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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
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