View
695
Download
1
Category
Tags:
Preview:
Citation preview
¿Qué sigue después de la identificación de los genes candidatos para las
enfermedades autoinmunes?Christopher J. Lessard, B.Sc., PhD
Investigador Asociado, Departamento de Artritis e Inmunología Clínica, Fundación para la Investigación Médica de Oklahoma, Oklahoma City, OK, E.U.
What's Next After Candidate Gene and Genome-wide Association
Studies in Autoimmunity
Christopher J. Lessard, Ph.D., B.Sc.Associate Research Scientist
Oklahoma Medical Research Foundation
2nd Colombian Autoimmune Symposium3 March 2011
Outline
• Identification of susceptibility loci– systemic lupus erythematosus (SLE)
• Question of missing heritability• Identification of causal variants• Functional studies• Epistasis• Future of genetics
Candidate Gene Studies in SLE
• History of SLE genetics began in 1970s
• C2, C4, C1q very rare but potent risk loci
• Human genome sequence 99.9% complete
• IRF5 most replicated SLE locus
• First genome-wide association study 2006
PAD14
PsorSLE1996
2006
2007
2010
2008
2009
2005
2004
2003
2001
MS CD RAT1DINS
IBD5SH2D2
ACARD1
5
CTLA4
PTPN22 PTPN22
IRF5IL2Ra IL7R
IL23RIFIH1 ICAM-1 FCRL3PHOX2
BATG16L
1
1st GWAS
Impact of Genome Wide Association Studies on Gene Discovery
Very few confirmed associations prior to 2006
PTPN22
PAD14
PsorSLE1996
2006
2007
2010
2008
2009
2005
2004
2003
2001
MS CD RAT1DINS
IBD5SH2D2
ACARD1
5
CTLA4
PTPN22 PTPN22
IRF5IL2Ra IL7R
IL23RIFIH1 ICAM-1 FCRL3PHOX2
BATG16L
1
C12orf30
KIAA350
ERBB3 CD226
Tenr-IL2 PTPN2
IL7R NKX2-3 10q21ATG16L
1PTPN2
IL12B 3p21IRGM 5p13
TRAF1STAT4
ITGAVTNFAIP
3CARD8
FOXJ1 IL2Ra CD58
IL7RADAM3
3IL12B
IL23R
PTPN21CLEC16
AC12orf
30BACH2
PRKCQ
IFIH1
PTPN11
ERBB3 K1F1B
PTPN1
JAK2
CTSH CTLA4
ORMDL3
IL23R
IRGMPTGER
4IL12B
PTPN2
NOD2ATG16L
1
STAT3
MST1
CDKAL1
ZNF365
TNFSF15
CCR6ITLN1
MUC19ICOSLG
C11orf30
PTPN22
OLIG3
NKX2-3
TRAF1
CD40
STAT4
TNFAIP3
ITGAM
PHRF1
SPP1
PXK
BLK
BANK1
DLK1 RIO3
MEG3 TYK2
C10orf59
IL2RA
SHBB3 IL27
LRRC18PTPN2
IL10
RTL1 INS
HIC2
C6orf173
GLIS3 UBASH3A BLK
CTLA4
IL2 CD69
IRF8
TNFRSF1A
TNFSF4
REL
STAT4CD6
ETS1CYP27B
1
JAZF1
IKZF1
UBE2L3
RASGRP3
ORMDL3
ATG5
PRDM1SLC15A
4
WDFY4
TNIP1
IL13
TNIP1TNFAIP
3
IL23A
STAT2
CCR6 AFF3
RBPJ IL2RA
ANKRD55 IRF5
IL6ST CCL21 STAT3 CBLB
SPRED2
METTL1 ETS1DQA1
CD40
6q21
5q33.3
ICA1
C8orf12
XKR6
SCUBE1
NMNAT2
8p23.1LYN 1q25.1
UHRF1BP1
IL10
7q11.23
CD44
IRF8
1st GWAS
Impact of Genome Wide Association Studies on Gene Discovery
PTPN22
2008: New SLE Genes Revealed by GWAS
Association between a SNP and disease status
DD 10/20 = .50 Dd 6/20 = .30 dd 4/20 = .20
DD 2/20 = .10 Dd 4/20 = .20 dd 14/20 = .70
Affecteds Controls
Test distributions for
a statistically significant difference
For “D” = disease risk allele:
MH
C
IRF5
TN
FAIP
3
MN GWAS Results Summary
431 SLE cases; 2155 controls
Graham et al. Nat Genet 2008
P=5x10-8 (genome-wide significant)
P=9x10-7 (suggestive)
Sig
nifi
canc
e le
vel
SNP Location (by chromosome then base pair)
Genome-wide - P=5x10-8
Suggestive - P=9x10-7
Many Genes, Fewer Genetic Pathways in SLE
Association of ~35 genes robustly confirmed, more on the way b((Modified from Moser et al, Gene & Immunity SLE Genetics Special Issue, 2009)
Innate Immune Response
Lymphocyte Activation/Functi
on
TLR/IFN signaling
STAT4
IRAK1
Pathways GenesDendritic cells
Macrophages
IRF5
Autoreactive T cells
Autoreactive B cells
Immune Complex
Clearance
TNF/NFκB signaling TNFAIP3
T Cell signaling
B Cell signaling
HLA-DR PTPN22
BLK BANK1
Macrophages
Neutrophils
Phagocytosis FcγR3A
FcγR2B
C4A C2
FcγR3B
C4B
IRAK1 TNFSF4
Other
PXK XKR6ICA1
ATG5
NMNAT2
MECP2
ITGAM
PDCD1
SCUBE1
UBE2L3
KIAA1542
CRP
Complement
STAT4
C1q
Apoptosis
Ubiquitination
DNA methylation
Unknown
Cellular adhesion
TNFAIP3
SPP1
HLA-DR
ITGAM
Cells
STAT4
LYN
TREX1
CD44
More Loci Identified in 2009-present
• Continued replication in European cohorts
• GWAS replication limited to top few hits– CD44 ranked ~2000– Ranking of SNPs by
p-value not optimal– Common or rare
variants hard to detect in small sample sizes
Asian GWAS completed in 2009
• First SLE GWAS in non-European population
• Evidence for susceptibility loci unique to Asians
• Some regions not evaluated in Europeans
Question of Missing Heritability
• Many risk loci, but marginal risk – odds ratios (OR) typically < 2.0– HLA and TNFAIP3 OR ≈ 2.5
• HLA and IRF5 account for ~1% of heritable risk for Europeans
• With all loci identified (~35), it is estimated that only 8-12% of heritable risk for Europeans has been identified
• Why?
GWAS to Date Limited
• Many of the causal variants yet to be identified
• Functional consequences remain elusive
• No subphenotype GWAS• Limited resequencing to find rare
variants• Methylation not comprehensively
studied • No whole transcriptome sequencing • No Amerindian and African-American
GWAS
Causal Variants Bottleneck
• Linkage Disequilibrium (LD)– Correlation between
variants– Aids identification of
association during GWAS (lowers cost)
– Makes causal variant localization difficult
CD44 Replication Dataset and Association Analysis
Analysis: Logistic regression (PLINK)
Compare distribution (frequencies) of SNP genotypes (DD, Dd, dd) between
cases and controls to identify a statistically significant difference
P-value thresholds for two levels of significance:
“Genome-wide” = p < 5 x 10-8
“Suggestive” = p-value < 1 x 10-4
GWAS identified 2 SNPs p=3x10-3
Cases Controls
Total
Europeans (EU) 3562 3491 7053
African Americans (AA)
1527 1811 3338
Asians (AS) 1265 1260 2525
Hispanics (H) 961 336 1297
Gullah (G) 152 123 275
7998 7492 15490
Replication of Association at 11p13 near CD44
meta-analysis
Results After Imputation
Association with Other Ethnic Groups
• Both AA and Asians associated with rs2732552:
- AA: P=5.0x10-3
- Asian: P=4.3x10-4
- Combined all Europeans, Asians and African-Americans: Pmeta=3.0x10-13
• rs387619 was associated with Asians, but not AA
• No evidence of association with Hispanics, Gullah or Amerindians
EuropeanAsian
African-American
Regulatory Region
• Haplotype contains several
regulatory elements:
- ~74 Kb telomeric of
CD44 and ~61 Kb
centromeric of PDHX
- Several regulatory sites
identified by mining
ENCODE ChIP-Seq data
CD44• Widely expressed in immunological cells
- Numerous splice variants
- Tissue specificity
- Variable exonic region
• Cell-surface glyoprotein involved in cell-cell
interactions, cell adhesion and migration
• Receptor for:
- Hyaluronic acid (HA), osteopontin,
collagens, matrix metalloproteinases
(MMPs)
• Accessory molecule in the synapse between
APC-T cell complex
~93Kb
CD44 and SLE
•Expression of protein widely studied
TNFAIP3 Putative Causal Variant
NF-B Signaling
TNFAIP3 encodes the protein A20
Vigo Heissmeyer & Anjana Rao, Nature Immunology 9, 227 - 229 (2008)
Attenuates TNF signaling
Association of TNFAIP3
• Identified a broad region of association
• No association in African-Americans
• Imputation and resequencing reveal putative causal variant
Europeans
Asians
Koreans
Imputation and Resequencing
• Imputation found a novel SNP in the 1000 Genomes dataset
• Resequencing identified a deletion/insertion polymorphism
• Together form TT>A putative causal polymorphism• Conditional analysis
Transcription Factors Sites Identified
• Chromatin immunopreciptitation followed by sequencing by ENCODE project
• Used this data to begin functional studies
Risk Variants Affect Biology
• Found difference in binding using electrophoretic mobility shift assays
• mRNA expression altered
• Protein expression altered
Epistasis
• Gene-gene interactions• Usually done statistically• No evidence for epistasis in regions found
in SLE work• TNFAIP3 and TNIP1 both associated with
SLE and do interact biologically
Future of Autoimmune Genetics
• Essential to recruit more subjects• Much larger GWAS:
– >50,000 subjects– More variants (>5 million)– Makes subphenotype studies possible
• Whole genome sequencing• Whole transcriptome sequencing• eQTL analysis with GWAS data• Epigenetic studies
Power to Detect Association
• Power influenced by:– Allele freqency– Odds ratio– Sample size
• Need large sample size for rare variants and recessive effects
USA LFRR Collaborators & Approved Users
Andrey ShawWashingtonU
Missouri
Joseph McCuneUniversity of Michigan
Michigan
Timothy BehrensUniversity of Minnesota
Minnesota
Michael SchneiderSouthern Illinois UTimothy NiewoldTammy UtsetUChicago
Illinois John B. HarleyCincinnati Children’s Hospital MCJane OlsonCase Western University
Ohio
J. Lee NelsonF. Hutchinson Cancer Res CtrGerald NopomVirginia Mason Research Ctr
Washington
Steve BindorBio-Rad LabsLisa BarcellosUC BerkeleyEvan HermelTouro UniversityLindsey CriswellUCSFBetty TsaoUCLAAnshu AgrawalUniv CaliforniaMariana IsraeliDan WallaceMichael WeismanCSMCChaim JacobUSC
California
Gary GilkesonDiane KamenMUSC
South Carolina
Bart HaynesDuke University
North Carolina
Andras PerlSUNYJane SalmonHospital for Special SurgeryNicholas ChiarazziCharles ChuPeter GregersonNorth Shore University Hospital
New York
Harold ChapmanHarvard Medical SchoolBrigette HuberTufts UniversityPatricia FraserBrigham & Women’s Hospital
Massachusetts
Mark ShriverPenn State UKathleen SullivanChildren’s Hospital of Philadelphia
Pennsylvania
Mark MamulaYale University
Connecticut
Jonathan ChairesJames Brown Cancer CenterBart HaynesDukes UniversityDama LaxminarayanaWake Forest University
Kentucky
OklahomaMorris FosterOUKathleen O’NeilOUHSC
OMRFMarta E. Alarcón-RiquelmeDarise FarrisBart FrankJudith JamesKen KaufmanBiji KurienJoan MerrillCourtney MontgomeryKathy MoserPatrick GaffneySwapan NathAmr SawalhaHal ScofieldEdward Wakeland
David KarpUT Southwestern MC
Texas
Robert KimberleyUAB
Alabama
International LFRR Collaborators & Approved Users
United Kingdom
Timothy VyseImperial College of Science, Technology & MedicineGrant GallagherUniversity of Glasgow
Puerto RicoAna QuinteroOMRF
SpainManual Ramos-CasalsBarcelona Hospital
South KoreaBao Sang-CheolUniversity of Seoul
DenmarkRuna NolsoeSteno Diabetes Center
JapanSachiko HirosaJuntonda U School of MedicineSumida TakayukiUniversity of Tsukuba
AustraliaCarola VinuesaAustralian National University
Juan-Manuel AnayaAdriana RojasUniversity del Rosario
Colombia
Ontario
Rose GoldsteinUniversity of OttawaAndrew PatersonUniversity of Toronto
Roland JonssonUniversity of Bergen
Marta AlarconUppsala University
Markus PerolaNational Public Health Institute
Norway
Sweden
Finland
SGENE: The Sjogren’s Genetics Network
International group interested in contributing DNA samples and clinical data for genetic studies
Currently:15 contributing sites (plus subsites) >2000 SS cases (AECG criteria), 2000 controlsDeveloping consistent clinical dataset
Goal:Continue to expand sitesOverall: 15,000+ cases
Primary cohort for Replication Studies
The LFRR always needs more participants
lupus-recruiters@lupus.omrf.orglupus.omrf.org
More Sjögren’s syndrome Patients Needed
lessardc@omrf.org
The Lupus Family Registry & Repository
Recommended