Revealing Genetic

Architecture of Schizophreniausing Bioinformatic Tools

Zeynep Sena Ağım

Boğaziçi University,

December 03, 20101

OUTLINE

• Introduction

– Schizophrenia

– Molecular Biology

– Molecular Genetic Studies

• Aim

• Methods

• Results

• Future Perspectives

2

Schizophrenia Facts

• one of the most common neuropsychiatric disorders in the world

• heritability is ~80%

• with a lifetime rate of

– ~1- 2% in the US (2.2 million)

– 0.5 - 1 % in Turkey (600.000)

• Gender differences (in the US)

– Males, earlier onset

• Males: 18-25

• Females: 25-30 & 40

– Males , poorer diagnosis

– Male / Female ratio: 1.4

McGrath JJ, Schizophr Bull (2005)

Cohen RZ, Am J Psychiatry (1999)3

Rorschach Inkblot Technique

4

Symptoms

PsychosisEmotional Regulation

Lack of

motivationVerbal

fluency

Social

withdrawal

Neurocognition

Hallucinations

Delusions

Memory

Attention

Learning

Sellin et al, CNS Spectr, 2008

Cellular and Molecular

Interactions in Schizophrenia

Gray JA, Mol Psychiat. 2007

• Glutamatergic

– NMDA receptors

– Glutamate receptors

• Neurodevelopmental

– Neuronal survival

– Growth

– Differentiation

• Dopaminergic

– Dopamine receptors

5

Molecular Genetic Studies

on Schizophrenia

• Linkage Studies

– Family based studies are extremely successful in detecting rare variants with strong effects on etiology of the disorder.

– Loci on 2q, 5q, 8p, 13q and 22q

• Genome-wide Association Studies (GWAS)

– have greater power than linkage studies to detect common risk alleles with small effects

6Nieratshker et al, 2010, Frontiers in Behav Neurosc

Low-frequency variant and

disease susceptibilty

7McCarthy, 2008, Nat. Rev. Genet.

AIM

• So far, studies on schizophrenia were not able to give consistent results.

• GWAS conclude the most significant variation among hundred thousands of SNP. (p<5 x 10-8)

• To investigate significant haplotypeblocks that are inherited together in schizophrenia cases.

8

METHODOLOGY

1. Gathering genes that are associated with schizophrenia

• NIH catalog

• SZGene Forum

• Pubmed

2. Pathway Analysis

• Ingenuity Pathway Analysis

3. Gathering GWA datasets

• NIH Catalog

• dbGAP

4. Examination of selected gene in GWA datasets

• PLINK

• Haploview

9

1. Gathering genes that are associated

with schizophrenia

10

NIH Catalog

SZGene

Pubmed

2839

11

Validation

Shared ones 46

http://www.genome.gov/gwastudies/index.cfm?pageid=26525384#searchFormhttp://www.schizophreniaforum.org/res/sczgene/default.aspwww.pubmed.com

2. Pathway AnalysisIngenuity Pathway Analysis

11http://www.ingenuity.com/

ERBB4 gene and protein level

• 1.1 mb

28 exons

1308 aa

• EGF receptor family

• Ligand: NRG family

• Mitogenesis

and differentiation

12

ERBB4 in Schizophrenia

• ERBB4 transgenic models show similiar, but not the same behavioural phenotypes of SCZ.

• Perturbations in NRG1-ERBB4 signalingcontribute to SCZ.

• Increase in mRNA of ERBB4 in SCZ postmortembrains

• Ligand-induced activation of ERBB4 in SCZ

13

First evidence of association of ERBB4 variantin human brain with schizophrenia

14

3. Gathering GWA datasets

15

Athanasiu

February 23, 2010

201 Caucasian cases, 305 European controls 2,663 European cases, 13,780

European controls

PLAA

ACSM1 ANK3

Affymetrix [572,888]

International

Schizophrenia Consortium

July 01, 2009

3,322 European descent cases, 3,587 European

descent controls

4,692 European descent cases, 15,493

European descent controls

MHC

TCF4

FXR1

PTBP2

Affymetrix [739,995]

Shi

July 01, 2009

2,681 European ancestry cases, 2,653 European

ancestry controls, 1,286 African American cases,

973 African American controls

5,327 European ancestry cases, 16,424

European ancestry controls

SLC17A1,

SLC17A3,

BTN3A2,

BTN2A2,

BTN3A1,

HIST1H2AG,

HIST1H2BJ,

PRSS16,

POM121L2,

ZNF184

HLA-DQA1

Affymetrix [up to 843,798]

Stefansson

July 01, 2009

2,663 European cases, 13,498 European controls 10,282 European cases, 21,093

European controls

MHC, PRSS16

NOTCH4

NRGN

TCF4

VRK2

SLCO6A1

Illumina [314,868]

Need

February 06, 2009

871 European ancestry cases, 863 European

ancestry controls

1,460 European ancestry cases, 12,995

European ancestry controls

NS Illumina [312,565]

O'Donovan

July 30, 2008

479 cases, 2,937 controls 6,666 cases, 9,897 controls ZNF804A Affymetrix [362,532]

Walsh

April 25, 2008

150 cases, 268 controls 83 children, 154 parents Pending Illumina [~550,000]

Sullivan

March 18, 2008

738 cases, 733 controls NR AGBL1

ACSM1,

BUCS1

Affymetrix [492,900]

Kirov

March 11, 2008

574 cases, 605 controls, 1,148 parents of cases NR CCDC60 Illumina [~550,000] (pooled)

Shifman

February 15, 2008

660 cases, 2,271 controls 2,274 cases, 4,401 controls RELN Affymetrix [510,552]

Lencz

March 20, 2007

178 cases, 144 controls NR CSF2RA,

IL3RA

Affymetrix [439,511]

First Author/Date Replication Sample Size PlatformReported

Gene(s)Initial Sample Size

CATIE

nonGAIN

GAIN

Schizophrenia Datasets

16

CATIE

EA

Female Male

GAIN

EA

Female Male

nonGAIN

EA

Female Male

MGS

EA

Female Male

Haploviewhttp://www.broadinstitute.org/haploview/haploview”

Haplotype Analysis

CATIE_EA

GAIN_EA

SS=0.6

HWE=0.05

MAF=0.05

4. Examination of ERBB4 in GWA datasets

nonGAIN_EA

AIM B

• Significance of haplotype blocks in ERBB4 gene are not consistent among three GWA datasets.

• It is very difficult to explain such a complex disease with a single SNP or mutation.

• To investigate statistical interactionamong genes in schizophrenia using three GWA datasets.

18

METHODOLOGY B

19

5. Filtering GWA datasets

• Plink

6. Deciding epistasis design

• Ingenuity

• Review articles

7. Epistasis

• Plink

8. Examination of interacting SNP/blocks

• Haploview

9. Features of blocks

• Exonic/intronic: dbSNP

• Transcription binding sites: fastSNP

• Alternative splicing: ASSP

5. Filtering GWA datasets

20

BEFORE AFTER

Courtesy of Melda Esendal

Age and Gender Distributions

in Cases and Controls

21

CATIE

Nu

mb

er o

f su

bje

cts

Age Interval

Nu

mb

er o

f in

div

idu

als

Nu

mb

er o

f in

div

idu

als

GAIN

nonGAIN

MGS

Nu

mb

er o

f su

bje

cts

Age Interval

BEFORE

Nu

mb

er o

f su

bje

cts

Age Interval

AFTER

Nu

mb

er o

f su

bje

cts

Age Interval

BEFORE

Nu

mb

er o

f su

bje

cts

Age Interval

AFTER

Nu

mb

er o

f su

bje

cts

Age Interval

BEFORE

Nu

mb

er o

f su

bje

cts

Age Interval

AFTER

Courtesy of Melda Esendal

6. Deciding epistasis design

22

7. Epistasis

23

processes Genes

1 NMDA GRIN2B

2 NMDA GRIK3

3 NMDA GRIK4

4 NMDA GRM3

5 NMDA GRM4

6 NMDA GRM7

7 PI3K/AKT signalling, growth factors and related ERBB4

8 PI3K/AKT signalling, growth factors and related NRG1

9 PI3K/AKT signalling, growth factors and related NRXN1

10 other related genes DISC1

11 other related genes PDE4B

12 other related genes DLG1

13 other related genes DLG2

14 other related genes DLG3

15 other related genes DLG4

16 other related genes DLG5

17 other related genes DLG6

18 other related genes DLG7

19 other related genes PICK1

20 other related genes ESR1

21 other related genes ESR2

22 other related genes AR

Gene 1 Gene 2Size of the Gene 2

Number of Significant Interaction (p<0.001)

ERBB4 DLG2 2172912 22

ERBB4 ESR1 472929 1

ERBB4 GRIK4 326162 5

ERBB4 GRIN2B 418910 3

ERBB4 GRM3 220977 2

ERBB4 GRM4 133540 1

ERBB4 GRM7 971530 37

ERBB4 NRG1 1124806 16

ERBB4 NRXN1 1114032 6

8. Examination of interacting SNP/blocks

24

ERBB4 - GRM7 INTERACTIONSSNP1 SNP2 P_COMBINED_C+G+NG

GRM7_rs12497688 0,000041539

GRM7_rs1878164 0,0005

GRM7_rs3749448 0,0003

GRM7_rs6443090 0,0003

GRM7_rs7621420 0,0002

GRM7_rs9814881 0,000094884

GRM7_rs11918634 0,0004

GRM7_rs41412146 0,0005

ERBB4_rs10203686 GRM7_rs9819314 0,0008

GRM7_rs12497688 0,0009

GRM7_rs13072518 0,0006

GRM7_rs4143516 0,0008

GRM7_rs6799329 0,0008

GRM7_rs9814881 0,0008

ERBB4_rs10497945 GRM7_rs576913 0,0006

GRM7_rs10510356 0,000095985

GRM7_rs17047073 0,0003

GRM7_rs41346444 0,0003

GRM7_rs13072518 0,000029052

GRM7_rs1499146 0,0004

GRM7_rs9311976 0,0003

GRM7_rs9812630 0,0005

GRM7_rs9860274 0,0009

GRM7_rs9875215 0,000065973

ERBB4_rs13419353 GRM7_rs4686148 0,0005

ERBB4_rs16847102 GRM7_rs4686101 0,0004

ERBB4_rs16847543 GRM7_rs4686101 0,0007

GRM7_rs1508724 0,0002

GRM7_rs1963265 0,000011619

GRM7_rs6769814 0,0002

ERBB4_rs1992029 GRM7_rs11706752 0,0008

GRM7_rs11131064 0,0002

GRM7_rs9881908 0,0009

ERBB4_rs3828242 GRM7_rs11706752 0,0008

GRM7_rs752298 0,0007

GRM7_rs752300 0,0009

GRM7_rs779699 0,0008

ERBB4_rs10165449

ERBB4_rs10175279

ERBB4_rs10206846

ERBB4_rs10932433

ERBB4_rs13025037

ERBB4_rs17744862

ERBB4_rs2371438

ERBB4_rs6435664

CONCLUSION

• So far, none of the ERBB4 variants havebeen shown to be significant in GWAS.

• Significant ERBB4 haplotype blocks arenot overlapping among three GWA datasets.

• By epistasis analysis, we showed that SNPsin two blocks in ERBB4 are in statisticalinteraction with four blocks in the GRM7 gene.

25

FUTURE PERSPECTIVES

26

8. EXAMINATION OF INTERACTING SNP/BLOCKS

•Haploview

9. FEATURES OF BLOCKS

•Exonic/intronic: dbSNP

•Transcription binding sites: fastSNP

•Alternative splicing: ASSP

Acknowledgements

27

Prof Dr Nazlı Başak

Dr Hilmi Özçelik

NDAL Members

Ozcelik Lab Group