GENOTIPI ED ENDOFENOTIPI NELLA

SCHIZOFRENIA

Ragalna, 26 Febbraio - 2 Marzo

Massimo GennarelliProfessore Associato, Sezione di Biologia e Genetica,

Dipartimento di Scienze Biomediche e Biotecnologiche, Università degli Studi di Brescia

Laboratorio di Genetica IRCCS-FBF, Brescia

“A Beautiful Mind”

Incidenza di malattia: 1%(Gottesman, 1991)

Sintomi Positivi o “psicotici”: deliri, allucinazioni

Sintomi Disorganizzati: pensiero e linguaggio confuso, comportamenti senza senso

Sintomi Negativi: piattezza emotiva o mancanza di espressione, incapacità di iniziare e portare a termine azioni, lacunosità dei contenuti del discorrere e la sua brevità, mancanza di piacere ed interesse per la vita.

SCHIZOFRENIA

Ereditabilità stimata: 80%

Dementia in the General Population

Based on 1991 Mortality Statistics, US Public Health Service

1000Live Births

32AD

156Alive, not Demented

32Other

Dementia

AGE 85:

780Dead of All Causes

Dead with AD

163Dead of

Other Causes

3AD

AGE 55:

834Alive, not Demented

APOE-e4/e4 and DementiaAPOE-e4/e4 and Dementia

1000

APOE-4/4

AGE 55:

835Alive, not Demented

163Dead

2Alzheimer's

AGE 85:

883Dead of All Causes

Dead with AD

102AD

13Alive, not

Demented

2Other

Dementia

SNP

(Single Nucleotide Polymorphism)

……A G C G G A T T A G T……..

……A G C G G G T T A T T……..

LAST UPDATE: May 25, 2006 1:38 PM .

OrganismNumber of

Submissions(ss#'s)

Number of RefSNP Clusters

(rs#'s) ( # validated)

Homo sapiens / geneReport

27,846,394 11,961,761 (5,646,244)

G/A

G

A

M SL SS LL

Individui LLIndividui SS/SL

INTERAZIONE SIGNFICATIVA FRA NUMERO DI EVENTI STRESSANTI E GENOTIPO SS/SL (P =

0,05)

Life events, first depression onset and the serotonin transporter gene

Wilhelm K, Mitchell PB, Niven H, Finch A, Wedgwood L, Scimone A, Blair IP, Parker G, Schofield PR.

Br J Psychiatry. 2006 Mar;188:210-215

Adverse life events had a significantly greater impact on the onset of depression for individuals with the s/s

genotype.

The 5-HTTLPR genotype is a significant predictor of onset of major depression following multiple adverse events

The Gene for…

Strategie per l’identificazione di geni di suscettibilità:

1.ANOMALIE CROMOSOMICHE: riarrangiamenti strutturali in famiglie selezionate

2. STUDI di LINKAGE: basati su famiglie numerose e multigenerazionali con membri affetti in quasi tutte le

generazioni

3. STUDI di ASSOCIAZIONE: - caso-controllo (pazienti e controlli )

- TDT (transmission disequilibrium test: genitori come controlli interni e 1 figlio affetto)

- SIB PAIR ANALYSIS (coppie di fratelli/sorelle)

Evidence from nearly a century of epidemiological research indicates that schizophrenia occurs in all populations with a prevalence in the range of 1.4 to 4.6 per 1000 and incidence rates in the range of 0.16-0.42 per 1000 population.

Multi-centre studies conducted by the World Health Organization have highlighted differences between 'Western' and 'Third World' populations as regards the course and outcome of the disorder, with a significantly better prognosis in the developing countries.

SCHIZOPHRENIA

Epidemiology of schizophrenia: the global burden of disease and disability.

Jablensky A. Eur Arch Psychiatry Clin Neurosci. 2000;250(6):274-85.

Ipotesi eziopatogenetiche

1. NEUROSVILUPPO: geni per fattori neurotrofici e proteine sinaptiche (BDNF, GSK3, GAP-43, SNAP 25,…)

2. INFIAMMATORIA: geni per citochine (TNF, IL-1, IL-1RA, IL-10 cluster,…..)

3. ALTERAZIONI DEI SISTEMI NEUROTRASMETTITORIALI:

– Sistema serotoninergico : 5-HT2A– Sistema dopaminergico: DRD3 – Sistema glutammatergico: GRIK3, GRIN1, GRIN2B

Strategies for the identification of susceptibility genes:

1.CHROMOSOMAL ABERRATION: anomalous rearrangements in selected families

2.2.LINKAGE STUDIESLINKAGE STUDIES: based on multigenerational : based on multigenerational numerous families with affected members in almost all the numerous families with affected members in almost all the

generations generations

3.3.ASSOCIATION STUDIESASSOCIATION STUDIES: : - - case-controlcase-control (patients and unrelated controls) (patients and unrelated controls)

- - TDTTDT (transmission disequilibrium test: parents as internal (transmission disequilibrium test: parents as internal controls and 1 affected child)controls and 1 affected child)

- - SIB PAIR ANALYSISSIB PAIR ANALYSIS (pairs of brothers/sisters) (pairs of brothers/sisters)

Strategies for the identification of susceptibility genes:

1.1.CHROMOSOMAL ABERRATIONCHROMOSOMAL ABERRATION: anomalous : anomalous rearrangements in selected familiesrearrangements in selected families

2.LINKAGE STUDIES: based on multigenerational numerous families with affected members in almost all the

generations

3.3.ASSOCIATION STUDIESASSOCIATION STUDIES: : - - case-controlcase-control (patients and unrelated controls) (patients and unrelated controls)

- - TDTTDT (transmission disequilibrium test: parents as internal (transmission disequilibrium test: parents as internal controls and 1 affected child)controls and 1 affected child)

- - SIB PAIR ANALYSISSIB PAIR ANALYSIS (pairs of brothers/sisters) (pairs of brothers/sisters)

Craddock et al, 2005

Loci di suscettibilità:

22q11-12

CANDIDATE GENES ON

FUNCTIONAL BASES

SUSCEPTIBILITY

LOCI

search for single nucleotide polymorphisms (SNPs) or mutations

Strategies for the identification of susceptibility genes:

1.1.CHROMOSOMAL ABERRATIONCHROMOSOMAL ABERRATION: anomalous : anomalous rearrangements in selected familiesrearrangements in selected families

2.2.LINKAGE STUDIESLINKAGE STUDIES: based on multigenerational : based on multigenerational numerous families with affected members in almost all the numerous families with affected members in almost all the

generationsgenerations

3.ASSOCIATION STUDIES: - case-control (patients and unrelated controls)

- TDT (transmission disequilibrium test: parents as internal controls and 1 affected child)

- SIB PAIR ANALYSIS (pairs of brothers/sisters)

compare the frequency of alleles or genotypes of a particular variant between disease cases and controls.

Case-control association

studies

To date: october 2006(from 1993)

Number of association studies on schizophrenia:

975Key words: “schizophrenia, gene, association, case-control, polymorphisms”

Allelic associations confirmed in many populations for psychiaric disorders

5HT2A: serotonin receptor 2A

DRD3: dopamine receptor D3

Susceptibility genes for schizophrenia Hashimoto R, Hattori S, Chiba S, et al.Psychiatry Clin Neurosci. 2006, suppl 1:S4-S10.

Molecular genetic studies of schizophrenia Riley B, Kendler KS. Eur J Hum Genetcs 2006. 14(6):669-80

DEFINIZIONE DI ENDOFENOTIPI

a partire da• marcatori biologici• caratteristiche morfologiche evidenziabili con

tecniche di neuroimaging (“hippocampal shape deformations”)

• parametri neurofisiologici (P50 “auditory evoked potential gating deficit”; “aberrant smooth-pursuit eye movement”)

misurabili anche nei parenti di I grado dei pazienti

STRATEGIE PER MIGLIORARE GLI STUDI DI ASSOCIAZIONE

CO

MT

PR

OD

H

NEUROPLASTICITA’

Sistemi Neurotrasmettitoriali Fattori Neurotrofici

Signaling Intracellulare

Others

FATTORI NEUROTROFICI

NGF, BDNF, NT-3, NT-4/5, GDNF, CNTF

Sviluppo, Mantenimento e Differenziazione neuronale, Plasticità

sinaptica

BDNF (Brain-Derived-Neurotrophic-Factor)

Ncbi, accession # x60201; Shintani, et al. 1992; Murer, et al. 2001; Mowla, et al. 2001

Chromosome 11

Promoter

5´

1 297 1040 1353 BP681468492

G492 A492

Val66 Met66

May be extracellularlyactive at TrkB receptors

ProBDNF (32 kDa)

Truncated proBDNF (28 kDa)Signalpeptide

Activity unknown

Val66 Met66

Mature BDNF (14 kDa)Signalpeptide

Essential role in development,survival and function of neurons

Val66 Met66

Cleaved in endoplasmicreticulum

Cleaved in trans-golgi networkand/or immature vesicles

Or

11p13 11p14

Stop codonStop codon

BDNF

TrkB

D.O.Bi.G. - Università di Genova

Pezewas et al. 2004

References SNPsCases

(n)Control

s (n)Results Populations

Numata et al 2006 Val66Met 159   yes age at onset Japan

Zhang et al 2006 G-712A; C– 270T;

Val66Met

84 250 no USA

Chen et al 2006 3 SNPS 560 576 no also gender China

Szczepankiewicz et al 2005

C270T 397 380 no also gender Poland

Dempster et al 2005 Val66Met 92 114 yes association with WMS-R scale UK

Neves-Pereira et al 2005

Val66Met; (GT)n

 321 350 yes haplotypes Scotland

Schumacher et al 2005

rs988748-(GT)n-

Val66Met

533 1097 yes haplotype Germany

Gourion et al 2005 Val66Met 210 99 no gene^gene interaction was associated with an earlier emergence of psychosis by 3 years.

France

de Krom et al 2005 C– 270T; Val66Met

273 580 no haplotypes Dutch

Galderisi et al 2005 C– 270T 106 111 no Italy

Skibinska et al 2004 Val66Met 336 375 no also stratification gender; age at onset Poland

Anttila et al 2005 C– 270T; Val66Met

94 98 no Finland 

Nanko et al 2003 A758GC-270T

178 332 noyes

Japan

Szekeres et al 2003 C-270T 101 68 yes Hungary

Virgos et al 2001 (GT)n 262 278 no Spain

Sasaki et al 1997 (GT)n 60 60 no Japan

BDNF: case-control studies in Schizophrenia

Frequenze alleliche e genotipiche del polimorfismo Val66Met del gene BDNF in 300 pazienti

schizofrenici e 424 controlli______________________________________________________Genotipi Pazienti ControlliVal/Val 178 (59,3%) 262 (61,8%)Val/Met 101 (33,7%) 137 (32,3%)Met/Met 21 ( 7,0%) 25 ( 5,9%)Total 300 424

Alleli Val 457 (76,2%) 661 (77,9%)Met 143 (23,8%) 187 (22,1%)______________________________________________________Alleli p=0.43Genotipi p=0.74

Ridotti livelli di espressione di BDNF e Ridotti livelli di espressione di BDNF e del suo recettore trkB in corteccia del suo recettore trkB in corteccia prefrontale di pazienti schizofreniciprefrontale di pazienti schizofrenici

Hashimoto et al. (2005) J. Neurosci. 25:372-383

Yun Long Tan, Dong Feng Zhou, Liou Yuan Cao, Xiang Yang Zhang

Geneslocalization

Schizop (n) Controls (n) Results References Populations

GRIA1 5q33     yes Magri et al 2006 Italian

GRIA4 11q22 372 392 no Guo et al 2004 China

GRIA4 11q22 100 100 yes Makino et al 2003 Japan

GRIK26q16.3-q21 100 100 no Shibata et al 2002 Japan

GRIK3 1p34-33 100/106 100/100 no Shibata et al 2006 Japan

GRIK3 1p34-33 160 160 no Lai et al 2005 China

GRIK3 1p34-33 99 116 yes Begni et al 2002 Italian

GRIK4 11q22.3 100/106 100/100 no Shibata et al 2006 Japan

GRIK5 19q13.2 100/106 100/100 no Shibata et al 2006 Japan

GRIN1 9q34.3 253 140 no Qin et al 2005 China

GRIN1 9q34.3 139 145 yes Begni et al 2003 Italian

GRIN1 9q34.3   91 no Martucci et al 2003Eur-American

GRIN1 9q34.3 120 94 no Hung et al 2002 China

GRIN2A 16p13.2 672 686 yesIwayama-Shigeno et al 2005 Japan

GRIN2B 12p12 253 140 no Qin et al 2005 China

GRIN2B 12p12 188 156 yes Di Maria et al 2004 Italian

GRIN2B 12p12 193 176 no Chiu et al 2003 China

GRIN2B 12p12 100 100 yes Miyatake et al 2002 Japan

GRIN2B 12p12 268 337 yes Ohtsuki et al 2001 Japan

GRIN2B 12p12 164 171 no Nishiguchi et al 2000 Japan

GRIN2D19q13.1-qter 200-201 219-221 yes Makino et al 2005 Japan

Genes localization Cases (n) Controls (n) Results References Populations

GRM2 3p21.1 213 220 no Joo et al 2001 Japan

GRM3 7q21.1-q21.2 674 716 no Norton et al 2005 UK, Ireland

GRM3 7q21.1-q21.2 752 752 yes Chen et al 2005 china

GRM3 7q21.1-q21.2 100 100 yes Fujii et al 2003 Japan

GRM3 7q21.1-q21.2 265/288 227/162 yes Marti et al 2002 German

GRM4 6p21.3 285 288 no Ohtsuki et al 2001 Japan

GRM511q14.2-q14.3     yes Devon et al 2001 UK

GRM7 3p26.1-p25.1     no Bolonna et al 2001 UK

GRM7 3p26.1-p25.1 181 182 no Bray et al 2000 UK

GRM8 7q31.3-q32.1 100 100 yes Takaki et al 2004 Japan

GRM8 7q31.3-q32.1     no Bolonna et al 2001 UK

Glutamate receptors subtypes: case-control studies in Schizophrenia

Gene Name Localization Endophenotypes References

PLXNB3 plexin B3 Xq28 - verbal performance - white matter volume

Rujescu et al 2007

PCM1 pericentriolar material 1 8p22 orbitofrontal gray matter volumetric deficits

Gurling et al 2006

- poor medial temporal lobe-related memory performance

- visuospatial dysfunction

Ho et al 2006

brain morphology Agartz et al 2006

BDNF Brain-Derived Neurotrophic Factor

11p13

variation of hippocampal volume Szeszko et al 2005 DISC1 Disrupted in schizophrenia 1 1q42.1 - working memory

- cognitive aging - decreased gray matter volume

in the prefrontal cortex - abnormalities in hippocampal

structure and function

Hennah et al 2006

G72/G30 13q34 cognitive impairment Goldberg et al 2006 RGS4 G-protein signaling subtype 4 1q23.3 dorsolateral prefrontal cortex

alteration Prasad KM,

DTNBP1 Dystrobrevin- binding protein 1

6p22.3 neurocognitive performance Donohoe et al 2007

cognitive improvement with atypical antipsychotics

Woodward et al 2007

prefrontal brain functioning Ehlis et al 2007 - prefrontal dysfunction

- working memory performance Bertolino et al 2006;

2004 morphological abnormalities Ohnishi et al 2006

COMT Catechol-O-methyltransferase

22q11.2

prefrontal cognition impairment Egan et al 2001 IL1RN interleukin-1 receptor

antagonist 2q14.2 ventricular volumetric changes Papiol et al 2005

IL-1beta interleukin 1-beta 2q14 structural brain alterations Meisenzahl et al 2001 APOE apolipoprotein e 19q13.2 hippocampal volume Hata et al 2002

NEUROCOGNITIVE ENDOPHENOTYPES (Gur et al 2007)

NEUROPHYSIOLOGICAL ENDOPHENOTYPES (Turetsky et al 2007)

Gene Name Localization Endophenotypes References

Houy et al 2004 Leonard et al 2002

Raux et al 2002

CHRNA7 nicotinic alpha 7 receptor

15q14 P50 sensory gating deficit

Dempster et al 2006 cPLA2 cytosolic

phospholipase A2 1p35 eye movement Rybakowski et al 2003

eye movement Rybakowski et al 2001 DRD3 dopamine D3 receptor 3q13.3 P300 amplitudes Mulert et al 2006

amplitude in centro-parietal area

Golimbet et al 2006

eye movement Rybakowski et al 2002 prefrontal P300 Gallinat et al 2003 prefrontal P300 Tsai et al 2003

COMT catechol-O-methyltransferase

22q11.2

eye tracking Thaker et al 2004 Hennah et al 2006

DISC1 disrupted at the

chromosome 1 breakpoint

1q42.1 reduction in the amplitude of the P300 event-related potential Blackwood, Muir, 2004

COSA VIENE

TESTATO

VANTAGGI

RISCHI

TEST GENETICI

GENETICA DELLA MALATTIADIAGNOSI/PROGNOSI

Malattiemendeliane

rare: mutazioniin singoli geni

Malattie complesse:geni di suscettibilità

MIGLIOR COMPRENSIONEDELLE PATOLOGIEE DELLE POSSIBILI

TERAPIE

Implicazioni etiche, legali e sociali

Date di introduzione di alcuni farmaci antipsicotici

1950 1960 1970 1980 1990 2000

CLORPROMAZINA

ALOPERIDOLO

CLOZAPINA

OLANZAPINA

RISPERIDONE

QUETIAPINA

TIPICI

ATIPICI

Enzimi del metabolismo di fase I (reazioni di attivazione):CYPs (famiglia del citocromo P450)

Enzimi del metabolismo di fase II (reazioni di Enzimi del metabolismo di fase II (reazioni di coniugazione):coniugazione): UGTs UGTs (uridine diphosphate glucuronyl-(uridine diphosphate glucuronyl-transferases)transferases) SULTs SULTs (sulfonyl transferases)(sulfonyl transferases)NAT2 NAT2 (N-acetyltransferases type II)(N-acetyltransferases type II) TPMTs TPMTs (thiopurine methyltransferases)(thiopurine methyltransferases)

FARMACOCINETICA

CLASSIFICATION OF METABOLIZER GROUPS

Heterozygous with one Heterozygous with one active allele (*1 or *2) active allele (*1 or *2)

and one deficient alleleand one deficient allele

INTERMEDIATE METABOLIZERS

(IM)

ULTRARAPID METABOLIZERS

(UM)

Heterozygous for duplicated *1 or Heterozygous for duplicated *1 or *2 + another active allele*2 + another active allele

POOR METABOLIZERS

(PM)

Homozygous or compound Homozygous or compound heterozygous with two heterozygous with two

deficient alleles (deficient alleles (*3,*4,*5,*6*3,*4,*5,*6))

EXTENSIVE METABOLIZERS

(EM)

Carriers of one duplicated Carriers of one duplicated active allele + deficient active allele + deficient

alleleallele

Impact of the CYP2D6 polymorphisms on the pharmacokinetics of antipsychotic drugs: dosage adaptation according to genotype.PM: poor metabolizerIM: intermediate metabolizerEM: extensive metabolizerUM: ultrarapid metabolizer

Kirchheiner J. Et al., Molecular Psychiatry 2004;1-32

Haloperidol Clozapine Quetiapine

Sertindole Ziprasidon

Olanzapine Risperidone

D1

D2

5-HT2a

5-HT1a

M1

1

2

H1

tipici

clozapinarisperidone

Atipici in generale

olanzapina

Non specificatoaloperidolo

Polimorfismi dei geni 5HT2A, 5HT2C, DRD3 e DRD4 e risposta alla clozapina ( Kirchheiner et al., 2004)

http://www.fda.gov/default.htm

Efficacy of rosiglitazone in a genetically defined population

with mild-to-moderate Alzheimer's diseasePharmacogenomics J. 2006 Jan 31

Risner ME, Saunders AM, Altman JF, Ormandy GC, Craft S, Foley IM, Zvartau-Hind ME, Hosford DA, Roses AD.

Mild-to-moderate AD patients were randomized to placebo or rosiglitazone (RSG) 2, 4 or 8 mg. Primary end points at Week 24 were mean change from baseline in AD

Assessment Scale-Cognitive (ADAS-Cog) and Clinician's Interview-Based Impression of Change Plus Caregiver Input global scores in the intention-to-treat population

(N=511), and results were also stratified by apolipoprotein E (APOE) genotype (n=323). No statistically significant differences on primary end points were detected between

placebo and any RSG dose. There was a significant interaction between APOE varepsilon4 allele status and ADAS-Cog (P=0.014). Exploratory analyses

demonstrated significant improvement in ADAS-Cog in APOE varepsilon4-negative patients on 8 mg RSG (P=0.024; not corrected for multiplicity). APOE

varepsilon4-positive patients did not show improvement and showed a decline at the lowest RSG dose (P=0.012; not corrected for multiplicity). Exploratory analyses suggested that APOE varepsilon4 non-carriers exhibited cognitive and functional

improvement in response to RSG, whereas APOE varepsilon4 allele carriers showed no improvement and some decline was noted. These preliminary

findings require confirmation in appropriate clinical studies.

Nature 437, 1299-1320 (27 October 2005)

A haplotype map of the human genomeThe International HapMap Consortium