VIEWS amp REVIEWS

Varinder S Alg BScReecha Sofat MScHenry Houlden PhDDavid J Werring PhD

Correspondence toDr Werringdwerringuclacuk

Supplemental data atwwwneurologyorg

Genetic risk factors for intracranialaneurysmsA meta-analysis in more than 116000 individuals

ABSTRACT

Objective There is an urgent need to identify risk factors for sporadic intracranial aneurysm (IA) devel-opment and rupture A genetic component has long been recognized but firm conclusions have beenelusive given the generally small sample sizes and lack of replication Genome-wide association studieshave overcome some limitations but the number of robust genetic risk factors for IA remains uncertain

Methods We conducted a comprehensive systematic review and meta-analysis of all geneticassociation studies (including genome-wide association studies) of sporadic IA conducted ac-cording to Strengthening the Reporting of Genetic Association Studies and Human Genome Epi-demiology Network guidelines We tested the robustness of associations using random-effectsand sensitivity analyses

Results Sixty-one studies including 32887 IA cases and 83683 controls were included Weidentified 19 single nucleotide polymorphisms associated with IA The strongest associationsrobust to sensitivity analyses for statistical heterogeneity and ethnicity were found for the fol-lowing single nucleotide polymorphisms on chromosome 9 within the cyclin-dependent kinaseinhibitor 2B antisense inhibitor gene (rs10757278 odds ratio [OR] 129 95 confidence inter-val [CI] 121ndash138 and rs1333040 OR 124 95 CI 120ndash129) on chromosome 8 near theSOX17 transcription regulator gene (rs9298506 OR 121 95 CI 115ndash127 andrs10958409 OR 119 95CI 113ndash126) and on chromosome 4 near the endothelin receptorA gene (rs6841581 OR 122 95 CI 114ndash131)

Conclusions Our comprehensivemeta-analysis confirms a substantial genetic contribution to sporadicIA implicatingmultiple pathophysiologic pathways mainly relating to vascular endothelial maintenanceHowever the limited data for IA compared with other complex diseases necessitates large-scale repli-cation studies in a full spectrum of populations with investigation of how genetic variants relate to phe-notype (eg IA size location and rupture status) Neurology 2013802154ndash2165

GLOSSARYACE 5 angiotensin-converting enzyme ANRIL 5 antisense noncoding RNA in the INK4 locus CI 5 confidence intervalCGAS 5 candidate gene association study EDNRA 5 endothelin receptor type A GWAS 5 genome-wide associationstudies IA 5 intracranial aneurysm IL 5 interleukin LD 5 linkage disequilibrium MMP 5 matrix metalloproteinase OR 5odds ratio SAH 5 subarachnoid hemorrhage SNP 5 single nucleotide polymorphism

Intracranial aneurysms (IAs) are present in 2ndash5 of the general population approximately07ndash19 of cases rupture causing subarachnoid hemorrhage (SAH)1 The annual incidenceof SAH is 8ndash9 in 100000 yet because of the high risk of death or severe disability2 and youngerage at onset compared with other stroke types (40ndash65 years)3 SAH has a disproportionatelylarge socioeconomic impact

SAH has been associated with commonmodifiable risk factors including hypertension smokingand alcohol intake4ndash6 However a genetic component has long been recognized for example first-degree relatives of patients with aneurysmal SAH have a 4- to 7-fold increased risk of being affectedcompared with the general population78 Candidate gene association studies (CGAS) of sporadic IAhave identified numerous potential risk loci but these studies have been limited by small sample

From the Department of Brain Repair and Rehabilitation Institute of Neurology (VSA DJW) Centre for Clinical Pharmacology Division ofMedicine (RS) and Department of Molecular Neuroscience Institute of Neurology (HH) University College London London UK

Go to Neurologyorg for full disclosures Funding information and disclosures deemed relevant by the authors if any are provided at the end of the article

This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use distribution andreproduction in any medium provided the original work is properly cited

2154 copy 2013 American Academy of Neurology

sizes and lack of replication More recentlygenome-wide association studies (GWAS) haveidentified novel genetic loci strongly associatedwith IAs9ndash14 but these still explain only a frac-tion of the genetic risk

Thus the number of true genetic risk factorsand the strength of their associations with IAsremain uncertain The pooling of data fromall genetic association studies allows risk esti-mates to be determined with more precisionthan is possible from any single study andallows assessment of robustness to statisticalheterogeneity and population ethnicity differ-ences across studies We therefore undertooka comprehensive systematic review and meta-analysis of all published CGAS and GWAS ofsporadic IA including sensitivity analyses forstatistical heterogeneity and ethnicity

METHODS Our meta-analysis was conducted in accordance

with the Human Genome Epidemiology Network guidelines15

and followed published recommendations to improve the quality

of meta-analyses of genetic association studies16 We assessed the

quality of reporting of genotyping on the basis of the Strength-

ening the Reporting of Genetic Association Studies statement17

Search strategy Electronic databases (PubMed EMBASE and

Google Scholar) were used to retrieve potentially relevant articles

on human genetic studies of IAs and SAH that had been published

up to December 2012 Search terms used under the Medical Subject

Headings were aneurysm(s) intracranial aneurysm(s) subarachnoid

hemorrhage genetics SNPs [single nucleotide polymorphisms] pol-

ymorphism GWAS and linkage and candidate gene(s)

Articles in all languages were searched (see figure 1 for a flow

diagram of the search strategy) and translated as necessary After

relevant articles were retrieved references were also checked for

other potentially relevant articles not found in the initial search

Selection criteria We included cohort or case-control studies

evaluating associations of genetic polymorphisms with proven

IA (using CTMR angiograms or digital subtraction angiogra-

phy) including unrelated individuals from CGAS and GWAS

in all ethnicities Where duplicate datasets or cohorts existed only

the largest study was included All single nucleotide polymor-

phisms (SNPs) evaluated in at least 2 published studies (CGAS

or GWAS) were included in meta-analysis Exclusion criteria

were a history of inherited conditions associated with IAs eg

adult polycystic kidney disease or Ehlers-Danlos syndrome

Data extraction Data were extracted by 2 of the authors (VSA

and DJW) and differences were resolved by consensus Where

available genotype frequencies for each SNP were recorded

Where they were unavailable we estimated the number of cases

per genotype category by using published information on risk

allele frequencies and odds ratios (ORs) for IA

Statistical analysis For each genetic polymorphism with more

than one publication meta-analysis was performed to determine a

pooledOR and 95 confidence intervals (CIs) by using a fixed-effect

model (Mantel-Haenszel method) Interstudy heterogeneity p valueswere measured fromCochran Q test and quantified by using Higgins

I2 statistic SNP associations showing significant interstudy heteroge-

neity (interstudy heterogeneity p values 005 and I2 50) were

examined under a random-effects (DerSimonian and Laird) model

and where appropriate subjected to sensitivity analysis to determine

the effect of individual studies on the pooled OR Ethnic subgroup

analysis was also performed where appropriate

All genetic models (dominant recessive and additive) were as-

sessed in studies recording genotype frequencies Bonferroni correc-

tion was applied for multiple testing (p 001) In studies not

recording genotype frequencies per-allele (additive) ORs were

pooled with calculation of CIs Controls were checked to be in

Hardy-Weinberg equilibrium for each study before meta-analysis

Publication bias was assessed by using the Egger regression

asymmetry test and visualization of funnel plots Review Manager

(RevMan) version 52 from the Cochrane Collaboration (London

UK) 2011 and Comprehensive Meta-Analysis (22) from Biostat

(Tampa FL) were used to create the forest plots and perform sen-

sitivity and publication bias analysis

RESULTS Our search identified 642 articles of which134 articles met initial inclusion criteria Two studiespublished in Chinese were translated After screeningfor duplication and eligibility data from 66 case-controlstudies (60 CGAS and 6 GWAS) were included

Figure 1 Flow chart illustrates the number of studies evaluated in this meta-analysis

GWAS 5 genome-wide association studies IA 5 intracranial aneurysm SNP 5 single nucle-otide polymorphism

Neurology 80 June 4 2013 2155

Genotype and allele frequencies were reported in 48publications the remaining 18 reported additive ORsIn total 32887 cases and 83683 control subjects wereincluded evaluating 41 polymorphisms in 29 genes (fig-ure 1) The studies encompassed mainly white Euro-pean Chinese and Japanese populations Risk allelefrequencies and control Hardy-Weinberg equilibriumstatus for each study of all SNPs meta-analyzed areshown in table e-1 on the Neurologyreg Web site atwwwneurologyorg

We identified 19 SNPs (from CGAS and GWAS)that were significantly associated with IAs in at leastone genetic model (table 1) The most robust associa-tions were seen in 11 of 12 SNPs discovered fromGWAS (table 1) with the strongest associations forloci on chromosome 9 (rs1333040 and rs10757278)chromosome 8 (rs9298506 and rs10958409) andchromosome 4 (rs6841581 figure 2) Other GWASloci variants associated with IAs included 9p213(rs2891168) 2q33 (rs1429412 and rs700651) 7q13(rs4628172) 12q22 (rs6538595) and 20p201(rs1132274 table 1 and figures e-1 and e-2)

Eight SNPs from CGAS were significantly associ-ated with IAs SERPINA3 (rs4934) and 2 variants relat-ing to collagen genes (COL1A2 [rs42524 GC] andCOL3A1 [rs1800255 GA]) showed the most robustassociations The remaining significantly associatedCGAS SNPs included heparan sulfate proteoglycan 2(rs3767137) versican (rs251124 and rs173686) angi-otensin-converting enzyme (ACE) ID and interleukin6 (IL6) G572C (table 1 figure 3 and figure e-2)

SNPs demonstrating statistical heterogeneity Nine ofthe 19 significantly associated SNPs (SERPINA3ACE ID IL6 G572C chondroitin sulfate proteogly-can 2 [rs173686 rs1333040 rs700651 rs9298506rs10958409 and rs4628172]) showed significant sta-tistical heterogeneity random-effects and sensitivityanalyses were possible only for the 7 variants reportedin more than 2 publications (table 2) After sensitivityanalysis the SERPINA3 ACE ID and IL6 G572CSNPs (figure 3F) derived from CGAS no longershowed an association with IA

Ethnicity analysis Nine SNPs (2 from CGAS and 7from GWAS) were subjected to subgroup analysis todetermine the effect of ethnicity (table 3 and figurese-3 to e-6) Associations with IA in Japanese Chineseand white populations were compared for each SNPafter which 5 of these 9 SNPs (all from GWAS) re-mained robustly associated with IA in all groups (table 3)

Publication bias Inspection of funnel plots and calcula-tion of Egger p values confirmed that none of the SNPs(CGAS or GWAS) associated with IAs (table 1) showedpublication bias See figures e-7 to e-11 for funnel plotsof all SNPs (CGAS and GWAS) associated with IA

CGAS not confirming associations with IAs Twenty-twocandidate SNPs reported in more than one study werenot associated with IAs after meta-analysis (table e-2)including the following endothelial nitric oxide syn-thase variable number tandem repeat 4a4b endo-thelial nitric oxide synthase T786C endothelialnitric oxide synthase G894T endoglin intron7 WtIns matrix metalloproteinase (MMP)-3 5A6A elastin intron 20TC elastin intron 23CTIL-1b C5111T IL-6 G174C plasminogin activatorinhibitor-1 4G5G glycoprotein IIIa A1A2 factorXIII valineleucine APOE e2 and e4 COL4A1 CT(rs3783107) fibrillin-2 CG (rs331079) MMP-2CT (rs243865) MMP-9 C1562T (rs3918242)COL3A1 (rs2138533CT and rs11887092AG)rs10757272CT and rs11661542AC

DISCUSSION This comprehensive systematic reviewand meta-analysis has identified 19 SNPs associatedwith IAs among 32887 cases and 83683 controlsThe variety of genes implicated by these SNPs sug-gests that multiple pathophysiologic pathwaysmainly involved in vascular endothelial maintenanceand extracellular matrix integrity are likely to con-tribute to IA development and rupture The mostrobust SNPs identified arose from variants on chro-mosomes 4 8 and 9 derived from GWAS and con-firmed by subsequent replication case-control studies

Genetic variants associated with IAs identified by GWAS

SNPs with potential roles in vascular endothelial mainte-

nance The SNP rs1333040 located on chromosome9p213 in the cyclin-dependent kinase inhibitor 2Bantisense gene was strongly associated with IAs Twomajor linkage disequilibrium (LD) blocks exist withinthe 9p213 locus one associated with vascular diseasesand the other associated with diabetes This locus isassociated with a wide spectrum of arterial disordersincluding coronary artery disease and abdominal aorticaneurysms18 raising the possibility that there are as yetunidentified common processes involved in all thesecommon cardiovascular pathologies The cyclin-dependent kinase inhibitor 2B antisense gene onintron 12 is associated with cell cycle signalingalthough its exact biological roles are not fully under-stood910 Association of the rs1333040 SNP with IAswas found to be independent of smoking and hyper-tension in one study19 In multivariable analysis thisSNP was preferentially associated with posterior com-municating artery aneurysms providing preliminaryevidence that aneurysm site could be related to geneticfactors19 However larger cohorts will need to be stud-ied in addition to scanning the entire region to checkwhich SNPs are in LD with the rs1333040-T alleleand whether they have any cumulative (epistatic) effecton IA phenotype including location

2156 Neurology 80 June 4 2013

Genotype and allele frequencies were reported in 48publications the remaining 18 reported additive ORsIn total 32887 cases and 83683 control subjects wereincluded evaluating 41 polymorphisms in 29 genes (fig-ure 1) The studies encompassed mainly white Euro-pean Chinese and Japanese populations Risk allelefrequencies and control Hardy-Weinberg equilibriumstatus for each study of all SNPs meta-analyzed areshown in table e-1 on the Neurologyreg Web site atwwwneurologyorg

We identified 19 SNPs (from CGAS and GWAS)that were significantly associated with IAs in at leastone genetic model (table 1) The most robust associa-tions were seen in 11 of 12 SNPs discovered fromGWAS (table 1) with the strongest associations forloci on chromosome 9 (rs1333040 and rs10757278)chromosome 8 (rs9298506 and rs10958409) andchromosome 4 (rs6841581 figure 2) Other GWASloci variants associated with IAs included 9p213(rs2891168) 2q33 (rs1429412 and rs700651) 7q13(rs4628172) 12q22 (rs6538595) and 20p201(rs1132274 table 1 and figures e-1 and e-2)

Eight SNPs from CGAS were significantly associ-ated with IAs SERPINA3 (rs4934) and 2 variants relat-ing to collagen genes (COL1A2 [rs42524 GC] andCOL3A1 [rs1800255 GA]) showed the most robustassociations The remaining significantly associatedCGAS SNPs included heparan sulfate proteoglycan 2(rs3767137) versican (rs251124 and rs173686) angi-otensin-converting enzyme (ACE) ID and interleukin6 (IL6) G572C (table 1 figure 3 and figure e-2)

SNPs demonstrating statistical heterogeneity Nine ofthe 19 significantly associated SNPs (SERPINA3ACE ID IL6 G572C chondroitin sulfate proteogly-can 2 [rs173686 rs1333040 rs700651 rs9298506rs10958409 and rs4628172]) showed significant sta-tistical heterogeneity random-effects and sensitivityanalyses were possible only for the 7 variants reportedin more than 2 publications (table 2) After sensitivityanalysis the SERPINA3 ACE ID and IL6 G572CSNPs (figure 3F) derived from CGAS no longershowed an association with IA

Ethnicity analysis Nine SNPs (2 from CGAS and 7from GWAS) were subjected to subgroup analysis todetermine the effect of ethnicity (table 3 and figurese-3 to e-6) Associations with IA in Japanese Chineseand white populations were compared for each SNPafter which 5 of these 9 SNPs (all from GWAS) re-mained robustly associated with IA in all groups (table 3)

Publication bias Inspection of funnel plots and calcula-tion of Egger p values confirmed that none of the SNPs(CGAS or GWAS) associated with IAs (table 1) showedpublication bias See figures e-7 to e-11 for funnel plotsof all SNPs (CGAS and GWAS) associated with IA

CGAS not confirming associations with IAs Twenty-twocandidate SNPs reported in more than one study werenot associated with IAs after meta-analysis (table e-2)including the following endothelial nitric oxide syn-thase variable number tandem repeat 4a4b endo-thelial nitric oxide synthase T786C endothelialnitric oxide synthase G894T endoglin intron7 WtIns matrix metalloproteinase (MMP)-3 5A6A elastin intron 20TC elastin intron 23CTIL-1b C5111T IL-6 G174C plasminogin activatorinhibitor-1 4G5G glycoprotein IIIa A1A2 factorXIII valineleucine APOE e2 and e4 COL4A1 CT(rs3783107) fibrillin-2 CG (rs331079) MMP-2CT (rs243865) MMP-9 C1562T (rs3918242)COL3A1 (rs2138533CT and rs11887092AG)rs10757272CT and rs11661542AC

DISCUSSION This comprehensive systematic reviewand meta-analysis has identified 19 SNPs associatedwith IAs among 32887 cases and 83683 controlsThe variety of genes implicated by these SNPs sug-gests that multiple pathophysiologic pathwaysmainly involved in vascular endothelial maintenanceand extracellular matrix integrity are likely to con-tribute to IA development and rupture The mostrobust SNPs identified arose from variants on chro-mosomes 4 8 and 9 derived from GWAS and con-firmed by subsequent replication case-control studies

Genetic variants associated with IAs identified by GWAS

SNPs with potential roles in vascular endothelial mainte-

nance The SNP rs1333040 located on chromosome9p213 in the cyclin-dependent kinase inhibitor 2Bantisense gene was strongly associated with IAs Twomajor linkage disequilibrium (LD) blocks exist withinthe 9p213 locus one associated with vascular diseasesand the other associated with diabetes This locus isassociated with a wide spectrum of arterial disordersincluding coronary artery disease and abdominal aorticaneurysms18 raising the possibility that there are as yetunidentified common processes involved in all thesecommon cardiovascular pathologies The cyclin-dependent kinase inhibitor 2B antisense gene onintron 12 is associated with cell cycle signalingalthough its exact biological roles are not fully under-stood910 Association of the rs1333040 SNP with IAswas found to be independent of smoking and hyper-tension in one study19 In multivariable analysis thisSNP was preferentially associated with posterior com-municating artery aneurysms providing preliminaryevidence that aneurysm site could be related to geneticfactors19 However larger cohorts will need to be stud-ied in addition to scanning the entire region to checkwhich SNPs are in LD with the rs1333040-T alleleand whether they have any cumulative (epistatic) effecton IA phenotype including location

2156 Neurology 80 June 4 2013

Table 1 SNPs associated with IAs

Genelocus SNPGeneticmodel No studies Cases Controls FE OR (95 CI)

pcorrected(Bonferroni) pHet (I2)

Robust associationafter randomeffectssensitivityanalyses

Potential biologicalmechanism ofassociation with IA

Genome-wide association study SNP associations

9p213 CT (rs1333040) Additive 129ndash1113141924 11949 29014 124 (12ndash129) 983 3 10208 540 3 10208 (80) Vascular endothelium

9p21 AG (rs10757278) Additive 718ndash2124 3394 17075 129 (121ndash138) 159 3 10213 084 (0) NA Vascular endothelium

8q11 AG (rs9298506) Additive 89101324 9246 26331 121 (115ndash127) 155 3 10213 190 3 10207 (84) Vascular endothelium

8q11 AG (rs10958409) Additive 89ndash11132024 9873 27029 120 (115ndash126) 178 3 10215 687 3 10205 (76) Vascular endothelium

4q3123 AG (rs6841581) Additive 612 4370 14181 122 (114ndash131) 195 3 1028 039 (4) NA Vascular endothelium

9p213 CG (rs2891168) Additive 21419 2076 1985 132 (121ndash144) 984 3 10210 098 (0) NA Vascular endothelium

2q33 GA (rs1429412) Additive 491124 2675 7632 120 (112ndash130) 107 3 10206 030 (18) NA Unknown

2q33 GA (rs700651) Additive 69132024 4283 13236 111 (106ndash118) 713 3 10205 117 3 10202 (66) Unknown

7q13 GT (rs4628172) Additive 21113 2443 6376 111 (103ndash119) 404 3 10203 471 3 10203 (87) NA Unknown

12q22 GA (rs6538595) Additive 612 4370 14181 116 (110ndash123) 112 3 1027 098 (0) NA Unknown

20p121 GA (rs1132274) Additive 512 4370 14181 119 (111ndash128) 829 3 1027 052 (0) NA Unknown

Candidate gene SNP associations

SERPINA3 AG (rs4934) Dominant 340ndash42 892 1029 222 (168ndash294) 000001 000001 (97) 3 Extracellular matrix

Recessive 080 (049ndash131) 025 004 (69)

Additive 127 (107ndash150) 00002 000001 (94)

COL1A2 GC (rs42524) Dominant 343ndash45 812 806 177 (114ndash275) 00009 009 (58) NA Extracellular matrix

Recessive 114 (016ndash794) 086 031 (4)

Additive 169 (111ndash257) 0001 006 (64)

COL3A1 GA (rs1800255) Dominant 24647 546 2235 155 (121ndash200) 000001 025 (23) NA Extracellular matrix

Recessive 131 (072ndash238) 025 070 (0)

Additive 140 (114ndash172) 00001 026 (21)

HSPG2 AG (rs3767137) Additive 23132 1316 1742 122 (108ndash139) 0002 0122 (58) NA Extracellular matrix

Versican (CSPG2) CT (rs251124) Additive 330ndash32 1489 1687 125 (11ndash141) 00007 0201 (38) NA Extracellular matrix

Versican (CSPG2) AG (rs173686) Additive 23031 857 879 123 (105ndash143) 0008 0013 (84) NA Extracellular matrix

ACE ID Dominant 548252 767 1172 082 (061ndash112) 066 0002 (76) 3 Vascular endothelium

Recessive 139 (105ndash184) 0002 001 (69)

Additive 123 (107ndash140) 0003 047 (0)

Continued

Neurology

80

June42

013

2157

The SNP rs10757278 on chromosome 9p213located in a noncoding RNA region called ANRIL(antisense noncoding RNA in the INK4 locus)showed robust association with IAs but its functionremains unclear Moreover the observed association ofrs10757278 with IA may arise frommoderate LDwiththe known IA risk variant rs1333040 SNP (r5 059)from which it is approximately 41 kbp upstream20

However these 2 SNPs were not in strong LD in aJapanese population raising the possibility of a smallerLD block associated with IA In a Japanese study19

haplotype analysis of both risk alleles of these SNPsdemonstrated significant association with IAs (OR156 95 CI 132ndash185 p 5 29 3 10207) Func-tional studies show altered ANRIL expression as aresult of variations within the 9p213 region14 withreduced expression of RNA encoded by CDKN2Aand CDKN2B in mice14 these 2 genes adjacent tothe 9p21 region encode tumor-suppressor proteins(p14ndash16) which are involved in cell proliferation21

Moreover p14 levels correlate with ANRIL activityand modulate MMP-3 levels which may influenceextracellular matrix repair21

In the most recent GWAS14 in a Caucasian popu-lation of familial and sporadic IA 6 SNPs also locatedin the 9p213 region were associated with IA one ofthese (rs6475606) achieved GWAS-level statistical sig-nificance (OR 134 p5 4293 10207) for associationwith sporadic and familial IAs Association with thers1333040 SNP was also confirmed in this study14

further strengthening association of this locus with IATwo SNPs on chromosome 8q (rs10958409 and

rs9298506) surrounding the SOX17 gene were studiedin European and Japanese populations rs10958409 wasassociated in both populations but the rs9298506 SNPwas associated with IA in the European but not Japanesepopulation SOX17 plays an important role in generationand maintenance of stem cells of endothelial and hema-topoietic lineages crucial in the formation and mainte-nance of vascular endothelium2223 Furthermore SOX17knockout mice show multiple vascular abnormalitiesreflecting defective endothelial remodeling22 Defectivestem cells involved in vascular maintenance are thusplausible targets in IA pathogenesis The heterogene-ity observed in the meta-analysis of these 2 SNPsmay partly be explained by the difference in risk allelefrequencies in the white population (table e-1)24

compared with the other populations studied(rs10958409 risk allele frequency 088 vs approxi-mately 02 rs9298506 risk allele frequency 017 vsapproximately 080) Further support implicatingchromosome 8 loci was shown in a recent GWAS14

in which a novel SNP also in the region of theSOX17 gene was significantly associated with IAs(rs1072737 OR 122 95 CI 107ndash139) inde-pendent of smoking For both SOX17 loci in the

Tab

le1

Con

tinu

ed

Gen

elocu

sSNP

Gen

etic

mod

elNo

stud

ies

Cas

esCon

trols

FE

OR

(95

CI)

pcorrecte

d(Bon

ferron

i)pHet(I2

)

Rob

ustas

sociat

ion

afte

rra

ndom

effe

ctsse

nsitivity

analys

es

Pot

ential

biological

mec

hanism

ofas

sociat

ionwithIA

IL6

G572C

Dom

inan

t5

363753ndash55

912

3410

068

(048ndash096)

0004

000001

(92

)3

Inflam

matorymed

iator

Rec

essive

047

(034ndash065)

000001

000001

(94

)

Add

itive

061

(049ndash075)

000001

000001

(95

)

Abbr

eviation

sACE

5an

giote

nsin-con

verting

enzy

me

CI5

conf

iden

ceinte

rvalCSPG

5ch

ondr

oitinsu

lfat

epro

teog

lyca

nFE

5fixe

d-effe

ctsmod

elHSPG

5he

par

ansu

lfat

epr

oteo

glyc

anI2

5Higgins

stat

isticdem

onstrating

degr

eeof

inte

rstu

dyhe

tero

gen

eity

IA5

intrac

ranial

aneu

rysm

IL5

inte

rleu

kin

NA

5no

tap

plicab

leO

R5

odds

ratio

p Het5

inte

rstu

dyhe

tero

gen

eity

pva

lue

SNP5

sing

lenu

cleo

tide

polymor

phism

5SNP

robu

stly

asso

ciat

edwithIA

follo

wingse

nsitivityan

dra

ndom

-effec

tsan

alys

is3

5SNPsno

tro

bustly

asso

ciat

edwithIA

follo

wingse

nsitivityan

alys

isan

drand

om-effec

tsan

alys

is

2158 Neurology 80 June 4 2013

Figure 2 Forest plots (additive model) for genome-wide association studies in all populations

(A) rs1333040 single nucleotide polymorphism (SNP) (B) rs10757278 SNP (C) rs9298506 (D) rs10958409 (E)rs6841581 CI 5 confidence interval IA 5 intracranial aneurysm

Neurology 80 June 4 2013 2159

Figure 3 Forest plots for candidate gene single nucleotide polymorphisms associated with intracranial aneurysms

(A) Dominant model forest plots for SERPINA3 single nucleotide polymorphism (SNP) (B) COL3A1 (rs1800255) dominant model forest plot (C) COL1A2(rs42524) dominant model forest plot (D)ACE ID recessive model forest plot (E) IL6G572C SNP recessive model forest plot before sensitivity analysis (F)IL6 G572C SNP recessive model forest plot after sensitivity analysis CI 5 confidence interval IA 5 intracranial aneurysm M-H 5 Mantel-Haenszel

2160 Neurology 80 June 4 2013

most recent GWAS14 smoking was an independentrisk factor acting in a multiplicative manner (chromo-some 8 rs1072737 OR 212 95 CI 176ndash256 andchromosome 9 rs6475606 OR 211 95 CI 177ndash252)

A further recent GWAS12 found the SNPrs6841581AG on chromosome 4q3123 coding forthe endothelin receptor type A (EDNRA) gene wassignificantly associated with IA in Dutch Finnish andJapanese populations (OR 122 95 CI 114ndash131

Table 2 Random-effects and sensitivity analyses of associated SNPs that showed significant statisticalheterogeneity under fixed-effects modeling

SNPNostudies

Studiesexcluded

FE OR (95 CI)before adjustment

Random-effectsOR (95 CI)

FE OR (95 CI) aftersensitivity analysis Genetic model

SERPINA3 3 Liu42 222 (168ndash294)a 212 (036ndash1231) 107 (074ndash153) Dominant

127 (107ndash150)a 125 (061ndash259) 097 (079ndash119) Additive

ACE ID 5 Slowik50 139 (105ndash184)a 144 (086ndash242) 120 (088ndash162) Recessive

IL6 G572C 5 Sun36 047 (034ndash065)a 149 (024ndash927) 103 (065ndash162) Recessive

061 (049ndash075)a 095 (031ndash285) 104 (079ndash139) Additive

rs1333040 11 Low13 123 (119ndash128)a 126 (115ndash139)a 131 (126ndash137)a Additive

rs700651 6 Low13 111 (106ndash118)a 114 (103ndash125)a 119 (111ndash128)a Additive

rs9298506 8 Low13 121 (115ndash127)a 124 (108ndash141)a 132 (124ndash139)a Additive

rs10958409 9 410112024b 120 (115ndash126)a 122 (11ndash134)a 123 (117ndash13)a Additive

Abbreviations ACE 5 angiotensin-converting enzyme CI 5 confidence interval FE 5 fixed-effects model IL 5 interleukinOR 5 odds ratio SNP 5 single nucleotide polymorphismaSignificant association with IAb Four studies including one white population and 3 Japanese populations were excluded

Table 3 Subgroup ethnicity analysis

SNPNostudies Ethnicity

FE OR all ethnicities(all studies)

FE OR (95 CI)ethnicity subanalysis pcorrected pHet (I2) Genetic model

ACE ID 45 White 139 (105ndash184) 142 (106ndash192)a 0002 0006 (76) Recessive

Japanese 120 (056ndash257) 054 NA Recessive

IL6 G572C 35 Chinese 030 (019ndash047) 041 (029ndash058)a 000001 00001 (95) Recessive

25 White 426 (092ndash197) 01 003 (78) Recessive

35 Chinese 058 (044ndash075) 050 (039ndash064)a 000001 000001 (97) Additive

25 White 138 (083ndash229) 01 019 (42) Additive

rs1333040 611 Japanese 124 (12ndash129) 115 (109ndash122)a 175 3 10206 306 3 10208 (88) Additive

511 White 130 (124ndash136)a 191 3 10206 078 (0) Additive

rs10757278 57 White 129 (121ndash138) 130 (12ndash143)a 12 3 10209 091 (0) Additive

27 Japanese 127 (113ndash141)a 26 3 10205 022 (33) Additive

rs9298506 58 White 121 (115ndash127) 135 (127ndash144)a 24 3 10204 052 (0) Additive

38 Japanese 103 (095ndash112) 045 654 3 10204 (86) Additive

rs10958409 59 White 12 (115ndash126) 131 (123ndash139)a 82 3 10202 0052 (62) Additive

49 Japanese 109 (102ndash117)a 0009 008 (55) Additive

rs700651 33 White 111 (106ndash118) 119 (109ndash129)a 482 3 10205 023 (31) Additive

33 Japanese 106 (094ndash131) 008 002 (75) Additive

rs6841581 46 White 122 (114ndash131) 125 (113ndash138)a 121 3 10205 018 (38) Additive

26 Japanese 12 (108ndash132)a 356 3 10204 087 (0) Additive

rs6538595 46 White 116 (110ndash123) 116 (108ndash124)a 213 3 10205 091 (0) Additive

26 Japanese 117 (106ndash128)a 149 3 10203 06 (0) Additive

Abbreviations ACE 5 angiotensin-converting enzyme CI 5 confidence interval FE 5 fixed-effects model I2 5 Higgins statistic demonstrating degree ofinterstudy heterogeneity IA5 intracranial aneurysm IL5 interleukin NA5 not applicable OR 5 odds ratio pHet 5 interstudy heterogeneity p value SNP 5

single nucleotide polymorphisma Significant association with IA

Neurology 80 June 4 2013 2161

p 5 195 3 1028) Another GWAS13 in a Japanesepopulation showed that the rs6842241 SNP located at4q3122 near the EDNRA gene was significantly asso-ciated with IA (OR 125 95 CI 116ndash134 p 5

9583 10209) EDNRA is a G-proteinndashcoupled recep-tor for endothelins which modulate vasoconstrictionand vasodilatation after hemodynamic insult Endo-thelin-1mdashthe predominant isoform in vascularsmooth-muscle cellsmdashactivates EDNRA Endothelinsignaling is activated at the site of vascular injury1225

causing cell proliferation indeed after SAH EDNRAand endothelin-1 protein levels are both increased inCSF26ndash28 Alternatively downregulation of EDNRAsignaling could lead to defective vascular repair allowinganeurysms to develop after injury a hypothesis sup-ported by functional analysis of EDNRA variants show-ing lower transcriptional activity for the rs6841581risk allele (G)13

SNPs with unknown function or not examined in more

than 2 studies Five SNPs from GWAS do not corre-spond to genes with any known functions relevant toIA development Two of these SNPs (rs1429412 andrs700651) at locus 2q33 flank the BOLL gene(involved in germ cell development) and the phospho-lipase Cndashlike 1 gene (involved in intracellular cascadereactions with abolished phospholipase C activity)Phospholipase Cndashlike 1 lies close to the vascular endo-thelial growth factor receptor-2 which is involved inCNS angiogenesis and is a marker for endothelial pro-genitor cells Three further SNPs on chromosome 2were associated with familial IAs rs11693075 (close tothe histone deacetylase complex SAP130 gene)rs1897472 and rs3769801 (both located within thephosphodiesterase-1 gene)14

One associated SNP located on chromosome 7(rs4628172 GT) is within the TMEM195 genewhich encodes transmembrane proteins possiblyinvolved in fatty acid biosynthesis and iron bind-ing1113 Further associated SNPs from the latestGWAS include variants on chromosome 12q22(rs6538595 rs11112585 and rs2374513) within theC12orf75 gene14 The rs1132274 GT SNP on chro-mosome 20p121 was also associated with IA10 How-ever none of these SNPs yet has known clearfunctional relevance to IA

Three further loci not included in this meta-analysis(because they were reported in only one study) showedsignificant association with IAs10 These loci included18q112 (rs11661542) 10q2432 (rs12413409) and13q132 (rs9315204) but their mechanisms of associ-ation remain unclear

Genetic variants associated with IAs identified by CGAS

Eight SNPs from CGAS were associated with IAs inour meta-analysis albeit with significant statisticalheterogeneity These SNPs relate to genes with

plausible relevance to IA biology including those impor-tant for extracellular matrix integrity inflammatorymedi-ators and vascular endothelium maintenanceExtracellular matrix gene variants may predispose to IAdevelopment by weakening arterial walls In our meta-analysis 6 such SNPs showed significant associationswith IA COL1A2 (rs42524) COL3A1 (rs1800255)SERPINA3 versican (rs251124 and rs173686) and hep-aran sulfate proteoglycan 2 (rs3767137) The COL1A2rs42524 SNP is located on chromosome 7q221 close tothe elastin genemdasha protein component of arterial wallspreviously associated with IAs in a linkage-associationstudy29 Collagen type 1 is usually found in the adventi-tial layer of cerebral arteries and provides tensile strengthmaking it a plausible factor in IA development Never-theless caution is needed in generalizing this result as all3 studies identified were in Far East populations forrobust association to be confirmed this SNP has to beexamined in other populations including Europebecause risk allele frequencies may differ Collagen type3 (COL 3A1) was investigated in 3 Chinese populationsrs1800255 (GA) was the only SNP significantly asso-ciated with IA but replication in other ethnicities andlarger cohorts is required to confirm these findings

We also found associations between 2 versicangenetic variants (rs251124 and rs173686) and IAsin Dutch Japanese and Chinese populations30ndash32

In a previous linkage study on a Japanese cohortchromosome 5q143 very close to the versican genewas implicated in IA30 In vivo studies on abdominalaortic aneurysm samples found reduced versican mes-senger RNA expression3334 which could reflect adecline in smooth-muscle cells associated with weak-ness in the vascular wall Moreover a GWAS35 onthoracic and aortic dissections showed significantassociation with the 5q13ndash14 locus correspondingto the versican gene

The proinflammatory cytokine IL6 G572C SNPhad an overall protective association with IAs in arecessive model However there was significant sta-tistical heterogeneity and sensitivity analysis revealedno association once one Chinese study36 wasexcluded which may partly reflect the greater riskallele frequency (079 vs 005ndash009) and inclusionof only unruptured IAs in this study36 Anothersource of heterogeneity may be sex because one otherstudy37 included only male controls and this may notbe appropriate because SAH has a female predomi-nance Therefore the overall association of this SNPwith IA is not robust Sustained abnormal vascularremodeling in association with inflammation ishypothesized to be a key mechanism in IA develop-ment22 IL-6 can injure vessel walls by inhibiting col-lagen synthesis thus increasing wall fragility andincreasing the risk of aneurysmal dilatation IL-6 lev-els are increased in the CSF and plasma after

2162 Neurology 80 June 4 2013

SAH3839 but whether this is directly related to theG572C SNP remains uncertain because in these stud-ies patients were not genotyped for the mutant allelemoreover IL-6 may be released as part of a systemicinflammatory response in the acute phase after SAH

Strengths of our study Our systematic review andmeta-analysis is the largest and most comprehensiveto date on genetic variants associated with IAs We as-sessed strength of association by using multiplegenetic models and carefully assessed the robustnessof all associations by performing random-effects sen-sitivity and ethnic subgroup analyses We conductedour analysis and reporting according to quality guide-lines for genetic association studies including HumanGenome Epidemiology Network15 and Strengtheningthe Reporting of Genetic Association Studies17

Limitations of our study Our assessment of interstudyheterogeneity by visual inspection of the forest plotsand sensitivity analyses only partly addresses potentialsources of heterogeneity between studies Furtherindividual patient data are required on other factorsthat could also contribute to heterogeneity such assex proportions age genotyping methods or errorsdisease phenotype (aneurysm size location and rup-ture status) and environmental risk factor status Ourmeta-analysis was only able to pool reported posi-tively associated SNPs from the 6 GWAS studiesbut data on many SNPs sequenced not reaching thethreshold for GWAS statistical significance were notavailable thus limiting our meta-analysis to 41 SNPsin total Indeed for many of the candidate-gene stud-ies pooling all available data from GWAS mightallow more robust risk estimates to be calculateddespite 8 candidate gene SNPs being associated withIAs on average only 27 publications were availablefor each variant

We have identified 19 candidate SNPs associatedwith IAs of which 16 were robust to random-effectsor sensitivity analyses The ORs suggest that thegenetic contribution to aneurysm development issubstantial but the variants identified implicate mul-tiple pathophysiologic mechanisms particularlyinvolving vascular endothelium maintenance Never-theless the amount of data on genetic associationswith IAs remains much smaller than for other com-plex diseases so further large replication studies in afull spectrum of populations are required ideallyusing GWAS or exome sequencing to identify rarerfunctional variants A limitation of most studiesincluded is that both ruptured and unruptured IAswere analyzed together because only a minority ofIAs are destined to rupture future studies shouldaddress genetic risk factors specifically related to rup-ture status Another important area for future researchis to explore how genetic factors are related to

aneurysm location size or morphology becausethere may be specific genetic associations for particu-lar aneurysm phenotypes (which may also relate torupture risk) Finally validation studies of risk modelsin prospective cohorts (eg of unruptured IAs fol-lowed up for rupture risk) would be helpful in deter-mining the relevance of genetic risk loci in addition towell-established risk factors for overall disease pheno-type to ultimately reduce the devastating burden ofaneurysmal SAH

AUTHOR CONTRIBUTIONSDr Alg designed the study performed the literature search acquired and

carried out the statistical analysis of the data and drafted the manuscript

Dr Sofat analyzed and interpreted the data and revised the manuscript

Dr Houlden supervised the study analyzed and interpreted the data and

provided revisions to the manuscript Dr Werring conceived and super-

vised the study checked the literature search interpreted the results and

provided critical revisions to the manuscript

STUDY FUNDINGSupported and funded by the Stroke Association (TSA 200708) Dr Alg

receives research support from the Stroke Association Dr Werring re-

ceives research support from the Department of HealthHigher Educa-

tion Funding Council for England (Clinical Senior Lectureship Award)

Part of this work was undertaken at UCLHUCL which received a pro-

portion of funding from the Department of Healthrsquos NIHR Biomedical

Research Centres funding scheme

DISCLOSUREThe authors report no disclosures relevant to the manuscript Go to

Neurologyorg for full disclosures

Received November 23 2012 Accepted in final form February 212013

REFERENCES1 Rinkel GJ Djibuti M Algra A van Gijn J Prevalence and

risk of rupture of intracranial aneurysms a systematic

review Stroke 199829251ndash256

2 Rinkel GJ Algra A Long-term outcomes of patients with

aneurysmal subarachnoid haemorrhage Lancet Neurol

201110349ndash356

3 Vlak MH Rinkel GJ Greebe P van der Bom JG Algra A

Trigger factors for rupture of intracranial aneurysms in

relation to patient and aneurysm characteristics J Neurol

20122591298ndash1302

4 Clarke M Systematic review of reviews of risk factors for

intracranial aneurysms Neuroradiology 200850653ndash664

5 Feigin VL Rinkel GJ Lawes CM et al Risk factors for

subarachnoid hemorrhage an updated systematic review of

epidemiological studies Stroke 2005362773ndash2780

6 Juvela S Risk factors for aneurysmal subarachnoid hemor-

rhage Stroke 2002332152ndash2153 author reply 2152ndash2153

7 Krischek B Inoue I The genetics of intracranial aneur-

ysms J Hum Genet 200651587ndash594

8 Bromberg JE Rinkel GJ Algra A et al Subarachnoid hae-

morrhage in first and second degree relatives of patients with

subarachnoid haemorrhage BMJ 1995311288ndash289

9 Bilguvar K Yasuno K Niemela M et al Susceptibility loci

for intracranial aneurysm in European and Japanese pop-

ulations Nat Genet 2008401472ndash1477

Neurology 80 June 4 2013 2163

The SNP rs10757278 on chromosome 9p213located in a noncoding RNA region called ANRIL(antisense noncoding RNA in the INK4 locus)showed robust association with IAs but its functionremains unclear Moreover the observed association ofrs10757278 with IA may arise frommoderate LDwiththe known IA risk variant rs1333040 SNP (r5 059)from which it is approximately 41 kbp upstream20

However these 2 SNPs were not in strong LD in aJapanese population raising the possibility of a smallerLD block associated with IA In a Japanese study19

haplotype analysis of both risk alleles of these SNPsdemonstrated significant association with IAs (OR156 95 CI 132ndash185 p 5 29 3 10207) Func-tional studies show altered ANRIL expression as aresult of variations within the 9p213 region14 withreduced expression of RNA encoded by CDKN2Aand CDKN2B in mice14 these 2 genes adjacent tothe 9p21 region encode tumor-suppressor proteins(p14ndash16) which are involved in cell proliferation21

Moreover p14 levels correlate with ANRIL activityand modulate MMP-3 levels which may influenceextracellular matrix repair21

In the most recent GWAS14 in a Caucasian popu-lation of familial and sporadic IA 6 SNPs also locatedin the 9p213 region were associated with IA one ofthese (rs6475606) achieved GWAS-level statistical sig-nificance (OR 134 p5 4293 10207) for associationwith sporadic and familial IAs Association with thers1333040 SNP was also confirmed in this study14

further strengthening association of this locus with IATwo SNPs on chromosome 8q (rs10958409 and

rs9298506) surrounding the SOX17 gene were studiedin European and Japanese populations rs10958409 wasassociated in both populations but the rs9298506 SNPwas associated with IA in the European but not Japanesepopulation SOX17 plays an important role in generationand maintenance of stem cells of endothelial and hema-topoietic lineages crucial in the formation and mainte-nance of vascular endothelium2223 Furthermore SOX17knockout mice show multiple vascular abnormalitiesreflecting defective endothelial remodeling22 Defectivestem cells involved in vascular maintenance are thusplausible targets in IA pathogenesis The heterogene-ity observed in the meta-analysis of these 2 SNPsmay partly be explained by the difference in risk allelefrequencies in the white population (table e-1)24

compared with the other populations studied(rs10958409 risk allele frequency 088 vs approxi-mately 02 rs9298506 risk allele frequency 017 vsapproximately 080) Further support implicatingchromosome 8 loci was shown in a recent GWAS14

in which a novel SNP also in the region of theSOX17 gene was significantly associated with IAs(rs1072737 OR 122 95 CI 107ndash139) inde-pendent of smoking For both SOX17 loci in the

Tab

le1

Con

tinu

ed

Gen

elocu

sSNP

Gen

etic

mod

elNo

stud

ies

Cas

esCon

trols

FE

OR

(95

CI)

pcorrecte

d(Bon

ferron

i)pHet(I2

)

Rob

ustas

sociat

ion

afte

rra

ndom

effe

ctsse

nsitivity

analys

es

Pot

ential

biological

mec

hanism

ofas

sociat

ionwithIA

IL6

G572C

Dom

inan

t5

363753ndash55

912

3410

068

(048ndash096)

0004

000001

(92

)3

Inflam

matorymed

iator

Rec

essive

047

(034ndash065)

000001

000001

(94

)

Add

itive

061

(049ndash075)

000001

000001

(95

)

Abbr

eviation

sACE

5an

giote

nsin-con

verting

enzy

me

CI5

conf

iden

ceinte

rvalCSPG

5ch

ondr

oitinsu

lfat

epro

teog

lyca

nFE

5fixe

d-effe

ctsmod

elHSPG

5he

par

ansu

lfat

epr

oteo

glyc

anI2

5Higgins

stat

isticdem

onstrating

degr

eeof

inte

rstu

dyhe

tero

gen

eity

IA5

intrac

ranial

aneu

rysm

IL5

inte

rleu

kin

NA

5no

tap

plicab

leO

R5

odds

ratio

p Het5

inte

rstu

dyhe

tero

gen

eity

pva

lue

SNP5

sing

lenu

cleo

tide

polymor

phism

5SNP

robu

stly

asso

ciat

edwithIA

follo

wingse

nsitivityan

dra

ndom

-effec

tsan

alys

is3

5SNPsno

tro

bustly

asso

ciat

edwithIA

follo

wingse

nsitivityan

alys

isan

drand

om-effec

tsan

alys

is

2158 Neurology 80 June 4 2013

Figure 2 Forest plots (additive model) for genome-wide association studies in all populations

(A) rs1333040 single nucleotide polymorphism (SNP) (B) rs10757278 SNP (C) rs9298506 (D) rs10958409 (E)rs6841581 CI 5 confidence interval IA 5 intracranial aneurysm

Neurology 80 June 4 2013 2159

Figure 3 Forest plots for candidate gene single nucleotide polymorphisms associated with intracranial aneurysms

(A) Dominant model forest plots for SERPINA3 single nucleotide polymorphism (SNP) (B) COL3A1 (rs1800255) dominant model forest plot (C) COL1A2(rs42524) dominant model forest plot (D)ACE ID recessive model forest plot (E) IL6G572C SNP recessive model forest plot before sensitivity analysis (F)IL6 G572C SNP recessive model forest plot after sensitivity analysis CI 5 confidence interval IA 5 intracranial aneurysm M-H 5 Mantel-Haenszel

2160 Neurology 80 June 4 2013

most recent GWAS14 smoking was an independentrisk factor acting in a multiplicative manner (chromo-some 8 rs1072737 OR 212 95 CI 176ndash256 andchromosome 9 rs6475606 OR 211 95 CI 177ndash252)

A further recent GWAS12 found the SNPrs6841581AG on chromosome 4q3123 coding forthe endothelin receptor type A (EDNRA) gene wassignificantly associated with IA in Dutch Finnish andJapanese populations (OR 122 95 CI 114ndash131

Table 2 Random-effects and sensitivity analyses of associated SNPs that showed significant statisticalheterogeneity under fixed-effects modeling

SNPNostudies

Studiesexcluded

FE OR (95 CI)before adjustment

Random-effectsOR (95 CI)

FE OR (95 CI) aftersensitivity analysis Genetic model

SERPINA3 3 Liu42 222 (168ndash294)a 212 (036ndash1231) 107 (074ndash153) Dominant

127 (107ndash150)a 125 (061ndash259) 097 (079ndash119) Additive

ACE ID 5 Slowik50 139 (105ndash184)a 144 (086ndash242) 120 (088ndash162) Recessive

IL6 G572C 5 Sun36 047 (034ndash065)a 149 (024ndash927) 103 (065ndash162) Recessive

061 (049ndash075)a 095 (031ndash285) 104 (079ndash139) Additive

rs1333040 11 Low13 123 (119ndash128)a 126 (115ndash139)a 131 (126ndash137)a Additive

rs700651 6 Low13 111 (106ndash118)a 114 (103ndash125)a 119 (111ndash128)a Additive

rs9298506 8 Low13 121 (115ndash127)a 124 (108ndash141)a 132 (124ndash139)a Additive

rs10958409 9 410112024b 120 (115ndash126)a 122 (11ndash134)a 123 (117ndash13)a Additive

Abbreviations ACE 5 angiotensin-converting enzyme CI 5 confidence interval FE 5 fixed-effects model IL 5 interleukinOR 5 odds ratio SNP 5 single nucleotide polymorphismaSignificant association with IAb Four studies including one white population and 3 Japanese populations were excluded

Table 3 Subgroup ethnicity analysis

SNPNostudies Ethnicity

FE OR all ethnicities(all studies)

FE OR (95 CI)ethnicity subanalysis pcorrected pHet (I2) Genetic model

ACE ID 45 White 139 (105ndash184) 142 (106ndash192)a 0002 0006 (76) Recessive

Japanese 120 (056ndash257) 054 NA Recessive

IL6 G572C 35 Chinese 030 (019ndash047) 041 (029ndash058)a 000001 00001 (95) Recessive

25 White 426 (092ndash197) 01 003 (78) Recessive

35 Chinese 058 (044ndash075) 050 (039ndash064)a 000001 000001 (97) Additive

25 White 138 (083ndash229) 01 019 (42) Additive

rs1333040 611 Japanese 124 (12ndash129) 115 (109ndash122)a 175 3 10206 306 3 10208 (88) Additive

511 White 130 (124ndash136)a 191 3 10206 078 (0) Additive

rs10757278 57 White 129 (121ndash138) 130 (12ndash143)a 12 3 10209 091 (0) Additive

27 Japanese 127 (113ndash141)a 26 3 10205 022 (33) Additive

rs9298506 58 White 121 (115ndash127) 135 (127ndash144)a 24 3 10204 052 (0) Additive

38 Japanese 103 (095ndash112) 045 654 3 10204 (86) Additive

rs10958409 59 White 12 (115ndash126) 131 (123ndash139)a 82 3 10202 0052 (62) Additive

49 Japanese 109 (102ndash117)a 0009 008 (55) Additive

rs700651 33 White 111 (106ndash118) 119 (109ndash129)a 482 3 10205 023 (31) Additive

33 Japanese 106 (094ndash131) 008 002 (75) Additive

rs6841581 46 White 122 (114ndash131) 125 (113ndash138)a 121 3 10205 018 (38) Additive

26 Japanese 12 (108ndash132)a 356 3 10204 087 (0) Additive

rs6538595 46 White 116 (110ndash123) 116 (108ndash124)a 213 3 10205 091 (0) Additive

26 Japanese 117 (106ndash128)a 149 3 10203 06 (0) Additive

Abbreviations ACE 5 angiotensin-converting enzyme CI 5 confidence interval FE 5 fixed-effects model I2 5 Higgins statistic demonstrating degree ofinterstudy heterogeneity IA5 intracranial aneurysm IL5 interleukin NA5 not applicable OR 5 odds ratio pHet 5 interstudy heterogeneity p value SNP 5

single nucleotide polymorphisma Significant association with IA

Neurology 80 June 4 2013 2161

p 5 195 3 1028) Another GWAS13 in a Japanesepopulation showed that the rs6842241 SNP located at4q3122 near the EDNRA gene was significantly asso-ciated with IA (OR 125 95 CI 116ndash134 p 5

9583 10209) EDNRA is a G-proteinndashcoupled recep-tor for endothelins which modulate vasoconstrictionand vasodilatation after hemodynamic insult Endo-thelin-1mdashthe predominant isoform in vascularsmooth-muscle cellsmdashactivates EDNRA Endothelinsignaling is activated at the site of vascular injury1225

causing cell proliferation indeed after SAH EDNRAand endothelin-1 protein levels are both increased inCSF26ndash28 Alternatively downregulation of EDNRAsignaling could lead to defective vascular repair allowinganeurysms to develop after injury a hypothesis sup-ported by functional analysis of EDNRA variants show-ing lower transcriptional activity for the rs6841581risk allele (G)13

SNPs with unknown function or not examined in more

than 2 studies Five SNPs from GWAS do not corre-spond to genes with any known functions relevant toIA development Two of these SNPs (rs1429412 andrs700651) at locus 2q33 flank the BOLL gene(involved in germ cell development) and the phospho-lipase Cndashlike 1 gene (involved in intracellular cascadereactions with abolished phospholipase C activity)Phospholipase Cndashlike 1 lies close to the vascular endo-thelial growth factor receptor-2 which is involved inCNS angiogenesis and is a marker for endothelial pro-genitor cells Three further SNPs on chromosome 2were associated with familial IAs rs11693075 (close tothe histone deacetylase complex SAP130 gene)rs1897472 and rs3769801 (both located within thephosphodiesterase-1 gene)14

One associated SNP located on chromosome 7(rs4628172 GT) is within the TMEM195 genewhich encodes transmembrane proteins possiblyinvolved in fatty acid biosynthesis and iron bind-ing1113 Further associated SNPs from the latestGWAS include variants on chromosome 12q22(rs6538595 rs11112585 and rs2374513) within theC12orf75 gene14 The rs1132274 GT SNP on chro-mosome 20p121 was also associated with IA10 How-ever none of these SNPs yet has known clearfunctional relevance to IA

Three further loci not included in this meta-analysis(because they were reported in only one study) showedsignificant association with IAs10 These loci included18q112 (rs11661542) 10q2432 (rs12413409) and13q132 (rs9315204) but their mechanisms of associ-ation remain unclear

Genetic variants associated with IAs identified by CGAS

Eight SNPs from CGAS were associated with IAs inour meta-analysis albeit with significant statisticalheterogeneity These SNPs relate to genes with

plausible relevance to IA biology including those impor-tant for extracellular matrix integrity inflammatorymedi-ators and vascular endothelium maintenanceExtracellular matrix gene variants may predispose to IAdevelopment by weakening arterial walls In our meta-analysis 6 such SNPs showed significant associationswith IA COL1A2 (rs42524) COL3A1 (rs1800255)SERPINA3 versican (rs251124 and rs173686) and hep-aran sulfate proteoglycan 2 (rs3767137) The COL1A2rs42524 SNP is located on chromosome 7q221 close tothe elastin genemdasha protein component of arterial wallspreviously associated with IAs in a linkage-associationstudy29 Collagen type 1 is usually found in the adventi-tial layer of cerebral arteries and provides tensile strengthmaking it a plausible factor in IA development Never-theless caution is needed in generalizing this result as all3 studies identified were in Far East populations forrobust association to be confirmed this SNP has to beexamined in other populations including Europebecause risk allele frequencies may differ Collagen type3 (COL 3A1) was investigated in 3 Chinese populationsrs1800255 (GA) was the only SNP significantly asso-ciated with IA but replication in other ethnicities andlarger cohorts is required to confirm these findings

We also found associations between 2 versicangenetic variants (rs251124 and rs173686) and IAsin Dutch Japanese and Chinese populations30ndash32

In a previous linkage study on a Japanese cohortchromosome 5q143 very close to the versican genewas implicated in IA30 In vivo studies on abdominalaortic aneurysm samples found reduced versican mes-senger RNA expression3334 which could reflect adecline in smooth-muscle cells associated with weak-ness in the vascular wall Moreover a GWAS35 onthoracic and aortic dissections showed significantassociation with the 5q13ndash14 locus correspondingto the versican gene

The proinflammatory cytokine IL6 G572C SNPhad an overall protective association with IAs in arecessive model However there was significant sta-tistical heterogeneity and sensitivity analysis revealedno association once one Chinese study36 wasexcluded which may partly reflect the greater riskallele frequency (079 vs 005ndash009) and inclusionof only unruptured IAs in this study36 Anothersource of heterogeneity may be sex because one otherstudy37 included only male controls and this may notbe appropriate because SAH has a female predomi-nance Therefore the overall association of this SNPwith IA is not robust Sustained abnormal vascularremodeling in association with inflammation ishypothesized to be a key mechanism in IA develop-ment22 IL-6 can injure vessel walls by inhibiting col-lagen synthesis thus increasing wall fragility andincreasing the risk of aneurysmal dilatation IL-6 lev-els are increased in the CSF and plasma after

2162 Neurology 80 June 4 2013

SAH3839 but whether this is directly related to theG572C SNP remains uncertain because in these stud-ies patients were not genotyped for the mutant allelemoreover IL-6 may be released as part of a systemicinflammatory response in the acute phase after SAH

Strengths of our study Our systematic review andmeta-analysis is the largest and most comprehensiveto date on genetic variants associated with IAs We as-sessed strength of association by using multiplegenetic models and carefully assessed the robustnessof all associations by performing random-effects sen-sitivity and ethnic subgroup analyses We conductedour analysis and reporting according to quality guide-lines for genetic association studies including HumanGenome Epidemiology Network15 and Strengtheningthe Reporting of Genetic Association Studies17

Limitations of our study Our assessment of interstudyheterogeneity by visual inspection of the forest plotsand sensitivity analyses only partly addresses potentialsources of heterogeneity between studies Furtherindividual patient data are required on other factorsthat could also contribute to heterogeneity such assex proportions age genotyping methods or errorsdisease phenotype (aneurysm size location and rup-ture status) and environmental risk factor status Ourmeta-analysis was only able to pool reported posi-tively associated SNPs from the 6 GWAS studiesbut data on many SNPs sequenced not reaching thethreshold for GWAS statistical significance were notavailable thus limiting our meta-analysis to 41 SNPsin total Indeed for many of the candidate-gene stud-ies pooling all available data from GWAS mightallow more robust risk estimates to be calculateddespite 8 candidate gene SNPs being associated withIAs on average only 27 publications were availablefor each variant

We have identified 19 candidate SNPs associatedwith IAs of which 16 were robust to random-effectsor sensitivity analyses The ORs suggest that thegenetic contribution to aneurysm development issubstantial but the variants identified implicate mul-tiple pathophysiologic mechanisms particularlyinvolving vascular endothelium maintenance Never-theless the amount of data on genetic associationswith IAs remains much smaller than for other com-plex diseases so further large replication studies in afull spectrum of populations are required ideallyusing GWAS or exome sequencing to identify rarerfunctional variants A limitation of most studiesincluded is that both ruptured and unruptured IAswere analyzed together because only a minority ofIAs are destined to rupture future studies shouldaddress genetic risk factors specifically related to rup-ture status Another important area for future researchis to explore how genetic factors are related to

aneurysm location size or morphology becausethere may be specific genetic associations for particu-lar aneurysm phenotypes (which may also relate torupture risk) Finally validation studies of risk modelsin prospective cohorts (eg of unruptured IAs fol-lowed up for rupture risk) would be helpful in deter-mining the relevance of genetic risk loci in addition towell-established risk factors for overall disease pheno-type to ultimately reduce the devastating burden ofaneurysmal SAH

AUTHOR CONTRIBUTIONSDr Alg designed the study performed the literature search acquired and

carried out the statistical analysis of the data and drafted the manuscript

Dr Sofat analyzed and interpreted the data and revised the manuscript

Dr Houlden supervised the study analyzed and interpreted the data and

provided revisions to the manuscript Dr Werring conceived and super-

vised the study checked the literature search interpreted the results and

provided critical revisions to the manuscript

STUDY FUNDINGSupported and funded by the Stroke Association (TSA 200708) Dr Alg

receives research support from the Stroke Association Dr Werring re-

ceives research support from the Department of HealthHigher Educa-

tion Funding Council for England (Clinical Senior Lectureship Award)

Part of this work was undertaken at UCLHUCL which received a pro-

portion of funding from the Department of Healthrsquos NIHR Biomedical

Research Centres funding scheme

DISCLOSUREThe authors report no disclosures relevant to the manuscript Go to

Neurologyorg for full disclosures

Received November 23 2012 Accepted in final form February 212013

REFERENCES1 Rinkel GJ Djibuti M Algra A van Gijn J Prevalence and

risk of rupture of intracranial aneurysms a systematic

review Stroke 199829251ndash256

2 Rinkel GJ Algra A Long-term outcomes of patients with

aneurysmal subarachnoid haemorrhage Lancet Neurol

201110349ndash356

3 Vlak MH Rinkel GJ Greebe P van der Bom JG Algra A

Trigger factors for rupture of intracranial aneurysms in

relation to patient and aneurysm characteristics J Neurol

20122591298ndash1302

4 Clarke M Systematic review of reviews of risk factors for

intracranial aneurysms Neuroradiology 200850653ndash664

5 Feigin VL Rinkel GJ Lawes CM et al Risk factors for

subarachnoid hemorrhage an updated systematic review of

epidemiological studies Stroke 2005362773ndash2780

6 Juvela S Risk factors for aneurysmal subarachnoid hemor-

rhage Stroke 2002332152ndash2153 author reply 2152ndash2153

7 Krischek B Inoue I The genetics of intracranial aneur-

ysms J Hum Genet 200651587ndash594

8 Bromberg JE Rinkel GJ Algra A et al Subarachnoid hae-

morrhage in first and second degree relatives of patients with

subarachnoid haemorrhage BMJ 1995311288ndash289

9 Bilguvar K Yasuno K Niemela M et al Susceptibility loci

for intracranial aneurysm in European and Japanese pop-

ulations Nat Genet 2008401472ndash1477

Neurology 80 June 4 2013 2163

Figure 2 Forest plots (additive model) for genome-wide association studies in all populations

(A) rs1333040 single nucleotide polymorphism (SNP) (B) rs10757278 SNP (C) rs9298506 (D) rs10958409 (E)rs6841581 CI 5 confidence interval IA 5 intracranial aneurysm

Neurology 80 June 4 2013 2159

Figure 3 Forest plots for candidate gene single nucleotide polymorphisms associated with intracranial aneurysms

(A) Dominant model forest plots for SERPINA3 single nucleotide polymorphism (SNP) (B) COL3A1 (rs1800255) dominant model forest plot (C) COL1A2(rs42524) dominant model forest plot (D)ACE ID recessive model forest plot (E) IL6G572C SNP recessive model forest plot before sensitivity analysis (F)IL6 G572C SNP recessive model forest plot after sensitivity analysis CI 5 confidence interval IA 5 intracranial aneurysm M-H 5 Mantel-Haenszel

2160 Neurology 80 June 4 2013

most recent GWAS14 smoking was an independentrisk factor acting in a multiplicative manner (chromo-some 8 rs1072737 OR 212 95 CI 176ndash256 andchromosome 9 rs6475606 OR 211 95 CI 177ndash252)

A further recent GWAS12 found the SNPrs6841581AG on chromosome 4q3123 coding forthe endothelin receptor type A (EDNRA) gene wassignificantly associated with IA in Dutch Finnish andJapanese populations (OR 122 95 CI 114ndash131

Table 2 Random-effects and sensitivity analyses of associated SNPs that showed significant statisticalheterogeneity under fixed-effects modeling

SNPNostudies

Studiesexcluded

FE OR (95 CI)before adjustment

Random-effectsOR (95 CI)

FE OR (95 CI) aftersensitivity analysis Genetic model

SERPINA3 3 Liu42 222 (168ndash294)a 212 (036ndash1231) 107 (074ndash153) Dominant

127 (107ndash150)a 125 (061ndash259) 097 (079ndash119) Additive

ACE ID 5 Slowik50 139 (105ndash184)a 144 (086ndash242) 120 (088ndash162) Recessive

IL6 G572C 5 Sun36 047 (034ndash065)a 149 (024ndash927) 103 (065ndash162) Recessive

061 (049ndash075)a 095 (031ndash285) 104 (079ndash139) Additive

rs1333040 11 Low13 123 (119ndash128)a 126 (115ndash139)a 131 (126ndash137)a Additive

rs700651 6 Low13 111 (106ndash118)a 114 (103ndash125)a 119 (111ndash128)a Additive

rs9298506 8 Low13 121 (115ndash127)a 124 (108ndash141)a 132 (124ndash139)a Additive

rs10958409 9 410112024b 120 (115ndash126)a 122 (11ndash134)a 123 (117ndash13)a Additive

Abbreviations ACE 5 angiotensin-converting enzyme CI 5 confidence interval FE 5 fixed-effects model IL 5 interleukinOR 5 odds ratio SNP 5 single nucleotide polymorphismaSignificant association with IAb Four studies including one white population and 3 Japanese populations were excluded

Table 3 Subgroup ethnicity analysis

SNPNostudies Ethnicity

FE OR all ethnicities(all studies)

FE OR (95 CI)ethnicity subanalysis pcorrected pHet (I2) Genetic model

ACE ID 45 White 139 (105ndash184) 142 (106ndash192)a 0002 0006 (76) Recessive

Japanese 120 (056ndash257) 054 NA Recessive

IL6 G572C 35 Chinese 030 (019ndash047) 041 (029ndash058)a 000001 00001 (95) Recessive

25 White 426 (092ndash197) 01 003 (78) Recessive

35 Chinese 058 (044ndash075) 050 (039ndash064)a 000001 000001 (97) Additive

25 White 138 (083ndash229) 01 019 (42) Additive

rs1333040 611 Japanese 124 (12ndash129) 115 (109ndash122)a 175 3 10206 306 3 10208 (88) Additive

511 White 130 (124ndash136)a 191 3 10206 078 (0) Additive

rs10757278 57 White 129 (121ndash138) 130 (12ndash143)a 12 3 10209 091 (0) Additive

27 Japanese 127 (113ndash141)a 26 3 10205 022 (33) Additive

rs9298506 58 White 121 (115ndash127) 135 (127ndash144)a 24 3 10204 052 (0) Additive

38 Japanese 103 (095ndash112) 045 654 3 10204 (86) Additive

rs10958409 59 White 12 (115ndash126) 131 (123ndash139)a 82 3 10202 0052 (62) Additive

49 Japanese 109 (102ndash117)a 0009 008 (55) Additive

rs700651 33 White 111 (106ndash118) 119 (109ndash129)a 482 3 10205 023 (31) Additive

33 Japanese 106 (094ndash131) 008 002 (75) Additive

rs6841581 46 White 122 (114ndash131) 125 (113ndash138)a 121 3 10205 018 (38) Additive

26 Japanese 12 (108ndash132)a 356 3 10204 087 (0) Additive

rs6538595 46 White 116 (110ndash123) 116 (108ndash124)a 213 3 10205 091 (0) Additive

26 Japanese 117 (106ndash128)a 149 3 10203 06 (0) Additive

Abbreviations ACE 5 angiotensin-converting enzyme CI 5 confidence interval FE 5 fixed-effects model I2 5 Higgins statistic demonstrating degree ofinterstudy heterogeneity IA5 intracranial aneurysm IL5 interleukin NA5 not applicable OR 5 odds ratio pHet 5 interstudy heterogeneity p value SNP 5

single nucleotide polymorphisma Significant association with IA

Neurology 80 June 4 2013 2161

p 5 195 3 1028) Another GWAS13 in a Japanesepopulation showed that the rs6842241 SNP located at4q3122 near the EDNRA gene was significantly asso-ciated with IA (OR 125 95 CI 116ndash134 p 5

9583 10209) EDNRA is a G-proteinndashcoupled recep-tor for endothelins which modulate vasoconstrictionand vasodilatation after hemodynamic insult Endo-thelin-1mdashthe predominant isoform in vascularsmooth-muscle cellsmdashactivates EDNRA Endothelinsignaling is activated at the site of vascular injury1225

causing cell proliferation indeed after SAH EDNRAand endothelin-1 protein levels are both increased inCSF26ndash28 Alternatively downregulation of EDNRAsignaling could lead to defective vascular repair allowinganeurysms to develop after injury a hypothesis sup-ported by functional analysis of EDNRA variants show-ing lower transcriptional activity for the rs6841581risk allele (G)13

SNPs with unknown function or not examined in more

than 2 studies Five SNPs from GWAS do not corre-spond to genes with any known functions relevant toIA development Two of these SNPs (rs1429412 andrs700651) at locus 2q33 flank the BOLL gene(involved in germ cell development) and the phospho-lipase Cndashlike 1 gene (involved in intracellular cascadereactions with abolished phospholipase C activity)Phospholipase Cndashlike 1 lies close to the vascular endo-thelial growth factor receptor-2 which is involved inCNS angiogenesis and is a marker for endothelial pro-genitor cells Three further SNPs on chromosome 2were associated with familial IAs rs11693075 (close tothe histone deacetylase complex SAP130 gene)rs1897472 and rs3769801 (both located within thephosphodiesterase-1 gene)14

One associated SNP located on chromosome 7(rs4628172 GT) is within the TMEM195 genewhich encodes transmembrane proteins possiblyinvolved in fatty acid biosynthesis and iron bind-ing1113 Further associated SNPs from the latestGWAS include variants on chromosome 12q22(rs6538595 rs11112585 and rs2374513) within theC12orf75 gene14 The rs1132274 GT SNP on chro-mosome 20p121 was also associated with IA10 How-ever none of these SNPs yet has known clearfunctional relevance to IA

Three further loci not included in this meta-analysis(because they were reported in only one study) showedsignificant association with IAs10 These loci included18q112 (rs11661542) 10q2432 (rs12413409) and13q132 (rs9315204) but their mechanisms of associ-ation remain unclear

Genetic variants associated with IAs identified by CGAS

Eight SNPs from CGAS were associated with IAs inour meta-analysis albeit with significant statisticalheterogeneity These SNPs relate to genes with

plausible relevance to IA biology including those impor-tant for extracellular matrix integrity inflammatorymedi-ators and vascular endothelium maintenanceExtracellular matrix gene variants may predispose to IAdevelopment by weakening arterial walls In our meta-analysis 6 such SNPs showed significant associationswith IA COL1A2 (rs42524) COL3A1 (rs1800255)SERPINA3 versican (rs251124 and rs173686) and hep-aran sulfate proteoglycan 2 (rs3767137) The COL1A2rs42524 SNP is located on chromosome 7q221 close tothe elastin genemdasha protein component of arterial wallspreviously associated with IAs in a linkage-associationstudy29 Collagen type 1 is usually found in the adventi-tial layer of cerebral arteries and provides tensile strengthmaking it a plausible factor in IA development Never-theless caution is needed in generalizing this result as all3 studies identified were in Far East populations forrobust association to be confirmed this SNP has to beexamined in other populations including Europebecause risk allele frequencies may differ Collagen type3 (COL 3A1) was investigated in 3 Chinese populationsrs1800255 (GA) was the only SNP significantly asso-ciated with IA but replication in other ethnicities andlarger cohorts is required to confirm these findings

We also found associations between 2 versicangenetic variants (rs251124 and rs173686) and IAsin Dutch Japanese and Chinese populations30ndash32

In a previous linkage study on a Japanese cohortchromosome 5q143 very close to the versican genewas implicated in IA30 In vivo studies on abdominalaortic aneurysm samples found reduced versican mes-senger RNA expression3334 which could reflect adecline in smooth-muscle cells associated with weak-ness in the vascular wall Moreover a GWAS35 onthoracic and aortic dissections showed significantassociation with the 5q13ndash14 locus correspondingto the versican gene

The proinflammatory cytokine IL6 G572C SNPhad an overall protective association with IAs in arecessive model However there was significant sta-tistical heterogeneity and sensitivity analysis revealedno association once one Chinese study36 wasexcluded which may partly reflect the greater riskallele frequency (079 vs 005ndash009) and inclusionof only unruptured IAs in this study36 Anothersource of heterogeneity may be sex because one otherstudy37 included only male controls and this may notbe appropriate because SAH has a female predomi-nance Therefore the overall association of this SNPwith IA is not robust Sustained abnormal vascularremodeling in association with inflammation ishypothesized to be a key mechanism in IA develop-ment22 IL-6 can injure vessel walls by inhibiting col-lagen synthesis thus increasing wall fragility andincreasing the risk of aneurysmal dilatation IL-6 lev-els are increased in the CSF and plasma after

2162 Neurology 80 June 4 2013

SAH3839 but whether this is directly related to theG572C SNP remains uncertain because in these stud-ies patients were not genotyped for the mutant allelemoreover IL-6 may be released as part of a systemicinflammatory response in the acute phase after SAH

Strengths of our study Our systematic review andmeta-analysis is the largest and most comprehensiveto date on genetic variants associated with IAs We as-sessed strength of association by using multiplegenetic models and carefully assessed the robustnessof all associations by performing random-effects sen-sitivity and ethnic subgroup analyses We conductedour analysis and reporting according to quality guide-lines for genetic association studies including HumanGenome Epidemiology Network15 and Strengtheningthe Reporting of Genetic Association Studies17

Limitations of our study Our assessment of interstudyheterogeneity by visual inspection of the forest plotsand sensitivity analyses only partly addresses potentialsources of heterogeneity between studies Furtherindividual patient data are required on other factorsthat could also contribute to heterogeneity such assex proportions age genotyping methods or errorsdisease phenotype (aneurysm size location and rup-ture status) and environmental risk factor status Ourmeta-analysis was only able to pool reported posi-tively associated SNPs from the 6 GWAS studiesbut data on many SNPs sequenced not reaching thethreshold for GWAS statistical significance were notavailable thus limiting our meta-analysis to 41 SNPsin total Indeed for many of the candidate-gene stud-ies pooling all available data from GWAS mightallow more robust risk estimates to be calculateddespite 8 candidate gene SNPs being associated withIAs on average only 27 publications were availablefor each variant

We have identified 19 candidate SNPs associatedwith IAs of which 16 were robust to random-effectsor sensitivity analyses The ORs suggest that thegenetic contribution to aneurysm development issubstantial but the variants identified implicate mul-tiple pathophysiologic mechanisms particularlyinvolving vascular endothelium maintenance Never-theless the amount of data on genetic associationswith IAs remains much smaller than for other com-plex diseases so further large replication studies in afull spectrum of populations are required ideallyusing GWAS or exome sequencing to identify rarerfunctional variants A limitation of most studiesincluded is that both ruptured and unruptured IAswere analyzed together because only a minority ofIAs are destined to rupture future studies shouldaddress genetic risk factors specifically related to rup-ture status Another important area for future researchis to explore how genetic factors are related to

aneurysm location size or morphology becausethere may be specific genetic associations for particu-lar aneurysm phenotypes (which may also relate torupture risk) Finally validation studies of risk modelsin prospective cohorts (eg of unruptured IAs fol-lowed up for rupture risk) would be helpful in deter-mining the relevance of genetic risk loci in addition towell-established risk factors for overall disease pheno-type to ultimately reduce the devastating burden ofaneurysmal SAH

AUTHOR CONTRIBUTIONSDr Alg designed the study performed the literature search acquired and

carried out the statistical analysis of the data and drafted the manuscript

Dr Sofat analyzed and interpreted the data and revised the manuscript

Dr Houlden supervised the study analyzed and interpreted the data and

provided revisions to the manuscript Dr Werring conceived and super-

vised the study checked the literature search interpreted the results and

provided critical revisions to the manuscript

STUDY FUNDINGSupported and funded by the Stroke Association (TSA 200708) Dr Alg

receives research support from the Stroke Association Dr Werring re-

ceives research support from the Department of HealthHigher Educa-

tion Funding Council for England (Clinical Senior Lectureship Award)

Part of this work was undertaken at UCLHUCL which received a pro-

portion of funding from the Department of Healthrsquos NIHR Biomedical

Research Centres funding scheme

DISCLOSUREThe authors report no disclosures relevant to the manuscript Go to

Neurologyorg for full disclosures

Received November 23 2012 Accepted in final form February 212013

REFERENCES1 Rinkel GJ Djibuti M Algra A van Gijn J Prevalence and

risk of rupture of intracranial aneurysms a systematic

review Stroke 199829251ndash256

2 Rinkel GJ Algra A Long-term outcomes of patients with

aneurysmal subarachnoid haemorrhage Lancet Neurol

201110349ndash356

3 Vlak MH Rinkel GJ Greebe P van der Bom JG Algra A

Trigger factors for rupture of intracranial aneurysms in

relation to patient and aneurysm characteristics J Neurol

20122591298ndash1302

4 Clarke M Systematic review of reviews of risk factors for

intracranial aneurysms Neuroradiology 200850653ndash664

5 Feigin VL Rinkel GJ Lawes CM et al Risk factors for

subarachnoid hemorrhage an updated systematic review of

epidemiological studies Stroke 2005362773ndash2780

6 Juvela S Risk factors for aneurysmal subarachnoid hemor-

rhage Stroke 2002332152ndash2153 author reply 2152ndash2153

7 Krischek B Inoue I The genetics of intracranial aneur-

ysms J Hum Genet 200651587ndash594

8 Bromberg JE Rinkel GJ Algra A et al Subarachnoid hae-

morrhage in first and second degree relatives of patients with

subarachnoid haemorrhage BMJ 1995311288ndash289

9 Bilguvar K Yasuno K Niemela M et al Susceptibility loci

for intracranial aneurysm in European and Japanese pop-

ulations Nat Genet 2008401472ndash1477

Neurology 80 June 4 2013 2163

Figure 3 Forest plots for candidate gene single nucleotide polymorphisms associated with intracranial aneurysms

(A) Dominant model forest plots for SERPINA3 single nucleotide polymorphism (SNP) (B) COL3A1 (rs1800255) dominant model forest plot (C) COL1A2(rs42524) dominant model forest plot (D)ACE ID recessive model forest plot (E) IL6G572C SNP recessive model forest plot before sensitivity analysis (F)IL6 G572C SNP recessive model forest plot after sensitivity analysis CI 5 confidence interval IA 5 intracranial aneurysm M-H 5 Mantel-Haenszel

2160 Neurology 80 June 4 2013

most recent GWAS14 smoking was an independentrisk factor acting in a multiplicative manner (chromo-some 8 rs1072737 OR 212 95 CI 176ndash256 andchromosome 9 rs6475606 OR 211 95 CI 177ndash252)

A further recent GWAS12 found the SNPrs6841581AG on chromosome 4q3123 coding forthe endothelin receptor type A (EDNRA) gene wassignificantly associated with IA in Dutch Finnish andJapanese populations (OR 122 95 CI 114ndash131

Table 2 Random-effects and sensitivity analyses of associated SNPs that showed significant statisticalheterogeneity under fixed-effects modeling

SNPNostudies

Studiesexcluded

FE OR (95 CI)before adjustment

Random-effectsOR (95 CI)

FE OR (95 CI) aftersensitivity analysis Genetic model

SERPINA3 3 Liu42 222 (168ndash294)a 212 (036ndash1231) 107 (074ndash153) Dominant

127 (107ndash150)a 125 (061ndash259) 097 (079ndash119) Additive

ACE ID 5 Slowik50 139 (105ndash184)a 144 (086ndash242) 120 (088ndash162) Recessive

IL6 G572C 5 Sun36 047 (034ndash065)a 149 (024ndash927) 103 (065ndash162) Recessive

061 (049ndash075)a 095 (031ndash285) 104 (079ndash139) Additive

rs1333040 11 Low13 123 (119ndash128)a 126 (115ndash139)a 131 (126ndash137)a Additive

rs700651 6 Low13 111 (106ndash118)a 114 (103ndash125)a 119 (111ndash128)a Additive

rs9298506 8 Low13 121 (115ndash127)a 124 (108ndash141)a 132 (124ndash139)a Additive

rs10958409 9 410112024b 120 (115ndash126)a 122 (11ndash134)a 123 (117ndash13)a Additive

Abbreviations ACE 5 angiotensin-converting enzyme CI 5 confidence interval FE 5 fixed-effects model IL 5 interleukinOR 5 odds ratio SNP 5 single nucleotide polymorphismaSignificant association with IAb Four studies including one white population and 3 Japanese populations were excluded

Table 3 Subgroup ethnicity analysis

SNPNostudies Ethnicity

FE OR all ethnicities(all studies)

FE OR (95 CI)ethnicity subanalysis pcorrected pHet (I2) Genetic model

ACE ID 45 White 139 (105ndash184) 142 (106ndash192)a 0002 0006 (76) Recessive

Japanese 120 (056ndash257) 054 NA Recessive

IL6 G572C 35 Chinese 030 (019ndash047) 041 (029ndash058)a 000001 00001 (95) Recessive

25 White 426 (092ndash197) 01 003 (78) Recessive

35 Chinese 058 (044ndash075) 050 (039ndash064)a 000001 000001 (97) Additive

25 White 138 (083ndash229) 01 019 (42) Additive

rs1333040 611 Japanese 124 (12ndash129) 115 (109ndash122)a 175 3 10206 306 3 10208 (88) Additive

511 White 130 (124ndash136)a 191 3 10206 078 (0) Additive

rs10757278 57 White 129 (121ndash138) 130 (12ndash143)a 12 3 10209 091 (0) Additive

27 Japanese 127 (113ndash141)a 26 3 10205 022 (33) Additive

rs9298506 58 White 121 (115ndash127) 135 (127ndash144)a 24 3 10204 052 (0) Additive

38 Japanese 103 (095ndash112) 045 654 3 10204 (86) Additive

rs10958409 59 White 12 (115ndash126) 131 (123ndash139)a 82 3 10202 0052 (62) Additive

49 Japanese 109 (102ndash117)a 0009 008 (55) Additive

rs700651 33 White 111 (106ndash118) 119 (109ndash129)a 482 3 10205 023 (31) Additive

33 Japanese 106 (094ndash131) 008 002 (75) Additive

rs6841581 46 White 122 (114ndash131) 125 (113ndash138)a 121 3 10205 018 (38) Additive

26 Japanese 12 (108ndash132)a 356 3 10204 087 (0) Additive

rs6538595 46 White 116 (110ndash123) 116 (108ndash124)a 213 3 10205 091 (0) Additive

26 Japanese 117 (106ndash128)a 149 3 10203 06 (0) Additive

Abbreviations ACE 5 angiotensin-converting enzyme CI 5 confidence interval FE 5 fixed-effects model I2 5 Higgins statistic demonstrating degree ofinterstudy heterogeneity IA5 intracranial aneurysm IL5 interleukin NA5 not applicable OR 5 odds ratio pHet 5 interstudy heterogeneity p value SNP 5

single nucleotide polymorphisma Significant association with IA

Neurology 80 June 4 2013 2161

p 5 195 3 1028) Another GWAS13 in a Japanesepopulation showed that the rs6842241 SNP located at4q3122 near the EDNRA gene was significantly asso-ciated with IA (OR 125 95 CI 116ndash134 p 5

9583 10209) EDNRA is a G-proteinndashcoupled recep-tor for endothelins which modulate vasoconstrictionand vasodilatation after hemodynamic insult Endo-thelin-1mdashthe predominant isoform in vascularsmooth-muscle cellsmdashactivates EDNRA Endothelinsignaling is activated at the site of vascular injury1225

causing cell proliferation indeed after SAH EDNRAand endothelin-1 protein levels are both increased inCSF26ndash28 Alternatively downregulation of EDNRAsignaling could lead to defective vascular repair allowinganeurysms to develop after injury a hypothesis sup-ported by functional analysis of EDNRA variants show-ing lower transcriptional activity for the rs6841581risk allele (G)13

SNPs with unknown function or not examined in more

than 2 studies Five SNPs from GWAS do not corre-spond to genes with any known functions relevant toIA development Two of these SNPs (rs1429412 andrs700651) at locus 2q33 flank the BOLL gene(involved in germ cell development) and the phospho-lipase Cndashlike 1 gene (involved in intracellular cascadereactions with abolished phospholipase C activity)Phospholipase Cndashlike 1 lies close to the vascular endo-thelial growth factor receptor-2 which is involved inCNS angiogenesis and is a marker for endothelial pro-genitor cells Three further SNPs on chromosome 2were associated with familial IAs rs11693075 (close tothe histone deacetylase complex SAP130 gene)rs1897472 and rs3769801 (both located within thephosphodiesterase-1 gene)14

One associated SNP located on chromosome 7(rs4628172 GT) is within the TMEM195 genewhich encodes transmembrane proteins possiblyinvolved in fatty acid biosynthesis and iron bind-ing1113 Further associated SNPs from the latestGWAS include variants on chromosome 12q22(rs6538595 rs11112585 and rs2374513) within theC12orf75 gene14 The rs1132274 GT SNP on chro-mosome 20p121 was also associated with IA10 How-ever none of these SNPs yet has known clearfunctional relevance to IA

Three further loci not included in this meta-analysis(because they were reported in only one study) showedsignificant association with IAs10 These loci included18q112 (rs11661542) 10q2432 (rs12413409) and13q132 (rs9315204) but their mechanisms of associ-ation remain unclear

Genetic variants associated with IAs identified by CGAS

Eight SNPs from CGAS were associated with IAs inour meta-analysis albeit with significant statisticalheterogeneity These SNPs relate to genes with

plausible relevance to IA biology including those impor-tant for extracellular matrix integrity inflammatorymedi-ators and vascular endothelium maintenanceExtracellular matrix gene variants may predispose to IAdevelopment by weakening arterial walls In our meta-analysis 6 such SNPs showed significant associationswith IA COL1A2 (rs42524) COL3A1 (rs1800255)SERPINA3 versican (rs251124 and rs173686) and hep-aran sulfate proteoglycan 2 (rs3767137) The COL1A2rs42524 SNP is located on chromosome 7q221 close tothe elastin genemdasha protein component of arterial wallspreviously associated with IAs in a linkage-associationstudy29 Collagen type 1 is usually found in the adventi-tial layer of cerebral arteries and provides tensile strengthmaking it a plausible factor in IA development Never-theless caution is needed in generalizing this result as all3 studies identified were in Far East populations forrobust association to be confirmed this SNP has to beexamined in other populations including Europebecause risk allele frequencies may differ Collagen type3 (COL 3A1) was investigated in 3 Chinese populationsrs1800255 (GA) was the only SNP significantly asso-ciated with IA but replication in other ethnicities andlarger cohorts is required to confirm these findings

We also found associations between 2 versicangenetic variants (rs251124 and rs173686) and IAsin Dutch Japanese and Chinese populations30ndash32

In a previous linkage study on a Japanese cohortchromosome 5q143 very close to the versican genewas implicated in IA30 In vivo studies on abdominalaortic aneurysm samples found reduced versican mes-senger RNA expression3334 which could reflect adecline in smooth-muscle cells associated with weak-ness in the vascular wall Moreover a GWAS35 onthoracic and aortic dissections showed significantassociation with the 5q13ndash14 locus correspondingto the versican gene

The proinflammatory cytokine IL6 G572C SNPhad an overall protective association with IAs in arecessive model However there was significant sta-tistical heterogeneity and sensitivity analysis revealedno association once one Chinese study36 wasexcluded which may partly reflect the greater riskallele frequency (079 vs 005ndash009) and inclusionof only unruptured IAs in this study36 Anothersource of heterogeneity may be sex because one otherstudy37 included only male controls and this may notbe appropriate because SAH has a female predomi-nance Therefore the overall association of this SNPwith IA is not robust Sustained abnormal vascularremodeling in association with inflammation ishypothesized to be a key mechanism in IA develop-ment22 IL-6 can injure vessel walls by inhibiting col-lagen synthesis thus increasing wall fragility andincreasing the risk of aneurysmal dilatation IL-6 lev-els are increased in the CSF and plasma after

2162 Neurology 80 June 4 2013

SAH3839 but whether this is directly related to theG572C SNP remains uncertain because in these stud-ies patients were not genotyped for the mutant allelemoreover IL-6 may be released as part of a systemicinflammatory response in the acute phase after SAH

Strengths of our study Our systematic review andmeta-analysis is the largest and most comprehensiveto date on genetic variants associated with IAs We as-sessed strength of association by using multiplegenetic models and carefully assessed the robustnessof all associations by performing random-effects sen-sitivity and ethnic subgroup analyses We conductedour analysis and reporting according to quality guide-lines for genetic association studies including HumanGenome Epidemiology Network15 and Strengtheningthe Reporting of Genetic Association Studies17

Limitations of our study Our assessment of interstudyheterogeneity by visual inspection of the forest plotsand sensitivity analyses only partly addresses potentialsources of heterogeneity between studies Furtherindividual patient data are required on other factorsthat could also contribute to heterogeneity such assex proportions age genotyping methods or errorsdisease phenotype (aneurysm size location and rup-ture status) and environmental risk factor status Ourmeta-analysis was only able to pool reported posi-tively associated SNPs from the 6 GWAS studiesbut data on many SNPs sequenced not reaching thethreshold for GWAS statistical significance were notavailable thus limiting our meta-analysis to 41 SNPsin total Indeed for many of the candidate-gene stud-ies pooling all available data from GWAS mightallow more robust risk estimates to be calculateddespite 8 candidate gene SNPs being associated withIAs on average only 27 publications were availablefor each variant

We have identified 19 candidate SNPs associatedwith IAs of which 16 were robust to random-effectsor sensitivity analyses The ORs suggest that thegenetic contribution to aneurysm development issubstantial but the variants identified implicate mul-tiple pathophysiologic mechanisms particularlyinvolving vascular endothelium maintenance Never-theless the amount of data on genetic associationswith IAs remains much smaller than for other com-plex diseases so further large replication studies in afull spectrum of populations are required ideallyusing GWAS or exome sequencing to identify rarerfunctional variants A limitation of most studiesincluded is that both ruptured and unruptured IAswere analyzed together because only a minority ofIAs are destined to rupture future studies shouldaddress genetic risk factors specifically related to rup-ture status Another important area for future researchis to explore how genetic factors are related to

aneurysm location size or morphology becausethere may be specific genetic associations for particu-lar aneurysm phenotypes (which may also relate torupture risk) Finally validation studies of risk modelsin prospective cohorts (eg of unruptured IAs fol-lowed up for rupture risk) would be helpful in deter-mining the relevance of genetic risk loci in addition towell-established risk factors for overall disease pheno-type to ultimately reduce the devastating burden ofaneurysmal SAH

AUTHOR CONTRIBUTIONSDr Alg designed the study performed the literature search acquired and

carried out the statistical analysis of the data and drafted the manuscript

Dr Sofat analyzed and interpreted the data and revised the manuscript

Dr Houlden supervised the study analyzed and interpreted the data and

provided revisions to the manuscript Dr Werring conceived and super-

vised the study checked the literature search interpreted the results and

provided critical revisions to the manuscript

STUDY FUNDINGSupported and funded by the Stroke Association (TSA 200708) Dr Alg

receives research support from the Stroke Association Dr Werring re-

ceives research support from the Department of HealthHigher Educa-

tion Funding Council for England (Clinical Senior Lectureship Award)

Part of this work was undertaken at UCLHUCL which received a pro-

portion of funding from the Department of Healthrsquos NIHR Biomedical

Research Centres funding scheme

DISCLOSUREThe authors report no disclosures relevant to the manuscript Go to

Neurologyorg for full disclosures

Received November 23 2012 Accepted in final form February 212013

REFERENCES1 Rinkel GJ Djibuti M Algra A van Gijn J Prevalence and

risk of rupture of intracranial aneurysms a systematic

review Stroke 199829251ndash256

2 Rinkel GJ Algra A Long-term outcomes of patients with

aneurysmal subarachnoid haemorrhage Lancet Neurol

201110349ndash356

3 Vlak MH Rinkel GJ Greebe P van der Bom JG Algra A

Trigger factors for rupture of intracranial aneurysms in

relation to patient and aneurysm characteristics J Neurol

20122591298ndash1302

4 Clarke M Systematic review of reviews of risk factors for

intracranial aneurysms Neuroradiology 200850653ndash664

5 Feigin VL Rinkel GJ Lawes CM et al Risk factors for

subarachnoid hemorrhage an updated systematic review of

epidemiological studies Stroke 2005362773ndash2780

6 Juvela S Risk factors for aneurysmal subarachnoid hemor-

rhage Stroke 2002332152ndash2153 author reply 2152ndash2153

7 Krischek B Inoue I The genetics of intracranial aneur-

ysms J Hum Genet 200651587ndash594

8 Bromberg JE Rinkel GJ Algra A et al Subarachnoid hae-

morrhage in first and second degree relatives of patients with

subarachnoid haemorrhage BMJ 1995311288ndash289

9 Bilguvar K Yasuno K Niemela M et al Susceptibility loci

for intracranial aneurysm in European and Japanese pop-

ulations Nat Genet 2008401472ndash1477

Neurology 80 June 4 2013 2163

most recent GWAS14 smoking was an independentrisk factor acting in a multiplicative manner (chromo-some 8 rs1072737 OR 212 95 CI 176ndash256 andchromosome 9 rs6475606 OR 211 95 CI 177ndash252)

A further recent GWAS12 found the SNPrs6841581AG on chromosome 4q3123 coding forthe endothelin receptor type A (EDNRA) gene wassignificantly associated with IA in Dutch Finnish andJapanese populations (OR 122 95 CI 114ndash131

Table 2 Random-effects and sensitivity analyses of associated SNPs that showed significant statisticalheterogeneity under fixed-effects modeling

SNPNostudies

Studiesexcluded

FE OR (95 CI)before adjustment

Random-effectsOR (95 CI)

FE OR (95 CI) aftersensitivity analysis Genetic model

SERPINA3 3 Liu42 222 (168ndash294)a 212 (036ndash1231) 107 (074ndash153) Dominant

127 (107ndash150)a 125 (061ndash259) 097 (079ndash119) Additive

ACE ID 5 Slowik50 139 (105ndash184)a 144 (086ndash242) 120 (088ndash162) Recessive

IL6 G572C 5 Sun36 047 (034ndash065)a 149 (024ndash927) 103 (065ndash162) Recessive

061 (049ndash075)a 095 (031ndash285) 104 (079ndash139) Additive

rs1333040 11 Low13 123 (119ndash128)a 126 (115ndash139)a 131 (126ndash137)a Additive

rs700651 6 Low13 111 (106ndash118)a 114 (103ndash125)a 119 (111ndash128)a Additive

rs9298506 8 Low13 121 (115ndash127)a 124 (108ndash141)a 132 (124ndash139)a Additive

rs10958409 9 410112024b 120 (115ndash126)a 122 (11ndash134)a 123 (117ndash13)a Additive

Abbreviations ACE 5 angiotensin-converting enzyme CI 5 confidence interval FE 5 fixed-effects model IL 5 interleukinOR 5 odds ratio SNP 5 single nucleotide polymorphismaSignificant association with IAb Four studies including one white population and 3 Japanese populations were excluded

Table 3 Subgroup ethnicity analysis

SNPNostudies Ethnicity

FE OR all ethnicities(all studies)

FE OR (95 CI)ethnicity subanalysis pcorrected pHet (I2) Genetic model

ACE ID 45 White 139 (105ndash184) 142 (106ndash192)a 0002 0006 (76) Recessive

Japanese 120 (056ndash257) 054 NA Recessive

IL6 G572C 35 Chinese 030 (019ndash047) 041 (029ndash058)a 000001 00001 (95) Recessive

25 White 426 (092ndash197) 01 003 (78) Recessive

35 Chinese 058 (044ndash075) 050 (039ndash064)a 000001 000001 (97) Additive

25 White 138 (083ndash229) 01 019 (42) Additive

rs1333040 611 Japanese 124 (12ndash129) 115 (109ndash122)a 175 3 10206 306 3 10208 (88) Additive

511 White 130 (124ndash136)a 191 3 10206 078 (0) Additive

rs10757278 57 White 129 (121ndash138) 130 (12ndash143)a 12 3 10209 091 (0) Additive

27 Japanese 127 (113ndash141)a 26 3 10205 022 (33) Additive

rs9298506 58 White 121 (115ndash127) 135 (127ndash144)a 24 3 10204 052 (0) Additive

38 Japanese 103 (095ndash112) 045 654 3 10204 (86) Additive

rs10958409 59 White 12 (115ndash126) 131 (123ndash139)a 82 3 10202 0052 (62) Additive

49 Japanese 109 (102ndash117)a 0009 008 (55) Additive

rs700651 33 White 111 (106ndash118) 119 (109ndash129)a 482 3 10205 023 (31) Additive

33 Japanese 106 (094ndash131) 008 002 (75) Additive

rs6841581 46 White 122 (114ndash131) 125 (113ndash138)a 121 3 10205 018 (38) Additive

26 Japanese 12 (108ndash132)a 356 3 10204 087 (0) Additive

rs6538595 46 White 116 (110ndash123) 116 (108ndash124)a 213 3 10205 091 (0) Additive

26 Japanese 117 (106ndash128)a 149 3 10203 06 (0) Additive

Abbreviations ACE 5 angiotensin-converting enzyme CI 5 confidence interval FE 5 fixed-effects model I2 5 Higgins statistic demonstrating degree ofinterstudy heterogeneity IA5 intracranial aneurysm IL5 interleukin NA5 not applicable OR 5 odds ratio pHet 5 interstudy heterogeneity p value SNP 5

single nucleotide polymorphisma Significant association with IA

Neurology 80 June 4 2013 2161

p 5 195 3 1028) Another GWAS13 in a Japanesepopulation showed that the rs6842241 SNP located at4q3122 near the EDNRA gene was significantly asso-ciated with IA (OR 125 95 CI 116ndash134 p 5

9583 10209) EDNRA is a G-proteinndashcoupled recep-tor for endothelins which modulate vasoconstrictionand vasodilatation after hemodynamic insult Endo-thelin-1mdashthe predominant isoform in vascularsmooth-muscle cellsmdashactivates EDNRA Endothelinsignaling is activated at the site of vascular injury1225

causing cell proliferation indeed after SAH EDNRAand endothelin-1 protein levels are both increased inCSF26ndash28 Alternatively downregulation of EDNRAsignaling could lead to defective vascular repair allowinganeurysms to develop after injury a hypothesis sup-ported by functional analysis of EDNRA variants show-ing lower transcriptional activity for the rs6841581risk allele (G)13

SNPs with unknown function or not examined in more

than 2 studies Five SNPs from GWAS do not corre-spond to genes with any known functions relevant toIA development Two of these SNPs (rs1429412 andrs700651) at locus 2q33 flank the BOLL gene(involved in germ cell development) and the phospho-lipase Cndashlike 1 gene (involved in intracellular cascadereactions with abolished phospholipase C activity)Phospholipase Cndashlike 1 lies close to the vascular endo-thelial growth factor receptor-2 which is involved inCNS angiogenesis and is a marker for endothelial pro-genitor cells Three further SNPs on chromosome 2were associated with familial IAs rs11693075 (close tothe histone deacetylase complex SAP130 gene)rs1897472 and rs3769801 (both located within thephosphodiesterase-1 gene)14

One associated SNP located on chromosome 7(rs4628172 GT) is within the TMEM195 genewhich encodes transmembrane proteins possiblyinvolved in fatty acid biosynthesis and iron bind-ing1113 Further associated SNPs from the latestGWAS include variants on chromosome 12q22(rs6538595 rs11112585 and rs2374513) within theC12orf75 gene14 The rs1132274 GT SNP on chro-mosome 20p121 was also associated with IA10 How-ever none of these SNPs yet has known clearfunctional relevance to IA

Three further loci not included in this meta-analysis(because they were reported in only one study) showedsignificant association with IAs10 These loci included18q112 (rs11661542) 10q2432 (rs12413409) and13q132 (rs9315204) but their mechanisms of associ-ation remain unclear

Genetic variants associated with IAs identified by CGAS

Eight SNPs from CGAS were associated with IAs inour meta-analysis albeit with significant statisticalheterogeneity These SNPs relate to genes with

plausible relevance to IA biology including those impor-tant for extracellular matrix integrity inflammatorymedi-ators and vascular endothelium maintenanceExtracellular matrix gene variants may predispose to IAdevelopment by weakening arterial walls In our meta-analysis 6 such SNPs showed significant associationswith IA COL1A2 (rs42524) COL3A1 (rs1800255)SERPINA3 versican (rs251124 and rs173686) and hep-aran sulfate proteoglycan 2 (rs3767137) The COL1A2rs42524 SNP is located on chromosome 7q221 close tothe elastin genemdasha protein component of arterial wallspreviously associated with IAs in a linkage-associationstudy29 Collagen type 1 is usually found in the adventi-tial layer of cerebral arteries and provides tensile strengthmaking it a plausible factor in IA development Never-theless caution is needed in generalizing this result as all3 studies identified were in Far East populations forrobust association to be confirmed this SNP has to beexamined in other populations including Europebecause risk allele frequencies may differ Collagen type3 (COL 3A1) was investigated in 3 Chinese populationsrs1800255 (GA) was the only SNP significantly asso-ciated with IA but replication in other ethnicities andlarger cohorts is required to confirm these findings

We also found associations between 2 versicangenetic variants (rs251124 and rs173686) and IAsin Dutch Japanese and Chinese populations30ndash32

In a previous linkage study on a Japanese cohortchromosome 5q143 very close to the versican genewas implicated in IA30 In vivo studies on abdominalaortic aneurysm samples found reduced versican mes-senger RNA expression3334 which could reflect adecline in smooth-muscle cells associated with weak-ness in the vascular wall Moreover a GWAS35 onthoracic and aortic dissections showed significantassociation with the 5q13ndash14 locus correspondingto the versican gene

The proinflammatory cytokine IL6 G572C SNPhad an overall protective association with IAs in arecessive model However there was significant sta-tistical heterogeneity and sensitivity analysis revealedno association once one Chinese study36 wasexcluded which may partly reflect the greater riskallele frequency (079 vs 005ndash009) and inclusionof only unruptured IAs in this study36 Anothersource of heterogeneity may be sex because one otherstudy37 included only male controls and this may notbe appropriate because SAH has a female predomi-nance Therefore the overall association of this SNPwith IA is not robust Sustained abnormal vascularremodeling in association with inflammation ishypothesized to be a key mechanism in IA develop-ment22 IL-6 can injure vessel walls by inhibiting col-lagen synthesis thus increasing wall fragility andincreasing the risk of aneurysmal dilatation IL-6 lev-els are increased in the CSF and plasma after

2162 Neurology 80 June 4 2013

SAH3839 but whether this is directly related to theG572C SNP remains uncertain because in these stud-ies patients were not genotyped for the mutant allelemoreover IL-6 may be released as part of a systemicinflammatory response in the acute phase after SAH

Strengths of our study Our systematic review andmeta-analysis is the largest and most comprehensiveto date on genetic variants associated with IAs We as-sessed strength of association by using multiplegenetic models and carefully assessed the robustnessof all associations by performing random-effects sen-sitivity and ethnic subgroup analyses We conductedour analysis and reporting according to quality guide-lines for genetic association studies including HumanGenome Epidemiology Network15 and Strengtheningthe Reporting of Genetic Association Studies17

Limitations of our study Our assessment of interstudyheterogeneity by visual inspection of the forest plotsand sensitivity analyses only partly addresses potentialsources of heterogeneity between studies Furtherindividual patient data are required on other factorsthat could also contribute to heterogeneity such assex proportions age genotyping methods or errorsdisease phenotype (aneurysm size location and rup-ture status) and environmental risk factor status Ourmeta-analysis was only able to pool reported posi-tively associated SNPs from the 6 GWAS studiesbut data on many SNPs sequenced not reaching thethreshold for GWAS statistical significance were notavailable thus limiting our meta-analysis to 41 SNPsin total Indeed for many of the candidate-gene stud-ies pooling all available data from GWAS mightallow more robust risk estimates to be calculateddespite 8 candidate gene SNPs being associated withIAs on average only 27 publications were availablefor each variant

We have identified 19 candidate SNPs associatedwith IAs of which 16 were robust to random-effectsor sensitivity analyses The ORs suggest that thegenetic contribution to aneurysm development issubstantial but the variants identified implicate mul-tiple pathophysiologic mechanisms particularlyinvolving vascular endothelium maintenance Never-theless the amount of data on genetic associationswith IAs remains much smaller than for other com-plex diseases so further large replication studies in afull spectrum of populations are required ideallyusing GWAS or exome sequencing to identify rarerfunctional variants A limitation of most studiesincluded is that both ruptured and unruptured IAswere analyzed together because only a minority ofIAs are destined to rupture future studies shouldaddress genetic risk factors specifically related to rup-ture status Another important area for future researchis to explore how genetic factors are related to

aneurysm location size or morphology becausethere may be specific genetic associations for particu-lar aneurysm phenotypes (which may also relate torupture risk) Finally validation studies of risk modelsin prospective cohorts (eg of unruptured IAs fol-lowed up for rupture risk) would be helpful in deter-mining the relevance of genetic risk loci in addition towell-established risk factors for overall disease pheno-type to ultimately reduce the devastating burden ofaneurysmal SAH

AUTHOR CONTRIBUTIONSDr Alg designed the study performed the literature search acquired and

carried out the statistical analysis of the data and drafted the manuscript

Dr Sofat analyzed and interpreted the data and revised the manuscript

Dr Houlden supervised the study analyzed and interpreted the data and

provided revisions to the manuscript Dr Werring conceived and super-

vised the study checked the literature search interpreted the results and

provided critical revisions to the manuscript

STUDY FUNDINGSupported and funded by the Stroke Association (TSA 200708) Dr Alg

receives research support from the Stroke Association Dr Werring re-

ceives research support from the Department of HealthHigher Educa-

tion Funding Council for England (Clinical Senior Lectureship Award)

Part of this work was undertaken at UCLHUCL which received a pro-

portion of funding from the Department of Healthrsquos NIHR Biomedical

Research Centres funding scheme

DISCLOSUREThe authors report no disclosures relevant to the manuscript Go to

Neurologyorg for full disclosures

Received November 23 2012 Accepted in final form February 212013

REFERENCES1 Rinkel GJ Djibuti M Algra A van Gijn J Prevalence and

risk of rupture of intracranial aneurysms a systematic

review Stroke 199829251ndash256

2 Rinkel GJ Algra A Long-term outcomes of patients with

aneurysmal subarachnoid haemorrhage Lancet Neurol

201110349ndash356

3 Vlak MH Rinkel GJ Greebe P van der Bom JG Algra A

Trigger factors for rupture of intracranial aneurysms in

relation to patient and aneurysm characteristics J Neurol

20122591298ndash1302

4 Clarke M Systematic review of reviews of risk factors for

intracranial aneurysms Neuroradiology 200850653ndash664

5 Feigin VL Rinkel GJ Lawes CM et al Risk factors for

subarachnoid hemorrhage an updated systematic review of

epidemiological studies Stroke 2005362773ndash2780

6 Juvela S Risk factors for aneurysmal subarachnoid hemor-

rhage Stroke 2002332152ndash2153 author reply 2152ndash2153

7 Krischek B Inoue I The genetics of intracranial aneur-

ysms J Hum Genet 200651587ndash594

8 Bromberg JE Rinkel GJ Algra A et al Subarachnoid hae-

morrhage in first and second degree relatives of patients with

subarachnoid haemorrhage BMJ 1995311288ndash289

9 Bilguvar K Yasuno K Niemela M et al Susceptibility loci

for intracranial aneurysm in European and Japanese pop-

ulations Nat Genet 2008401472ndash1477

Neurology 80 June 4 2013 2163

p 5 195 3 1028) Another GWAS13 in a Japanesepopulation showed that the rs6842241 SNP located at4q3122 near the EDNRA gene was significantly asso-ciated with IA (OR 125 95 CI 116ndash134 p 5

9583 10209) EDNRA is a G-proteinndashcoupled recep-tor for endothelins which modulate vasoconstrictionand vasodilatation after hemodynamic insult Endo-thelin-1mdashthe predominant isoform in vascularsmooth-muscle cellsmdashactivates EDNRA Endothelinsignaling is activated at the site of vascular injury1225

causing cell proliferation indeed after SAH EDNRAand endothelin-1 protein levels are both increased inCSF26ndash28 Alternatively downregulation of EDNRAsignaling could lead to defective vascular repair allowinganeurysms to develop after injury a hypothesis sup-ported by functional analysis of EDNRA variants show-ing lower transcriptional activity for the rs6841581risk allele (G)13

SNPs with unknown function or not examined in more

than 2 studies Five SNPs from GWAS do not corre-spond to genes with any known functions relevant toIA development Two of these SNPs (rs1429412 andrs700651) at locus 2q33 flank the BOLL gene(involved in germ cell development) and the phospho-lipase Cndashlike 1 gene (involved in intracellular cascadereactions with abolished phospholipase C activity)Phospholipase Cndashlike 1 lies close to the vascular endo-thelial growth factor receptor-2 which is involved inCNS angiogenesis and is a marker for endothelial pro-genitor cells Three further SNPs on chromosome 2were associated with familial IAs rs11693075 (close tothe histone deacetylase complex SAP130 gene)rs1897472 and rs3769801 (both located within thephosphodiesterase-1 gene)14

One associated SNP located on chromosome 7(rs4628172 GT) is within the TMEM195 genewhich encodes transmembrane proteins possiblyinvolved in fatty acid biosynthesis and iron bind-ing1113 Further associated SNPs from the latestGWAS include variants on chromosome 12q22(rs6538595 rs11112585 and rs2374513) within theC12orf75 gene14 The rs1132274 GT SNP on chro-mosome 20p121 was also associated with IA10 How-ever none of these SNPs yet has known clearfunctional relevance to IA

Three further loci not included in this meta-analysis(because they were reported in only one study) showedsignificant association with IAs10 These loci included18q112 (rs11661542) 10q2432 (rs12413409) and13q132 (rs9315204) but their mechanisms of associ-ation remain unclear

Genetic variants associated with IAs identified by CGAS

Eight SNPs from CGAS were associated with IAs inour meta-analysis albeit with significant statisticalheterogeneity These SNPs relate to genes with

plausible relevance to IA biology including those impor-tant for extracellular matrix integrity inflammatorymedi-ators and vascular endothelium maintenanceExtracellular matrix gene variants may predispose to IAdevelopment by weakening arterial walls In our meta-analysis 6 such SNPs showed significant associationswith IA COL1A2 (rs42524) COL3A1 (rs1800255)SERPINA3 versican (rs251124 and rs173686) and hep-aran sulfate proteoglycan 2 (rs3767137) The COL1A2rs42524 SNP is located on chromosome 7q221 close tothe elastin genemdasha protein component of arterial wallspreviously associated with IAs in a linkage-associationstudy29 Collagen type 1 is usually found in the adventi-tial layer of cerebral arteries and provides tensile strengthmaking it a plausible factor in IA development Never-theless caution is needed in generalizing this result as all3 studies identified were in Far East populations forrobust association to be confirmed this SNP has to beexamined in other populations including Europebecause risk allele frequencies may differ Collagen type3 (COL 3A1) was investigated in 3 Chinese populationsrs1800255 (GA) was the only SNP significantly asso-ciated with IA but replication in other ethnicities andlarger cohorts is required to confirm these findings

We also found associations between 2 versicangenetic variants (rs251124 and rs173686) and IAsin Dutch Japanese and Chinese populations30ndash32

In a previous linkage study on a Japanese cohortchromosome 5q143 very close to the versican genewas implicated in IA30 In vivo studies on abdominalaortic aneurysm samples found reduced versican mes-senger RNA expression3334 which could reflect adecline in smooth-muscle cells associated with weak-ness in the vascular wall Moreover a GWAS35 onthoracic and aortic dissections showed significantassociation with the 5q13ndash14 locus correspondingto the versican gene

The proinflammatory cytokine IL6 G572C SNPhad an overall protective association with IAs in arecessive model However there was significant sta-tistical heterogeneity and sensitivity analysis revealedno association once one Chinese study36 wasexcluded which may partly reflect the greater riskallele frequency (079 vs 005ndash009) and inclusionof only unruptured IAs in this study36 Anothersource of heterogeneity may be sex because one otherstudy37 included only male controls and this may notbe appropriate because SAH has a female predomi-nance Therefore the overall association of this SNPwith IA is not robust Sustained abnormal vascularremodeling in association with inflammation ishypothesized to be a key mechanism in IA develop-ment22 IL-6 can injure vessel walls by inhibiting col-lagen synthesis thus increasing wall fragility andincreasing the risk of aneurysmal dilatation IL-6 lev-els are increased in the CSF and plasma after

2162 Neurology 80 June 4 2013

SAH3839 but whether this is directly related to theG572C SNP remains uncertain because in these stud-ies patients were not genotyped for the mutant allelemoreover IL-6 may be released as part of a systemicinflammatory response in the acute phase after SAH

Strengths of our study Our systematic review andmeta-analysis is the largest and most comprehensiveto date on genetic variants associated with IAs We as-sessed strength of association by using multiplegenetic models and carefully assessed the robustnessof all associations by performing random-effects sen-sitivity and ethnic subgroup analyses We conductedour analysis and reporting according to quality guide-lines for genetic association studies including HumanGenome Epidemiology Network15 and Strengtheningthe Reporting of Genetic Association Studies17

Limitations of our study Our assessment of interstudyheterogeneity by visual inspection of the forest plotsand sensitivity analyses only partly addresses potentialsources of heterogeneity between studies Furtherindividual patient data are required on other factorsthat could also contribute to heterogeneity such assex proportions age genotyping methods or errorsdisease phenotype (aneurysm size location and rup-ture status) and environmental risk factor status Ourmeta-analysis was only able to pool reported posi-tively associated SNPs from the 6 GWAS studiesbut data on many SNPs sequenced not reaching thethreshold for GWAS statistical significance were notavailable thus limiting our meta-analysis to 41 SNPsin total Indeed for many of the candidate-gene stud-ies pooling all available data from GWAS mightallow more robust risk estimates to be calculateddespite 8 candidate gene SNPs being associated withIAs on average only 27 publications were availablefor each variant

We have identified 19 candidate SNPs associatedwith IAs of which 16 were robust to random-effectsor sensitivity analyses The ORs suggest that thegenetic contribution to aneurysm development issubstantial but the variants identified implicate mul-tiple pathophysiologic mechanisms particularlyinvolving vascular endothelium maintenance Never-theless the amount of data on genetic associationswith IAs remains much smaller than for other com-plex diseases so further large replication studies in afull spectrum of populations are required ideallyusing GWAS or exome sequencing to identify rarerfunctional variants A limitation of most studiesincluded is that both ruptured and unruptured IAswere analyzed together because only a minority ofIAs are destined to rupture future studies shouldaddress genetic risk factors specifically related to rup-ture status Another important area for future researchis to explore how genetic factors are related to

aneurysm location size or morphology becausethere may be specific genetic associations for particu-lar aneurysm phenotypes (which may also relate torupture risk) Finally validation studies of risk modelsin prospective cohorts (eg of unruptured IAs fol-lowed up for rupture risk) would be helpful in deter-mining the relevance of genetic risk loci in addition towell-established risk factors for overall disease pheno-type to ultimately reduce the devastating burden ofaneurysmal SAH

AUTHOR CONTRIBUTIONSDr Alg designed the study performed the literature search acquired and

carried out the statistical analysis of the data and drafted the manuscript

Dr Sofat analyzed and interpreted the data and revised the manuscript

Dr Houlden supervised the study analyzed and interpreted the data and

provided revisions to the manuscript Dr Werring conceived and super-

vised the study checked the literature search interpreted the results and

provided critical revisions to the manuscript

STUDY FUNDINGSupported and funded by the Stroke Association (TSA 200708) Dr Alg

receives research support from the Stroke Association Dr Werring re-

ceives research support from the Department of HealthHigher Educa-

tion Funding Council for England (Clinical Senior Lectureship Award)

Part of this work was undertaken at UCLHUCL which received a pro-

portion of funding from the Department of Healthrsquos NIHR Biomedical

Research Centres funding scheme

DISCLOSUREThe authors report no disclosures relevant to the manuscript Go to

Neurologyorg for full disclosures

Received November 23 2012 Accepted in final form February 212013

REFERENCES1 Rinkel GJ Djibuti M Algra A van Gijn J Prevalence and

risk of rupture of intracranial aneurysms a systematic

review Stroke 199829251ndash256

2 Rinkel GJ Algra A Long-term outcomes of patients with

aneurysmal subarachnoid haemorrhage Lancet Neurol

201110349ndash356

3 Vlak MH Rinkel GJ Greebe P van der Bom JG Algra A

Trigger factors for rupture of intracranial aneurysms in

relation to patient and aneurysm characteristics J Neurol

20122591298ndash1302

4 Clarke M Systematic review of reviews of risk factors for

intracranial aneurysms Neuroradiology 200850653ndash664

5 Feigin VL Rinkel GJ Lawes CM et al Risk factors for

subarachnoid hemorrhage an updated systematic review of

epidemiological studies Stroke 2005362773ndash2780

6 Juvela S Risk factors for aneurysmal subarachnoid hemor-

rhage Stroke 2002332152ndash2153 author reply 2152ndash2153

7 Krischek B Inoue I The genetics of intracranial aneur-

ysms J Hum Genet 200651587ndash594

8 Bromberg JE Rinkel GJ Algra A et al Subarachnoid hae-

morrhage in first and second degree relatives of patients with

subarachnoid haemorrhage BMJ 1995311288ndash289

9 Bilguvar K Yasuno K Niemela M et al Susceptibility loci

for intracranial aneurysm in European and Japanese pop-

ulations Nat Genet 2008401472ndash1477

Neurology 80 June 4 2013 2163

SAH3839 but whether this is directly related to theG572C SNP remains uncertain because in these stud-ies patients were not genotyped for the mutant allelemoreover IL-6 may be released as part of a systemicinflammatory response in the acute phase after SAH

Strengths of our study Our systematic review andmeta-analysis is the largest and most comprehensiveto date on genetic variants associated with IAs We as-sessed strength of association by using multiplegenetic models and carefully assessed the robustnessof all associations by performing random-effects sen-sitivity and ethnic subgroup analyses We conductedour analysis and reporting according to quality guide-lines for genetic association studies including HumanGenome Epidemiology Network15 and Strengtheningthe Reporting of Genetic Association Studies17

Limitations of our study Our assessment of interstudyheterogeneity by visual inspection of the forest plotsand sensitivity analyses only partly addresses potentialsources of heterogeneity between studies Furtherindividual patient data are required on other factorsthat could also contribute to heterogeneity such assex proportions age genotyping methods or errorsdisease phenotype (aneurysm size location and rup-ture status) and environmental risk factor status Ourmeta-analysis was only able to pool reported posi-tively associated SNPs from the 6 GWAS studiesbut data on many SNPs sequenced not reaching thethreshold for GWAS statistical significance were notavailable thus limiting our meta-analysis to 41 SNPsin total Indeed for many of the candidate-gene stud-ies pooling all available data from GWAS mightallow more robust risk estimates to be calculateddespite 8 candidate gene SNPs being associated withIAs on average only 27 publications were availablefor each variant

We have identified 19 candidate SNPs associatedwith IAs of which 16 were robust to random-effectsor sensitivity analyses The ORs suggest that thegenetic contribution to aneurysm development issubstantial but the variants identified implicate mul-tiple pathophysiologic mechanisms particularlyinvolving vascular endothelium maintenance Never-theless the amount of data on genetic associationswith IAs remains much smaller than for other com-plex diseases so further large replication studies in afull spectrum of populations are required ideallyusing GWAS or exome sequencing to identify rarerfunctional variants A limitation of most studiesincluded is that both ruptured and unruptured IAswere analyzed together because only a minority ofIAs are destined to rupture future studies shouldaddress genetic risk factors specifically related to rup-ture status Another important area for future researchis to explore how genetic factors are related to

aneurysm location size or morphology becausethere may be specific genetic associations for particu-lar aneurysm phenotypes (which may also relate torupture risk) Finally validation studies of risk modelsin prospective cohorts (eg of unruptured IAs fol-lowed up for rupture risk) would be helpful in deter-mining the relevance of genetic risk loci in addition towell-established risk factors for overall disease pheno-type to ultimately reduce the devastating burden ofaneurysmal SAH

AUTHOR CONTRIBUTIONSDr Alg designed the study performed the literature search acquired and

carried out the statistical analysis of the data and drafted the manuscript

Dr Sofat analyzed and interpreted the data and revised the manuscript

Dr Houlden supervised the study analyzed and interpreted the data and

provided revisions to the manuscript Dr Werring conceived and super-

vised the study checked the literature search interpreted the results and

provided critical revisions to the manuscript

STUDY FUNDINGSupported and funded by the Stroke Association (TSA 200708) Dr Alg

receives research support from the Stroke Association Dr Werring re-

ceives research support from the Department of HealthHigher Educa-

tion Funding Council for England (Clinical Senior Lectureship Award)

Part of this work was undertaken at UCLHUCL which received a pro-

portion of funding from the Department of Healthrsquos NIHR Biomedical

Research Centres funding scheme

DISCLOSUREThe authors report no disclosures relevant to the manuscript Go to

Neurologyorg for full disclosures

Received November 23 2012 Accepted in final form February 212013

REFERENCES1 Rinkel GJ Djibuti M Algra A van Gijn J Prevalence and

risk of rupture of intracranial aneurysms a systematic

review Stroke 199829251ndash256

2 Rinkel GJ Algra A Long-term outcomes of patients with

aneurysmal subarachnoid haemorrhage Lancet Neurol

201110349ndash356

3 Vlak MH Rinkel GJ Greebe P van der Bom JG Algra A

Trigger factors for rupture of intracranial aneurysms in

relation to patient and aneurysm characteristics J Neurol

20122591298ndash1302

4 Clarke M Systematic review of reviews of risk factors for

intracranial aneurysms Neuroradiology 200850653ndash664

5 Feigin VL Rinkel GJ Lawes CM et al Risk factors for

subarachnoid hemorrhage an updated systematic review of

epidemiological studies Stroke 2005362773ndash2780

6 Juvela S Risk factors for aneurysmal subarachnoid hemor-

rhage Stroke 2002332152ndash2153 author reply 2152ndash2153

7 Krischek B Inoue I The genetics of intracranial aneur-

ysms J Hum Genet 200651587ndash594

8 Bromberg JE Rinkel GJ Algra A et al Subarachnoid hae-

morrhage in first and second degree relatives of patients with

subarachnoid haemorrhage BMJ 1995311288ndash289

9 Bilguvar K Yasuno K Niemela M et al Susceptibility loci

for intracranial aneurysm in European and Japanese pop-

ulations Nat Genet 2008401472ndash1477

Neurology 80 June 4 2013 2163

10 Yasuno K Bilguvar K Bijlenga P et al Genome-wide

association study of intracranial aneurysm identifies three

new risk loci Nat Genet 201042420ndash425

11 Akiyama K Narita A Nakaoka H et al Genome-wide

association study to identify genetic variants present in Jap-

anese patients harboring intracranial aneurysms J Hum

Genet 201055656ndash661

12 Yasuno K Bakircioglu M Low SK et al Common variant

near the endothelin receptor type A (EDNRA) gene is

associated with intracranial aneurysm risk Proc Natl Acad

Sci USA 201110819707ndash19712

13 Low SK Takahashi A Cha PC et al Genome-wide asso-

ciation study for intracranial aneurysm in the Japanese

population identifies three candidate susceptible loci and

a functional genetic variant at EDNRA Hum Mol Genet

2012212102ndash2110

14 Foroud T Koller DL Lai D et al Genome-wide associ-

ation study of intracranial aneurysms confirms role of An-

ril and SOX17 in disease risk Stroke 2012432846ndash2852

15 Little JH The HuGENettrade HuGE Review Handbook

version 10 HuGENet 200659 Available at httpwww

hugenetca Accessed February 1 2012

16 Minelli C Thompson JR Abrams KR Thakkinstian A

Attia J The quality of meta-analyses of genetic association

studies a review with recommendations Am J Epidemiol

20091701333ndash1343

17 Little J Higgins JP Ioannidis JP et al Strengthening the

reporting of genetic association studies (STREGA) an

extension of the STROBE Statement Hum Genet

2009125131ndash151

18 Helgadottir A Thorleifsson G Magnusson KP et al The

same sequence variant on 9p21 associates with myocardial

infarction abdominal aortic aneurysm and intracranial

aneurysm Nat Genet 200840217ndash224

19 Nakaoka H Takahashi T Akiyama K et al Differential

effects of chromosome 9p21 variation on subphenotypes

of intracranial aneurysm site distribution Stroke 201041

1593ndash1598

20 Hashikata H Liu W Inoue K et al Confirmation of an

association of single-nucleotide polymorphism rs1333040

on 9p21 with familial and sporadic intracranial aneurysms

in Japanese patients Stroke 2010411138ndash1144

21 Olsson S Csajbok LZ Jood K Nylen K Nellgard B

Jern C Association between genetic variation on chromo-

some 9p21 and aneurysmal subarachnoid haemorrhage

J Neurol Neurosurg Psychiatry 201182384ndash388

22 Hashimoto T Meng H Young WL Intracranial aneur-

ysms links among inflammation hemodynamics and vas-

cular remodeling Neurol Res 200628372ndash380

23 Hashimoto T Pittet JF Angiopoietin-2 modulator of vascu-

lar permeability in acute lung injury PLoS Med 20063e113

24 Deka R Koller DL Lai D et al The relationship between

smoking and replicated sequence variants on chromosomes

8 and 9 with familial intracranial aneurysm Stroke 2010

411132ndash1137

25 Wang X Douglas SA Louden C Vickery-Clark LM

Feuerstein GZ Ohlstein EH Expression of endothelin-

1 endothelin-3 endothelin-converting enzyme-1 and

endothelin-A and endothelin-B receptor mRNA after angi-

oplasty-induced neointimal formation in the rat Circ Res

199678322ndash328

26 Suzuki H Sato S Suzuki Y Takekoshi K Ishihara N

Shimoda S Increased endothelin concentration in CSF

from patients with subarachnoid hemorrhage Acta Neurol

Scand 199081553ndash554

27 Kraus GE Bucholz RD Yoon KW Knuepfer MM

Smith KR Jr Cerebrospinal fluid endothelin-1 and endothe-

lin-3 levels in normal and neurosurgical patients a clinical

study and literature review Surg Neurol 19913520ndash29

28 Itoh S Sasaki T Asai A Kuchino Y Prevention of delayed

vasospasm by an endothelin ETA receptor antagonist BQ-

123 change of ETA receptor mRNA expression in a

canine subarachnoid hemorrhage model J Neurosurg

199481759ndash764

29 Onda H Kasuya H Yoneyama T et al Genomewide-

linkage and haplotype-association studies map intracranial

aneurysm to chromosome 7q11 Am J Hum Genet 2001

69804ndash819

30 Sun H Zhang D Zhao J Chondroitin sulfate proteogly-

can 2 (CSPG2) gene polymorphisms rs173686 and

rs251124 are not associated with intracranial aneurysms

in Chinese Han nationality Ups J Med Sci 2007112

289ndash295

31 Ruigrok YM Rinkel GJ Wijmenga C The versican gene

and the risk of intracranial aneurysms Stroke 200637

2372ndash2374

32 Ruigrok YM Rinkel GJ Wijmenga C et al Association

analysis of genes involved in the maintenance of the integ-

rity of the extracellular matrix with intracranial aneurysms

in a Japanese cohort Cerebrovasc Dis 200928131ndash134

33 Yang F Zhao P Zhang Y et al Relationship between

chondroitin sulfate proteoglycan and coronary atheroscle-

rosis in the youth Chin Med J (Engl) 1996109162ndash167

34 Handley CJ Samiric T Ilic MZ Structure metabolism

and tissue roles of chondroitin sulfate proteoglycans Adv

Pharmacol 200653219ndash232

35 Breimer LH Genetics of aneurysmal disease the dogs that

did not bark Angiology 201061233ndash237

36 Sun H Zhang D Zhao J The interleukin-6 gene

-572GC promoter polymorphism is related to intracra-

nial aneurysms in Chinese Han nationality Neurosci Lett

20084401ndash3

37 Morgan L Cooper J Montgomery H Kitchen N

Humphries SE The interleukin-6 gene-174GC and

-572GC promoter polymorphisms are related to cerebral

aneurysms J Neurol Neurosurg Psychiatry 200677915ndash917

38 Gaetani P Tartara F Pignatti P et al Cisternal CSF levels

of cytokines after subarachnoid hemorrhage Neurol Res

199820337ndash342

39 Osuka K Suzuki Y Tanazawa T et al Interleukin-6 and

development of vasospasm after subarachnoid haemor-

rhage Acta Neurochir (Wien) 1998140943ndash951

40 Slowik A Borratynska A Turaj W et al Alpha1-antichy-

motrypsin gene (SERPINA3) AT polymorphism as a risk

factor for aneurysmal subarachnoid hemorrhage Stroke

200536737ndash740

41 Krischek B Akagawa H Tajima A et al The alaninethreonine

polymorphism of the alpha-1-antichymotrypsin (SERPINA3)

gene and ruptured intracranial aneurysms in the Japanese

population Cerebrovasc Dis 20072346ndash49

42 Liu W Zhu Y Ge M Pang Q Yu Y Polymorphism rs4934

of SERPINA3 and sporadic intracranial aneurysms in the

Chinese population Cerebrovasc Dis 20102968ndash72

43 Yoneyama T Kasuya H Onda H et al Collagen type I

alpha2 (COL1A2) is the susceptible gene for intracranial

aneurysms Stroke 200435443ndash448

2164 Neurology 80 June 4 2013

44 Zhu Y Li W Ge M et al Polymorphism rs42524 of

COL1A2 and sporadic intracranial aneurysms in the Chi-

nese population J Neurosurg 20081091060ndash1064

45 Joo SP Kim TS Lee IK et al The role of collagen type I

alpha2 polymorphisms intracranial aneurysms in Koreans

Surg Neurol 20097248ndash53

46 Hua T Zhang D Zhao YL Wang S Zhao JZ Correla-

tion of COL3A1 gene with type III collagen stability in

intracranial aneurysm [in Chinese] Zhonghua Yi Xue Za

Zhi 200888445ndash448

47 Chen J Zhu Y Jiang Y et al A functional variant of the

collagen type III alpha1 gene modify risk of sporadic intra-

cranial aneurysms Hum Genet 20121311137ndash1143

48 Takenaka K Yamakawa H Sakai H et al Angiotensin

I-converting enzyme gene polymorphism in intracranial

saccular aneurysm individuals Neurol Res 199820

607ndash611

49 Keramatipour M McConnell RS Kirkpatrick P et al The

ACE I allele is associated with increased risk for ruptured

intracranial aneurysms J Med Genet 200037498ndash500

50 Slowik A Borratynska A Pera J et al II genotype of the

angiotensin-converting enzyme gene increases the risk for

subarachnoid hemorrhage from ruptured aneurysm Stroke

2004351594ndash1597

51 Pannu H Kim DH Seaman CR Van Ginhoven G

Shete S Milewicz DM Lack of an association between

the angiotensin-converting enzyme insertiondeletion poly-

morphism and intracranial aneurysms in a Caucasian pop-

ulation in the United States J Neurosurg 200510392ndash96

52 Staalso JM Nielsen M Edsen T et al Common variants

of the ACE gene and aneurysmal subarachnoid hemor-

rhage in a Danish population a case-control study

J Neurosurg Anesthesiol 201123304ndash309

53 Fontanella M Rainero I Gallone S et al Interleukin 6

gene polymorphisms are not associated with aneurysmal

subarachnoid haemorrhage in an Italian population

J Neurol Neurosurg Psychiatry 200879471ndash473

54 Zhang G Tu Y Feng W Huang L Li M Qi S Association

of interleukin-6-572GC gene polymorphisms in the Can-

tonese population with intracranial aneurysms J Neurol Sci

201130694ndash97

55 Liu Y Sun J Wu C Cao X He M You C The inter-

leukin-6-572GC gene polymorphism and the risk of

intracranial aneurysms in a Chinese population Genet

Test Mol Biomarkers 201216822ndash826

Neurology 80 June 4 2013 2165

DOI 101212WNL0b013e318295d7512013802154-2165 Neurology

Varinder S Alg Reecha Sofat Henry Houlden et al individuals

Genetic risk factors for intracranial aneurysms A meta-analysis in more than 116000

This information is current as of June 3 2013

ServicesUpdated Information amp

httpnneurologyorgcontent80232154fullincluding high resolution figures can be found at

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Subspecialty Collections

httpnneurologyorgcgicollectionsubarachnoid_hemorrhageSubarachnoid hemorrhage

httpnneurologyorgcgicollectionassociation_studies_in_geneticsAssociation studies in genetics e

httpnneurologyorgcgicollectionall_cerebrovascular_disease_strokAll Cerebrovascular diseaseStrokefollowing collection(s) This article along with others on similar topics appears in the

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rights reserved Print ISSN 0028-3878 Online ISSN 1526-632X1951 it is now a weekly with 48 issues per year Copyright copy 2013 American Academy of Neurology All

reg is the official journal of the American Academy of Neurology Published continuously sinceNeurology

DOI 101212WNL0b013e318295d7512013802154-2165 Neurology

Varinder S Alg Reecha Sofat Henry Houlden et al individuals

Genetic risk factors for intracranial aneurysms A meta-analysis in more than 116000

This information is current as of June 3 2013

ServicesUpdated Information amp

httpnneurologyorgcontent80232154fullincluding high resolution figures can be found at

Supplementary Material httpnneurologyorgcontentsuppl2013060180232154DC1

Supplementary material can be found at

References httpnneurologyorgcontent80232154fullref-list-1

This article cites 54 articles 18 of which you can access for free at

Citations httpnneurologyorgcontent80232154fullotherarticles

This article has been cited by 1 HighWire-hosted articles

Subspecialty Collections

httpnneurologyorgcgicollectionsubarachnoid_hemorrhageSubarachnoid hemorrhage

httpnneurologyorgcgicollectionassociation_studies_in_geneticsAssociation studies in genetics e

httpnneurologyorgcgicollectionall_cerebrovascular_disease_strokAll Cerebrovascular diseaseStrokefollowing collection(s) This article along with others on similar topics appears in the

Permissions amp Licensing

httpwwwneurologyorgaboutabout_the_journalpermissionsits entirety can be found online atInformation about reproducing this article in parts (figurestables) or in

Reprints

httpnneurologyorgsubscribersadvertiseInformation about ordering reprints can be found online

rights reserved Print ISSN 0028-3878 Online ISSN 1526-632X1951 it is now a weekly with 48 issues per year Copyright copy 2013 American Academy of Neurology All

reg is the official journal of the American Academy of Neurology Published continuously sinceNeurology