doi:10.

1093/brain/awv078

BRAIN 2015: 138; 15181530

| 1518

An ImmunoChip study of multiple sclerosis risk

in African Americans
Noriko Isobe,1,2 Lohith Madireddy,1 Pouya Khankhanian,1 Takuya Matsushita,1,3
Stacy J. Caillier,1 Jayaji M. More,1 Pierre-Antoine Gourraud,1 Jacob L. McCauley,4
Ashley H. Beecham,4 International Multiple Sclerosis Genetics Consortium, Laura Piccio,5
Joseph Herbert,6 Omar Khan,7 Jeffrey Cohen,8 Lael Stone,8 Adam Santaniello,1
Bruce A. C. Cree,1 Suna Onengut-Gumuscu,9 Stephen S. Rich,9 Stephen L. Hauser,1
Stephen Sawcer10,* and Jorge R. Oksenberg1,*
*These authors contributed equally to this work.

1 Department of Neurology, School of Medicine, University of California, San Francisco, CA 94158, USA
2 Division of Neurology, Department of Internal Medicine, Saga University Faculty of Medicine, Saga, Saga 849-8501, Japan
3 Department of Neurological Therapeutics, Neurological Institute, Graduate School of Medical Sciences, Kyushu University,
Fukuoka, Fukuoka 812-8582, Japan
4 John P. Hussman Institute for Human Genomics and The Dr John T Macdonald Foundation Department of Human Genetics,
University of Miami, Miller School of Medicine, Miami, FL 33136, USA
5 Department of Neurology, Washington University School of Medicine, St. Louis, MO 63108, USA
6 Department of Neurology, New York University School of Medicine, New York, NY 10016, USA
7 Multiple Sclerosis Centre and The Sastry Foundation Advanced Imaging Laboratory, Department of Neurology, Wayne State
University School of Medicine, Detroit, MI 48201, USA
8 Mellen Centre for Multiple Sclerosis Treatment and Research, Cleveland Clinic, Cleveland, OH 44195, USA
9 Centre for Public Health Genomics, University of Virginia, Charlottesville, VA 22908, USA
10 Department of Clinical Neurosciences, Cambridge Biomedical Campus, Hills Road, Cambridge CB2 0QQ, UK

Received November 20, 2014. Revised January 5, 2015. Accepted January 26, 2015. Advance Access publication March 28, 2015
The Author (2015). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved.
For Permissions, please email: journals.permissions@oup.com

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The aims of this study were: (i) to determine to what degree multiple sclerosis-associated loci discovered in European populations
also inuence susceptibility in African Americans; (ii) to assess the extent to which the unique linkage disequilibrium patterns in
African Americans can contribute to localizing the functionally relevant regions or genes; and (iii) to search for novel African
American multiple sclerosis-associated loci. Using the ImmunoChip custom array we genotyped 803 African American cases with
multiple sclerosis and 1516 African American control subjects at 130 135 autosomal single nucleotide polymorphisms. We conducted association analysis with rigorous adjustments for population stratication and admixture. Of the 110 non-major histocompatibility complex multiple sclerosis-associated variants identied in Europeans, 96 passed stringent quality control in our
African American data set and of these, 470% (69) showed over-representation of the same allele amongst cases, including 21
with nominally signicant evidence for association (one-tailed test P 5 0.05). At a further eight loci we found nominally signicant
association with an alternate correlated risk-tagging single nucleotide polymorphism from the same region. Outside the regions
known to be associated in Europeans, we found seven potentially associated novel candidate multiple sclerosis variants (P 5 10 4),
one of which (rs2702180) also showed nominally signicant evidence for association (one-tailed test P = 0.034) in an independent
second cohort of 620 African American cases and 1565 control subjects. However, none of these novel associations reached
genome-wide signicance (combined P = 6.3  10 5). Our data demonstrate substantial overlap between African American and
European multiple sclerosis variants, indicating common genetic contributions to multiple sclerosis risk.

ImmunoChip in African Americans with MS

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| 1519

Correspondence to: Jorge R. Oksenberg, PhD,

Department of Neurology, School of Medicine,
University of California,
San Francisco,
675 Nelson Rising Lane,
Room 215C,
San Francisco,
CA 94158, USA
E-mail: Jorge.Oksenberg@ucsf.edu

Keywords: multiple sclerosis; African Americans; ImmunoChip; linkage disequilibrium

Abbreviations: GWAS = genome-wide association study; IMSGC = International Multiple Sclerosis Genetics Consortium;
MHC = major histocompatibility complex; SNP = single nucleotide polymorphism; WTCCC2 = Wellcome Trust Case Control
Consortium 2

Introduction

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Multiple sclerosis is a chronic, inammatory disease of the

CNS and a common cause of neurological disability in
young adults (Hauser and Goodin, 2012). Its modest heritability reects complex polygenic effects and, most likely,
gene-environment interactions [Simon et al., 2011;
International Multiple Sclerosis Genetics Consortium
(IMSGC), 2013a; Sawcer et al., 2014]. The results from
genome-wide association studies (GWAS) have claried important aspects of multiple sclerosis pathogenesis and provided strong empirical support for a model of inheritance
driven primarily by allelic variants that are relatively
common in the general population [Oksenberg and
Baranzini, 2010; IMSGC and Wellcome Trust Case
Control Consortium 2 (WTCCC2), 2011]. The strongest
susceptibility signal genome-wide maps to HLA-DRB1 in
the class II region of the major histocompatibility complex
(MHC, 6p21.3) and explains up to 10.5% of the genetic
variance underlying risk. The HLA association implies that
mechanistically, multiple sclerosis clusters with other antigen-specic autoimmune diseases, a hypothesis supported
by the observation that the non-MHC associated variants
appear to locate predominantly in or near genes inuencing
the function of the adaptive immune system (IMSGC and
WTCCC2, 2011). Interestingly, some of the non-MHC allelic variants associated with multiple sclerosis have also
emerged in GWAS of other autoimmune diseases (IMSGC
and WTCCC2, 2011; Cotsapas et al., 2011), suggesting
that common underlying risk mechanisms might exist
across multiple immune-related conditions. To better
describe this overlap and rene the regions of interest in
susceptibility loci, a mega-consortium was established to
conduct cost-effective candidate loci association studies
across multiple autoimmune diseases using a common,
high-coverage single nucleotide polymorphisms (SNPs)
array known as the ImmunoChip (Cortes and Brown,
2011). The chip was designed in 2010 and 207 728 variants were considered for inclusion, of which 196 524
passed manufacturing quality control (192 402 autosomal,

1595 X-linked, 1735 Y-linked, 791 pseudoautosomal and

one mitochondrial). The multiple sclerosis input to the content came from two sources: an early analysis of a wellpowered GWAS (IMSGC and WTCCC2, 2011) and a
meta-analysis of previously published smaller GWAS
(Patsopoulos et al., 2011).
Typing the ImmunoChip in a new independent data set
identied 48 novel multiple sclerosis susceptibility variants
with genome-wide signicance (IMSGC, 2013b). These results considerably enhanced the roster of validated risk loci
and demonstrated the discovery power of this array that
has been similarly effective in other autoimmune diseases
(Trynka et al., 2011; Cooper et al., 2012; Eyre et al., 2012;
Jostins et al., 2012; Juran et al., 2012; Liu et al., 2012;
Tsoi et al., 2012; Hinks et al., 2013). However, the utility
of this platform in non-Europeans remains to be addressed.
In addition, consistent with their longer evolutionary history, populations of African origin are known to have, on
average, characteristically smaller blocks of linkage disequilibrium compared to populations with European ancestry
(Tishkoff and Kidd, 2004), implying that the study of
populations of African origin could help to narrow the regions of interest and assist in identifying causative variants
(Buyske et al., 2012; Gong et al., 2013).
Notwithstanding difculties in surveillance, multiple
sclerosis is almost non-existent in black Africans and
early estimates suggested that the disease was signicantly
less prevalent in African Americans than in European
Americans (relative risk of 0.64; Wallin et al., 2004).
However, contemporary studies are challenging the longheld belief that African Americans are at a reduced risk
for developing multiple sclerosis (Wallin et al., 2012;
Langer-Gould et al., 2013). Furthermore, compared with
whites, African Americans are more likely to have a more
severe disease course, which at least in part appears to be
genetically determined (Buchanan et al., 2004; Cree et al.,
2004, 2009; Boster et al., 2009; Kimbrough et al., 2014).
Here, we applied the ImmunoChip to a well-curated
African American multiple sclerosis data set to investigate:
(i) whether European multiple sclerosis-associated variants

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| BRAIN 2015: 138; 15181530

are also associated with the disease in African Americans;

(ii) whether the smaller haplotype blocks, characteristic of
African American genomes, can contribute to better mapping of the functionally relevant variants driving the association; and (iii) whether the array can identify novel
multiple sclerosis-associated loci in African American
patients.

Materials and methods

Patients and control subjects

SNP genotyping and quality control

SNP genotyping was conducted using the ImmunoChip, an
Illumina Innium HD custom array at the Wellcome Trust
Sanger Institute, Hinxton, Cambridge, UK for the cases and
at the Centre for Public Health Genomics, University of
Virginia, Charlottesville, VA, USA for the controls. Standard
quality control measures were implemented using PLINK
v1.07 (Purcell et al., 2007). SNPs with missing rates higher
than 2%, Hardy-Weinberg proportion test P 5 10 5 in controls and P 5 10 8 in cases, and distinct missing proportion
between cases and controls with P 5 10 3 were excluded. For
the further analysis, 130 248 autosomal SNPs remained,
including 96 of 110 SNPs known to be associated in
Europeans (IMSGC, 2013b). Samples were excluded for missing genotyping rates exceeding 2%, extreme autosomal heterozygosity of 43 standard deviations (SD), or excessive Identity
By Descent (IBD) with PI_HAT 40.20.

Population stratification
Principal component (PC) analyses were used to assess ancestry and control for the effects of population stratication.
Principal component analysis was conducted using pruned
autosomal non-MHC SNPs with minor allele frequency
41% and pairwise r2 5 0.1 with a window size of 100
SNPs (25 408 SNPs). The scree plot indicated that PC1 explained the vast majority of the variance in the African
American data set (Supplementary Fig. 1A). According to the
plot, PC1 were used to remove two outlier samples with the
values outside  6 SD and all following association analyses
were conducted using PC1 as a covariate. PC1 values were
highly correlated with the previously reported percentage of
African ancestry (r = 0.99, P 5 2.2  10 16) (Reich et al.,

2005; Isobe et al., 2013). When the PC1 components of individual samples were compared with each other, the control
samples from SLEGEN had higher PC1 values compared to
the UCSF cases and controls (P = 2.53  10 27 and
1.77  10 16, respectively), suggesting the proximity of
SLEGEN controls to African ancestry (Supplementary Fig.
1B). Thus, to eliminate association signals derived from the
different population admixture levels between the two control
groups, association was analysed between the two with PC1 as
a covariate, which identied 113 SNPs with P-values 510 3
to be removed. Finally, 130 135 autosomal SNPs remained for
the following analysis. Another principal component analysis
was conducted including samples from the 1000 Genome
Project (The 1000 Genomes Project Consortium, 2010) as a
reference, with commonly available autosomal SNPs pruned
with the same criteria as above (24 994 SNPs). From this
principal component analysis, an additional 22 samples
located far from the relevant reference populations were
removed (Fig. 1). Ultimately, 803 African American multiple
sclerosis cases and 1516 healthy African American control
subjects remained for further analysis. Principal component
analyses were performed using the R package SNPRelate
(Zheng et al., 2012).

Association analysis
Following quality control analyses, association tests were conducted assuming the additive effect of the allele for the affectation status with PC1 as a covariate to control for population
stratication and admixture. First, the replication status of 96
European multiple sclerosis SNPs (IMSGC, 2013b) was evaluated using one-tailed tests. For each multiple sclerosis variant,
the Cochrane Heterogeneity Q Test was also performed to test
effect size differences between African Americans and
Europeans. Additionally, SNPs with association P-values
510 4 locating outside 2 Mb (1 Mb centromeric and 1 Mb
telomeric) anking the European multiple sclerosis-associated
SNPs were nominated as candidates for novel multiple sclerosis-associated variants in African Americans. All association
tests for genotyped SNPs were conducted using PLINK (v1.07)
(Purcell et al., 2007). Power calculations were performed using
Bioconductors GeneticsDesign package version 1.28.0
(Warnes et al., 2010).

Fine mapping with imputation

For multiple sclerosis SNPs, regardless of evidence of replication
in African Americans, we assessed whether there was a more
strongly associated risk-tagging SNP in the region anking the
multiple sclerosis SNP by testing for association amongst the
genotyped SNPs from these regions, including those assessable
by imputation. The regions of multiple sclerosis SNPs were
dened as the range of chromosomal positions where SNPs
in linkage disequilibrium around the multiple sclerosis
SNPs with r2 4 0.5 locate in the European populations of the
1000 Genome Project. Imputation was performed using
IMPUTE2 (v2.3.0) (Howie et al., 2009) and the 1000
Genomes Phase 1 integrated haplotypes (released in September
2013) were used as a reference panel. In addition to the
previously conducted quality control measures, those AT/GC
SNPs with failed alignment were removed. Missing genotypes
for the genotyped SNPs were not imputed. To increase the

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The core screening data set consists of 842 de-identied DNA

samples from African American cases and 498 African
American controls. All multiple sclerosis subjects met established diagnostic criteria (McDonald et al., 2001; Polman
et al., 2011). Ascertainment protocols and clinical and demographic characteristics have been summarized elsewhere (Cree
et al., 2004; Oksenberg et al., 2004). All study participants are
self-reported African Americans. Additionally, data from 1114
African American control subjects were provided by the
International Consortium on the Genetics of Systemic Lupus
Erythematosus (SLEGEN), totalling 842 cases and 1612 controls. The University of California at San Francisco
Institutional Review Board approved this study.

N. Isobe et al.

ImmunoChip in African Americans with MS

BRAIN 2015: 138; 15181530

| 1521

Replication study of unreported

multiple sclerosis variants

Figure 1 Principal component analysis of the African

American Immunochip data set with reference to samples
from the 1000 Genome Project. African American (AfAm)
case/control samples are shown as + and reference samples from
the 1000 Genome Project are shown as closed circles. YRI and
LWK = Africans; ASW = African Americans; CEU, TSI, FIN, GBR
and IBS = Europeans; MXL, PUR, CLM = Central-South Americans;
CHB, CHS and JPN = Asians.

Results
Replication study of the multiple
sclerosis-associated SNPs in
non-MHC regions
We screened 130 135 autosomal SNPs in 803 African
American multiple sclerosis cases and 1516 African
American control subjects. Figure 2 shows a Circos plot
summarizing the results from this screen; as anticipated,
the strongest association was observed in the MHC
region on chromosome 6p21.3 (P = 2.75  10 8). In
Europeans 110 SNPs from 103 discrete loci outside the
MHC region have been established as risk variants in multiple sclerosis (IMSGC, 2013b); in our African American
screen, results passing stringent quality control were available for 96 of these SNPs (including rs3190930 a proxy
SNP for rs802734, Supplementary Table 1). Amongst these
96 we found that 470% (69/96) had the same allele overrepresented in cases as in European multiple sclerosis cases,
a highly signicant excess of concordance (one-tailed binomial test P = 1.07  10 5). For 21 of these 69 the excess
frequency in cases was nominally signicant (one-tailed test
P 5 0.05) (Table 1); for all of these the effect sizes in
African Americans were statistically indistinguishable from
those observed in Europeans (heterogeneity test P 4 0.05,
Supplementary Table 1). Even including unreplicated multiple sclerosis SNP, the obtained effect sizes of multiple
sclerosis variants in African Americans were generally correlated with those in Europeans (Supplementary Fig. 2). To
estimate the level of concordance that might be expected if
effects were the same in African Americans as in
Europeans, we estimated for each of the 96 SNPs the
power of a study with 803 cases and 1516 control subjects

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imputation accuracy we did not pre-phase genotypes and the

number of Markov chain Monte Carlo iteration (-iter) was
increased to 60 with burnin 20. Association tests were conducted with the frequentist test using SNPTEST (v2.5b) assuming an additive effect of each SNP (Marchini and Howie,
2010). Similar to the analysis in the genotyped SNPs, the PC1
component was included as a covariate in the analysis. SNPs
were removed when they had poor imputation accuracy with
the INFO score obtained from IMPUTE2 lower than 0.7,
minor allele frequency 51%, or a Hardy-Weinberg
Equilibrium test violation (P 5 10 5 in controls and
P 5 10 8 in cases). Regions were also considered replicated
when a neighbouring SNP had an association with false discovery rate (FDR) P 5 0.05 corrected with P-values of all the
SNPs, both genotyped and imputed, within the region. To calculate linkage disequilibrium parameters between the original
European multiple sclerosis SNP and the optimal risk-tagging
SNP of African Americans, we used the 1000 Genome Project
population data set (The 1000 Genomes Project Consortium,
2010) for Europeans and our case-control data set for African
Americans. GTOOL (v0.7.5) (Freeman and Marchini, 2007)
was used to convert post-imputed genetic data to PLINKformat data to be ready to calculate linkage disequilibrium in
PLINK. Imputation was also performed for the linkage disequilibrium region of the candidate novel multiple sclerosis variants
using a maximum range of positions where proxy SNPs
(r2 4 0.5 in African Americans) of the candidate SNPs locate
to capture the possible causative variants. ANNOVAR
(Wang et al., 2010), SNPnexus (Dayem Ullah et al., 2013),
and RegulomeDB (Boyle et al., 2012) were used to annotate
obtained variants. We plotted the association test results
using LocusZoom (Pruim et al., 2010) with needed
modications.

For the candidate multiple sclerosis loci previously unreported

in Europeans, a replication study was conducted on an independent African American group consisting of 620 multiple
sclerosis cases and 1565 controls by genotyping the top
SNPs after imputation in the region of interest. SNP genotyping was completed in the replication data set using predesigned
and custom TaqMan SNP Genotyping Assays. TaqMan
SNP genotyping assays were conducted in 384-well plates on
an ABI 7900HT Sequence Detection System using SDS 2.3
software. Association P-values were provided with one-tailed
test. We also performed meta-analysis under a xed-effects
model with effect sizes and standards errors from the African
American ImmunoChip (discovery) data set and the replication
study. Here a SNP was considered to have replicated when the
replication P 5 0.05 (one-tailed test) and the combined P-value
of meta-analysis is more signicant than the discovery P-value.
For the replication study, no adjustment of population admixture was conducted.

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N. Isobe et al.

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Figure 2 Circos plot of ImmunoChip in African Americans. The outermost track shows the autosomal chromosomes. The second track
indicates the genes closest to the non-MHC multiple sclerosis-associated variants. Gene names of replicated loci in African Americans with
identical SNPs as Europeans are shown in bold black, replicated loci with alternate variants are shown in bold blue, and unreplicated loci are
shown in grey. The replicated novel multiple sclerosis locus in African Americans is indicated in bold red. The innermost track indicates log10(P)
(two-tailed test) of association tests for each ImmunoChip SNP from African Americans (dark blue) and Europeans (light blue). The range of y-axis
is 08, excluding the peaks of Europeans with higher significance. The dark red line in the middle of the plot represents log10(P) = 4.

to identify nominally signicant association (one-tailed test

P 5 0.05 or half the power to observe two-tailed test
P 5 0.1), assuming effect sizes equivalent to those seen in
the European screen (IMSGC, 2013b) and the risk allele
frequencies observed in our African American control
population (Supplementary Table 1). Across the 96 variants
we found that the average power was 18.6%, with values

ranging from 5.5% (at rs2028597) to 49.9% (at

rs6677309). Based on this average value we would anticipate seeing nominally signicant association (one-tailed test
P 5 0.05) at between 12 and 24 SNPs with the same risk
allele as in Europeans. Our observation of 21 such SNPs is
thus entirely consistent with these variants exerting equivalent effect in African Americans and Europeans.

rs6677309
rs12927355
rs11554159
rs71624119
rs917116
rs1920296
rs2050568
rs1800693
rs2688608
rs2104286
rs7238078
rs2248359
rs2028597
rs201847125
rs7552544
rs2255214
rs1843938
rs4976646
rs7120737
rs9282641
rs34536443

1
16
19
5
7
3
1
12
10
10
18
20
3
7
1
3
7
5
11
3
19

117080166
11194771
18285944
55440730
28172739
121543577
157770241
6440009
75658349
6099045
56384192
52791518
105558837
50325567
101240893
121770539
3113034
176788570
47702395
121796768
10463118

Position

CD58
CLEC16A
IFI30
ANKRD55
JAZF1
IQCB1
FCRL1
TNFRSF1A
(C10orf55)
IL2RA
MALT1
(CYP24A1)
CBLB
(IKZF1)
(VCAM1)
(CD86)
(CARD11)
RGS14
AGBL2
CD86
TYK2

Genea

Intronic
Intronic
Exonic
Intronic
Intronic
Intronic
Intronic
Intronic
Intergenic
Intronic
Intronic
Intergenic
Intronic
Intergenic
Intergenic
Intergenic
Intergenic
Intronic
Intronic
Utr5
Exonic

Function

A
G
G
G
C
C
G
G
A
A
A
G
G
G
A
C
A
G
G
G
C

RA

0.879
0.678
0.730
0.755
0.205
0.644
0.534
0.398
0.549
0.722
0.770
0.599
0.920
0.695
0.558
0.518
0.438
0.340
0.145
0.919
0.951

RAF
(cont.)
1.34
1.21
1.15
1.12
1.12
1.14
1.08
1.14
1.07
1.21
1.05
1.07
1.04
1.11
1.08
1.11
1.08
1.13
1.13
1.12
1.28

(1.271.41)
(1.171.26)
(1.111.20)
(1.081.17)
(1.071.16)
(1.111.18)
(1.051.12)
(1.111.18)
(1.031.10)
(1.161.26)
(1.021.10)
(1.031.10)
(0.981.11)
(1.071.15)
(1.051.12)
(1.081.15)
(1.051.12)
(1.091.17)
(1.081.18)
(1.051.19)
(1.181.40)

OR (95% CI)

Europeansb

1.5  10
8.2  10
2.6  10
2.7  10
2.1  10
6.8  10
1.3  10
6.9  10
6.4  10
7.6  10
6.3  10
9.8  10
1.8  10
2.9  10
3.7  10
1.7  10
2.2  10
1.0  10
7.6  10
5.9  10
1.2  10

08

04

08

12

06

10

06

08

01

05

03

23

05

16

06

15

08

09

13

27

28

RAF
(cont.)
0.471
0.747
0.755
0.935
0.702
0.700
0.156
0.362
0.254
0.933
0.743
0.354
0.962
0.893
0.800
0.712
0.411
0.537
0.266
0.956
0.994

RAF
(cases)
0.547
0.787
0.795
0.943
0.715
0.728
0.202
0.399
0.307
0.939
0.777
0.399
0.972
0.901
0.810
0.726
0.435
0.558
0.286
0.966
0.996

African Americans

1.23
1.28
1.27
1.40
1.20
1.19
1.21
1.16
1.17
1.33
1.17
1.15
1.46
1.24
1.18
1.15
1.13
1.13
1.14
1.35
2.12

(1.091.39)
(1.101.48)
(1.091.47)
(1.081.82)
(1.041.37)
(1.041.36)
(1.031.42)
(1.021.31)
(1.021.34)
(1.031.71)
(1.021.35)
(1.011.30)
(1.022.08)
(1.011.53)
(1.011.38)
(1.001.32)
(1.001.29)
(1.001.27)
(0.991.30)
(0.971.88)
(0.875.18)

OR (95% CI)

4.7  10
5.3  10
8.7  10
6.1  10
6.2  10
7.1  10
1.1  10
1.1  10
1.3  10
1.4  10
1.5  10
1.7  10
1.9  10
1.9  10
2.0  10
2.4  10
2.8  10
3.0  10
3.2  10
3.7  10
4.9  10

02

02

02

02

02

02

02

02

02

02

02

02

02

02

02

03

03

03

04

04

04

P
(one-tailed)

2.2  10
4.8  10
2.1  10
1.0  10
3.8  10
5.7  10
1.8  10
8.2  10
2.2  10
4.8  10
1.4  10
2.9  10
6.6  10
2.9  10
2.8  10
6.5  10
4.8  10
9.6  10
9.3  10
2.8  10
2.7  10

het. Pd

01

01

01

01

01

01

01

01

02

01

01

01

01

01

01

01

01

01

01

01

01

0.499
0.421
0.307
0.116
0.256
0.307
0.120
0.329
0.126
0.223
0.090
0.140
0.055
0.138
0.131
0.225
0.171
0.314
0.275
0.096
0.080

Power

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Position is based on human genome 19 and dbSNP 137.

a
When SNPs locate intergenic, the closest genes are shown in brackets.
b
Results of Europeans are originated from IMSGC, 2013b.
c
SNPs with association P-values 5 0.05 (one-tailed test) were shown.
d
Cochrane Heterogeneity Q test.
AA = African Americans; Chr = choromosome; CI = confidence interval; cont. = controls; het. = heterogeneity; RA = risk allele; RAF = risk allele frequency.

rsID

Chr

Table 1 Replicated 21 SNPs in African Americans out of 96 non-MHC multiple sclerosis susceptibility variants of Europeans

ImmunoChip in African Americans with MS

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| BRAIN 2015: 138; 15181530

evidence for association (Table 2, Figs 2 and 3, and

Supplementary Fig. 3). One of the variants (rs1861842)
in the PVT1/MIR1208 locus shows modest linkage disequilibrium (r2 = 0.409) with the corresponding lead European
SNP (rs759648) in the European population but rather
little linkage disequilibrium with that SNP in African
Americans (r2 = 0.142), suggesting that these two SNPs
(rs759648 and rs1861842) tag the same signal in
Europeans while only rs1861842 is correlated with the
signal in African Americans, consistent with this SNP
being a better tag for the functionally relevant variant
(Fig. 3A).
Taking advantage of the unique linkage disequilibrium
patterns in the African American genome enabled us to
possibly narrow two additional disease-association regions,
MMEL1 at 1p36 and ZFP36L1 at 14q22-q24. In the
MMEL1 locus, the linkage disequilibrium block in
African Americans (r2 4 0.5) anking rs111375644
(lowest P-value in African Americans) is 2 494 816
2 728 455 bp and is 3.5 kb smaller than linkage disequilibrium block in Europeans anking rs3748817 (lowest
P-value in Europeans), excluding LOC115110 from the
candidate disease-associated genes (Fig. 3B). Furthermore,
the narrow linkage disequilibrium region (r2 4 0.8) around
rs3748817 spreads across 237 kb in Europeans and includes ve genes, whereas the size of the high linkage disequilibrium region in African Americans for rs111375644
was 16 kb and includes a single gene (TNFRSF14). In the
ZFP36L1 locus, the linkage disequilibrium region in
African Americans around the most signicantly associated
SNP rs8011424, was 25.6 kb smaller (69 265 911
69 310 210 bp) than that in Europeans anking the established multiple sclerosis SNP (rs2236262), highlighting the
upstream region of ZFP36L1 (Supplementary Fig. 3D).
However, as these variants are monomorphic or show no
signicant linkage disequilibrium with their respective
European lead SNP even in the European population,
they may represent additional risk alleles rather than successful ne mapping of European signals. Lastly, in the
IRF8 locus, the optimal SNP in African Americans after
imputation (rs13333054) coincides with the previously reported SNP in the 2011 GWAS (IMSGC and WTCCC2,
2011) rather than the ImmunoChip (IMSGC, 2013b)
(Supplementary Fig. 3E).
In this African American cohort 4 of 96 European lead
SNPs showed nominally signicant evidence of association
with the alternate allele to that seen in Europeans
(Supplementary Table 1), raising the possibility that these
variants might be exerting different, even opposite effects in
this population. However, this number of seemingly opposite effects is consistent with that expected to result from
random sampling variation overwhelming genuine but
modest signals. In a study of this size and considering 96
variants, we would anticipate seeing up to ve apparently
reversed effects by chance alone. A similar low frequency of
apparently reversed signals was seen in our previous
African American study (Isobe et al., 2013) and also in

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Unsurprisingly, the two SNPs with the most signicant association in the African Americans were those with the
strongest effects in Europeans, rs6677309 (CD58) and
rs12927355 (CLEC16A).
Among the 21 replicated variants, two exonic multiple
sclerosis SNPs in IFI30 (rs11554159) and TYK2
(rs34536443), respectively, are predicted as probably
damaging. For rs34536443 in TYK2, the protective allele
drives T lymphocyte differentiation towards a Th2 phenotype (Couturier et al., 2011). This variant was also found
to be associated with juvenile idiopathic arthritis, primary
biliary cirrhosis, psoriasis and rheumatoid arthritis (Eyre
et al., 2012; Liu et al., 2012; Tsoi et al., 2012; Hinks
et al., 2013). Other replicated SNPs included rs1800693
in TNFRSF1A, which functionality mimics the effect of
TNF blocking drugs (Gregory et al., 2012), and
rs2104286 in IL2RA, which seems to increase the ratio
of soluble/membrane IL2RA and inhibits IL2 signalling
(Maier et al., 2009). Both rs1800693 (TNFRSF1A) and
rs2104286 (IL2RA) accounted for 450% of posterior
probability of association in Europeans (IMSGC, 2013b).
In a previous study using an overlapping, albeit larger
sample set of African Americans, we reported signicant
associations for 8 of 74 tested non-MHC multiple sclerosis
risk SNPs with a two-tailed test P 5 0.01 threshold,
whereas (coincidentally) 21 variants exceeded the onetailed test P 5 0.05 threshold (Isobe et al., 2013). When
compared to this study, all of these variants had the same
direction of association despite the limited statistical power
of both studies (Supplementary Table 2). However, associations in this study did not reach statistical signicance in
seven loci due most likely to the relatively low effect sizes
of these variants. Additionally, lower minor allele frequency
of the updated multiple sclerosis SNPs compared to the
previous SNPs prevented replication for two loci
(MMEL1 and IRF8). On the other hand, the TYK2 locus
was replicated, this time with a different risk-tagging SNP
from our previous study (rs8112449, not replicated; Isobe
et al., 2013).
Given the possibility of allelic heterogeneity across ancestral groups, we reasoned that SNPs lying close to the
European lead SNP have increased prior odds even if not
in linkage disequilibrium. To look for such effects we
searched in the African American data set for nominally
associated SNPs within the intervals anking each of the
110 European SNPs (with boundaries of the anking intervals dened by the most distant SNP in linkage disequilibrium with the European lead SNP; r2 4 0.5 in the 1000
Genome data set of Europeans). We considered rst the
21 intervals containing European lead SNPs that showed
nominal evidence of association, and found more signicantly associated SNPs in 20 (data not shown). Among
them only eight were in linkage disequilibrium (r2 4 0.5)
with the European lead SNP. In the remaining 89 regions
( = 110
21), after correction for independent testing at
each locus, and setting a FDR of 0.05, we found eight
regions that contained SNPs showing nominally signicant

N. Isobe et al.

0.96

IRF8 (dist = 38273),

LOC146513
(dist = 325553)
TNFSF14
1.02

1.06

1.10

4.3  10

3.7  10

8.3  10

7.3  10

5.8  10

01

01

02

01

01

rs12150912

rs13333054

rs8011424

rs1861842

rs79281846

rs78553800

rs115126543

MIR1208 (dist = 46226),

LINC00977
(dist = 1020053)
ZFP36L1 (dist = 9296),
ACTN1
(dist = 68584)
IRF8 (dist = 54822),
LINC01082
(dist = 218754)
TNFSF14 (dist = 3775),
C3 (dist = 3472)

TNFRSF14
(dist = 15959),
FAM213B (dist = 6963)
EVI5 (dist = 10883),
RPL5 (dist = 28750)
TRIM71 (dist = 53040),
CCR4 (dist = 6255)
PXT1

RA

0.651

0.255

0.253

0.392

0.080

0.956

0.022

0.065

cases

RAF

1.92 (1.432.58)

3.06 (1.765.33)
1.58 (1.22-2.06)
1.57 (1.222.03)
1.30 (1.141.48)

1.35 (1.161.57)

1.36 (1.171.58)

1.24 (1.071.45)

0.039

0.008
0.926
0.057
0.328

0.214

0.204

0.629

cont.

OR (95%CI)

05

05

5.2  10

6.0  10

8.4  10

8.5  10

5.5  10

03

05

05

05

04

4.8  1004

7.9  10

1.5  10

raw p

2.5  10

8.7  10

1.7  10

4.3  10

2.4  10

4.8  10

2.4  10

9.7  10

03

03

02

03

02

02

02

03

(19)

(228)

(371)

(499)

(98)

(268)

(2697)

(664)

FDR P (No. P)d

0.344

0.036

0.019

0.409

0.003

0.002

EUR

0.99

1.00

1.00

0.99

0.77

1.00

0.071

0.008

0.124

0.142

0.011

0.032

0.000

0.022

R2

AfAm

1.00

1.00

1.00

0.63

1.00

0.96

0.85

1.00

LD info (between A and B)e

BRAIN 2015: 138; 15181530

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a
Regions are defined as range of positions where SNPs in linkage disequilibrium (r2 4 0.5) with the multiple sclerosis-associated SNPs in Europeans of the 1000 Genome Project locate. Chromosome number and genomic positions (human genome 19,
kb) are shown.
b
One-tailed test.
c
Imputed SNPs are shown in italics.
d
False discovery rate P-values are shown with the number of SNP P-values in linkage disequilibrium regions in brackets.
e
Linkage disequilibrium information in Europeans originated from European data set of the 1000 Genome Project and that in African Americans are from our African American data set.
f
Top SNP in linkage disequilibrium region (B) is monomorphic in Europeans.
AfAm = African Americans; Chr = chromosome; CI = confidence interval; dist = distance; EUR = Europeans; LD = linkage disequilibrium; MAF = minor allele frequency; NA = not available; OR = odds ratio; RA = risk allele in Europeans;
SE = standard error.

rs1077667
(19: 66626676)

rs35929052
(16: 8599486018)

rs2236262
(14: 6923369302)

2.0  10

1.08

PVT1 (dist = 45446),

MIR1208
(dist = 3417)
ZFP36L1

NA

CCR4 (dist = 17080),

GLB1 (dist = 24617)
PXT1

0.96

01

rs111375644

EVI5

1.05

2.4  10

rs41286801
(1: 9268093373)
rs4679081
(3: 3297133014)
rs941816
(6: 36346-36384)
rs759648
(8: 129155129222)

01

MMEL1

Gene

rs3748817
(1: 24842721)

OR

rsIDc

AfAm

Gene

rsID (LD regiona)

RA

(B) Top SNP in African Americans

(A) European multiple sclerosis-associated SNPs

Table 2 Alternate SNPs in replicating known susceptibility regions

ImmunoChip in African Americans with MS

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| BRAIN 2015: 138; 15181530

N. Isobe et al.

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Figure 3 Narrowing in the causative region using African American data set. Comparative association plots for the loci of (A) PVT1/
MIR1208 and (B) MMEL1 of (i) African Americans after fine mapping with imputation; and (ii) the discovery data set of European ImmunoChip.
SNPs with the top association P-values are shown in purple with SNP IDs. For the plots of African Americans, genotyped SNPs are shown in
closed circles and imputed ones in closed triangles. Colours of the marks represent linkage disequilibrium (r2) with the top SNP in each
population. Chromosomal positions are based on human genome 19. Note differences in the scales of y-axis.

1.05
1.05
1.01
1.10
1.01
1.01
A
A
G
G
C
G
183512840
119577577
6197182
2325978
101584223
21853100
03

03

04

02

rs789176
rs11123495
rs6986146
rs11062158
rs12313149
rs2180439
01

05

5.3  10
1.1  10
3.6  10
2.0  10
2.7  10
8.0  10
01

01

01

01

01

3.4  10
9.9  10
8.9  10
2.7  10
5.9  10
7.8  10

02

1.23
1.09
1.12
1.23
1.17
1.15

(1.111.36)
(0.981.21)
(1.011.25)
(1.11.37)
(1.061.30)
(1.041.27)

0.71
0.28
0.22
0.21
0.28
0.31
06

06

06

05

0.74
0.25
0.21
0.22
0.28
0.30
1.31
1.40
1.35
1.42
1.40
1.43
0.70
0.26
0.20
0.21
0.24
0.29

cases cont.

0.74
0.32
0.26
0.28
0.29
0.36
T
A
T
G
T
G
SMG7
(EN1)
(LOC100287015)
CACNA1C
SLC5A8
(PAX1)
rs2702180
rs1438856
rs6986146i
rs10848633
rs10860696
rs6047667

Gened

RA RAF

OR (95% CI)

(1.141.50)
(1.211.62)
(1.161.56)
(1.231.64)
(1.211.62)
(1.231.66)

Pe

1.3  10
6.2  10
6.3  10
6.9  10
7.2  10
2.6  10

04

cases cont.

1.15
0.83
0.90
1.05
0.98
0.95
183517971
119570839
6197182
2316019
101559080
21827351
1
2
8
12
12
20

OR Pg
RAFf
Position
Chr rsIDc

(n = 2) for
02

Position is based on human genome 19 and dbSNP 137. List of genotyped top SNPs with association P 5 10 4 are shown in Supplementary Table 3. Assay failed in one of the seven candidate loci.
a
Fixed model is applied.
b
Regions are defined as range of positions where SNPs in linkage disequilibrium (r2 4 0.5) with the top SNPs after imputation in African Americans of the 1000 Genome Project locate.
c
Imputed SNPs are shown in italics.
d
When SNPs locate intergenic, the closest genes are shown in brackets.
e
SNPTEST was used for the association test in the imputed data set while PLINK was used for the test in the genotyped data set before imputation. Here, P-values from SNPTEST were shown.
f
Risk alleles are those in the discovery data set.
g
One-tailed test.
h
When multiple correction was conducted for the SNPs available in each linkage disequilibrium region with FDR methods, the FDR P-values (no. SNPs in each locus) were P = 7.7  10 01 (n = 133) for rs789176, P = 2.1  10
rs11123495, P = 6.1  10 01 (n = 1) for rs6986146, P = 8.3  10 01 (n = 78) for rs11062158, P = 7.3  10 01 (n = 3) for rs12313149, and P = 5.0  10 01 (n = 1) for rs2180439.
i
As replication of the top SNP after imputation, rs6986480 failed, the top genotyped SNP was used for the replication study.
AA = African Americans; Chr = chromosome; CI = confidence interval; cont. = control; LD = linkage disequilibrium; OR = odds ratio; RA = risk allele; RAF = risk allele frequency.

01

01

02

01

02

5.6  10
2.0  10
6.1  10
4.1  10
6.8  10
5.0  10
(1.001.10)
(1.011.09)
(0.981.04)
(1.001.21)
(0.971.06)
(0.981.05)

Ph
RA OR (95% CI)
rsID
OR (95% CI)

Position

Regional top SNP in European ICb

Replication study
Top SNPs after imputation

Table 3 Replication of candidate novel multiple sclerosis-associated loci in African Americans

The recent completion of the ImmunoChip project raised to

110 the number of non-MHC multiple sclerosis risk DNA
variants in Europeans (IMSGC, 2013b). In aggregate, the
proportion of the genetic variance accounting for disease
risk explained by these polymorphisms, including the
MHC, is roughly 27% (IMSGC, 2013b). Our main goal
was to assess the transferability of this updated multiple
sclerosis genetic map to African Americans. The number
of replicated variants (21 of 96) was within the range of
expectation given the power of our study, suggesting that
most, if not all of the multiple sclerosis risk SNPs discovered in Europeans are also relevant in African
Americans and possibly in other non-white populations as
well. An excess of concordant direction for allelic effects of
the European multiple sclerosis SNPs in African Americans
is consistent with this generalization.
For several of the established loci even though we failed
to see evidence for signicant association with the
European lead SNP, we did nd evidence of association
with independent anking variants. Most of these new variants were uncorrelated with the European lead SNP in both

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Discussion

Meta-anlaysisa

Exploring potential association

outside the established multiple
sclerosis-associated loci

06

our study of European multiple sclerosis variants in an

Indian data set (Pandit et al., 2011). Although such allele
ipping could theoretically have resulted from unusual
population-based differences in allele frequencies (Lin
et al., 2007; Zaykin and Shibata, 2008; Clarke and
Cardon, 2010), no such differences are apparent in the
1000 Genomes data set (The 1000 Genomes Project
Consortium, 2010), where the minor allele for these four
SNPs is the same in both populations.

Recognizing the limited power of the data set, we nevertheless explored the evidence for association seen at SNPs
mapping outside the designated 110 multiple sclerosis loci
and outside the MHC region. In this analysis we identied
seven regions containing at least one SNP with P 5 10 4
(Supplementary Table 3). Only one of these (rs11123495)
showed nominally signicant association in the European
ImmunoChip data set (P = 1.98  10 2) but the direction
of the association was opposite from that in African
Americans (IMSGC, 2013b). When analysing these seven
regions in an independent replication cohort (620 African
American cases with multiple sclerosis and 1565 control
subjects), we found evidence of association for only one
variant (rs2702180 in SMG7) (one-tailed test P = 0.034,
Table 3). However, in a combined analysis across both
African American data sets, this SNP failed to reach
genome-wide signicance (P = 6.3  10 5).

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02

ImmunoChip in African Americans with MS

1528

| BRAIN 2015: 138; 15181530

(Gateva et al., 2009; Cunninghame Graham et al., 2011;

Jacob et al., 2012) but a multi-ethnic study pointed out that
SLE-associated variants located in NCF2 were signicantly
associated with the expression of SMG7 (Kim-Howard
et al., 2014). Additional studies will be required to validate
the association in an independent African American data
set with larger sample size and to determine if the association with this locus can also be observed in European
populations. The SMG7 region locates outside the highest
peak in a genome-wide admixture scan, which may partially explain why the multiple sclerosis association with
SMG7/NCF2 was found in African Americans but not in
Europeans (Reich et al., 2005).
In conclusion, we show the extensive replication of
European multiple sclerosis variants in African Americans,
consistent with a shared genetic architecture for multiple
sclerosis susceptibility across these different populations.
However, as the ImmunoChip design was mainly based
on reference European populations (Cortes and Brown,
2011), the utility of the array to genotype non-European
populations, potentially lacking tag SNPs for some haplotypes and the full range of cross-ancestral genetic pleiotropy, remains unknown. Our results suggest that
ImmunoChip-like platforms have substantial potential to
ne-map regions of interest by taking advantage of different haplotypic structures, but the need for very large
sample sizes and functional studies is still evident. Even
with arrays capable of tagging variation in both populations, very large sample sizes would be necessary to exclude
any modest effect of a European variant in an African
American population and vice versa. Trans-ancestral studies are also likely to help in the discovery of new genes
and pathways vital to disease susceptibility (Diabetes
Genetics Replication and Meta-analysis Consortium et al.,
2014). In addition, the clinical expression of multiple sclerosis, including its severity, is known to have a genetic basis,
but to date no disease modiers have been convincingly
identied. The severe clinical course and treatment-resistance typical of multiple sclerosis in African Americans
highlights an additional opportunity, i.e. to identify modiers of disease severity and progression that could lead to
much-needed therapeutic opportunities.

Supplementary material
Supplementary material is available at Brain online.

Acknowledgements
The authors thank the multiple sclerosis patients and
healthy controls who participated in this study. The authors acknowledge the contributions of H. Mousavi and
R. Guerrero (UCSF) for sample processing and management, and the genotyping teams at the Wellcome Trust
Sanger Institute, UK, the Cambridge NIHR Biomedical

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the African American and European populations, suggesting that these must be different effects although the possibility of artefacts cannot be excluded. However, for one
variant (rs1861842 adjacent to MIR1208) we did see linkage disequilibrium in the European population but not in
the African American population, indicating that the original disease risk signal has been successfully ne mapped
beyond what was possible within the European population.
Altogether, our data conrm that the potential of less extensive linkage disequilibrium structure present in African
Americans to aid in ne mapping is only likely to be advantageous if the study has adequate power to demonstrate
signicant genome-wide association.
The signal we identied in the region of MMEL1, locates
telomeric to MMEL1, between FAM213B and TNFRSF14,
whereas the European signal locates to the 18th intron of
MMEL1 itself. This increases the evidence supporting a
role for these other genes. FAM213B is associated with
biosynthesis of prostaglandin F2 and cyclooxygenase pathway whereas TNFRSF14, a member of the TNF receptor
superfamily ts well within the activatory/inhibitory inammation pathways mechanistically associated with multiple
sclerosis (IMSGC and WTCCC2, 2011). Mutations in
TNFRSF14 have also been associated with B cell lymphoma (Morin et al., 2011; Lohr et al., 2012), consistent with
an increasing appreciation that disordered B cell function is
intimately associated with multiple sclerosis pathogenesis
(Hauser and Goodin, 2012). Interestingly, a recent network-based pathway analysis also ranked TNFRSF14
higher compared to MMEL1 as a multiple sclerosis susceptibility locus (IMSGC, 2013a). Similarly, for EVI5 on
chromosome 1, which is a well-established risk locus with
relatively large effect size (odds ratio = 1.20) (IMSGC,
2013b), the reported top risk-tagging SNP in Europeans
locates at the 3 untranslated region (UTR) of EVI5,
while in African Americans, the associated SNP
(rs115126543) identied in this study locates 11 kb upstream of the gene itself within transcription factor binding
sites and a DNase I hypersensitive site (Bernstein et al.,
2012), suggesting a role for transcriptional regulatory
mechanisms mediating risk. However, it is notable that
risk allele frequencies for both these variants are low and
therefore so is power. As neither is identied with clear
signicance additional studies will be required to conrm
the relevance of these observations.
The screen identied seven novel regions containing at
least one SNP with suggestive evidence of association, of
which only one (rs2702180 in SMG7 on chromosome 1)
replicated in an independent data set using relatively lenient
but predetermined replication criteria. SMG7 encodes a
protein that is essential for nonsense-mediated mRNA
decay, a process linked to autoimmunity (Bachmann
et al., 2006). Interestingly, the risk-tagging SNP was reported to be associated with the expression level of
SMG7 in brain tissues (Gibbs et al., 2010) and in lymphoblastoid cells (Stranger et al., 2007). In SLE, NCF2 adjacent
to SMG7 is reported to be associated with the disease

N. Isobe et al.

ImmunoChip in African Americans with MS

Research Centre, UK and the Centre for Public Health

Genomics, University of Virginia, Charlottesville, VA,
USA. This manuscript is dedicated to the memory of
Joseph Herbert, in recognition of his contributions and
leadership in multiple sclerosis research, and committed
dedication to people aficted with the disease.

Funding
This study is supported by grants from the National
Institute of Health (R01NS076492, R01NS046297 and
R01NS049477) and the UK Multiple Sclerosis Society
(898/08). Recruitment of study participants and sample acquisition was supported by National Multiple Sclerosis
Society (RG2899-D11 and RC2 GM093080). N.I. was supported by Postdoctoral Fellowship for Research Abroad
from Japan Society for the Promotion of Science (JSPS)
and is currently a JSPS Research Fellow. L.P. is a Harry
Weaver Neuroscience Scholar of the National Multiple
Sclerosis Society (JF2144A2/1).

Bachmann MP, Bartsch H, Gross JK, Maier SM, Gross TF,

Workman JL, et al. Autoimmunity as a result of escape from
RNA surveillance. J Immunol 2006; 177: 1698707.
Bernstein BE, Birney E, Dunham I, Green ED, Gunter C, Snyder M.
An integrated encyclopedia of DNA elements in the human genome.
Nature 2012; 489: 5774.
Boster AL, Endress CF, Hreha SA, Caon C, Perumal JS, Khan OA.
Pediatric-onset multiple sclerosis in African-American black and
European-origin white patients. Pediatr Neurol 2009; 40: 313.
Boyle AP, Hong EL, Hariharan M, Cheng Y, Schaub MA,
Kasowski M, et al. Annotation of functional variation in personal
genomes using RegulomeDB. Genome Res 2012; 22: 17907.
Buchanan RJ, Martin RA, Zuniga M, Wang S, Kim M. Nursing home
residents with multiple sclerosis: comparisons of African American
residents to white residents at admission. Mult Scler 2004; 10:
6607.
Buyske S, Wu Y, Carty CL, Cheng I, Assimes TL, Dumitrescu L, et al.
Evaluation of the metabochip genotyping array in African
Americans and implications for ne mapping of GWAS-identied
loci: the PAGE study. PLoS One 2012; 7: e35651.
Clarke GM, Cardon LR. Aspects of observing and claiming allele ips
in association studies. Genet Epidemiol 2010; 34: 26674.
Cooper JD, Simmonds MJ, Walker NM, Burren O, Brand OJ, Guo H,
et al. Seven newly identied loci for autoimmune thyroid disease.
Hum Mol Genet 2012; 21: 52028.
Cortes A, Brown MA. Promise and pitfalls of the Immunochip.
Arthritis Res Ther 2011; 13: 101.
Cotsapas C, Voight BF, Rossin E, Lage K, Neale BM, Wallace C, et al.
Pervasive sharing of genetic effects in autoimmune disease. PLoS
Genet 2011; 7: e1002254.
Couturier N, Bucciarelli F, Nurtdinov RN, Debouverie M, LebrunFrenay C, Defer G, et al. Tyrosine kinase 2 variant inuences T
lymphocyte polarization and multiple sclerosis susceptibility. Brain
2011; 134: 693703.
Cree BA, Khan O, Bourdette D, Goodin DS, Cohen JA, Marrie RA,
et al. Clinical characteristics of African Americans vs Caucasian
Americans with multiple sclerosis. Neurology 2004; 63: 203945.

| 1529

Cree BA, Reich DE, Khan O, De Jager PL, Nakashima I, Takahashi T,

et al. Modication of Multiple Sclerosis Phenotypes by African
Ancestry at HLA. Arch Neurol 2009; 66: 22633.
Cunninghame Graham DS, Morris DL, Bhangale TR, Criswell LA,
Syvanen AC, Ronnblom L, et al. Association of NCF2, IKZF1,
IRF8, IFIH1, and TYK2 with systemic lupus erythematosus. PLoS
Genet 2011; 7: e1002341.
Dayem Ullah AZ, Lemoine NR, Chelala C. A practical guide for the
functional annotation of genetic variations using SNPnexus. Brief
Bioinform 2013; 14: 43747.
Diabetes Genetics Replication And Meta-analysis (DIAGRAM)
Consortium, Asian Genetic Epidemiology Network Type 2
Diabetes (AGEN-T2D) Consortium, South Asian Type 2 Diabetes
(SAT2D) Consortium, Mexican American Type 2 Diabetes
(MAT2D) Consortium, and Type 2 Diabetes Genetic Exploration
by Next-generation sequencing in multi-Ethnic Samples (T2DGENES) ConsortiumGenome-wide trans-ancestry meta-analysis
provides insight into the genetic architecture of type 2 diabetes
susceptibility. Nat Genet 2014; 46: 23444.
Eyre S, Bowes J, Diogo D, Lee A, Barton A, Martin P, et al. Highdensity genetic mapping identies new susceptibility loci for rheumatoid arthritis. Nat Genet 2012; 44: 133640.
Freeman C, Marchini J. GTOOL. 2007. http://www.well.ox.ac.uk/~
cfreeman/software/gwas/gtool.html
Gateva V, Sandling JK, Hom G, Taylor KE, Chung SA, Sun X, et al. A
large-scale replication study identies TNIP1, PRDM1, JAZF1,
UHRF1BP1 and IL10 as risk loci for systemic lupus erythematosus.
Nat Genet 2009; 41: 122833.
Gibbs JR, van der Brug MP, Hernandez DG, Traynor BJ, Nalls MA,
Lai SL, et al. Abundant quantitative trait loci exist for DNA methylation and gene expression in human brain. PLoS Genet 2010; 6:
e1000952.
Gong J, Schumacher F, Lim U, Hindorff LA, Haessler J, Buyske S,
et al. Fine Mapping and Identication of BMI Loci in African
Americans. Am J Hum Genet 2013; 93: 66171.
Gregory AP, Dendrou CA, Atteld KE, Haghikia A, Xifara DK,
Butter F, et al. TNF receptor 1 genetic risk mirrors outcome of
anti-TNF therapy in multiple sclerosis. Nature 2012; 488: 50811.
Hauser SL, Goodin DS. Multiple Sclerosis and other demyelinating
diseases. In: Longo DL, Fauci AS, Kasper DL, Hauser SL,
Jameson JL, Loscalzo J, editors. Harrisons principle of internal medicine. 18th edn. New York: McGraw Hill; 2012.
p. 3395409.
Hinks A, Cobb J, Marion MC, Prahalad S, Sudman M, Bowes J, et al.
Dense genotyping of immune-related disease regions identies 14
new susceptibility loci for juvenile idiopathic arthritis. Nat Genet
2013; 45: 6649.
Howie BN, Donnelly P, Marchini J. A exible and accurate genotype
imputation method for the next generation of genome-wide association studies. PLoS Genet 2009; 5: e1000529.
International Multiple Sclerosis Genetics Consortium (IMSGC)
Network-based multiple sclerosis pathway analysis with GWAS
data from 15,000 cases and 30,000 controls. Am J Hum Genet
2013a; 92: 85465.
International Multiple Sclerosis Genetics Consortium (IMSGC)
Analysis of immune-related loci identies 48 new susceptibility variants for multiple sclerosis. Nat Genet 2013b; 45: 135360.
International Multiple Sclerosis Genetics Consortium (IMSGC),
Wellcome Trust Case Control Consortium 2 (WTCCC2). Genetic
risk and a primary role for cell-mediated immune mechanisms in
multiple sclerosis. Nature 2011; 476: 2149.
Isobe N, Gourraud PA, Harbo HF, Caillier SJ, Santaniello A,
Khankhanian P, et al. Genetic risk variants in African Americans
with multiple sclerosis. Neurology 2013; 81: 21927.
Jacob CO, Eisenstein M, Dinauer MC, Ming W, Liu Q, John S, et al.
Lupus-associated causal mutation in neutrophil cytosolic factor 2
(NCF2) brings unique insights to the structure and function of
NADPH oxidase. Proc Natl Acad Sci USA 2012; 109: E5967.

Downloaded from by guest on June 15, 2015

References

BRAIN 2015: 138; 15181530

1530

| BRAIN 2015: 138; 15181530

Polman CH, Reingold SC, Banwell B, Clanet M, Cohen JA, Filippi M,

et al. Diagnostic criteria for multiple sclerosis: 2010 revisions to the
McDonald criteria. Ann Neurol 2011; 69: 292302.
Pruim RJ, Welch RP, Sanna S, Teslovich TM, Chines PS, Gliedt TP,
et al. LocusZoom: regional visualization of genome-wide association
scan results. Bioinformatics 2010; 26: 23367.
Purcell S, Neale B, Todd-Brown K, Thomas L, Ferreira MA, Bender D,
et al. PLINK: a tool set for whole-genome association and population-based linkage analyses. Am J Hum Genet 2007; 81: 55975.
Reich D, Patterson N, De Jager PL, McDonald GJ, Waliszewska A,
Tandon A, et al. A whole-genome admixture scan nds a candidate
locus for multiple sclerosis susceptibility. Nat Genet 2005; 37:
11138.
Sawcer S, Franklin RJ, Ban M. Multiple sclerosis genetics. Lancet
Neurol 2014; 13: 7009.
Simon KC, Munger KL, Ascherio A. XVI European Charcot
Foundation lecture: nutrition and environment: can MS be prevented? J Neurol Sci 2011; 311: 18.
Stranger BE, Nica AC, Forrest MS, Dimas A, Bird CP, Beazley C, et al.
Population genomics of human gene expression. Nat Genet 2007;
39: 121724.
The 1000 Genomes Project ConsortiumA map of human genome variation from population-scale sequencing. Nature 2010; 467:
106173.
Tishkoff SA, Kidd KK. Implications of biogeography of human populations for race and medicine. Nat Genet 2004; 36: S217.
Trynka G, Hunt KA, Bockett NA, Romanos J, Mistry V, Szperl A,
et al. Dense genotyping identies and localizes multiple common and
rare variant association signals in celiac disease. Nat Genet 2011;
43: 1193201.
Tsoi LC, Spain SL, Knight J, Ellinghaus E, Stuart PE, Capon F, et al.
Identication of 15 new psoriasis susceptibility loci highlights the
role of innate immunity. Nat Genet 2012; 44: 13418.
Wallin MT, Culpepper WJ, Coffman P, Pulaski S, Maloni H,
Mahan CM, et al. The Gulf War era multiple sclerosis cohort: age
and incidence rates by race, sex and service. Brain 2012; 135:
177885.
Wallin MT, Page WF, Kurtzke JF. Multiple sclerosis in US veterans of
the Vietnam era and later military service: race, sex, and geography.
Ann Neurol 2004; 55: 6571.
Wang K, Li M, Hakonarson H. ANNOVAR: functional annotation of
genetic variants from high-throughput sequencing data. Nucleic
Acids Res 2010; 38: e164.
Warnes G, Duffy D, Man M, Qiu W, Lazarus R. GeneticsDesign:
Functions for designing genetics studies. R package version 1.28.0.
2010. http://www.bioconductor.org/packages/release/bioc/html/Gen
eticsDesign.html
Zaykin DV, Shibata K. Genetic ip-op without an accompanying
change in linkage disequilibrium. Am J Hum Genet 2008; 82:
7946.
Zheng X, Levine D, Shen J, Gogarten SM, Laurie C, Weir BS. A highperformance computing toolset for relatedness and principal component analysis of SNP data. Bioinformatics 2012; 28: 33268.

Downloaded from by guest on June 15, 2015

Jostins L, Ripke S, Weersma RK, Duerr RH, McGovern DP, Hui KY,
et al. Host-microbe interactions have shaped the genetic architecture
of inammatory bowel disease. Nature 2012; 491: 11924.
Juran BD, Hirscheld GM, Invernizzi P, Atkinson EJ, Li Y, Xie G,
et al. Immunochip analyses identify a novel risk locus for primary
biliary cirrhosis at 13q14, multiple independent associations at four
established risk loci and epistasis between 1p31 and 7q32 risk variants. Hum Mol Genet 2012; 21: 520921.
Kimbrough DJ, Sotirchos ES, Wilson JA, Al-Louzi O, Conger A,
Conger D, et al. Retinal damage and vision loss in AfricanAmerican multiple sclerosis patients. Ann Neurol 2014; 77: 22836.
Kim-Howard X, Sun C, Molineros JE, Maiti AK, Chandru H,
Adler A, et al. Allelic heterogeneity in NCF2 associated with systemic lupus erythematosus (SLE) susceptibility across four ethnic
populations. Hum Mol Genet 2014; 23: 165668.
Langer-Gould A, Brara SM, Beaber BE, Zhang JL. Incidence of multiple sclerosis in multiple racial and ethnic groups. Neurology 2013;
80: 17349.
Lin PI, Vance JM, Pericak-Vance MA, Martin ER. No gene is an
island: the ip-op phenomenon. Am J Hum Genet 2007; 80:
5318.
Liu JZ, Almarri MA, Gaffney DJ, Mells GF, Jostins L, Cordell HJ,
et al. Dense ne-mapping study identies new susceptibility loci for
primary biliary cirrhosis. Nat Genet 2012; 44: 113741.
Lohr JG, Stojanov P, Lawrence MS, Auclair D, Chapuy B, Sougnez C,
et al. Discovery and prioritization of somatic mutations in diffuse
large B-cell lymphoma (DLBCL) by whole-exome sequencing. Proc
Natl Acad Sci USA 2012; 109: 387984.
Maier LM, Anderson DE, Severson CA, Baecher-Allan C, Healy B,
Liu DV, et al. Soluble IL-2RA levels in multiple sclerosis subjects
and the effect of soluble IL-2RA on immune responses. J Immunol
2009; 182: 15417.
Marchini J, Howie B. Genotype imputation for genome-wide association studies. Nat Rev Genet 2010; 11: 499511.
McDonald WI, Compston A, Edan G, Goodkin D, Hartung HP,
Lublin FD, et al. Recommended diagnostic criteria for multiple sclerosis: guidelines from the International Panel on the diagnosis of
multiple sclerosis. Ann Neurol 2001; 50: 1217.
Morin RD, Mendez-Lago M, Mungall AJ, Goya R, Mungall KL,
Corbett RD, et al. Frequent mutation of histone-modifying genes
in non-Hodgkin lymphoma. Nature 2011; 476: 298303.
Oksenberg JR, Baranzini SE. Multiple sclerosis geneticsis the glass
half full, or half empty? Nat Rev Neurol 2010; 6: 42937.
Oksenberg JR, Barcellos LF, Cree BA, Baranzini SE, Bugawan TL,
Khan O, et al. Mapping multiple sclerosis susceptibility to the
HLA-DR locus in African Americans. Am J Hum Genet 2004; 74:
1607.
Pandit L, Ban M, Sawcer S, Singhal B, Nair S, Radhakrishnan K, et al.
Evaluation of the established non-MHC multiple sclerosis loci in an
Indian population. Mult Scler 2011; 17: 13943.
Patsopoulos NA, Esposito F, Reischl J, Lehr S, Bauer D, Heubach J,
et al. Genome-wide meta-analysis identies novel multiple sclerosis
susceptibility loci. Ann Neurol 2011; 70: 897912.

N. Isobe et al.