GWAS of SADB

MRI-WMH

Stphanie Debette
MCU-PH; Epidmiologie et Neurologie Hpital Lariboisire et Inserm U740; Universit Paris 7 - DHU Neurovasc Sorbonne Paris-Cit Chaire dExcellence ANR Universit de Versailles St Quentin en Yvelines - Inserm U708
Adjunct Associate Professor Department of Neurology; Boston University School of Medicine; Framingham Heart Study; USA

Genome-Wide Association Studies of Small Artery Disease of the Brain

MRI-defined White Matter Hyperintensities

Stphanie Debette
MCU-PH; Epidmiologie et Neurologie Hpital Lariboisire et Inserm U740; Universit Paris 7 - DHU Neurovasc Sorbonne Paris-Cit Chaire dExcellence ANR Universit de Versailles St Quentin en Yvelines - Inserm U708
Adjunct Associate Professor Department of Neurology; Boston University School of Medicine; Framingham Heart Study; USA

GWAS of SADB
Introduction To SADB and white matter hyperintensities (WMH) To GWAS GWAS of WMH Summary of findings Follow-up and extension Interpretation Unsolved mysteries of SADB genetics Missing heritability of WMH? Genetics of other expressions of SADB?

GWAS of SADB
Introduction To SADB and white matter hyperintensities (WMH) To GWAS GWAS of WMH Summary of findings Follow-up and extension Interpretation Unsolved mysteries of SADB genetics Missing heritability of WMH? Genetics of other expressions of SADB?

Small Artery Disease of the Brain can be

Overt (Stroke)
Small subcortical ischemic stroke Deep ICH

Covert (MRI-defined SADB)

White matter hyperintensities

Small subcortical brain infarct

Cerebral microbleeds

Small Artery Disease of the Brain can be

Overt (Stroke)
Small subcortical ischemic stroke Deep ICH

Covert (MRI-defined SADB)

White matter hyperintensities

Small subcortical brain infarct

Cerebral microbleeds

Accelerated cognitive decline Risk of Stroke, Dementia and Death

Debette & Markus; BMJ 2010

Small Artery Disease of the Brain can be

Overt (Stroke)
Small subcortical ischemic stroke Deep ICH

Covert (MRI-defined SADB)

White matter hyperintensities

Small subcortical brain infarct

Cerebral microbleeds

Rationale for GWAS of WMH burden

Heritability of WMH volume is high: 55-80%
Framingham Heart Study; general population over 3 generations: 55% World War II veteran twins: 71% GENOA; hypertensive sibships: 80%

Can be studied non-invasively and quantitatively on large population-based samples Assumption that endophenotypes such as WMH reflect a less complex genetic architecture than more downstream clinical disorders (e.g. stroke or dementia)
Morrison; Stroke 2000 Atwood; Stroke 2004 Turner; Hypertension 2004 Preston; Dialogues Clin Neurosci 2005

White matter hyperintensities

White matter hyperintensities are indicators of cerebral small artery disease
Lipohyalinosis, Arteriolosclerosis, Atherosclerosis at the origin of arterioles, or Cerebral Amyloid Angiopathy

Pathological correlates include myelin pallor, tissue rarefaction with myelin and axonal loss, and mild gliosis

Diffuse loss of myelin, Cystic lacunar lesions

Lipohyalinosis; Enlarged perivascular spaces Schmidt, Acta Neuropathol 2011; Pantoni, Stroke 2007

White matter hyperintensities

The pathophysiology of WMH is imperfectly understood WMH are often considered to reflect areas of incomplete infarction
Absence of anastomoses in the vascular architecture of deep brain regions increasing vulnerabality to chronic hypoperfusion

Dysfunction of cerebral arteriolar and capillary endothelium

Blood-brain barrier disruption

chronic leakage of plasma fluid components migration of cells into the vessel wall, with subsequent tissue damage loss of autoregulatory ability and arteriolar thrombosis.
Wardlaw, Lancet Neurol 2013 De Reuck, Acta Neurol Belg 1972 Pantoni, Stroke 2007

White matter hyperintensities

Few risk factors are known Age and hypertension are currently deemed the most important predictors of WMH burden
Midlife BP measurements and cumulative BP measured over several years appear to be better predictors of WMH progression than single late-life measurements
A significant association of increasing BP with subtle white matter changes is already seen in young adults in their thirties

Not all individuals with WMH are hypertensive, and other risk factors, including genetic, are likely involved
Maillard; Lancet Neurol 2012 Debette; Neurology 2011 De Leeuw; Brain 2002 Dufouil; Neurology 2001

Tools in genetic epidemiology

Linkage studies (family-based):
Simultaneously examine transmission across generations of both disease phenotype and marker alleles Genetic association studies (population-based): Compare frequency of genetic variants; between patients and controls; or across the spectrum of a continuous trait more powerful for complex diseases

Risch; Science 1996

Candidate Gene Studies

Test association of disease with candidate genetic variants
Based on a priori hypotheses about pathophysiology

Thousands have been published; with very few convincingly replicated loci
Main methodological flaws
small samples absence of pre-planned replication limited screening of genetic variation wrong candidate...

Hirschhorn; Genet Med 2002

Candidate Gene Studies of WMH burden

Genes Apolipoprotein E Methylenetetrahydrofolate reductase Angiotensin-converting enzyme Angiotensinogen Matrix metalloproteinase 3 Matrix metalloproteinase 9 Angiotensin II receptor 1 Adducin 1 Endothelial nitric oxide synthase Aldosterone synthase Intercellular adhesion molecule 1 Kinesin light-chain 1 Paraoxanase 1 Cholesteryl ester transfer protein Factor 3 Kit Ligand Calpain10 Matrix metalloproteinase 2 Interleukin-6 Glucocorticoid receptor Polymorphism rs7412; rs429358 2;3;4 rs1801133 C677T rs1799752 I/D Reference Bronge, 1999; de Leeuw, 2004; Gurol, 2006; Lunetta, 2007; Hogh, 2007; Paternoster, 2009

Kohara, 2003; Hassan, 2004; Paternoster, 2009 Amar, 1998; Hassan, 2002; Sierra, 2002; Paternoster, 2009 rs699 M235T Gormley, 2007; Schmidt, 2001; van Rijn, 2007; Paternoster, 2009 rs679620 K45E Fornage, 2007 rs2250889 P574R Fornage, 2007 rs5186 A1166 C Henskens, 2005; van Rijn, 2007 rs4961 Gly460Trp van Rijn, 2006 rs1799983 Henskens, 2005 rs1799998 -344C/T Verpillat, 2001 rs5498 K469E Han, 2005 rs8702 Szolnoki, 2007 rs854560 Schmidt, 2000; Hadjigeorgiou, 2007 rs1800775 C-629A Qureischie, 2009 rs3917643 Smith, 2009 rs995029 Smith, 2009 rs7571442 Smith, 2009 rs9928731 Smith, 2009 rs1800795 -174G/C Fornage, 2008 rs6190 R23K van Rossum, 2008

Genome-Wide Association Studies

Genotype a very large number of single nucleotide polymorphisms (SNPs) distributed across the chromosomes
Assuming NO a priori hypothesis on loci of interest

Microarrays of 500,000 to 5,000,000 variants

Imputation of additional variants using a reference panel
HapMap: 2,500,000 variants 1000G Phase I v3: 38,000,000 variants
Feero; NEJM 2010 Zeggini; Nature Genet 2005 The 1000 Genomes Project Consortium; Nature 2012

GWAS have identified thousands of novel loci associated with various traits major progress in understanding of pathophysiology

NHGRI GWA Catalog www.genome.gov/GWAStudies

Genome-Wide Association Studies

Candidate genes (11 or 18 SNPs)

Large sample size required:

>> 1,000 to > 10,000 More if rare variant; if small relative risk

GWAS (500,000 or 300,000 SNPs)

Zondervan; Nature Protocols 2007

GWAS of SADB
Introduction To SADB and white matter hyperintensities (WMH) To GWAS GWAS of WMH Summary of findings Follow-up and extension Interpretation Unsolved mysteries of SADB genetics Missing heritability of WMH? Genetics of other expressions of SADB?

 SCI

CHARGE Consortium
(Cohorts for Heart and Aging Research in Genomic Epidemiology)

Rotterdam Study I; II Cardiovascular Health Study (CHS) Framingham Heart Study (FHS) Atherosclerosis Risk in Communities Study (ARIC) Age Gene-Environment Susceptibility / Reykjavik Study (AGES) Austrian Stroke Prevention Study (ASPS)

+ Collaborating Study: 3C-Dijon Study

Psaty, Circ Cardiovasc Genet 2009

GWAS of WMH burden -- Methods

9,361 individuals of European descent (7 cohorts) Mean age: 69.5 years Men: 42.6% HTN: 55.9% Exclusion of participants with stroke or TIA at time of MRI Brain MRI on 0.5-1.5T scanners; T1, T2, Flair / PD Quantification of WMH volume using (Semi-)automated segmentation algorithm (AGES, ASPS, FHS, RS) Visual scoring on 10-point scale (ARIC, CHS)

GWAS of WMH burden -- Methods

Genotyping platforms: Illumina 370-Duo, Affymetrix 6.0, Illumina 610 , Affymetrix 500K + 50K Gene Focused Panel, Illumina 550 Imputation to the 2.5 Million autosomal SNPs from HapMap2 CEU panel

Linear regression, additive genetic model: Log (WMH+1) ~ + 1 * SNP + 2 * age + 3 * sex + 4 * ICV
Fixed effects meta-analysis of results from the 7 cohorts Effective sample size weighted method (different scales)

GWAS of WMH burden -- Results

8

P= x-8 10-8 5 x5 10

-Log10 (P-value)

Chromosome

Fornage, Debette, et al, Ann Neurol 2011

GWAS of WMH burden -- Results

8

rs3744028, p=4x10-9
P= x-8 10-8 5 x5 10

17q25

-Log10 (P-value)

Chromosome

Fornage, Debette, et al, Ann Neurol 2011

GWAS of WMH burden -- Results

rs3744028 p = 4x10-9

r2 = 0.5

rs1055129 p = 4x10-8

MRPL38 FBF1 UNC13D WBP2 TRIM47 TRIM65 ACOX1

Fornage, Debette, et al, Ann Neurol 2011

GWAS of WMH burden -- Replication

Replication of association with chr17q25 locus in initial paper
In Europeans from the general population (AGES + 3C-Dijon, N=3,024) rs3744028: p=2.0x10-7 rs1055129 : p=5.9x10-4 NOT in African-Americans (ARIC, N=807)

GWAS of WMH burden -- Replication

Replication of association with chr17q25 locus in initial paper
In Europeans from the general population (AGES + 3C-Dijon, N=3,024) rs3744028: p=2.0x10-7 rs1055129 : p=5.9x10-4 NOT in African-Americans (ARIC, N=807)

Subsequent replication in 3 independent papers

Population-based European METASTROKE (stroke patients) European

Healthy adults Japanese

GWAS of WMH burden -- Interpretation

rs3744028 p = 4x10-9

What do these mean?

rs1055129 p = 4x10-8

MRPL38 FBF1 UNC13D WBP2 TRIM47 TRIM65 ACOX1

Fornage, Debette et al, Ann Neurol 2011

GWAS of WMH burden -- Interpretation

rs3744028 p = 4x10-9

rs1055129 p = 4x10-8

- rs3744208 intronic in TRIM65 & associated with mRNA levels of TRIM47 in lymphoblastoid cell lines - rs1055129 intronic in TRIM47

MRPL38 FBF1 UNC13D WBP2 ACOX1

TRIM47

TRIM65

Fornage, Debette et al, Ann Neurol 2011

TRIM47 and TRIM65

TRIM = Tripartite Motif Containing 47 and 65
Ring-finger B-box coiled-coil proteins involved in apoptosis, innate immunity, cell cycle regulation, neuroprotection

TRIM47 and TRIM65 are two E3 ubiquitin ligases (Ring finger)

The ubiquitin-proteasome system (UPS) targets numerous cellular proteins for degradation Complex process involving multiple enzymes, including E3 ligases Regulates many basic cellular processes

Ciechanover, Neuron 2004

TRIM47 and TRIM65

TRIM = Tripartite Motif Containing 47 and 65
Ring-finger B-box coiled-coil proteins involved in apoptosis, innate immunity, cell cycle regulation, neuroprotection

TRIM47 and TRIM65 are two E3 ubiquitin ligases (Ring finger)

Ubiquitin aggregate formation rapidly after onset of reperfusion in mouse model of focal ischemia / reperfusion (brain) Formation of ubiquitin aggregates may be associated with tissue survival in postischemic period Hochrainer, Stroke 2012

Inflammatory responses to ischemia/reperfusion (kidney) may be controlled by a balance between ubiquitination and deubiquitination
Luong, Circ Res 2013

 Genetic associations do NOT necessarily imply causality

Indirect association (observed) Linkage disequilibrium

Phenotype
Direct association (unobserved)

chromosome

A Typed variant

B Unobserved causal variant

Non-coding variants may be involved in transcriptional regulation, but sometimes of more distant genes
Identification of causal variant and gene requires additional experiments: sequencing / fine mapping, functional studies

Ioannidis, Nat Rev Genet 2009

GWAS of WMH burden -- Results

rs3744028 p = 4x10-9

rs1055129 p = 4x10-8

MRPL38 FBF1 UNC13D WBP2 ACOX1 TRIM47 TRIM65

unc-13 homolog D: AR Familial hemophagocytic lymphohistiocytosis (MIM 608698)

acyl-CoA oxidase 1; palmitoyl: pseudoneonatal adrenoleukodystrophy (MIM 264470)

 Phenotype refinement Pleiotropy with other diseases Gene-environment interaction

Chr17q25
Phenotype Genotype

WMHV

Bioinformatics Targeted sequencing Gene expression Function studies, in vitro / animal

Chr17q25 locus and SADB characteristics

It is unclear whether the chr17q25 locus is also associated with other MRI-markers of SVD, especially lacunar infarcts.
Two studies have examined the association of the chr17q25 locus with MRI-defined lacunar infarcts with negative findings Japanese healthy adults Ischemic stroke patients from the Metastroke project

Chr17q25 locus and other MRI-markers of SADB

Association of lacunar infarcts and dilated perivascular spaces (dPVS) with chr17q25 variants in the 3C-Dijon MRI-study French population-based study of non-institutionalized community persons aged 65+ yrs
rs3744028 rs1055129

Lacunar infarcts Lenticular dPVS Subcortical dPVS

N OR 95% CI * 132/1626 1.10 (0.80-1.50)

154/1416 1.27 (0.95-1.69) 324/1416 0.87 (0.69-1.08)

p 0.56
0.10 0.20

OR 95% CI * 1.35 (1.03-1.77)

1.32 (1.03-1.71) 0.98 (0.81-1.19)

p 0.029
0.030 0.83

* Adjusted for age, gender and principal components (and total intracranial volume for dPVS)
Unpublished

Chr17q25 locus and SADB characteristics

It is unclear whether the chr17q25 locus is also associated with other MRI-markers of SVD, especially lacunar infarcts.
Two studies have examined the association of the chr17q25 locus with MRI-defined lacunar infarcts with negative findings Japanese healthy adults Ischemic stroke patients from the Metastroke project

Moreover, no study has so far examined the relationship of the chr17q25 locus with WMH characteristics.

Deep subcortical

Periventricular

Frontal dominance Occipital dominance

Zhu, J Neurol 2012

Chr17q25 locus and WMH characteristics

Association of WMH characteristics with chr17q25 variants in the 3C-Dijon MRI-study (N=1,572)
rs3744028 SE / OR(95%CI) WMHV Periventricular WMHV Deep WMHV Occipital dominance* 0.0906 0.0228 0.0944 0.0238 0.0402 0.0160 0.67 (0.53-0.83) p 7.05x10-5 7.08x10-5 0.012 3.00x10-4 rs1055129 SE / OR(95%CI) 0.0624 0.0199 0.0707 0.0207 0.0242 0.0140 0.72 (0.59-0.87) p 1.71x10-3 6.53x10-4 0.084 9.00x10-4

* Occipital dominance = Top vs bottom tertile of the occipital-frontal gradient (occipital frontal WMHV) Adjusted for age, gender and principal components (to account for population structure)
Unpublished

Chr17q25 locus and SADB characteristics

It is unclear whether the chr17q25 locus is also associated with other MRI-markers of SVD, especially lacunar infarcts.
Two studies have examined the association of the chr17q25 locus with MRI-defined lacunar infarcts with negative findings Japanese healthy adults Ischemic stroke patients from the Metastroke project

Moreover, no study has so far examined the relationship of the chr17q25 locus with WMH characteristics. The association of chr17q25 variants with WMH progression is unknown

Schmidt, Ann Neurol 2005

Chr17q25 locus and WMH progression

Association of chr17q25 (rs3744028) with 4-yr WMHV progression, overall and stratified on median BP, in 3C-Dijon MRI-study
N All Delta-WMHV SBP < 149.5 mmHg Delta-WMHV SBP > 149.5 mmHg Delta-WMHV 440 464 495 -0.0537 0.0343 0.0853 0.0291 -0.0262 0.0312 0.12 0.0036 0.40 519 0.0920 0.0273 0.0008 <0.0001 959 0.0238 0.0214 0.27 Beta SE P p (inter)

DBP < 83.5 mmHg

Delta-WMHV DBP > 83.5 mmHg Delta-WMHV 0.0009

Delta-WMHV = annualized change (WMHV2 WMHV1) / time Adjusted for age, gender and principal components

Unpublished

GWAS of SADB
Introduction To SADB and white matter hyperintensities (WMH) To GWAS GWAS of WMH Summary of findings Follow-up and extension Interpretation Unsolved mysteries of SADB genetics Missing heritability of WMH? Genetic risk factors of other expressions of SADB? Do they overlap?

Heritability of WMHV = 55-80%

Variance explained by chr17q25 <2% of WMH heritability

Perspectives to decipher genetics of WMH

More GWAS
Increase sample size to increase power Include non European ethnicities Account for interaction with hypertension

Explore rare variants

Exome Chip genotyping Whole exome / genome (or targeted) sequencing

Well-conducted candidate gene / pathway studies can be useful

Genes involved in other expressions of SADB: APOE Genes involved in monogenic causes of SADB NOTCH3 CADASIL pathway genes

 42 cross-sectional or longitudinal studies (N=29,965) Including unpublished data from 3 population-based studies APOE4 associated with WMH burden Z-score meta-analysis: APOE4+: p=0.0034 APOE44: P=0.0030 APOE2 associated with WMH burden Z-score meta-analysis: APOE2+: p=0.00053
Schilling et al, Neurology 2013

Common variants at the NOTCH3 gene increase the risk of age-related white matter lesions in hypertensives
Association of NOTCH3 variants with WMH in ASPS sample (discovery)

Replication of finding in CHARGE consortium

Schmidt et al., Brain 2011

Contribution of genes of CADASIL and CARASIL disease pathways to common non-Mendelian SADB?

Genetics other expressions of SADB?

Ischemic stroke NINJ2 Intracerebral hemorrhage APOE2,4

Dissection

PITX2; ZFHX2 9p21; HDAC9

Atrial fibrillation
Large artery atheroma

Covert SADB

?
Autres

SADB ?
?

Diabte Obesit Tabac HTA Hyperchol

Chr 17q25

Fornage; Ann Neurol 2010 Traylor; Lancet Neurol 2012 Biffi; Ann Neurol 2011

AGES-Reykjavik: Lenore Launer, Albert V. Smith, Vilmundur Gudnason, Gudny Eiriksdottir, Tamara Harris Atherosclerosis Risk in Communities Study: Thomas Mosley, Myriam Fornage, Eric Dr. Myriam Fornage Boerwinkle, Jan Bressler

Neuro-CHARGE group - stroke

Austrian Study of Stroke Prevention: Reinhold Schmidt, Helena Schmidt, KatjaElisabeth Petrovic, Franz Fazekas, Margherita Cavalieri Cardiovascular Health Study: Josh Bis, Will Longstreth, Annette Fitzpatrick, Oscar Lopez, Bruce Psaty, Thomas Lumley, Kenneth Rice, Lewis Kuller, James Becker, Nicole Glazer, Jerome Rotter, Erin Wallace

Neuro-CHARGE working group

ERF: Cornelia van Duijn, Maaike Schuur, Carla Ibrahim-

Dr. Sudha Seshadri

Framingham Heart Study: Philip Wolf, Anita L. DeStefano, Stephanie Debette, Qiong Yang, Charles DeCarli, Larry Atwood, Alexa Beiser, Rhoda Au, Yi-Chen Hsieh Rotterdam Study: Monique Breteler, Cornelia van Duijn, M. Arfan Ikram, Ben Verhaaren, Aad Van der Lugt

Neuro-CHARGE Working Group

Collaborating Studies: EUROSPAN, LBC, Health ABC, 3C study, ROS/MAP, WHICAP TASCOG, Group Health, EADI, GERAD, ADGC, ISGC and METASTROKE Fundaci ACE, PROSPER, Generation Scotland, AAA, GEMS, GENOA

Department of Neurology of Lariboisire Hospital, Paris

Pr. Hugues Chabriat

Pr. Marie-Germaine Bousser

Inserm Unit U740, Paris 7 University

Dr. Anne Joutel Pr. Elisabeth Tournier-Lasserve

Dr. Ganesh Chauhan

Dr. Sara Kaffashian

Dr. Acha Soumar

Sabrina Schilling

Guillaume Faure

Pr. Christophe Tzourio & Inserm Unit U708