Regione Emilia-RomagnaProgramma di ricerca Regione-Università 2010-2012

Area 1 - Strategic Programs

Recognition, diagnosis and therapy of Recognition, diagnosis and therapy of mitochondrial disorders in neurological services mitochondrial disorders in neurological services

of the Emilia-Romagna region (ER-MITO)of the Emilia-Romagna region (ER-MITO)

Strategic Program Scientific Coordinator:Dr. Valerio Carelli

IRCCS Istituto delle Scienze Neurologiche di Bologna

Salvatore Di Mauro 2013

1988 – 2014 Medicina Mitocondriale1988 – 2014 Medicina Mitocondriale

EEmilia-milia-RRomagna-omagna-MITOMITOcondri (condri (ER-MITOER-MITO))

Un programma finalizzato a:• creare un network regionale di specialisti • definire la prevalenza minima di 4 fenotipi frequenti

di malattia mitocondriale nella regione Emilia-Romagna

• definire un percorso diagnostico clinico/moleculare e delle indicazioni terapeutiche condivisi tra i servizi di neurologia

• identificare nuovi difetti moleculari di malattia mitocondriale

• valutare possibili biomarkers predittivi• porre le basi per futuri studi clinici sulle nuove terapie

ER-MITO: Work-packages

1. WP1. Clinical phenotype 1: Mitochondrial hereditary optic Mitochondrial hereditary optic neuropathies (LHON and DOA) – Dr. Valerio Carellineuropathies (LHON and DOA) – Dr. Valerio Carelli

2. WP2. Clinical phenotype 2: Ptosis and chronic progressive Ptosis and chronic progressive external ophthalmoplegia (CPEO) – Prof. Rocco Liguoriexternal ophthalmoplegia (CPEO) – Prof. Rocco Liguori

3. WP3. Clinical phenotype 3: Acute adult mitochondrial Acute adult mitochondrial encephalomyopathies – Prof. Paolo Frigio Nichelliencephalomyopathies – Prof. Paolo Frigio Nichelli

4. WP4. Clinical phenotype 4: Pediatric mitochondrial Pediatric mitochondrial encephalomyopaties – Dr. Elvio Della Giustinaencephalomyopaties – Dr. Elvio Della Giustina

5. WP5. Laboratory: Molecular diagnosis and biomarkers of Molecular diagnosis and biomarkers of mitochondrial diseases – Dr. Maria Lucia Valentinomitochondrial diseases – Dr. Maria Lucia Valentino

6. WP6. Epidemiology: Epidemiology of mitochondrial diseases in Epidemiology of mitochondrial diseases in the Emilia-Romagna region – Prof. Roberto Dthe Emilia-Romagna region – Prof. Roberto D’’AlessandroAlessandro

WP1. Clinical phenotype 1: Mitochondrial hereditary optic neuropathiesMitochondrial hereditary optic neuropathies

Coordinator Dr. Valerio Carelli (Bologna)Coordinator Dr. Valerio Carelli (Bologna)

Leber’s hereditary optic neuropathy (LHON)>mtDNA ND1, ND4, ND6

Dominant optic atrophy (DOA)>OPA1

Primary objectives will be: 1) Ascertainment of patients with mitochondrial hereditary optic neuropathies (LHON and DOA), defined by clinical criteria and/or molecular diagnosis.

Secondary objectives will be: 1) Establishment of therapeutic and follow-up protocols in acute and chronic LHON patients and DOA patients 2) Identification of the molecular basis for optic neuropathy cases with hallmark of mitochondrial pathogenesis who tested negative for one of the three LHON primary mutations and screening of the OPA1 gene 3) Observational studies of possibly useful biomarkers.

CRITERI DI INCLUSIONECRITERI DI INCLUSIONEThe categories of patients we aim to ascertain are:1. Acute/dynamic LHON: patients observed within 1 year from onset of rapid loss of central vision (monolateral or bilateral) (these patients will enter the fast-track diagnostic pathway)2. Chronic LHON: patients observed later than 1 year from onset of rapid loss of central vision with bilateral established optic atrophy3. DOA: patients with defective central vision present since childhood or patients unaware of low vision with evidence of optic nerve pathologyTo be enrolled in the present protocol, these patients must satisfy the following inclusion/exclusion criteria:Inclusion criteria1. Painless loss of central vision (subacute/acute for LHON or slowly progressive for DOA)2. Loss of temporal fibers (papillo-macular bundle) or temporal pallor or diffuse pallor at fundus examination3. At least one of:o central or cecocentral scotoma at computerized visual fieldso thinning of the RNFL thickness prevalent on the infero-temporal quadrants for chronic LHON and DOA, or thickening of the RNFL on the inferior and superior quadrants with thinning of the temporal quadrant for acute LHON cases, at OCTExclusion criteria1. Complete resolution of the visual defect within one month from onset, spontaneous or after steroid therapy2. Evidence of compressive lesions on brain MRI

WP2. Clinical phenotype 2: Ptosis and Chronic Progressive External Ptosis and Chronic Progressive External

Ophthalmoplegia (CPEO)Ophthalmoplegia (CPEO)Coordinator Prof. Rocco Liguori (Bologna)Coordinator Prof. Rocco Liguori (Bologna)

Molecular basis:-mtDNA single deletion (sporadic)-mtDNA point mutation (maternally inherited)-mtDNA multiple deletions (AR, AD, de novo sporadic) associated with a nuclear gene mutation (POLG1 and 2, ANT1, Twinkle, OPA1, etc…..)

The primary objective of this WP will be: 1. Ascertainment of patients with mitochondrial ptosis, with or without chronic progressive external ophthalmoplegia (CPEO) diagnosed on the basis of one or more of the following: identified molecular defect (mtDNA point mutation or single deletion or multiple deletions, or nDNA mutation), or evidence of mitochondrial myopathy at muscle biopsy, or biochemical OXPHOS defect in molecularly undefined cases.

Secondary objectives will be: 1) Establishment of therapeutic and follow-up protocol in mitochondrial ptosis/CPEO patients. 2) Identification of new nuclear genes causing mitochondrial ptosis/CPEO phenotype. 3) Observational studies of possibly useful biomarkers in mitochondrial myopathy

CRITERI DI INCLUSIONECRITERI DI INCLUSIONE

The patients we aim to ascertain are any case of isolated or syndromic, sporadic or familial, ptosis/CPEO. To be enrolled in the present protocol these patients must satisfy the following criteria: Inclusion criteria 1. Age over 18 years 2. Either one or both of the following clinical signs, for at least 6 months of duration: Ptosis: monolateral or bilateral External Ophthalmoplegia: partial or complete with or without diplopia Exclusion criteria 1. Clinical evaluation detecting positivity of the ice-pack test 2. Clinical evaluation detecting pupillary involvement without a defined etiology 3. Clinical evaluation detecting ocular pain or exophthalmus 4. Positive AchR antibodies 5. Laboratory evidence of thyroid dysfunction and positivity of anti-thyroid antibodies associated to signs of extraocular muscles involvement at orbit MRI 6. Positive repetitive nerve stimulation (3-5 Hz ) of the facial nerve showing decremental response 7. Presence of cranial nerves, brainstem or extraocular muscles lesions at brain and orbital MRI.

WP3. Clinical phenotype 3: Acute adult mitochondrial encephalomyopathies Acute adult mitochondrial encephalomyopathies Coordinator Prof. Paolo Frigio Nichelli (Modena)Coordinator Prof. Paolo Frigio Nichelli (Modena)

MitochondrialEncephalomyopathyLacticAcidosisStroke-like

MELAS syndromeA3243G/tRNALeu mtDNApoint mutation

WP3. Clinical phenotype 3: Unit on status epilepticus Unit on status epilepticus

Prof. Tinuper (Bologna) Prof. Tinuper (Bologna)

Primary objective will be: 1. Ascertainment of patients with acute adult mitochondrial encephalomyopathy, as defined by the occurrence of stroke-like episodes and/or occipital status epilepticus in patients from 18 to 40 years of age.

Secondary objectives will be: 2. Establishment of therapeutic and follow-up protocols in patients diagnosed with acute adult mitochondrial encephalomyopathy 3. Identification of new molecular defects in the patients remaining without genetic diagnosis. 4. Observational studies of possibly useful biomarkers.

CRITERI DI INCLUSIONECRITERI DI INCLUSIONEThe categories of patients we aim to ascertain are: Patients with cryptogenic ischemic stroke and/or occipital status epilepticus of 18-40 years of age, respectively recruited through two different health care pathways: - Stroke Units - Intensive Care Units. To ascertain these patients we set the following inclusion criteria: 1. Age between 18 and 40 years 2. At least one of the following features: a. Cryptogenic stroke-like lesions of any location not conforming the distribution of major arteries at cerebral MRI, b. Occipital status epilepticus as evaluated by EEG and defined as paroxysmal or slow activity over the posterior regions, including: - seizures with a main involvement of the parieto-temporo-occipital regions (visual symptoms, clonic head and/or eyes deviation with or without secondary generalization) - ictal EEG with periodic lateralized epileptiform discharges or prolonged seizure discharges over the parieto-temporo-occipital regions - post-ictal EEG with paroxysmal or slow activity over the parieto-temporo-occipital regions

c. Status epilepticus with prominent lateralized clonic component and continuous EEG discharges on the central regions Exclusion criteria: 1. Juvenile ischemic stroke with a definite cause (cardioembolism, cervicocerebral artery dissection, thrombophilia, vasculitis). 2. Status epilepticus with a definite cause (traumatic or neoplastic or other identifiable not MitD causes).

Participating adult Neuro-Services in ERParticipating adult Neuro-Services in ER

WP4. Clinical phenotype 4: Pediatric mitochondrial encephalomyopaties Pediatric mitochondrial encephalomyopaties Coordinator Dr. Della Giustina (Reggio Emilia)Coordinator Dr. Della Giustina (Reggio Emilia)

Encephalopaties in pediatric patients with simmetrical bilateral cerebral lesions with or without white matterinvolvement/cavitation

Primary objective will be: 1.Ascertainment of patients with subtypes of pediatric mitochondrial encephalomyopathies in the ERR diagnosed on the basis of one of the following: - identified molecular defect (mtDNA or nDNA mutation), or - evidence of mitochondrial myopathy at muscle biopsy, or - biochemical OXPHOS defect (muscle and/or fibroblasts) in molecularly undefined cases.

Secondary objectives will be: 1. Establishment of therapeutic and follow-up protocols in patients diagnosed with pediatric mitochondrial encephalomyopathies. 2. Identify the molecular basis of patients who tested negative for the known genetic defects. 3. Observational studies to identify possibly useful biomarkers in pediatric mitochondrial encephalomyopathies.

CRITERI DI INCLUSIONECRITERI DI INCLUSIONEThe goal of the research methodology in this WP is to screen and ascertain MitD patients with defined clinical subtypes (Leigh and Leigh-like syndromes, Alpers syndrome, etc.), for which the minimal prevalence in ERR will be estimated. To this aim, we will take into particular consideration some neuro-imaging markers, suggestive of these MitD subtypes, such as the evidence of brain bilateral lesions or leukoencephalopathy or both, evidence of cerebellar atrophy or evidence of stroke-like lesions. Thus, to be enrolled in the present protocol, patients must satisfy the following inclusion/exclusion criteria: Inclusion criteria 1.Elevated plasma (arterial) or cerebrospinal fluid (CSF) lactate levels2.Defined brain magnetic resonance imaging features (at least one): a. bilateral symmetrical lesions in the cerebral hemispheres and/or basal ganglia and/or cerebellum and/or brainstem. b. Leukoencephalopathy. c. Cerebellar atrophy. d. Stroke-like lesions. Exclusion criteria1. Inborn errors of metabolism other than OXPHOS defects.2. Pre and perinatal hypoxic-ischemic encephalopathy.3. Encephalopathy due intoxication (i.e., carbon monoxide).4. Acute Disseminated Encephalo-Myelitis (ADEM).

Participating pediatric Neuro-Services in Participating pediatric Neuro-Services in ERER

WP5. LaboratoryMolecular diagnosis and biomarkers of Molecular diagnosis and biomarkers of

mitochondrial diseases mitochondrial diseases Coordinator Dr. Maria Lucia Valentino (Bologna)

Diagnosis (WP5 and collaborating centers):Diagnosis (WP5 and collaborating centers):

- biochemistry (blood cells/muscle biopsy/fibroblasts/cybrids)

- genetics (blood/urinary cells/muscle/fibroblasts) - mtDNA: mutation screening and direct

sequencing- nDNA: candidate gene sequencing and NGS

panels

Research (WP5 and collaborating centers):Research (WP5 and collaborating centers):

- new mutations in known genes associated to new phenotypes- new genes in monogenic phenotypes: genome wide scanning, homozygosity mapping, exome sequencing in collaboration with centers of excellence for mito-disease (Besta Institute of Neurology)

- biomarkers: phosphorylated neurofilament heavy chain (Guy et al. Mol Vis 2009), lactate/alanine/creatine (Shaham et al. PNAS 2009), mitochondrial DAMPs (MTDs)/free mtDNA in plasma (Zhang et al. Nature 2010), serum FGF21 (fibroblast growth factor 21) (Suomalainen et al. Lancet Neurol 2011)

- bank of biological material: fibroblast cell lines and cybrids, muscle biopsies, plasma and serum, DNA samples from different tissues

WP6. EpidemiologyEpidemiology of mitochondrial diseases in the Emilia-Epidemiology of mitochondrial diseases in the Emilia-

Romagna regionRomagna regionCoordinator Prof. D’Alessandro (Bologna)

19 Regional neurological services

4 Clinical WPs WP6 Epidemiology

WP5 Laboratory

CRF case alert case discussion

diagnostic workout confirmed diagnosis

1 12

334 4 4

Epidemiology Epidemiology (WP6, Coordinator Prof. D(WP6, Coordinator Prof. D’’Alessandro):Alessandro):

• Centralized database (case collection, periodic contact and recall)

• Meeting organization and contact among WPs

• Intensive prevalence study in the Bologna province

• Minimal prevalence rate estimation in ER

Le fasi del ProgrammaLe fasi del Programma

• Prima fase (sei mesi):Prima fase (sei mesi):- Definire lo stato dell’arte delle patologie neuro-mitocondriali in RER

- Organizzare la rete dei servizi di neurologia della RER

• Seconda fase (due anni):Seconda fase (due anni):- Reclutamento attivo dei pazienti

- Diagnosi moleculari

- Organizzazione della biobanca; definizione di biomarcatori

• Terza fase (sei mesi)Terza fase (sei mesi)- Consolidamento del database; stime di prevalenza

- Analisi dei dati clinici e di laboratorio da parte di ciascun WP

TempogrammaTempogrammaGià fatto:

• Programma approvato dal PRR-U il 5 luglio 2012

• Approvazione del Comitato Etico coordinatore

• Approvazione di tutti i CE della RER

• Ricognizione dei contratti e delle convenzioni tra enti da attivare

• Database e web

Da fare:

• Accreditamento ECM

• Struttura gestionale del Programma

• Avvio del Programma (Ottobre 2014)

Organizzazione del Programma - Organizzazione del Programma - PropostaProposta

• Comitato scientifico: Responsabili WP e Direzione scientifica Irccs. Compiti: gestione del protocollo (es.: emendamenti); politica editoriale.

• Comitato esecutivo: Responsabili di area (una Area= un CE) e Responsabile Programma; Compiti: gestione organizzativa (es. Presentazione protocollo al CE); coordinamento locale rete professionisti.

• Segreteria organizzativa: presso il WP epidemiologico.

• Sito web come strumento di comunicazione interna ed esterna.

• Mailing list per comunicazioni periodiche (o social network?)

Thanks for your attention Thanks for your attention and to the many collaborators:and to the many collaborators:• Alfredo A. Sadun, USC, Los AngelesAlfredo A. Sadun, USC, Los Angeles• Rubens Belfort Jr, UNIFESP, Sao PaoloRubens Belfort Jr, UNIFESP, Sao Paolo• Carla Giordano, University “La Sapienza”, RomeCarla Giordano, University “La Sapienza”, Rome• Palmiro Cantatore, University of Bari, BariPalmiro Cantatore, University of Bari, Bari• Anna Ghelli, University of Bologna, BolognaAnna Ghelli, University of Bologna, Bologna• Pio D’Adamo, University of Trieste, TriestePio D’Adamo, University of Trieste, Trieste• Vamsi Mootha, Harvard, BostonVamsi Mootha, Harvard, Boston• Massimo Zeviani, MRC, CambridgeMassimo Zeviani, MRC, Cambridge• Patrick Chinnery and Patrick Chinnery and Patrick Yu-Wai-Man, Patrick Yu-Wai-Man, University of Newcastle, NewcastleUniversity of Newcastle, Newcastle• Piero Barboni, San Raffaele, MilanoPiero Barboni, San Raffaele, Milano

• ………………and may lab:and may lab: