Sclerosi Laterale Amiotrofica: Clinica, Genetica, Nuove Prospettive Terapeutiche

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

Ravits et al., 2007 Diffusione

Clinical syndromes of ALS Syndrome Main clinical features Prognosis Classic ( Charcot ) ALS Progressive bulbar palsy (PBP) Progressive muscular atrophy (PMA) Primary Lateral Sclerosis (PLS) Limb onset (spinal); bulbar involvement usual; UMN + LMN signs; M:F ratio 3:2. 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). Almost always limb onset; > 50% develop UMN signs; 85% develop bulbar symptoms; heterogeneous condition but majority are ALS; M:F ratio 3-4. Clinically progressive pure UMN syndrome; after few yrs may convert to ALS. 60-70% of all cases at presentation; median survival 3-4 yrs. 20% of all case at presentation; median survival 2-3 yrs. 10% all cases at presentation; overlap with flail arm and flail leg syndromes; median survival 5 yrs; more long survivors (>10 yrs). 10 yrs or more.

Clinical syndromes of ALS (cont.) Syndrome Main clinical features Prognosis Flail arm syndrome ; man in a barrel syndrome; Vulpian- Bernhard syndrome Flail leg syndrome ; pseudopolyneuritic form of ALS; Patrikios syndrome Syndrome of predominantly LMN weakness of both arms; UMN signs develop in 50-70%; often slow progression; pathology is that of ALS. Syndrome of progressive leg weakness, predominantly LMN. About 10% of all cases; M:F ratio 9:1; prognosis better than in ALS syndrome more common in African and Asian patients. Rare; slow progression; DD difficult. Monomelic forms of ALS ALS-dementia syndrome (ALS-D) 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. 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. Juvenile onset form progressive over months or several yrs and then stabilises; does not generalises; pathology unknown. Usually 2 to 5 yrs.

( 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

REGIONS Bulbar Cervical Thoracic Abdominal Lombar UMN + + +/- + LMN + + + + 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

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 200 uv

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 1mV

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 Hirayama et al., 1987 Dorsal interosseous muscles

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 Strong et al., et 2009 al., 2099 5% dei Pazienti FTD hanno segni clinici o subclinici di sofferenza LMN

Anomalie neuropsicologiche nella SLA Phukan et al., 2007

2011

Terapia della SLA Sintomatica Nutrizione Respirazione Fisioterapia Palliazione

NUTRIZIONE II MN (V, VII, IX, XII) Disfagia +/- 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? 1. When NIV reachs its limit Tracheostomy is still used 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.

Rat Terapia Genica? Determine Antisense oligo Distribution in CNS Following ICV Administration = Completed Human 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 ASO Medicinal Chemistry SOD1 A4V Identification of Human SOD-1 ASO Candidates 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 Primate PK & Toxicology

3, 145-1456 Cova and Silani Stem cells

SLA: malattia extramotoneuronale? Ilieva et al., 2009 Tg SOD1

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% Fattori Genetici + Fattori Ambientali Malattia multifattoriale con eziopatogenesi ignota h 2 = 0.38 0.78 FALS 10% Geni sconosciuti FUS TDP43 Altri Altri geni SOD1 SOD1 SOD1 Malattia monogenica mendeliana con eziopatogenesi nota

Perché studiare la SLA familiare? Invecchiamento Fattori genetici? SALS Tossine ambientali?? Modello animale (SOD1, TARDBP ) MODELLO PATOGENETICO TERAPIA DELLA SLA FALS Mutazione in un singolo gene

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 AD 2 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 HSP 27 Senataxin SOD1 NF-L Dynactin (DCTN1) Glycyl trna synthetase VAPB HSP 22 SMN1 PON IGHMBP2 Seipin (BSCL2) Androgen receptor ALS OPTN FIG4 PLS ALSIN Spastin HSP/SPG Paraplegin Atlastin NIPA1 KIF5A HSP 60 Spartin Pure UMN

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 GSH perossidasi 2H + + O catalasi - 2 H 2 O 2 H 2 O + ½ O 2 SOD-Cu 1+ SOD-Cu2 + (ridotta) (ossidata) O 2 O - 2 ~150 mutazioni >>> mutazioni missenso, AD Correlazione genotipo/fenotipo Non correlazione tra stabilità/attività dell enzima mutato e fenotipo clinico

GAIN OF FUNCTION: I topi transgenici per SOD1 enzimaticamente attiva (hsod G93A ) e inattiva (hsod1 G85R ) sviluppano la malattia I topi msod1 -/- non sviluppano la malattia La delezione di msod1 non modifica la progressione nel topo hsod1 G85R I topi che iperesprimono hsod wt sono sani L iperespressione di hsod1 wt nel topo hsod1 G85R non modifica la progressione di malattia Mutazioni di SOD1: effetti biologici STRESS OSSIDATIVO DISFUNZIONE MITOCONDRIALE ECCITOTOSSICITÀ GLUTAMMATERGICA DISFUNZIONE DEL TRASPORTO ASSONALE RIDUZIONE DI FATTORI TROFICI DISFUNZIONE GLIALE ATTIVAZIONE DELLE CASPASI AGGREGAZIONE PROTEICA Dimero SOD1 Proteine Cellulari Proteasoma Mutazioni Oligomerizazzione Aggregati Proteici Chaperone Citoscheletro Mitochondria

Coorte studiata: FALS 18/156 11% (25 Pz.) SALS 6/566 1% SLA familiare SLA sporadica A4V L84F L144F G93D V5M A95G G12R F45C V47F D101G G41S A4V L144F L84F Q22R F45C A95T V97L I113T D90A D90A G93D F45C

Mutazioni di SOD1: correlazioni genotipo-fenotipo PENETRANZA Completa Incompleta A4V, G41S, H43R, H46R, L84F, L84V, D90Ahom, E100G, L144F A4T, L8Q, N19S, E21G, N65S, D76Y, D90Ahet, G93S, I113T SITO DI ESORDIO Spinale Bulbare Variabile G37R, H46R, D76V, L84F, L84V, D90Ahom, E100K, E100G A4T, C6G, L8Q, D76Y, V148I, I151T A4V, G41S, N86S, D90Ahet, I113T, L144F DECORSO DI MALATTIA Rapido Medio Lento Variabile 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

Mutazione D90A - 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 - 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

Effetto fondatore della D90Ahom SLA D90Ahet: 1. Un gene malattia 2. Fenotipo variabile 3. Più aggressiva 4. In popolazioni outbred SLA D90Ahom: 1. Due geni malattia 2. Un fenotipo uniforme 3. Progressione lenta 4. In popolazioni isolate ( inbred ) Mutazione D90A originale (895 generazioni fa) 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 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

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

GFP-TDP-25 GFP-TDP-43 GFP-TDP-25 GFP-TDP-43 Flag-TDP-43 Flag-DTP-43 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 Hoechst GFP Flag Merge C-terminal fragments are toxic to cells and increase apoptosis Hoechst GFP Caspase-3 Merge 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 DsRed-TDP wt GFP-TDP 162-414 DsRed-TDP wt GFP-TDP 218-414 C-terminal fragments may impair TDP-43 nuclear localization and function 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 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-43 A315T 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 TFIID RNA Pol II Nuclear hormone receptors SYQG-rich YB-1 SFRS2 NF-kB RGG RGG TASR1/2 RRM ZnF CBP RNA ssdna dsdna

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%) 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 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 UN PAZIENTE CON ALS-FTD

R521C

CTRL FALS N2A SKNAS Effects of FUS/TLS mutations 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 FALS CTRL GFP-FUS(WT) GFP-FUS(R521G) WT H517Q R521G WT H517Q R521G NeuN FUS DAPI 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 Unknown SOD1 positive TDP-43 positive FUS positive ALS2 (alsin) ALS1 (SOD1) SALS non-sod1 FALS ALS10 (TARDBP) ALS6 (FUS) ALS4 (SETX) ALS5 (SPG11) ALS8 (VAPB) ALS9 (ANG) ALS12 (OPTN) OPTN positive?

FTLD Neuropathology FTLD-tau FTLD-U Pick s disease 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 statistica Conferma Schymick 2007 USA 276 271 no n/a n/a Dunckley 2007 USA 386 (901) Van Es 2007 Svezia, Belgio Olanda Van Es 2007 Svezia, Belgio Olanda, USA Cronin 2008 Irlanda, USA, Olanda 461 (876) Chiò 2009 USA, Italia 553 (2160) Landers 2009 USA, Francia UK, Olanda Van Es 2009 Europa, USA 2323 (2532) 542 (1025) 450 (906) FGGY Sì? No ITPR2 Sì? No 1767 1916 DPP6 Sì Dubbia 958 932 DPP6 No Dubbia 2338 (3008) SUNC1 No n/a 1821 2258 KIFAP3 Sì No 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 unpublished data 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 hsod1 G93A 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) KIF3A KIF3 KIFAP3 KIF3B

KIFAP-3 CC TC SNP rs1541160 TT Courtesy Orsetti et al., 2011

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 Ca 2+ -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

COHORT STUDIED: PON1 and FALS -1 st step (direct sequencing) 260 FALS (US and Italian) 188 SALS 188 CTRLs -2 nd step (genotyping) 996 SALS 971 CTRLs 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

COHORT STUDIED: PON2-3 and FALS -1 st step (direct sequencing) 166 FALS (US and Italian) -2 nd step (genotyping) 996 SALS 971 CTRLs PON2 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 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)

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 Pathogenic 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 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 Screening di geni candidati 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 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 Rapido 40.000 USD per genoma 15 Gb per microarray 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 2009 2 mutazione nel gene DHODH 14 18 Jan-Apr May-Aug Sept-Dec Jan-Apr May 2010 2011 7 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