Epilessia, disabilità intellettiva e dismorfismi: le nuove frontiere della genetica

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1 Varese 14 Dicembre 2012 Epilessia, disabilità intellettiva e dismorfismi: le nuove frontiere della genetica Rosario Casalone, MD, PhD SSD Genetica Dipartimento Materno Infantile Azienda Ospedaliera-Polo Universitario Ospedale di Circolo e Fondazione Macchi Varese TOMA, Busto Arsizio, 11 maggio 2012

2 Eziologia delle Epilessie tipi eziologia

3 Rilevanza clinica delle nuove tecnologie

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5 Epi4k http// International collaborative effort to sequence epilepsy genomes in 4000 patients First project 500 patients with Lennox-Gastaut syndrome and their parents Second project 1500 patients first degree relatives with GGE and NAFE 300families with three or more affected Third project investigate CNVs and compare them to exome sequence data to better understand the contribution of CNVs to epilepsy risk.

6 Tecnologia rivoluzionaria permette lo screening in un unico esperimento di tutto il genoma per l identificazione di anomalie cromosomiche quantitative in forma di microdelezioni o duplicazioni di dimensioni inferiori alle kb Comparative Genomic Hybridization Oligonucleotidi Array: griglia di sonde

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10 Copy Number Variations (CNV)/aberrations(CNA)/Variants uncertain significance(vous) Segmenti di DNA con estensione maggiore di 1 Kb presenti nel genoma con un numero variabile di copie. 12% DEL GENOMA DELETION 360Mb Contribuiscono: Se di dimensioni superiori a kb Identificano riarrangiamenti patogenetici alle differenze fenotipiche tra gli individui alla patogenesi di malattie, sia monogeniche che multigeniche

11 from probably 7-10 millions years ago (Kumar et al,2005 )

12 Review letteratura:first step! 33 studi : pazienti con RM/DD/ID/ASD testati con Array-CGH Anomalie patogenetiche riscontrate nel 12.2% dei casi di media (35-5.5% a seconda delle piattaforme usate). Cioè il 10% in più di quanto riscontrato con la indagine citogenetica (2-3%) e di quanto riscontrato con FISH pretelomeriche (2-3%). Risoluzione Array-CGH 100kb di media, sull intero genoma ( paia basi) 20-50kb, in regioni targeted piu affollate di probes Citogenetica 3-5Megabasi (3-5 milioni di paia di basi, kb)

13 Array-CGH è già oggi il test genetico piu rilevante per i pazienti affetti da RM/dismorfie/autismo/malformazioni congenite -Nella identificazione di anomalie predisponenti a malattie comuni

14 ANALISI CGH-ARRAY ANALISI SNPs Delezione 11 Mb 4q13.1 4q13.3 Malformazioni tipo Charge syndrome Cromosoma 8 NORMALE Genitori normali

15 ANALISI CGH-ARRAY Sordità, lieve ID, epilessia Delezione 0,39 Mb 22q Geni: TMEM184B, CSNK1E KCNJ4,KDELR3, DDX17, DMC1.

16 Stiamo mettendo a punto delle Linee guida interpretative, protocolli e consensi informati

17 Molte regioni coinvolte in microriarrangiamenti Array-CGH le analizza tutte in un solo esperimento

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19 Del(15)(q13.3)

20 GENE REVIEW,NCBI Dimensioni 2Mb, 6 genes, de novo nel 25% dei casi Ereditata da genitori normali nel 75% dei casi. Primo caso riportato da Sharp et al,nat Genet 2008 La delezione è riscontrata nel 1-2% dei pazienti con epilessia generalizzata. Un terzo dei portatori di del 15q13.3 presenta epilessia. Forme di epilessia: Juvenile myoclonic epilepsy Childhood absence epilepsy Juvenile absence epilepsy Tipi di crisi epilettiche:absence, myoclonic, generalized tonic-clonic.

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22 A Mesurel Paulet et al, Clin Genet 2010 N Hoppman-Chaney et al Clin Genet 2012 ID and seizures in all omozygotes CHRNA7 is a Cholinergic receptor, member of nicotinic acethylcholine receptor superfamily, mediator of signal transmission at Synapses. Variabilità elevata, penetranza ridotta?

23 H.Muhler et al over 570 pediatric patients with epilepsy identified 4 cases with Del15q13.3 (2 de novo, 1 inherited, 1 not known) All 4 patient had absence seizures with various degree of ID Variable phenotypic expression and penetrance

24 Del (16)(p13.11)

25 Am J Hum Genet 86, , 2010 Del 16p13.11 >100 kb is a disease risk factor or CNV predisponent Schizophrenia, Mental retardation Autism 3812 patients with diverse spectrum of epilepsy syndromes and 1299 controls Deletion>100kb ( 6 genes) found 23 patients (0.6%) 0 in controls

26 Del 16p13.11 Phenotype Highly variable phenotypes, no refractory, no particular seizure type Patogenetic mechanism Haploinsufficiency- reduced expression of some genes NGS no ( recessive acting) mutations in genes included Origin Frequently trasmitted by unaffected mothers! Gene NDE1 gene loss is candidate for disease mechanisms ( gene involved in cortical development, but it remain not clear)

27 Dup(15)(q11-13)

28 Larissa Stewart et al. 2011

29 A.Battaglia Orphanet J, su nati 75% of cases having -Ipotonia -Developmental delay -ID -Autistic behavior -Epilepsy severe and refractory with very poor outcome -Minor dysmophic features (down slanting palpebral fissure, low set ears skin pigmentary changes)

30 Microdeletions / duplications 15q D.T.Miller et al. JMG, 2008 Del 15q ARRAY CGH SNP Dup 15q13

31 -Wide variety of seizures -Onset between 6months to 9 years -Infantile spasms associated with hypsarrhytmic EEG -Lennox-Gastaut syndrome -Seizures where difficult to control in all -EEG not specific

32 Microrearrangement in chromosome 2 and epilepsy

33 Duplication 2q24 (9 cases) Heron et al 2010 familial neonatal case with seizure and ID caused by microduplication 2q24.3 Total : 9 cases 3 de novo 4 had a parent with the same abnormality in 2q24 are mapped 5 voltage gated sodium channel genes SCN1A, SCN2A, SCN3A, SCN7A, SCN9A

34 -Seizure onset in neonatal period -Seizure were refractory to multiple anticonvulsivant agents in 5/9 -stopped during the first 2 years of life in most patients (7 / 9) -no dysmorphic features -MRI: no pathologic findings in the majority of the patients

35 18 patients reported Deletion size from 200kb to 5.5 Mb including part of MBD5 gene to 15 other genes Features: ID with pronounced speech delay (18/18) Additional: coarsal facies short stature microcephaly (9/18) stereotypic repetitive behavior disturbed sleep pattern MRI abnormalities (9/17) seizures (17/18) generalized or partial onset between 1 to 12 years old resistant to therapy The deletion size do not correlate with the severity

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39 Phenotypic overlap in literature cases include: neonatal or infantile seizures mental retardation, language deficits, autism spectrum disorders In deleted region at least two included genes (KCNQ2 and CHRNA4) are Known associated with epilepsy Heather et. Al reported a case with del 20q13.33 masquerading as Pyridoxine -dependent Epilepsy clinical utility of Array-CGH

40 Chromosome Xq28 Long face Midfacies hypoplasia Large ears

41 82 patients with drug resistant epilepsy and comorbidity (ID, dysmorphism, family history of epilepsy or neuropsichiaric disorders) -Platform 135k -Results 15.6% of CNV pathogenetic (10-30% in literature epilepsy +ID) Del 15q11.2q13.1 Del 15q11.1q13.1 Del15q13.2q13.3 Del16p11.2 Del16p13.11 (2 cases) 3% of generalized epilepsy showed recurrent: del15q13.3, del16p13.11, del 15q11.2

42 cases with epilepsy + comorbidity (ID,Autism, congenital abnormalities) 73 patients (29.6%) had CNV 37% had CNV likely causative (10.9% of the total 247 cases) Del1p36 Del2q Dup7q11.23 Dup22q11.2 CNV probably pathogenetic (de novo, containing genes) : 20 cases (4.9% of total, 16.4% Of positive cases) : delxp22.13(cdkl5), dupxq28(mecp2),del1p and 4q26, Dup4q24.3-q31.1 (SCN1A,SCN2A), del2q , del3q25.32-q26.1, del6p22.3-p24.1, Dup7p22.1+dup17q12,del14q23.3 (Gephyrin), ring 14, del16p13.3(tbcid24)

43 Girirajan et al. NEJM- October % of affected Maternal origin

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45 WHOLE EXOME SEQUENCING Efficient strategy tha involves target capture and sequencing of gene exomes Corbett et al.,2012 Reported and mapped in a large family in 16p13.3 a form of focal Epilepsy and ID. They sequencing genes in the mapped interval (> 3 Mb) and identified a pathogenetic mutation in TBC1D24 gene of unknown function Veeramah et al,2012 A case of sporadic epileptic encephalopathy and autism+ ID Found 114 de novo mutations, 48 where novel variants The single novel de novo mutation: a missense mutation in SCNB8A encoding the Voltage gated sodium channel Nav 1.6 (first reported associated to epilepsy)

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49 Conclusions - Genetic testing array-cgh and NGS -are important diagnostic tools in different rare epileptic conditions, particularly affecting children -provides essential information to dissect complex phenotypes -pharmacogenomics will possibly guide treatment in the future

50 del paziente (o del feto) con malattia genetica

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