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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

Une leçon du Docteur Charcot à la Salpêtrière – André Brouillet, 1887

Charcot, J. M. & Joffory, A.

Deux cas d’atrophie musculaire progressive avec

lesions de la substance grise et des faisceaux antero-

lateraux de la moelle epiniere.

Arch. Physiol. Neurol. Pathol. 2, 744–754 (1869).

Sclerosi Laterale Amiotrofica

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

Bertram L and Tanzi RE, The Journal of Clinical Investigation 2005

SLA e le altre malattie neurodegenerative

SLA e Malattie

del Motoneurone

Una malattia da vulnerabilità

selettiva di un sistema

Mitocondrio

Transporto Assonale

Motoneurone e vulnerabilità

The majority of patients with

adult-onset motor neuron

disease will be found to have

IDIOPATHIC ALS

Eziopatogenesi

Ravits et al., 2009

Diffusione

Focalità all’ esordio

Ravits et al., 2007

Diffusione

Clinical syndromes of ALS

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, thenprogressive speach and swallowingdifficulties;limb involvement follows (months oryrs);M:F ratio: 1:1 (PBP > common inolder 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 butmajority 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 UMNsyndrome;after few yrs may convert to ALS.

10 yrs or more.

Syndrome Main clinical features Prognosis

Clinical syndromes of ALS (cont.)

“Flail arm syndrome”; man in abarrel syndrome; Vulpian-Bernhard syndrome

Syndrome of predominantly LMNweakness of both arms;UMN signs develop in 50-70%;often slow progression;pathology is that of ALS.

About 10% of all cases;M:F ratio 9:1;prognosis better than in ALSsyndrome more common in Africanand Asian patients.

“Flail leg syndrome”;“pseudopolyneuritic form” ofALS; Patrikios syndrome

Syndrome of progressive legweakness, predominantly LMN.

Rare;slow progression;DD difficult.

Monomelic forms of ALS Rare variants of ALS with slowlyprogressive focal (upper > lowerlimb UMN and LMN syndrome);Distinct LMN form most common inAsia (monomelic juvenile onsetamyotrophy; Hirayama’s syndrome);DD with multifocal motorneuropathy.

Juvenile onset form progressiveover months or several yrs and thenstabilises;does not generalises;pathology unknown.

ALS-dementia syndrome(ALS-D)

Dementia of fronto-temporal typepresent in 5% of all ALS cases;20-40% ALS patients have subtlecognitive changes of “frontal” type;ALS-D may present first withdementia or ALS progressing todementia, or with combination ofboth; about 50% familial.

Usually 2 to 5 yrs.

Syndrome Main clinical features Prognosis

(“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 Ascertain possible ALS-related syndromes.

5. Neuropathologic examinations Ascertain findings confirming/excluding ALS

6. Repetition of clinical and EMG Ascertain evidence of progression

(6 months apart)

Mitsumoto et al, 1998, 2006

CRITERI CLINICI

Trofismo

Tono

Stenia

ROT

Segni patologici

UMN

LMN

REGIONS

Bulbar Cervical Thoracic

Abdominal Lombar

+

+

+

+ + +

+ +/-

Clinically definite ALS

Criteri di El Escorial

Neurofisiologia

EMG

ENG

TMS-MEPs

EMG

• 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

200 msec L 100uV 200 msec L 100uV

EMG





• Massima Contrazione – Reclutamento PUM

200 msec L 200 uV

EMG





• Massima Contrazione – Reclutamento PUM <

200 msec L 1mV

SEGNI DI DENERVAZIONE IN FASE ATTIVA

• Potenziali di fibrillazione.

• Onde positive appuntite o sharp waves.

EMG

NUOVI CRITERI ELETTROMIOGRAFICI

DI EL ESCORIAL PER LA DIAGNOSI DI LMN

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.

EMG



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.





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.



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

• 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).

MRI

Filippini et al., Neurology, 2010

Neuroimaging

Diagnosis flow for ALS patients

1) First consultation • Hearing

• Neurological exam

2) Exclusion of other dubious diseases

(hospitalization)

• Blood Biochemistry

• Needle EMG (electromyogram)

• Nerve conduction study

•ＭR

• C.S.F.

• (Muscle biopsy)

3) ALS diagnosis

• Diagnosis Standard of ＷＦＮ (El

Escorial)

• Therapy (riluzole)

• Therapeutical Plan (Rare Disease)

• Follow-up visit in 1-2 months

6) Progression of Disease

• ALSFRS-R

• BMI

• FVC/ Pulmonary functional test/ Blood

gas test

5) Confirmation of Disease

• After 3-6 months

• Second opinion

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


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


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)

• 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)

DD: 1 in 35 patients initially diagnosed as having ALS may have SBMA

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)

“ patients are not demented and cognition is spared “

1874 Jean-Martin Charcot,

SLA e Disturbi Cognitivi

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

Strong et al., 2009

5 to 15%

25 to 50%

Strong et al., 2099

5% dei Pazienti FTD hanno segni clinici o subclinici di sofferenza LMN

Phukan et al., 2007

Anomalie neuropsicologiche nella SLA

Phukan et al., 2007

Terapia della SLA

Sintomatica Nutrizione

Respirazione

Fisioterapia

Palliazione

II° MN

(V, VII, IX, XII)

+/-

perdita

innervazione I°

Disfagia

PERDITA DI PESO & MALNUTRIZIONE

NUTRIZIONE

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

• 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

HOW DETECT DYSPHAGIA?

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

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)

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

What are the limits of NIV ?

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


• Pseudobulbar emotional lability

– Dextromethorphan and quinidine (IA)

– Fluvoxamine

– Amitriptyline

– Citalopram

– Dopamine

– Lithium


• Cramps

– Quinine sulphate 200 mg x 2 and vitamin E (I)

– Physiotherapy

– Carbamazepine

– Diazepam

– Phenytoin

– Verapamil

– Gabapentin


• 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


• Depression, anxiety, and insomnia

– Amitriptyline

– Sertraline

– Fluoxetine

– Paroxetine

– Zolpidem

– Diazepam

– Sub-lingual lorazepam


• 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

Eziologica Farmacologica

Terapia genica?

Cellule staminali?

“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

Traynor et al., 2006

Imputed placebo decline

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%

KNS-760704 (dexpramipexole)

Includes all study deaths to Week 28.

Log rank test: p = 0.0708

KNS-760704: Survival estimates

Terapia Genica?

Determine Antisense oligo Distribution in

CNS Following ICV Administration

= Completed

Identification

of Human

SOD-1 ASO Candidates

Human

Test in Human Fibroblasts

from A4V Patients

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

Rat

Identification of

Rat SOD-1 ASOs

Demonstrate SOD-1

Inhibition in Liver Following

Systemic Administration

Demonstrate SOD-1

Inhibition in CNS Following

ICV Administration

Examine Dose

Schedule Requirements,

PK & Histopathology

Primate PK & Toxicology

ASO Medicinal

Chemistry

SOD1 A4V

Cova and Silani

3, 145-1456

Stem cells

Tg SOD1

Ilieva et al., 2009

SLA: malattia extramotoneuronale?

2010

Human Clinical

Trials (2010) Chen et al., 2007

Chew et al.,

2007

Mazzini et al., 2004-

2008

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%

FALS 10% SOD1 SOD1 FUS

TDP43

Altri

SOD1 Altri

geni

Fattori Genetici

+

Fattori Ambientali

Geni sconosciuti

Malattia

multifattoriale con

eziopatogenesi ignota

h2 = 0.38 – 0.78

Malattia monogenica

mendeliana con

eziopatogenesi nota

Perché studiare la SLA familiare?

Fattori

genetici

Invecchiamento

Tossine

ambientali

? ?

?

Mutazione in un

singolo gene

SALS

FALS

MODELLO

PATOGENETICO

Modello animale

(SOD1, TARDBP…)

TERAPIA

DELLA SLA

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

Genetica della SLA Familiare

Pure

UMN

Pure

LMN

PLS PMA

ALS HSP/SPG HMN CMT

SOD1 ALSIN

Dynactin (DCTN1)

HSP 27

Glycyl tRNA synthetase

Senataxin

VAPB

NF-L Spastin

Paraplegin

Atlastin

NIPA1

HSP 60

KIF5A

Spartin

HSP 22

Seipin (BSCL2)

SMN1

IGHMBP2

Androgen receptor

SBMA

FTD

CHMP2B

MAPT

VCP

FUS

TARDBP

OPTN

FIG4 PON

~150 mutazioni

>>> mutazioni missenso, AD

Correlazione genotipo/fenotipo

Non correlazione tra

stabilità/attività dell’enzima

mutato e fenotipo clinico

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

SOD-Cu1+ SOD-Cu2+

(ridotta) (ossidata)

2H+ + O2- H2O2 H2O + ½ O2

GSH perossidasi catalasi

O2 O2-

Mutazioni di SOD1: effetti biologici

Proteine Cellulari

Proteasoma Chaperone

Mitochondria

Citoscheletro Dimero SOD1

Mutazioni

Aggregati Proteici

Oligomerizazzione

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

A4V

L84F

G93D F45C

L144F

G41S

D90A

SLA familiare A4VL84FL144FG93DV5MA95GG12RF45CV47FD101G

SLA sporadica

Q22RF45CA95TV97LI113TD90A

Coorte studiata: FALS 18/156 11% (25 Pz.) SALS 6/566 1%

Mutazioni di SOD1: correlazioni genotipo-fenotipo

Completa PENETRANZA

DECORSO DI MALATTIA

SITO DI ESORDIO

Incompleta

Spinale

Bulbare

Variabile

Rapido

Medio

Lento

Variabile

A4V, G41S, H43R, H46R, L84F, L84V, D90Ahom, E100G, L144F

A4T, L8Q, N19S, E21G, N65S, D76Y, D90Ahet, G93S, I113T

G37R, H46R, D76V, L84F, L84V, D90Ahom, E100K, E100G

A4T, C6G, L8Q, D76Y, V148I, I151T

A4V, G41S, N86S, D90Ahet, I113T, L144F

A4T, A4V, C6F, C6G, V7E, L8Q, G10V, G41S, G93A, I112T G127X

G85R, G93R, G93V, E100G, D101G, G108D, L126X

G41D, H46R, D76V, A89V, D90Ahom, G93D, E100K

E21G, G37R, L38V, D76Y, L84F, D90Ahet, G93R, I113T, L144F

- 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

AD

Mutazione D90A

- 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

AR

SLA D90Ahom:

1. Due geni malattia

2. Un fenotipo uniforme

3. Progressione lenta

4. In popolazioni isolate (”inbred”)

SLA D90Ahet:

1. Un gene malattia

2. Fenotipo variabile

3. Più aggressiva

4. In popolazioni ”outbred”

Effetto fondatore della D90Ahom

Mutazione D90A originale (895 generazioni fa)

pazienti SLA D90Ahet

pazienti SLA D90Ahom in

Scandinavia e Russia

pazienti SLA D90Ahom

in Francia e Italia

(43-45 generazioni fa)

Allele fondatore D90Ahom

con fattore modificatore

“protettivo” in cis

(promotore?)

(63 generazioni fa)

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 HCIs

Skein-like Lewy body-like

80-100% SALS ~100% SALS less specific

Cystatin-C 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

2006

TDP-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

TAR DNA binding protein 43

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

Ticozzi et al., CNS&ND-DT 2010

• Cytoplasmic redistribution

• Aggregate formation

GAIN OF FUNCTION vs LOSS OF FUNCTION

Effects of TARDBP mutations: gain of function?

WT

Q331K

M337V

G294A

Johnson et al, J Biol Chem 2009

Nonaka et al, Hum Mol Genet 2009

TDP-43 is intrinsecally aggregation prone

in vitro

ALS-associated TARDBP mutants accelerate

aggregation in vitro

ALS-associated TARDBP mutants increase

aggregation and toxicity in cell models

Hoechst GFP Caspase-3 Merge

GF

P-T

DP

-25 G

FP

-TD

P-4

3

Hoechst GFP Flag Merge

GF

P-T

DP

-25 G

FP

-TD

P-4

3

Fla

g-T

DP

-43

F

lag-D

TP

-43

Zhang et al, PNAS 2009

Effects of TDP-43 aggregation: gain of function?

Full lenght TDP-43 is not recruited into cytoplasmic

aggregates and its nuclear function is not

impaired

C-terminal fragments are toxic to cells and increase apoptosis

DsRed-TDP wt

GFP-TDP wt DsRed-TDP wt

GFP-TDP 162-414

DsRed-TDP wt

GFP-TDP 218-414

1 162 218 274 315 414

GFP WT 162

414

218

414

274

414

315

414 1

314

1

273

1

217

1

161

360 bp

177 bp

Full lenght TDP-43 may be recruited into

cytoplasmic aggregates of C-terminal fragments

C-terminal fragments may impair TDP-43

nuclear localization and function

Effects of TDP-43 aggregation: loss of function?

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)

• 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

FUsed in Sarcoma

Mackenzie et al., Lancet Neurology 2010

FUS/TLS biological activities

SYQG-rich

RNA Pol II

Nuclear hormone receptors

TFIID

NF-kB

YB-1

SFRS2

TASR1/2

RGG

RGG

RRM

ZnF

RNA

dsDNA

ssDNA

Transcriptional regulation and start-site recognition Splicing regulation mRNA maturation

Nucleo-cytoplasmic RNA shuttling mRNA transport Genome stability

CBP

FUS/TLS and genome stability

Wang et al, Nature 2008

• 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)

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

94 Pazienti FALS SOD1, TARDBP e ANG negativi

4 mutazioni identificate in 5 Pazienti (5.3%)

2 mutazioni in NLS R521G, R521C

2 nuove mutazioni missenso G156E (SYQG-rich domain)

R234L (G-rich domain)

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

J Med Genet, in press

964 Pazienti SALS 45 Pazienti FALS

6 mutazioni identificate in 7 SALS (0.6%) 2 mutazioni identificate in 2 FALS (4.4%)

1 mutazione in NLS R521C

6 nuove mutazioni missenso G191S, R216C, G225V, G230C, R234C

(G-rich domain) G507D

(RGG-rich domain)

CONFERMA DEL FENOTIPO COMUNE NEI DUE PAZIENTI CON p.R521C

• 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

Effects of FUS/TLS mutations

NeuN FUS DAPI Merge

C

TR

L

F

AL

S

FALS CTRL

N

2A

S

KN

AS

GFP-FUS(WT) GFP-FUS(R521G)

WT H517Q R521G WT H517Q R521G

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

• 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?

trans-Acting Splicing Disorders

RNA metabolism in neurodegeneration

ALS Neuropathology

UBIs

SOD1 positive TDP-43 positive

ALS1 (SOD1) SALS

ALS10 (TARDBP)

ALS6 (FUS)

FUS positive

ALS9 (ANG)

ALS12 (OPTN)

non-SOD1 FALS

Unknown

ALS2 (alsin)

ALS4 (SETX)

ALS5 (SPG11)

ALS8 (VAPB)

OPTN positive?

FTLD Neuropathology

FTLD-tau FTLD-U

Pick’s disease

PSP

CBD

AGD

MSTD

TDP-43 positive TDP-43 negative

Type 1

Type 2

Type 3

Type 4

SD

bvFTD, FTD-MND

bvFTD, PNFA (GRN)

FTD-VCP

FUS positive FUS negative

aFTLD-U

NIFID

BIBD

FTD3 - CHMP2B

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

statistica

Conferma

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

KIF3A KIF3B

KIFAP3

KIF3 KIFAP3 è parte del complesso KIF3 (kinesina II)

Trasporto di organelli cellulari verso l’estremità positiva del microtubulo

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

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

(p=0.017) unpublished data

CC

TC

TT

Courtesy Orsetti et al., 2011

SNP rs1541160

KIFAP-3

Cromosoma 9

Consorzio SLAGEN CENTRI FONDATORI:

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

CENTRI PARTECIPANTI: 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

Nucleotide Mutation Position FALS (260)

c.55>G N19D Ex 1 2

c.74+3>G Splicing Int 1 1

c.124T>G C42R Ex 2 1

c.269T>C L90P Ex 4 1

c.437T>G M127R Ex 5 2

c.438G>T M127I Ex 5 1

c.602C>T A201V Ex 6 4

c.943C>A P315T Ex 9 1

SALS (1184) CTRL (1159)

6 3

0 0

0 0

1 0

6 2

0 0

3 3

0 0

Total 13 5

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

COHORT STUDIED:

-1st step (direct sequencing) 166 FALS (US and Italian)

-2nd step (genotyping) 996 SALS

971 CTRLs

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

PON2

PON3

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)

PON2-3 and FALS

Novel PON variants: Disease specific mutations?

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

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

• 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

Pathogenic mutations?

• 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

PON mutations - Multiplicity of PON substrates – Properties shared by mutated PONs :

Possible relations to ALS pathogenesis

“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

• 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

• 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)

VANTAGGI SVANTAGGI

Exome Sequencing: proof of concept

4 individui affetti da Sindrome di Freeman-Sheldon (artrogriposi distale 2A)

Mutazione nel gene MYH3

2009

Jan-Apr May-Aug Sept-Dec Jan-Apr May

2010 2011

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

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

Exome Sequencing: stato dell’arte

2

14

18

7

mutazione nel gene

DHODH

ExomeFALS - Dati preliminari

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)

Partnership Istituto Auxologico - Istituto Besta - Università del Massachusetts

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


sinonimi 4,640 328 7.1

OMOZIGOSI 5,069 35 0.7


sinonimi 2,755 15 0.5

ExomeFALS: Dati preliminari

25 Individui sequenziati

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

Laboratorio di Neuroscienze

Antonia Ratti

Claudia Colombrita

Clarissa Colciago

Lidia Cova

Valentina Diana

Maura Figini

Elisa Onesto

Jenny Sassone

Cinzia Tiloca

Unità Operativa di Neurologia

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

sclerosi laterale amiotrofica - univacalabria.it · sclerosi laterale amiotrofica: clinica,...

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