
•
•
•
•
•
•
•
Chr 21q22.1 - 5 esoni
Enzima citoplasmatico Cu/Zn dipendente
Omodimero di 32 kDa
Monomero di 153 amminoacidi
Otto β-foglietti disposti a cilindro
Espressione costitutiva e ubiquitaria
Catalizza la trasformazione del radicale
superossido in ossigeno molecolare e
perossido di idrogeno
2H+
 +O
2
SOD-Cu1+
(ridotta)
(ossidata)
H2O2
GSH perossidasi
catalasi
H2O + ½ O2
SOD-Cu2+
O2
O
2
~150 mutazioni
>>> mutazioni missenso, AD
Correlazione genotipo/fenotipo
Non correlazione tra
stabilità/attività dell’enzima
mutato e fenotipo clinico
Mutazioni di SOD1:
effetti biologici
GAIN OF FUNCTION:
• I topi transgenici per SOD1
enzimaticamente attiva
(hSODG93A) e inattiva
(hSOD1G85R) sviluppano la
malattia
• I topi mSOD1 -/- non
sviluppano la malattia
• La delezione di mSOD1 non
modifica la progressione nel
topo hSOD1G85R
• I topi che iperesprimono
hSODwt sono sani
• L’iperespressione di hSOD1wt
nel topo hSOD1G85R non
modifica la progressione di
malattia
STRESS OSSIDATIVO
DISFUNZIONE MITOCONDRIALE
ECCITOTOSSICITÀ GLUTAMMATERGICA
DISFUNZIONE DEL TRASPORTO ASSONALE
RIDUZIONE DI FATTORI TROFICI
DISFUNZIONE GLIALE
ATTIVAZIONE DELLE CASPASI
AGGREGAZIONE PROTEICA
Mutazioni
Dimero
SOD1
Aggregati
Proteici
Citoscheletro
Oligomerizazzione
Proteine
Cellulari
Mitochondria
Proteasoma
Chaperone
Coorte studiata:
FALS 18/156 11%
SALS 6/566 1%
SLA familiare
A4V
L84F
L144F
G93D
V5M
A95G
G12R
F45C
V47F
D101G
(25 Pz.)
L144F
G41S
L84F
A4V
SLA sporadica
Q22R
F45C
A95T
V97L
I113T
D90A
D90A
G93D
F45C
Mutazioni di SOD1:
correlazioni genotipo-fenotipo
PENETRANZA
SITO DI
ESORDIO
DECORSO DI
MALATTIA
Completa
A4V, G41S, H43R, H46R, L84F, L84V, D90Ahom, E100G, L144F
Incompleta
A4T, L8Q, N19S, E21G, N65S, D76Y, D90Ahet, G93S, I113T
Spinale
G37R, H46R, D76V, L84F, L84V, D90Ahom, E100K, E100G
Bulbare
A4T, C6G, L8Q, D76Y, V148I, I151T
Variabile
A4V, G41S, N86S, D90Ahet, I113T, L144F
Rapido
A4T, A4V, C6F, C6G, V7E, L8Q, G10V, G41S, G93A, I112T G127X
Medio
G85R, G93R, G93V, E100G, D101G, G108D, L126X
Lento
G41D, H46R, D76V, A89V, D90Ahom, G93D, E100K
Variabile
E21G, G37R, L38V, D76Y, L84F, D90Ahet, G93R, I113T, L144F
Mutazione D90A
AD
- eterozigote (pochi), anche in casi SALS
- fenotipo molto variabile e più aggressivo
- progressione rapida della malattia
- mutazioni D90A descritte in Francia, UK, Belgio, Bielorussia, USA
- penetranza variabile
AR
- omozigote o composta (D96N)
- fenotipo caratteristico e uniforme (inizio con paresi agli arti inferiori)
- progressione lenta della malattia e lungo tempo di sopravvivenza (14 anni)
- allele D90A molto frequente nella popolazione della Scandinavia del Nord (2.5%)
- pazienti D90A omozigoti descritti anche in Italia, Germania, Francia, Russia
- penetranza completa
Effetto fondatore della D90Ahom
SLA D90Ahet:
1. Un gene malattia
2. Fenotipo variabile
3. Più aggressiva
4. In popolazioni ”outbred”
Mutazione D90A originale
(895 generazioni fa)
SLA D90Ahom:
1. Due geni malattia
2. Un fenotipo uniforme
3. Progressione lenta
4. In popolazioni isolate (”inbred”)
Allele fondatore D90Ahom
con fattore modificatore
“protettivo” in cis
(promotore?)
(63 generazioni fa)
pazienti SLA D90Ahom in
Scandinavia e Russia
(43-45 generazioni fa)
pazienti SLA D90Ahet
pazienti SLA D90Ahom
in Francia e Italia
Distribuzione dell’allele SOD1 D90A
Neuropathology of ALS and TDP-43
•
•
•
•
•
Extensive loss of anterior horn cells
Degeneration of Betz cells and other pyramidal neurons in the primary motor
cortex
Degeneration of corticospinal tracts
Reactive gliosis in the motor cortex and spinal cord
Presence of various inclusion bodies in degenerating neurons and surrounding
astrocytes
Bunina Bodies
UBIs
Skein-like
80-100% SALS
Cystatin-C
Lewy body-like
~100% SALS
HCIs
less specific
neurofilaments
Ubiquitinated TDP-43 in ALS and FTLD
• TDP-43 is the major protein
component of UBIs in SALS,
non-SOD1 FALS and FTLD-U
• Biochemical signature:
– Disease specific
hyperphosphorylated protein
at ~45 kDa
– Ubiquitinated HMW smear
– Truncated C-terminal
fragments at ~25 kDa
• Clearing of nuclear TDP-43
from UBI-bearing neurons
ALS AD PD C
AP+
P anti-TDP
TDP UBI
Neumann et al, Science 2006
TDP-43
2006
TAR DNA binding protein 43
• TDP-43 is encoded by the TARDBP gene on chromosome 1
• TDP-43 belongs to the hnRNP family
• TDP-43 known functions
–
–
–
–
Trascriptional regulation (HIV-1 TAR DNA element, mouse SP-10 promoter)
Splicing regulation (CFTR exon 9, Apo A-II exon 3, SMN2 exon 7)
mRNA stabilization (hNFL) and transport
mRNA translation and SG formation
Mackenzie et al., Lancet Neurology 2010
Coorte studiata:
FALS 6/125 4.8%
SALS 12/541 2.2%
Upper limb onset (Millecamps et al., 2010)
• 149 French FTLD-MND (71 familial – 78 sporadic)
• 3 variants in 9 patients
first evidence of pathogenic mutation as causative of behavioural
variant of FTD without MND – 74 y/o - bvFTD
TDP-43 toxicity: key events
• Cytoplasmic redistribution
• Aggregate formation
GAIN OF FUNCTION vs LOSS OF FUNCTION
Ticozzi et al., CNS&ND-DT 2010
Effects of TARDBP mutations:
gain of function?
TDP-43 is intrinsecally
aggregation prone
in vitro
WT
Q331K
M337V
G294A
ALS-associated TARDBP
mutants accelerate
aggregation
in vitro
Johnson et al, J Biol Chem 2009
ALS-associated TARDBP
mutants increase
aggregation and toxicity
in cell models
Nonaka et al, Hum Mol Genet 2009
Effects of TDP-43 aggregation:
gain of function?
Merge
Hoechst
GFP
Caspase-3
Merge
GFP-TDP-25
Flag-TDP-43
GFP-TDP-43
Flag-DTP-43
Flag
GFP-TDP-43
C-terminal fragments
are toxic to cells and
increase apoptosis
GFP
GFP-TDP-25
Full lenght TDP-43 is not
recruited into cytoplasmic
aggregates and its
nuclear function is not
impaired
Hoechst
Zhang et al, PNAS 2009
Effects of TDP-43 aggregation:
loss of function?
Full lenght TDP-43 may
be recruited into
cytoplasmic aggregates
of C-terminal fragments
DsRed-TDP wt
GFP-TDP wt
C-terminal fragments
may impair TDP-43
nuclear localization
and function
1
DsRed-TDP wt
GFP-TDP 162-414
162
218
274
DsRed-TDP wt
GFP-TDP 218-414
315
414
360 bp
177 bp
GFP
WT
162
414
218
414
274
414
315
414
1
314
1
273
1
217
1
161
Nonaka et al, Hum Mol Genet 2009
Loss of function - other evidences
• Flies lacking the Drosophila TDP-43 homolog TDBH present
deficient locomotor behaviors, reduced life span and anatomical
defects at the neuromuscular junctions. The expression of human
TDP-43 rescues the phenotype (Feiguin et al., FEBS Lett 2009)
• Prp-TDP-43A315T transgenic mice develop a disorder reminiscent of
ALS and FTLD-U, with formation of UBIs, but cytoplasmic aggergates
are NOT positive for TDP-43 (Wegorzewska et al., PNAS 2009)
• Loss of TDP-43 leads to CCDK6 activation and phosphorylation of
pRb resulting in deformation of the nuclear membrane,
dysregulation of the cell cycle and apoptosis (Iguchi et al., J Biol Chem 2009)
• The knockdown of TDP-43 in N2A cells inactivates Rho-GTPases,
inhibits neurite outgrowth and causes cell death (Ayala et al., PNAS 2008)
FUS/TLS (2009)
FUsed in Sarcoma
• FUS/TLS belongs to a family of DNA/RNA binding
proteins (TET)
–
–
–
–
–
cancer-associated fusion genes
highly conserved structure
N-terminal transactivating domain
RNA binding domain (GGUG)
C-terminal NLS
Mackenzie et al., Lancet Neurology 2010
FUS/TLS biological activities
Transcriptional regulation and start-site recognition
Splicing regulation
mRNA maturation
Nucleo-cytoplasmic RNA shuttling
mRNA transport
Genome stability
NF-kB
RNA
Pol II
TFIID
YB-1
RGG
SYQG-rich
Nuclear
hormone
receptors
RNA
RGG
RRM
ZnF
dsDNA
SFRS2
TASR1/2
CBP
ssDNA
FUS/TLS and genome stability
•
High-level of chromosomal instability in FUS -/- mice (Hicks et al, Nat Genet 2000)
•
Male FUS -/- mice are sterile and display defects in meiotic process, increased
sensitivity of fibroblasts to ionizing radiations (Kuroda et al, EMBO J 2000)
•
FUS is a target of ATM (Gardiner et al, Biochem J 2008)
•
FUS promotes DNA repair after double-stand breaks (Baechtold et al, J Biol Chem 1999)
•
FUS inhibites CBP/p300-mediated histone acetylation in response to DNA
damage signals (Wang et al, Nature 2008)
Wang et al, Nature 2008
FUS/TLS activities in CNS
• FUS is involved in mRNAs translocation to the dendritic spines for
local translation and may play a role in synaptic plasticity:
– FUS is recruited and accumulated in mouse dendritic spines of
excitatory post-synaptic sites
– FUS is localized in RNA -containing particles and associates with
actin-stabilizing protein Nd1-L mRNA
– FUS colocalizes with NMDAR complexes in mice brain tissue
– mGluR5 activation reversibly increases FUS recruitment and
accumulation
– FUS -/- mice show an abnormal dendrite morphology and reduced
spine density
(Fujii et al, Cell Biol 2005 and Fujii et al, J Biol Chem 2005))
• FUS is a major nuclear aggregate-interacting protein in HD
– FUS binds polyQ aggregates in vivo and in vitro
– FUS colocalizes with polyQ aggregates in HD human brain tissues
– SYQG-rich domain is essential for binding
(Doi et al, J Biol Chem 2008)
FUS/TLS in Italian FALS
FTD
J Med Genet, in press
94 Pazienti FALS
SOD1, TARDBP e ANG negativi
4 mutazioni identificate in 5 Pazienti (5.3%)
964 Pazienti SALS
45 Pazienti FALS
2 mutazioni in NLS
6 mutazioni identificate in 7 SALS (0.6%)
2 mutazioni identificate in 2 FALS (4.4%)
R521G, R521C
1 mutazione in NLS
2 nuove mutazioni missenso
R521C
G156E (SYQG-rich domain)
R234L (G-rich domain)
6 nuove mutazioni missenso
IDENTIFICAZIONE DI UN FENOTIPO COMUNE:
Esordio prossimale simmetrico
Coinvolgimento precoce della muscolatura
assile
Prevalenza di segni di interessamento di LMN
UN PAZIENTE CON ALS-FTD
G191S, R216C, G225V, G230C, R234C
(G-rich domain)
G507D
(RGG-rich domain)
CONFERMA DEL FENOTIPO COMUNE NEI DUE
PAZIENTI CON p.R521C
R521C
Effects of FUS/TLS mutations
CTRL
N2A
FALS
SKNAS
• Mutations cause FUS redistribution from nuceus to cytoplasm
• Mutations cause aggregates in neural cell lines
• Mutations in NLS do not alter FUS RNA binding properties
GFP-FUS(R521G)
CTRL
FALS
GFP-FUS(WT)
WT
NeuN
FUS
DAPI
H517Q
R521G
WT
H517Q
R521G
Merge
Kwiatkowski et al, Science 2009
Splicing defects and
Neurodegenerative diseases
• Alternative splicing is highly abundant in brain
relative to other tissues, where it allows cells to
modulate their protein composition in response
to different stimuli.
• Alternative splicing patterns are dependent on
the interaction between different RNA binding
proteins and common regulatory elements in the
pre-mRNAs.
• Disrupting the function of a single RNA binding
protein can affect many alternatively spliced
transcripts, a phenomenon that is increasingly
recognized as having a role in human diseases.
cis-Acting Splicing Disorders
• Neurofibromatosis type I,
Ataxia-Teleangiectasia
– 50% of mutations are associated with pre-mRNA
splicing defects
• Muscular Dystrophy
– some mutations induce exon skipping
• Frontotemporal Dementia with Parkinsonism – 17
– alternative splicing of exon 10 regulates relative levels
of tau isoforms (4R – 3R)
– several mutations are clustered around exon 10
• Spinal Muscular Atrophy
trans-Acting Splicing Disorders
• Disruption of Spliceosome assembly
– Spinal Muscular Atrophy
• Lack of SMN leads to defective assembly of snRNPs
– Retinitis Pigmentosa
• Mutations in genes encoding snRNPs-associated factors
• Indirect Targeting of RNA binding proteins
– Myotonic Dystrophy type 1 and 2
• CUG/CCUG expanded mRNAs bind and sequester alternative
splicing modulators MBNL and CUG-BP1
• Alterations in splicing of CLCN1, NMDAR1, MAPT and APP
– Fragile-X-associated Tremor Ataxia Syndrome
• sequestration of MBNL and hnRNP A1
• Direct Targeting of RNA binding proteins: ALS? FTLD-U?
RNA metabolism in neurodegeneration
ALS Neuropathology
UBIs
SOD1 positive
TDP-43 positive
FUS positive
Unknown
ALS2 (alsin)
ALS4 (SETX)
ALS1 (SOD1)
SALS
ALS6 (FUS)
ALS5 (SPG11)
non-SOD1 FALS
ALS8 (VAPB)
ALS10 (TARDBP)
ALS9 (ANG)
ALS12 (OPTN)
OPTN positive?
FTLD Neuropathology
FTLD-tau
Pick’s disease
FTLD-U
TDP-43 positive
TDP-43 negative
PSP
CBD
Type 1
bvFTD, PNFA (GRN)
FUS positive
FUS negative
AGD
Type 2
SD
MSTD
Type 3
bvFTD, FTD-MND
aFTLD-U
FTD3 - CHMP2B
Type 4
FTD-VCP
NIFID
BIBD
The ALS – FTLD Continuum
Seelar et al., JNNP 2010
Genetica della SLA Sporadica
Genome-wide Association Studies
Ricerca varianti rare
Whole Genome Association Studies (GWA)
Lavoro
Anno
Paese
SALS
CTRL
Associazione
Significatività Conferma
statistica
Schymick
2007
USA
276
271
no
n/a
n/a
Dunckley
2007
USA
386
(901)
542
(1025)
FGGY
Sì?
No
Van Es
2007
Svezia,
Belgio
Olanda
461
(876)
450
(906)
ITPR2
Sì?
No
Van Es
2007
Svezia,
Belgio
Olanda, USA
1767
1916
DPP6
Sì
Dubbia
Cronin
2008
Irlanda, USA,
Olanda
958
932
DPP6
No
Dubbia
Chiò
2009
USA, Italia
553
(2160)
2338
(3008)
SUNC1
No
n/a
Landers
2009
USA, Francia
UK, Olanda
1821
2258
KIFAP3
Sì
No
Van Es
2009
Europa, USA
2323
(2532)
9013
(5940)
UNC13A
Sì
Si
Shatunov
2010
Europa, USA
4312
8425
9p21
Sì
Si
1821 SALS e 2258 controlli (US e Europa)
288,357 SNP
Associazione con rs1541160 (p=1.84x10-8)
Incremento di sopravvivenza di 14 mesi per genotipo CC
rs1541160 (introne 8): non varianti in regioni codificanti
rs1541160 in LD con rs522444 nel promotore di KIFAP3
Creazione sito Sp1 (allele C)
Ridotta espressione di KIFAP3 (~40%)
Kinesin-Associated Protein 3
STUDIO DI REPLICA:
273 SALS Italiani
CC=3.83 yrs. (22)
CT=2.75 yrs. (111)
TT=2.29 yrs. (140)
AUMENTO DELLA SOPRAVVIVENZA
18.5 mesi
unpublished data
(p=0.017)
KIF3
KIFAP3 è parte del complesso KIF3 (kinesina II)
Trasporto di organelli cellulari verso l’estremità positiva
del microtubulo
KIFAP3
Eterotrimero: 2 subunità motorie (KIF3A e KIF3B) ed
una subunità di legame per il cargo (KIFAP3)
KIFAP3 lega mutSOD1, ma non wtSOD1
KIFAP3 è presente negli aggregati neuronali nel topo
hSOD1G93A
KIF3A
KIF3B
KIFAP-3
CC
SNP rs1541160
TC
TT
Courtesy Orsetti et al., 2011
Cromosoma 9
Consorzio SLAGEN
CENTRI FONDATORI:
CENTRI PARTECIPANTI:
IRCCS Istituto Auxologico Italiano
IRCCS Istituto Neurologico Besta
IRCCS Istituto Neurologico Mondino
Università degli Studi del Piemonte Orientale
A.O. Ospedale Niguarda
IRCCS Ospedale Maggiore Policlinico
Centro Clinico NEMO
Università degli Studi di Padova
Università degli Studi di Pisa
Università degli Studi di Brescia
CNR di Cosenza
Università degli Studi di Ferrara
Università degli Studi di Firenze
Università Federico II di Napoli
Università La Sapienza di Roma
OBIETTIVO:
WGAS su 2000 SALS di origine Italiana e 2000 controlli
Human660W-Quad
550.000 SNP
100.000 CNV
Suscettibilità
Età di esordio
Sito di esordio
Sopravvivenza
SALS: genetic risk factors
Paraoxonases
9 exons, 354-5 residues
Homology between PONs >80%
Six-bladed b-propeller (6 x 4 b-sheets)
Three a-helix regions
Ca2+-dependent enzyme
Expression modified by genetic and
environmental factors
(drugs, diet, smoke, alcoohl, Pb)
Paraoxonases and SALS
Five independent reports showed an association between haplotypes in
the PON cluster and SALS susceptibility…
...HOWEVER
No association from GWAs
Metanalysis was negative
(Wills et al. 2009)
Other studies were negative
PON1 and FALS
COHORT STUDIED:
-1st step (direct sequencing)
260 FALS (US and Italian)
188 SALS
188 CTRLs
-2nd step (genotyping)
996 SALS
971 CTRLs
Nucleotide
Mutation
Position
FALS (260)
SALS (1184)
CTRL (1159)
c.55>G
N19D
Ex 1
2
6
3
c.74+3>G
Splicing
Int 1
1
0
0
c.124T>G
C42R
Ex 2
1
0
0
c.269T>C
L90P
Ex 4
1
1
0
c.437T>G
M127R
Ex 5
2
6
2
c.438G>T
M127I
Ex 5
1
0
0
c.602C>T
A201V
Ex 6
4
3
3
c.943C>A
P315T
Ex 9
1
0
0
Total
13
5
PON2-3 and FALS
COHORT STUDIED:
-1st step (direct sequencing)
166 FALS (US and Italian)
-2nd step (genotyping)
996 SALS
971 CTRLs
PON2
PON3
Nucleotide
Mutation
Position
FALS (166)
SALS (1184)
CTRL (1159)
c.95G>A
C42Y*
Ex 2
1
0
0
c.286delA
R96GfsX5
Ex 4
1
6
4
c.361G>A
D121N
Exon 4
1
1
0
c.688G>A
D230N
Exon 6
2
1
0
c.971G>A
G324D
Exon 9
2
1
3
Total
7
4
* Mutation homozygous in a proband whose parents were
asymptomatic first cousins (suggesting AR)
Novel PON variants:
Disease specific mutations?
In total, from 9 FALS and 3 SALS, 8 coding sequence mutations
present in PON genes but not in controls
-mutation in an AR pedigree
Gene
FALS
SALS
CTRL
N
%
N
%
N
%
PON1
5/260
1.9
1/1184
0.1
0/1159
0.0
PON2
1/166
0.6
0/1184
0.0
0/1159
0.0
PON3
3/166
1.8
2/1184
0.2
0/1159
0.0
Total
9
4.3
3
0.3
0
0.0
Pathogenic mutations?
• PON mutations affect highly conserved residues
• In silico analysis predicts that mutations are deleterious
• C42 residue is mutated both in PON1 and PON2 (cysteine bond)
• homozygous C42Y mutation in progeny of first-cousin marriage
• three mutations are present in unrelated FALS cases
PON mutations - Multiplicity of PON substrates
– Properties shared by mutated PONs :
Possible relations to ALS pathogenesis
•
Altered metabolism of xenobiotics:
– Reduced metabolism of organophosphate compounds and/or
other neurotoxins
– Altered activity for specific substrates
•
Loss of physiological properties:
–
–
–
–
Loss of antioxidant activity is neurotoxic
Increased lipoperoxidation of cell membranes
Increased ER-stress
Acceleration of motor neuron aging
“Geni mancanti”:
Approcci classici allo studio delle malattie mendeliane
Linkage analysis
• whole-genome analysis
• rapida ed efficace
• relativamente economica
• SOD1, ALS2, SETX, VAPB, OPTN, FUS
MA:
• necessarie famiglie con numerosi
individui affetti in più generazioni
• difficoltosa in malattie ad esordio adulto
e rapido decorso come la SLA
Screening di geni candidati
• possibile in piccole famiglie o coorti di Pz.
• TARDBP, ANG, PON, FIG4
MA:
• analisi lenta e costosa
• non whole-genome
• scarsi risultati (selection bias)
IMPOSSIBILE STUDIARE VARIANTI
RARE SU SCALA GENOMICA IN
COORTI NUMEROSE
Next Generation Sequencing



Pyrosequencing
(Genome Sequencer FLX System –
454 LifeSciences, Roche)
Sequencing by Ligation
(SOLiD System – Applied Byosystem)
Sequencing by synthesis,
reversible chain termination methods
(Solexa – Genome Analyzer, Illumina)


Miglior rapporto qualità/prezzo

40.000 USD per genoma

15 Gb per microarray
Rapido


7 giorni per microarray
Disponibili molti software per l’analisi
bioinformatica dei dati
Problemi:
Sequenziare l’intero genoma in una coorte
di pazienti è ancora troppo costoso
Il whole-genome sequencing produce
“troppi” dati, difficili da interpretare con i
modelli esistenti
Exome Sequencing
L’ESOMA è la parte del genoma formata da
esoni, cioè da quelle porzioni di geni che sono
espresse e che forniscono il modello genetico
utilizzato nella sintesi di proteine e di altri
prodotti genici funzionali. È la parte
funzionalmente più rilevante del genoma, con
maggiori probabilità di contribuire al fenotipo di
un organismo.





L’esoma rappresenta circa l’1% del genoma umano (30 Mb su 3Gb)
La maggior parte (>85%) delle malattie mendeliane sono causate da mutazioni
nell’esoma
Le nostre consocenze attuali sulle conseguenze funzionali delle mutazioni al di fuori
dell’esoma sono molto limitate
L’exome sequencing è molto più economico del whole genome sequencing
L’esoma è quindi una regione ideale per la ricerca di mutazioni rare con alta
penetranza in coorti numerose
Exome Sequencing e malattie mendeliane

Malattie monogeniche

Generalmente malattie rare, ma 200.000 affetti negli USA e 35.000 in Italia

7.000 malattie mendeliane descritte

mutazione patogenetica sconosciuta in >2.000
VANTAGGI
• l’esoma rappresenta l’1% del genoma
• la maggior parte delle malattie
mendeliane sono causate da mutazioni in
regioni codificanti
• costi 10 volte inferiori al whole-genome
sequencing
SVANTAGGI
• non individua mutazioni in regioni non
codificanti
• ogni individuo ha ~600 nuovi SNPs
codificanti non precedentemente descritti
• necessari metodi di “filtraggio” per
identificare mutazioni patogenetiche
(+ individui)
Exome Sequencing: proof of concept
4 individui affetti da Sindrome di Freeman-Sheldon (artrogriposi distale 2A)
Mutazione nel gene MYH3
Exome Sequencing: stato dell’arte
Freeman-Sheldon sy
Bartter sy
Miller sy
Fowler sy
Perrault sy
Kabuki sy
Severe brain malformation
Sesenbrenner sy
Hyperphosphatasia MRS
Retinal-renal ciliopathy
Van Den Ende-Gupta sy
Anal atresia
Carnevale sy
Severe hypercholesterolemia
Familial hypolipidemia
Complex I deficiency
SCA
FAD deficiency
VCP-ALS
mutazione nel gene
DHODH
18
14
7
2
Jan-Apr
2009
May-Aug
2010
Sept-Dec
Jan-Apr
May 
2011
Seckel sy
Retinitis pigmentosa
Familial hypercolesterolemia
Intractable IBD
CMT
Dilated cardiomiopathy
Osteogenesis imperfecta
Haidu-Cheney sy
Failure of tooth eruption
Hereditary hypotrichosis
X-linked leucoencephalopathy
Acne inversa
Ochoa sy
Novel skeletal dysplasia
Non-syndromic MRS
Primary limphoedema
Primary microcephaly
Distal artrogriposis
HSP
HSN - dementia - hearing loss
Hereditary progeroid sy
Chondrodysplasia
Amelogenesis imperfecta
Infantile mt cardiomiopathy
Mosaic variegated aneuploidy
ExomeFALS - Dati preliminari
Partnership Istituto Auxologico - Istituto Besta - Università del Massachusetts
Tra il 1995 e il 2010 è stata raccolta un’ampia casistica di DNA di
pazienti Italiani, fenotipicamente caratterizzati:
200 FALS
1300 SALS
Con il Partner americano, il consorzio EXOMEFALS dispone di:
450 FALS
3000 SALS
Tale coorte FALS è fino ad oggi la più grande raccolta al mondo
Lo studio di questa coorte ha prodotto informazioni essenziali
sull’epidemiologia genetica della SLA in Italia
(SOD1, ANG, TARDBP, FUS, PON, OPTN, VCP)
ExomeFALS: Dati preliminari
25 Individui sequenziati
SNPs
Totale
Nuovi
%
TUTTI
13,805
946
6.8
non sinonimi
6,411
603
9.4
sinonimi
7,394
343
4.6
ETEROZIGOSI
8,736
911
10.4
non sinonimi
4,096
583
14.2
sinonimi
4,640
328
7.1
OMOZIGOSI
5,069
35
0.7
non sinonimi
2,315
21
0.9
sinonimi
2,755
15
0.5
numerose varianti in alcuni
“geni malattia” (?)
Exome sequencing come
“controllo” di precedenti
studi di genetica medica
Necessità di creare
Database condivisi
Istituto Auxologico Italiano
Università degli Studi di Milano
Unità Operativa di Neurologia
Laboratorio di Neuroscienze
Vincenzo Silani
Laura Adobbati
Luca Campana
Andrea Ciammola
Barbara Corrà
Alberto Doretti
Riccardo Doronzo
Carolina Lombardi
Luca Maderna
Niccolò Mencacci
Stefano Messina
Claudia Morelli
Barbara Poletti
Davide Sangalli
Federico Verde
Antonia Ratti
Claudia Colombrita
Clarissa Colciago
Lidia Cova
Valentina Diana
Maura Figini
Elisa Onesto
Jenny Sassone
Cinzia Tiloca