Genomic studies of schizophrenia: mapping madness?

1. Genomic studies of schizophrenia: mapping madness? Mike Owen, Department of Psychological Medicine, Wales College of Medicine, Cardiff University, UK.

2. Genetic epidemiology of schizophrenia • Familial, λs =10 • Individual differences in liability are largely genetic • Heritability 0.6-0.9 • Non-genetic factors also important • Multi-locus model consistently supported by analysis of family data • Genes with population λs > 3 unlikely • Number of susceptibility loci, degree of risk conferred by each and degree of interaction all unknown

3. Disease gene D18S21 Finding genes for schizophrenia • Linkage • Chromosomal abnormalities • Association • Convergent genomics

4. 6p24-p22 8p22-p21 1q21-q22 5q21-q33 6q21-25 1q42 1 2 3 4 5 6 7 8 10p15-p11 10q 25-26 13q32-q34 9 10 11 12 13 14 15 16 17p11-q25 22q11-q22 17 18 19 20 21 22 Y X Genome wide significant Multiple positives Published linkage data 2006

5. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 Chromosome Cardiff schizophrenia sib-pair genome screen VCFS CNP 351 microsatellite markers (10.3 cM) in 354 affected sib-pairs 179 (UK), 134 (Sweden), 41 (USA) Williams et al. Am J Hum Genet 2003

6. Data consistent with observed recurrence risks in relatives Meta-analysis suggests some consensus Many underpowered studies on uncertain value Larger studies needed for more genes, stronger evidence, interactions and better location 800-1000 ASPs optimal A number of groups are now seeking genes in currently positive regions Linkage studies of schizophrenia 2006

7. Positional genetics and schizophrenia • Positional Candidates • NRG1, 8p 1 • DTNBP1, 6p 2 • G72 (DAOA), 13q3 • Multiple positive studies as well as negative • No clear pattern of associated alleles or haplotypes • Risk variants not identified 1. Stefansson et al. 2002; 2. Straub et al. 2002; 3. Chumakov et al. 2002;

8. 6p24-p22 8p22-p21 DTNBP1 1q21-q22 DRD3 NRG1 RGS4 5q21-q33 6q21-25 1q42 2 1 3 4 5 6 7 8 10p15-p11 HTR2A 13q32-q34 10q 25-26 DAO G72/G30 10 9 11 12 13 14 15 16 17p11-q25 22q11-q22 18 17 19 20 21 22 Y X Positional candidate genes for schizophrenia

9. NRG1 association with schizophrenia Tosato, Dazzan and Collier, Schiz. Bull. 2005.

10. DTNBP1: complex pattern of association findings in schizophrenia Williams, O’Donovan and Owen, Schiz Bull, 2005,

11. DTNBP1 and schizophrenia • Multiple studies suggest that variation in DTNBP is associated with schizophrenia (11/14 positive). • Protection may be mediated by IQ. • No individual SNPs or haplotypes have consistently been implicated in susceptibility. • No systematic study aimed at detecting all common genetic variation. • More studies needed to identify risk variants. • Reduced expression of message and protein in schizophrenia –cause or compensation? • Cis-acting elements regulate DTNBP expression. • Can we relate specific haplotypes to gene expression?

12. DTNBP1 risk haplotype is associated with reduced expression • Allele ratios at SNP rs1047631, stratified by heterozygosity for the defined schizophrenia risk haplotype. • Schizophrenia risk haplotype tags one or more cis-acting variants that result in a relative reduction in DTNBP1 mRNA expression in human cerebral cortex. • Further analyses suggest that risk haplotypes identified in other Caucasian samples also index lower DTNBP1 expression. • Ties risk haplotypes to altered function • Suggests explanation for some of the discrepancies between studies Bray et al, Human Molecular Genetics, 2005.

13. 6p24-p22 8p22-p21 DTNBP1 1q21-q22 DRD3 NRG1 RGS4 5q21-q33 6q21-25 1q42 2 1 3 4 5 6 7 8 10p15-p11 HTR2A 13q32-q34 DAO G72/G30 10 9 11 12 13 14 15 16 PRODH COMT 22q11-q22 18 17 19 20 21 22 Y X Chromosomal abnormalities and schizophrenia

14. VCFS and Schizophrenia • 25+% of adults with VCFS develop schizophrenia (Murphy et al 2000) • Does a gene on 22q11 predispose to schizophrenia in the general population? • Some claims (COMT, PRODH, ZDHHC8, ARVCF) but none convincing

15. DISC1 and schizophrenia (Porteous, Millar, Blackwood, St Clair et al) • Possibility of position effect • Evidence for haplotype association with SZ • Associations with visual and working memory deficits • t (1,16) disrupts PDE4B (binding partner of DISC1) in family with psychosis (Millar et al 2005) • DISC1 complex protein associated with numerous cytoskeletal proteins involved in centrosomal and microtubule function, and with cell migration, neurite outgrowth, and membrane trafficking of receptors and possibly mitochondrial function.

16. Candidate genes for schizophrenia • NRG1 and DTNBP1: multiple positive studies. • DAOA(G72): some positives for both schizophrenia and bipolar disorder • DISC1: highly promising. • Effect sizes small-moderate (OR 1.3-2) • RGS4: some positives but support weakening. • COMT, PRODH, ZDHHC8, PPP3CC, CAPON, CHRNA7, TRAR4 and others: not yet convincing. • Cannot be explained by variants with manifest functional consequences. • Presumably effects on expression, splicing etc. • Remain cautious until risk variants identified. • Inconsistencies between studies. • Lack of power • Genotyping error • Stratification • Incomplete genetic information from indirect studies • NB dysbindin • Susceptibility variants on different backgrounds?? • Allelic heterogeneity/complexity • Heterogeneity: differences in case definition and ascertainment • It’s epidemiology now!

17. Emil Kraepelin, 1896, splits psychosis. • “crystallized dementia praecox and manic-depressive illness from an amorphous mass of madness” (Brockington & Leff, 1979). • Organic • Functional • Dementia Praecox • Manic-depressive insanity

18. Deconstructing Psychosis: Challenges to the Kraepelinian Dichotomy. • No “point of rarity” • Risk factors in common (Murray et al 2004) • Familial co-occurrence of SZP, SA and BP • Cardno et al twin study • Overlapping linkage regions • 13q, 22q, 6q • New genetic studies confirm this and suggest association with clinical syndromes.

19. Studying candidate genes across the Kraepelinian divide: Dysbindin. • No association between BP and the Cardiff haplotype in DTNBP1. • Suggestive evidence for association with BP with predominant psychosis. Upward trend: p = 0.014 Raybould et al, Biological Psychiatry, 57: 696-701, 2005.

20. Studying candidate genes across the Kraepelinian divide: Neuregulin. • NRG1 HAPICE confers risk to illness with both schizophrenia and mood features. • Effect size of NRG1 HAPICE increases with preponderance of mood-incongruent psychotic symptoms (sign test p=0.002). Green et al, Archives of General Psychiatry, 2005.

21. Studying candidate genes across the Kraepelinian divide: G72 (DAOA). • Some positive replications of G72 but no clear associated alleles or haplotypes. • Independent support for association with Bipolar disorder in several studies. • G72 probably strongest candidate gene for BP. • Is this a gene for both disorders?

22. DAOA (G72) in schizophrenia and bipolar disorder • Significant whole gene association in BP (n=706, p=0.045) but not SZ (n=709) vs controls (n=1416). • Significant whole gene association in “Mood” (n= 1153, p=0.0086) and in schizophrenia-mood (n=112, p=0.02) but not psychosis (n=818). • DAOA is probably a susceptibility locus for mood disorder rather than schizophrenia per se. • Extent to which association seen in schizophrenia depends upon clinical characteristics of sample. (Williams et al Archives of General Psychiatry, 2006).

23. DISC1 is associated with broad psychosis and mood phenotype. • t(1:11) segregates with Sz, BP and UP. • Linkage to SA • Hamshere et al 2005. • Evidence for haplotype association • Hennah et al 2003 • Hodgkinson et al 2004 • Schizophrenia, SA and BP • Thomson et al 2005 • SZ and BP.

24. Using genetics to dissect psychosis Craddock, O’Donovan and Owen, Schizophrenia Bulletin, 2006.

25. Do genetic findings in psychosis point to a common mechanism? • The genes most clearly implicated (NRG1, DTNBP1, G72) all code for proteins that potentially impact, directly or indirectly, on the function of glutamate synapses. Harrison and Owen, Lancet, 2003. • But caution required! • proteins implicated poorly understood • multiple processes implicated for NRG1 and DTNBP1

26. White matter abnormalities in schizophrenia (Mt. Sinai Conte Center) • Imaging studies • Defects of connectivity • DTI • Multiple gene expression studies in postmortem schizophrenia brains have found significant reduction of expression of myelin and oligodendrocyte related genes(e.g. Hakak et al., 2001) • Quantitative anatomical analyses have demonstrated decreased oligodendrocyte numbers in prefrontal cortex (Hof et al 2002, 2003). • Cause or effect?

27. CNP is marker for oligodendrocytes • Message and protein show reduced brain expression in schizophrenia • Located at 17q21.2 • rs2070106 is associated with CNP expression (P=0.001). • the lower-expressing allele was significantly associated with schizophrenia (P=.04) in a case-control sample. • All affected individuals in a linked pedigree were homozygous for the lower-expression allele (P=.03). Archives of General Psychiatry (2006) 63: 18-24.

28. Oligodendrocyte Lineage Transcription Factor 2 (OLIG2): a master regulator of all stages of oligodendrocyte lineage. • Basic helix–loop–helix transcription factor central to oligodendrocyte development • Down-regulated in schizophrenia(Tchakev et al, 2003; Katsel et al, 2005; Iwamoto et al, 2005) • Centrality in OL allows for a primary change responsible for several others (parsimony)

29. OLIG2 associated with schizophrenia: non redundant (r2<0.9) positives (submitted). Meff = 9 (all 16 pooled SNPs) gene corrected p = 0.0009 Experiment-wide corrections p = 0.013 (primary 14 genes) p = 0.038 (all 44 genes) CONSERVATIVE

30. Conclusions • Some highly promising findings (NRG1, DTNBP1, G72/DAOA, DISC1) • Need to establish risk nucleotides/mechanisms • And this might not be easy without functional readout (endophenotypic, animal, cellular) • Needs more detailed studies, collaboration, re-sequencing • Expect allelic heterogeneity, effects of ascertainment • Refining the associated phenotype by iteration (Symptoms, Course and outcome, Cognitive function, Imaging). NB samples. • Curation of association data (it’s epidemiology) • Quality standards • Meta-analysis • Need to support genetic associations with biology • DTNBP1 and NRG1 expression studies

31. Future • There are more significant linkages to account for. • Few if any exhaustive fine mapping studies • WGAs • Depend on CDCV hypothesis • Need very large samples • Sample characteristics will be crucial • Data handling and statistical challenges are huge • WTCCC • CNV analysis • Candidate pathway analysis (GxG) • Inclusion of E in genetic studies (GxE) • WGS (CDRV) • Understanding gene regulation (identifying regulatory sequences, miRNA, chromatin effects etc)

32. N Williams N Norton H Williams N Bray A Preece J Wilkinson S Dwyer Elaine Green Rachel Raybould Detelina Grozeva T Peirce B Glaser L Carroll M O’Donovan N Craddock G Kirov I Jones M Hamshere, P Holmans. S Macgregor, V Moskvina, Acknowledgements