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The challenging reality of schizophrenia gene3cs
Professor Bryan Mowry, MD, FRANZCP Queensland Brain Ins3tute
The University of Queensland &
Director, Gene3cs Program Queensland Centre for Mental Health
Research, Brisbane
May 2010
Gene3cs of schizophrenia
• Substan(al gene(c component (family, twin, adop(on)
• However, complex inheritance pa>ern (i.e. non-‐Mendelian) – Spectrum of disease: Mendelian diseases -‐ common diseases
• Mul(ple SZ variants – common and rare in the popula(on – Most of small/modest effect size
• Interac(ng with environmental/other gene(c factors
• Heterogeneity – allelic – locus – popula(on – phenotypic
Most SZ variants occur frequently and confer small effects on risk
SZ gene3c variants
Wright, Gen Biol, 01
There are different classes of gene3c variant
Frazer et al, Nat Rev Genet, 09
Vast majority hypothesised to be neutral
Recent Molecular Gene3c Technological Landmarks
• 2001: Two reference versions of human genome – Human Genome Sequencing Consor(um, reflec(ng assembly of sequences derived from numerous individuals
– Celera Genomics: consensus sequence derived from 5 individuals
• 2005-‐07: Interna(onal HapMap Project • 2007: Encode Pilot Project • 2007: First complete human genome sequence
– J. Craig Venter
• 2009: 1000 Genomes Project – To catalogue human varia(on including rare variants
Genome-‐wide associa3on (GWA) studies • Inves(gates associa(on between common gene(c varia(on and disease
• Hypothesis-‐free -‐ no bias or presump(ve list of candidate genes being tested
• This type of analysis requires: – a dense set of SNP markers (500,000 – >1 million) that capture a substan(al
propor(on of common varia(on across the genome – large numbers of study par(cipants (many 1000’s)
• Detects differences in allele frequencies between cases and controls at each marker
• >80 phenotypes (from all disease categories), GWA studies have provided: – compelling sta(s(cal associa(ons for >650 loci across genome – new understanding of molecular underpinnings & pathways of many diseases
Published Genome-‐Wide Associa3ons through 12/2009, 658 published GWA at p<5x10-‐8
NHGRI GWA Catalog www.genome.gov/GWAStudies
MGS GWAS and meta-‐analysis overview
MGS GWAS
META-‐ANALYSIS
Genomewide significant associa3on 7 SNPs on chromosome 6p22.1 with P<5 x 10-‐8
Lowest p-‐value = 9.54 x 10-‐9
MAIN RESULT
MGS Samples
Affimetrix 6.0 GWAS Array No MGS finding achieved genomewide sta(s(cal significance
Shi et al, Nature, 09
Common variants are associated with SZ 9394 cases / 12462 controls
Replica3on experiment (50 SNPs) underway: Includes AUS dataset
PGC SZ GWAS Collabora3on, WCPG, Nov 09
Where is the culprit?
Ioannidis et al, Nat Rev Genet, 09
How many variants do we expect to find (e.g. T2D)?
18 variants associated with T2D (MAF range: 0.073-‐0.50; OR: 1.05 – 1.15) These explain <4% total liability (small frac(on of heritability): lesson from GWAS
Assuming undiscovered variants have similar MAFs and ORs to these 18, and Es(ma(ng 40% heritability, > 800 variants are required
If assume undiscovered variants are rare, with MAFs ~x10 smaller (0.0073-‐0.050) and ORs ~10 larger (1.63 – 4.05) than those iden(fied, ~85 variants are required
Frazer et al, Nat Rev Genet, 09
Large (>3 Mb), rare chromosomal dele3ons are associated with SZ
ISC, Nature, 2008
1q21.1: replicates Stefansson et al, 08
15q13.3: replicates Stefansson et al, 08 CHRNA7 loca3on
22q11.2: overlaps with VCFS dele3ons
3391 SZ cases/3181 ethnically matched controls
The Road Ahead: moving from robust sta3s3cal associa3ons to knowledge of causal variants
• Refining the phenotype • Transethnic mapping
• Pathway & Network analyses • eQTL and gene expression studies • Next genera(on sequencing • Func(onal studies
• “Pure” clinical phenotype: e.g. SZ only, not SA • Sta(s(cally derived symptom dimensions or clusters (factor analysis/latent class analysis)
• Endophenotypes (measurable components along the pathway between disease [clinical phenotype] and distal genotype [Go>esman & Gould, 03])
– Neurophysiological, neuroimaging, neurocogni(ve
• Overlap with related disorders – Bipolar Disorder
Refining the SZ phenotype
Tamil Nadu SZ gene3c study
1999: Established study
2001-‐2014: NHMRC Support 2001-‐03: family recruitment 2003-‐05: linkage 2010-‐14: case/control recruitment (GWAS)
Clinical characteris3cs of linkage sample (n=262)
A sta3s3cally significant linkage result was observed in our Indian SZ family study
Genomewide significant
Genomewide sugges(ve
Corrected genomewide P=0.01
Nominal p = 0.01
Nominal p = 0.05
Holliday et al, Am J Psych, 2008
Small sample size: yet popula(on homogeneity, pure SZ phenotype, zero substance abuse, homogeneous environment
LCA of 606 Taiwanese SZ Affected Sibling Pairs >1200 SZ cases
Holliday et al, Arch Gen Psych, 09
Chr1q23-‐25 suscep3bility locus for a SZ Subtype resembling Deficit SZ iden3fied by LCA
Peak LOD=3.78, 189 cM from p-‐ter (1q23-‐25), P=0.012; SZ subtype
Original Chr 10q peak using SZ phenotype
Holliday et al, Arch Gen Psych, 09
Gene3c variants overlap for common diseases
Frazer et al, Nat Rev Genet, 09
SZ & BP partly share a common gene3c cause (1) Popula(on-‐based Swedish study (>2 million nuclear families)
Lichenstein et al, Lancet, 09
SZ & BP partly share a common gene3c cause (2) Common polygenic varia3on
Interna3onal SZ Consor3um GWAS (3327 cases; 3587 controls)
• Evaluated whether small effect, common variants collec(vely account for a substan(al propor(on of varia(on in SZ risk
• H1: Increasing propor(ons of small effect variants will be detected at increasingly liberal thresholds PT
• Varia(on summarised into profile scores
• LR of SZ case-‐control status on profile score
• Scores predicted c-‐c status in independent samples – SZ, BP, but not non-‐psych diseases
ISC, Nature, 09
The Road Ahead: moving from robust sta3s3cal associa3ons to knowledge of causal variants
• Refining the phenotype • Trans-‐ethnic mapping
• Pathway & Network analyses • eQTL and gene expression studies • Next genera(on sequencing • Func(onal studies
Popula(ons sampled in GWAS to date
Rosenberg et al, Nat Rev Genet, 2010
No single popula(on is sufficient for uncovering variants underlying disease in all popula(ons There are differences in: disease-‐allele frequency and LD pa>erns
phenotypic prevalence effect size rare variants
McCarthy, Nat Genet, 08
Trans-‐ethnic mapping will help iden3fy alleles of cosmopolitan effect (e.g. KCNQ1 in T2D)
KCNQ1 risk alleles in East Asians: MAF ~40%; Power to detect >80%
KCNQ1 risk alleles in Europeans: MAF ~5%; Power to detect <1%
Trans-‐ethnic mapping may also reveal new popula3on-‐specific loci
Barreo et al, Psychol Med, 2005
1997: Study commenced
NHMRC support: 1999-‐2003: recruit sample
2008-‐2010: conduct linkage & associa(on studies (GWAS)
The Iban of Sarawak cluster with other Asian popula3ons: Principal components analysis of popula3on structure
Xing et al, Gen Res, 2009
Xing et al, Gen Res, 2009
The Iban of Sarawak are a moderately isolated popula3on
Iban pedigree sample
Barreo et al, 05
Pedigree sample: Primary pedigree; 25 smaller mul(plex pedigrees; 14 pedigrees with demonstrated genealogical connec(ons to other pedigrees; 85 addi(onal pedigrees with proband plus available 1st, 2nd degree rela(ves
174 individuals with psycho(c disorders (SZ, SA, BRP, BP, MDD)
GWAS recently completed: linkage and associa(on analyses underway
Within this moderately isolated popula(on:
Extended, eight-‐genera(onal pedigree containing 168 individuals (23 affected)
DNA available for 51 individuals (11 affected)
The Road Ahead: moving from robust sta3s3cal associa3ons to knowledge of causal variants
• Refining the phenotype • Transethnic mapping
• Pathway & Network analyses • eQTL and gene expression studies • Next genera(on sequencing • Func(onal studies
Pathway Analysis -‐e.g. Convergence of two pathways, DISC1 & NRG1-‐ErbB4 that are disturbed in SZ
Jaaro-‐Peled, TINS, 09
(a) Neuronal progenitor cells: DISC1 regulates Wnt Pathway; in nucleus, encounters intracell. domains of NRG1 & ErbB4
(b) Postmito(c neurones (pre/perinatally): DISC1 interacts with PCM1 & BBS in dynein motor complex -‐ role in migra(on, arborisa(on
(c) Postnatal brains: at PSD of pyram. neurones, together with NMDA-‐type glutamate receptor, DISC1 & NRG1-‐ErbB4 cascades are likely to converge; may also converge in nucleus, media(ng gene transcrip(on
The Road Ahead: moving from robust sta3s3cal associa3ons to knowledge of causal variants
• Refining the phenotype • Transethnic mapping
• Pathway & Network analyses • eQTL and gene expression studies • Next genera(on sequencing • Func(onal studies
Cookson et al, Nat Rev Genet, 09
eQTL and Gene Expression studies – transcript abundance as a quan3ta3ve trait
The Road Ahead: moving from robust sta3s3cal associa3ons to knowledge of causal variants
• Refining the phenotype • Transethnic mapping
• Pathway analysis • eQTL and gene expression studies • Next genera(on sequencing • Func(onal studies
Next-‐gen sequencing can detect rare variants a component of gene3c “dark maoer”
Antanorakis et al, Nature, 2010
Geographic map of five sequenced (personal) genomes
Kim et al, Nature, 09
• Cost US $200K with 6 week total run (me using 3 next-‐gen sequencers vs Sanger: >$1million
• 1000 Genomes Project
• Need further reduc(on in: cost false-‐pos, false-‐neg rates
Next-‐genera3on sequencing: applica3ons
Uses in human and model organisms: • Comprehensive SNP, CNV and muta3on discovery • Quan3fica3on of gene expression • MicroRNA profiling • Genomewide methyla3on • Protein-‐DNA mapping
Two NG-‐Seq applica3ons
Targeted resequencing of candidate genes
• Resequenced exons/splice sites of 10 candidate genes (31Kb) for T1D (480 cases/480 controls)
• Tested for associa(on in 30000 par(cipants
• Discovered four rare variants that lowered T1D risk independently of each other
Complete genome sequencing in families
• Analysed whole genome sequences of family of four (2 siblings, both parents)
• Iden(fied recombina(on sites precisely (genomic inheritance analysis)
• Iden(fied very rare SNPs • Narrowed the number of
candidate genes for both Mendelian disorders in this family
Nejenski et al, Science, 09 Roach et al, Science, 2010
The Road Ahead: moving from robust sta3s3cal associa3ons to knowledge of causal variants
• Refining the phenotype • Transethnic mapping
• Pathway analysis • eQTL and gene expression studies • Next genera(on sequencing • Func(onal studies
The puzzle of gene deserts e.g. 8q24 region and suscep3bility to various cancers
Ioannidis et al, Nat Rev Genet, 09 335 Kb upstream of closest characterised gene (MYC oncogene)
8q24 gene desert: further inves3ga3ons of compelling sta3s3cal associa3on between rs6983267 and Colorectal Cancer (CRC)
• Need to be confident that finding is robust – Func(onal studies are (me-‐consuming and costly
• CRC risk locus containing rs6983267 has in vitro and in vivo proper(es of an enhancer and shows long-‐range physical interac(on with MYC (Using CHiP assays, luciferase reporter assays, chromosome conforma(on capture) (Pomerantz et al, Nat Genet, 09)
• rs6983267 confers poten(al to enhanced Wnt signalling (using CNV analyses, imputa(on & associa(on, in silico methods, transcrip(on factor binding assays, EMSA, luciferase reporter assays, CHiP-‐Seq, microarray gene expression) (Tuupanen et al, Nat Genet, 09)
• In vivo assessment in model organisms • Help tremendously to decipher the basic biological mechanisms
Celniker et al, Nature, 09
Unlocking the secrets of the genome
Caveat: because model organisms lack the genomic variability of humans, unable to fully explain human mul(factorial traits
Clinical transla3on of gene3c findings
McCarthy et al, Nature, 08
Acknowledgements Perth Carolyn Graham Jeremy Hyde
Hobart Ken Kirkby Ivor Jones
QCMHR Deborah Nertney Duncan McLean Elizabeth Holliday Patricia Nolan John McGrath Michael Theodoros
QBI Vikki Marshall Denis Bauer Jake Graoen Heather Smith Cheryl Filippich
QIMR Dale Nyholt Stuart MacGregor Peter Visscher
Iban of Sarawak Robert Barreo Edward Jerah Peter Loa Derek Nancarrow
Tamil Nadu, India Rangaswamy Thara Sujit John Tirupa3 Srinivasan Giri Krishnan Betsy Sajish Ayankaran Ramadas Anita Muthuraj Sowandari Kooeswaran Mangala Ramamur3 Padma Ramachandran Arthi Ganesh Meiya Vargheese Priya Thirunavakarasu Manimala Anuradha Vivek Bhuvaneshwari Anne Lavanya
ASRB Vaughan Carr Ulrich Schall Rodney Scoo Assen Jablensky Patricia Michie Stanley Caos Christos Pantelis Frans Henskens
Aus Twin Study Christos Pantelis Alex Fornito Jonathan Chalk Stephen Rose Dominique Hannah Stephen Wood Karen Shaw Liz Leeton Lauren Hoiles Carlyn Muir
Diaman3na Inst, PAH Maohew Brown Patrick Danoy
Univ. of Utah Lynn Jorde Scoo Watkins Tatum Simonson David Witherspoon
MGS Consor3um Pablo Gejman Douglas Levinson Alan Sanders Jubao Duan Nancy Buccola Robert Freedman Farooq Amin Donald Black Jeremy Silverman William Byerley Raymond Crowe C Robert Cloninger Maria Mar3nez Brian Suarez
PG SZ Consor3um Stephan Ripke
