PUBLIC HEALTH
ORIGINAL RESEARCH ARTICLE
published: 06 August 2014
doi: 10.3389/fpubh.2014.00112
Genetic associations with plasma B12, B6, and folate levels
in an ischemic stroke population from theVitamin
Intervention for Stroke Prevention (VISP) trial
Keith L. Keene1,2,Wei-Min Chen1,3, Fang Chen1, Stephen R.Williams1, Stacey D. Elkhatib1, Fang-Chi Hsu4,
Josyf C. Mychaleckyj 1,3, Kimberly F. Doheny 5, ElizabethW. Pugh5, Hua Ling5, Cathy C. Laurie6,
Stephanie M. Gogarten6, Ebony B. Madden7, Bradford B.Worrall 3,8* and Michele M. Sale1,3,9 on behalf of
the GARNET Collaborative Research Group
1 Center for Public Health Genomics, University of Virginia, Charlottesville, VA, USA
2 Department of Biology, Center for Health Disparities, East Carolina University, Greenville, NC, USA
3 Department of Public Health Sciences, University of Virginia, Charlottesville, VA, USA
4 Department of Biostatistical Sciences,Wake Forest School of Medicine,Winston Salem, NC, USA
5 Center for Inherited Disease Research, Johns Hopkins University School of Medicine, Baltimore, MD, USA
6 Department of Biostatistics, University ofWashington, Seattle, WA, USA
7 National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, USA
8 Department of Neurology, University of Virginia, Charlottesville, VA, USA
9 Department of Biochemistry & Molecular Genetics, University of Virginia, Charlottesville, VA, USA
Edited by:
Zhigang Li, Dartmouth College, USA
Reviewed by:
Konstantin G. Arbeev, Duke
University, USA
Raghib Ali, University of Oxford, UK
Jinyoung Byun, Dartmouth College,
USA
*Correspondence:
Bradford B.Worrall , Department of
Neurology, University of Virginia
Health System, Charlottesville, PO
Box 800394, VA 22908, USA
e-mail: bbw9r@virginia.edu
Background: B vitamins play an important role in homocysteine metabolism, with vitamin
deficiencies resulting in increased levels of homocysteine and increased risk for stroke.
We performed a genome-wide association study (GWAS) in 2,100 stroke patients from the
Vitamin Intervention for Stroke Prevention (VISP) trial, a clinical trial designed to determine
whether the daily intake of high-dose folic acid, vitamins B6, and B12 reduce recurrent
cerebral infarction.
Methods: Extensive quality control (QC) measures resulted in a total of 737,081 SNPs for
analysis. Genome-wide association analyses for baseline quantitative measures of folate,
Vitamins B12, and B6 were completed using linear regression approaches, implemented in
PLINK.
Results: Six associations met or exceeded genome-wide significance (P ≤5×10−08). For
baseline Vitamin B12, the strongest association was observed with a non-synonymous
SNP (nsSNP) located in the CUBN gene (P =1.76×10−13). Two additional CUBN intronic
SNPs demonstrated strong associations with B12 (P =2.92×10−10 and 4.11×10−10),
while a second nsSNP, located in theTCN1 gene, also reached genome-wide significance
(P =5.14×10−11). For baseline measures ofVitamin B6, we identified genome-wide signif-
icant associations for SNPs at the ALPL locus (rs1697421; P =7.06×10−10 and rs1780316;
P =2.25×10−08). In addition to the six genome-wide significant associations, nine SNPs
(two for Vitamin B6, six for Vitamin B12, and one for folate measures) provided suggestive
evidence for association (P ≤10−07).
Conclusion: Our GWAS study has identified six genome-wide significant associations, nine
suggestive associations, and successfully replicated 5 of 16 SNPs previously reported to
be associated with measures of B vitamins. The six genome-wide significant associations
are located in gene regions that have shown previous associations with measures of B
vitamins; however, four of the nine suggestive associations represent novel finding and
warrant further investigation in additional populations.
Keywords:VISP, association, GWAS, one-carbon metabolism, B12, B6, folate
INTRODUCTION
The B vitamins constitute a group of water-soluble vitamins that
play an important role in human health and cellular functions
including growth and development (1). Vitamins B6 (pyridiox-
ine), B9 (folic acid or folate), and B12 (cobalamin) have garnered
extensive attention for their putative impacts on human health and
diseases, ranging from cardiovascular disease and stroke to neu-
rocognitive function and depression. Specifically, these B vitamins
are critical for the maintenance of red blood cells (2), compo-
nents of the nervous (3), and immune systems (4). Vitamin B6
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Keene et al. GWAS of plasma B12, B6, and folate in VISP
deficiency, most common in the elderly, has been associated with
conditions such as anemia, and neurological abnormalities such
as depression, cognitive dysfunction, and neuropathy (5). Vitamin
B12 deficiency can result in irreversible brain and nervous system
damage and may be responsible for common symptoms such as
fatigue and poor memory (6, 7). Folate (Vitamin B9) is critical for
fetal growth and brain development, therefore folate deficiencies
during pregnancy can result in neural tube defects in babies (8).
In addition, Vitamins B6, B9, and B12 serve as important factors in
homocysteine metabolism, with vitamin deficiencies resulting in
increased levels of homocysteine (9, 10). Although controversial,
elevated homocysteine levels are thought to increase risk for stroke
(11) and vascular disease (12, 13).
Multiple factors contribute to variations in B vitamin levels in
humans. A balanced diet is one approach to help minimize the
detrimental effects of B vitamin deficiency. In January 1998, the
United States Food and Drug Administration required manufac-
turers to fortify bread and grain products with folic acid to help
prevent neural tube defects due to Vitamin B9 deficiency. These
efforts have proven somewhat successful, with estimates from the
2002–2006 National Health and Nutrition Examination Survey
(NHANES) reporting that most Americans are receiving adequate
amounts of folate (14). In contrast, for Vitamin B12, data suggest
that 5–15% of elderly patients are Vitamin B12 deficient, including
data from the Centers for Disease Control and Prevention (CDC)
and the NHANES study (15–17). Poor dietary intake, malabsorp-
tion from food, and genetic predisposition may all cause vitamin
deficiencies. Polymorphisms in genes involved in B vitamin metab-
olism and processing, transport, absorption, and excretion are
logical candidate genes that can influence B vitamin levels. Two
such examples include human conditions Imerslund–Grasbeck
syndrome (IGS) and megaloblastic anemia-1. IGS, a rare auto-
somal recessive disorder caused by mutations in cubilin (CUBN )
and/or amnionless (AMN ), was first characterized in the 1960s
(18, 19) and results in megaloblastic anemia during childhood as
a result of selective malabsorption of Vitamin B12. Additionally,
genetic variants in the CUBN and AMN genes are responsible
for the Finnish and Norwegian types of megaloblastic anemia-1,
respectively (20, 21).
Understanding the genetic factors contributing to vitamin defi-
ciencies offers opportunities for screening and identification of
high-risk individuals before the presentation of any clinical man-
ifestations. To date, several large-scale genome-wide association
studies (GWAS) testing for association with Vitamin B6, B12, and
folate have been published, resulting in more than 10 confirmed
loci for these traits (22–25). Our group has conducted a GWAS for
Vitamin B6, B12, and folate in an effort both to identify novel asso-
ciations and replicate previously reported associations for these
traits in a population of ischemic stroke patients from the Vita-
min Intervention for Stroke Prevention (VISP) clinical trial, an
NIH-funded, multi-center, double-blind, randomized, controlled
clinical trial designed to determine whether the daily intake of
high-dose folic acid, Vitamins B6, and B12 reduced recurrent cere-
bral infarction and a combined vascular endpoint. Unlike the
previous GWAS, the VISP study population represents an ethni-
cally diverse population of older patients that present with elevated
baseline homocysteine levels in the top quartile, have suffered a
stroke, and thus, more closely represent the elderly population
that is most prone to vitamin B deficiency and stroke.
MATERIALS AND METHODS
SUBJECTS
The VISP trial was a multi-center, double-blind, randomized, and
controlled clinical trial that enrolled patients aged 35 or older
with homocysteine levels above the 25th percentile at screening
and a non-disabling cerebral infarction (NDCI) within 120 days
of randomization (26, 27). NDCI was defined as an ischemic brain
infarction not due to embolism from a cardiac source, charac-
terized by the sudden onset of a neurological deficit. The deficit
must have persisted for at least 24 h, or if not, an infarction in
the part of the brain corresponding to the symptoms must have
been demonstrated by CT or MRI imaging. The trial was designed
to determine if daily intake of a multivitamin tablet with high-
dose folic acid, vitamin B6, and vitamin B12 reduced recurrent
cerebral infarction and non-fatal myocardial infarction (MI) or
mortality. Subjects were randomly assigned to receive daily doses
of the high-dose formulation (n= 1,827), containing 25 mg pyri-
doxine (B6), 0.4 mg cobalamin (B12), and 2.5 mg folic acid; or
the low-dose formulation (n= 1,853), containing 200µg pyri-
doxine, 6µg cobalamin, and 20µg folic acid. Enrollment in VISP
began in August 1997, and was completed in December 2002, with
3,680 participants enrolled from 55 clinic sites across the U.S.
and Canada and one site in Scotland. All human research was
approved by the relevant institutional review boards (IRBs), and
conducted according to the Declaration of Helsinki. The VISP
study protocol was approved by the IRBs of Wake Forest School
of Medicine (coordinating center) and the University of North
Carolina at Chapel Hill School of Medicine (statistical center).
The local IRB for each of the individual recruiting sites approved
the VISP protocol and all participants provided written, informed
consent. VISP data analysis by the Genomics and Randomized
Trial Network (GARNET) was approved by University of Virginia
School of Medicine IRB.
GENOME-WIDE ASSOCIATION STUDY IN VISP
A subset of VISP participants provided consent for inclusion in
genetic studies. These participants were included in the GWAS
component of VISP, supported by the National Human Genome
Research Institute (NHGRI), Grant U01 HG005160, as part of the
Genomics and Randomized Trials Network (GARNET); dbGaP
Study Accession: phs000343.v3.p1. Samples were genotyped at
the Johns Hopkins Center for Inherited Disease Research (CIDR),
using the Illumina HumanOmni1-Quad_v1-0_B BeadChip (Illu-
mina, San Diego, CA, USA). Individuals were excluded if they
were unexpected duplicates or had gender discrepancies. A total
of 2,100 individuals were included in the final genetic analyses;
summary statistics are provided in Table 1. These subjects con-
sisted of 1,725 individuals of European descent, 258 individuals of
African descent, and 117 individuals classified as others.
BIOMARKER MEASUREMENTS IN VISP
As previously described (28), basal levels of folate and Vitamin
B12 were determined by the central laboratory at Oregon Regional
Primate Research Center using single radioassays of folate and Vit-
amin B12 (Bio Rad Quantaphase II, Bio Rad Diagnostics, Hercules,
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Keene et al. GWAS of plasma B12, B6, and folate in VISP
Table 1 | Demographic summary statistics.
Number of individuals (EA/AA/other) 2100 (1725/258/117)
Age (years)
Mean±SD 67.2±10.8
Range 35–89
% Female participants (N) 37.4 (785)
Current smokers (%) 15.6
Hypertension (%) 71.0
Diabetes mellitus (%) 27.1
B Vitamin baseline measures
Vitamin B6 (pm/mL)±SD 42.45±37.38
Median 33.49
Vitamin B12 (pg/mL)±SD 358.79±181.91
Median 326
Folate (ng/mL)±SD 25.86±15.91
Median 22.67
CA, USA). For measures of Vitamin B6, EDTA plasma samples
were analyzed using a commercially available method for plasma
Pyridoxine 5′ Phosphate (ALPCO Inc Windham, NH, USA). The
principle of the assay is as follows: 3H-tyrosine is decarboxylated
by the vitamin B6 dependent enzyme tyrosine apodecarboxlase to
3H-tyramine. The activity of tyrosine apodecarboxlase is quanti-
tatively dependent on the amount of PLP present in the reaction
mixture. The 3H-tyramine thus produced is selectively extracted
into the scintillation cocktail and can be measured by liquid scin-
tillation counting. The excess 3H-tyrosine remains in the aqueous
phase and is not measured.
STATISTICAL ANALYSES
Extensive quality control (QC) measures were performed, result-
ing in a total of 737,081 SNPs for analysis. QC measures included
filtering SNPs based on missing call rate, Mendelian errors in
control trios, deviation from Hardy–Weinberg equilibrium in con-
trols, discordant calls in duplicate samples, sex differences in allele
frequency or heterozygosity, and minor allele frequency (MAF)
(29). Briefly, samples were clustered and genotypes determined
using GenomeStudio (version 2010.2). For initial QC, SNPs meet-
ing one or more of the following criteria were excluded: call
rate <85%, more than one replicate HapMap error, or cluster
separation <0.2. Genotype calls for individual chromosomes in
samples with large chromosomal anomalies (>10 Mb) or miss-
ing call rate >5% were filtered out. Furthermore, samples with
overall missing call rates >5% and SNPs with call rates <95%
and Hardy–Weinberg P-values ≤10−4 were excluded from sub-
sequent analyses. Multidimensional Scaling (MDS), utilizing the
software KING (30), was performed to address confounders due
to population substructure. Genome-wide association analyses for
baseline quantitative measures of folate, and Vitamins B12 and B6
were performed using linear regression approaches, assuming an
additive model, as implemented in PLINK; using age, sex, and
the top 10 principal components as covariates. Inverse normal
transformation was performed for each of the quantitative traits,
prior to analysis. Inverse normal transformations were used to
maintain ranks of the trait for each individual and minimize the
impact of outliers while also allowing for sufficient power. Regres-
sion coefficients (beta), coefficient T-statistic (STAT), andP-values
(asymptotic P-value for T -statistic) were calculated for the tested
(minor) allele. The proportion of total variance explained (h2) was
calculated as h2=Beta2× 2×MAF× (1-MAF). Our GWAS scan
results showed no evidence for inflation (GC lambda≤ 1.013 in
all scans.).
RESULTS
We identified six associations that meet or exceed genome-wide
significance (P ≤ 5× 10−08; Table 2; Figure S1 in Supplemen-
tary Material). Of these six associations, four were for Vitamin
B12, while the remaining two were for Vitamin B6. The strongest
evidence of association was observed for baseline Vitamin B12
(P = 1.76× 10−13; beta=−0.22) with a non-synonymous SNP
(nsSNP), located on chromosome 10 in the CUBN gene. Two
additional CUBN intronic SNPs (Figure 1) were also strongly
associated with Vitamin B12 (P = 2.92× 10−10; beta=−0.19 and
4.11× 10−10; beta=−0.18). A second nsSNP, located on chro-
mosome 11 in the transcobalamin 1 (TCN1) gene (Figure 2),
was also associated with baseline measures of Vitamin B12
(P = 5.14× 10−11; beta=−0.29). The two genome-wide signifi-
cant associations for Vitamin B6 measures (Figure 3) were located
in the alkaline phosphatase (ALPL), liver/bone/kidney gene
region (rs1697421; P = 7.06× 10−10, beta= 0.173 and rs1780316;
P = 2.25× 10−08; beta=−0.325). Although not reaching the
genome-wide significance threshold, our GWAS study has iden-
tified nine additional SNPs with suggestive (P ≤ 10−07) evidence
of association, two for measures of Vitamin B6, six for measures
of Vitamin B12, and one for measures of folate (Table 2). A clus-
ter of suggestive associations for Vitamin B12 was observed on
chromosome 19, near the fucosyltransferase 2 (FUT2) gene. P-
Values for the four associated SNPs ranged from 9.33× 10−07 to
2.67× 10−07. Individually, each associated SNP explains only a
small amount of the variance (h2) for each trait (ranging from
0.009 to 0.021).
In addition to our genome-wide analyses, we performed a look-
up of 16 SNPs previously associated with measures of vitamin B6
(n= 1) (22), vitamin B12 (n= 12) (22–24, 31), and folate (n= 3)
(22, 23, 25). Of the 16 SNPs previously reported in the literature,
genotype data were available for 10 of the reported SNPs, while
surrogate SNPs in high linkage disequilibrium (LD) (r2 > 0.9) or
the most significant SNP within 100 kb of the reported SNP were
reported for the remaining six SNPs. Accounting for multiple test-
ing (P = 0.05/16), five of the 16 SNPs, located in ALPL, MS4A3,
TCN1, CUBN, and FUT2, were successfully replicated (P ≤ 0.003)
in our study (Table 3; Table S1 in Supplementary Material). For
comparison, association results for the untransformed B vitamin
measures are reported in Table S2 in Supplementary Material.
DISCUSSION
We performed a GWAS for measures of Vitamin B12, Vitamin
B6, and folate by evaluating 737,081 SNPs in 2,100 participants
from the Vitamin Intervention for Stroke Prevention (VISP) clin-
ical trial. We observed six associations that reached genome-wide
significance (P ≤ 10−08), an additional nine SNPs with sugges-
tive (P ≤ 10−07) evidence of association, while replicating five
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Keene et al. GWAS of plasma B12, B6, and folate in VISP
Table 2 | Association results for SNPs with genome-wide (P ≤5×10−08) significance or suggestive evidence (P ≤10−07) for association.
SNP Chromosome Location
(bp)*
Minor
allele
Minor
allele
frequency
Beta STAT Variance
explained
P -value
(bold indicates
P <5×10−08)
Gene
(bold indicates
novel finding)
VITAMIN B6
rs12118362 1 21644584 A 0.213 0.172 5.228 0.010 1.91×10−07 NBPF3
rs1697421 1 21695879 T 0.471 0.173 6.197 0.015 7.06×10−10 ALPL (nearest)
rs1780316 1 21762222 T 0.060 −0.325 −5.616 0.012 2.25×10−08 ALPL
rs2267739 7 31103422 G 0.153 −0.241 −4.910 0.015 9.92×10−07 ADCYAP1R1
VITAMIN B12
rs7893634 10 17121145 A 0.415 0.143 4.972 0.010 7.22×10−07 CUBN
rs11254363 10 17170699 C 0.258 0.155 4.918 0.009 9.48×10−07 CUBN
rs12261966 10 17183006 A 0.311 −0.185 −6.336 0.015 2.92×10−10 CUBN
rs1801222 10 17196157 A 0.316 −0.218 −7.419 0.021 1.76×10−13 CUBN
rs11254375 10 17199198 G 0.312 −0.184 −6.282 0.015 4.11×10−10 CUBN
rs34324219 11 59379954 A 0.104 −0.292 −6.604 0.016 5.14×10−11 TCN1
SNP19-53897957⇓ 19 53897957 C 0.483 0.142 5.162 0.010 2.69×10−07 FUT2
rs516246 19 53897984 A 0.482 0.142 5.164 0.010 2.67×10−07 FUT2
rs492602 19 53898229 C 0.482 0.143 5.193 0.010 2.29×10−07 FUT2
rs2287921 19 53920084 C 0.471 0.140 4.921 0.010 9.33×10−07 RASIP1
FOLATE
rs12611820 2 2462633 C 0.245 −0.169 −4.913 0.011 9.75×10−07 MYT1L (nearest)
* Based on hg18
⇓Corresponds to SNP rs516316
FIGURE 1 | LocusZoom (49) association plot for single SNP associations with Vitamin B12 at the CUBN locus. The SNP position and -LOG (P -value) are
plotted on the X andY axis, respectively.
previously reported SNP associations. The most convincing asso-
ciations were observed for measures of Vitamin B12 at the CUBN
and TCN1 loci and the ALPL locus for measures of Vitamin B6.
Although we did not observe any genome-wide significant associ-
ations for folate, we did detect suggestive evidence for association
(P = 9.75× 10−07) near theMYT1L gene, located on chromosome
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Keene et al. GWAS of plasma B12, B6, and folate in VISP
FIGURE 2 | LocusZoom association plot for single SNP associations with Vitamin B12 at theTCN1 locus. The SNP position and -LOG (P -value) are plotted
on the X andY axis, respectively.
FIGURE 3 | LocusZoom association plot for single SNP associations with Vitamin B6 at theALPL locus. The SNP position and -LOG (P -value) are plotted
on the X andY axis, respectively.
2. Interestingly, genetic variations at this locus have been associ-
ated with depression (32) and schizophrenia (33–35). This locus
may help explain the recent data positively correlating serum folate
levels with cognitive test scores in children (36); suggesting further
evaluation of the effects of folate levels in the elderly are warranted.
The most robust associations for Vitamin B12 levels were
observed at the CUBN, FUT2, and TCN1 loci (Table 2). A clus-
ter of five SNPs spanning the CUBN gene provided evidence for
association with Vitamin B12 measures (P-values ranging from
9.48× 10−07 to 1.75× 10−13). The most significantly associated
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Keene et al. GWAS of plasma B12, B6, and folate in VISP
Table 3 | Significant associations of 16 previously reported associations of Vitamin B6,Vitamin B12, and folate.
SNP Chromosome Gene Literature P -value Reference Surrogate SNP VISP P -value
VITAMIN B6
rs1256335 1 ALPL 1.40×10−15 (22) – 3.41×10−05
VITAMIN B12
rs1801222 10 CUBN 2.87×10−09 (22) – 1.76×10−13
rs526934 11 TCN1 2.25×10−10 (22) – 3.38×10−06
rs2298585 11 MS4A3 2.64×10−15 (24) rs7929589 8.67×10−04
rs1047781 19 FUT2 3.62×10−36 (24) rs516246 2.67×10−07
SNP in this region, rs1801222, was a non-synonymous variant
resulting in a missense mutation, Phenylalanine to Serine. These
results were not surprising considering rs1801222 was previ-
ously associated with Vitamin B12 measures (22) and the protein
expressed by CUBN forms a receptor complex responsible for Vit-
amin B12 internalization in the ileum (37). Furthermore, genetic
variants in CUBN are responsible for the Finnish type of mega-
loblastic anemia-1 (38) in humans and more broadly for IGS in
canines as well (39, 40). A second cluster of suggestive associa-
tions near FUT2 gene were consistent with previously reported
associations in this region (24, 25, 41).
A second missense mutation (rs34324219), located in the Vit-
amin B12 binding protein, TCN1 gene was associated with base-
line measures of Vitamin B12 (P = 5.148× 10−11). The nsSNP,
rs34324219, results in an Aspartic acid to Tyrosine substitution and
represents the second most significant association in our study. In
the same VISP population, our group previously detected asso-
ciations between genetic variants of the related gene, TCN2, and
recurrent stroke risk (42). Although TCN1 is a logical candidate
gene influencing Vitamin B12 measures in this region, associa-
tions with variants in the nearby (~200 kb) MS4A3 gene (24)
suggest that multiple genes in this region may impact Vitamin
B12 levels. In an attempt to replicate the associations observed in
MS4A3 by Lin et al. (24) (rs2298585), we detected modest evi-
dence of association for the surrogate SNP, rs7929589 (r2= 0.39;
P = 8.67× 10−04; Table 3). The protein encoded by MS4A3 has
been proposed to function as a hematopoietic cell cycle regulator
(43), another potential link to the anemia observed in individuals
with Vitamin B12 deficiency (44).
For measures of Vitamin B6, associations at the ALPL locus
were most robust. Two variants at this locus reached genome-
wide significance (rs1697421; P = 7.06× 10−10 and rs1780316;
P = 2.25× 10−08). GWAS associations for variants near ALPL
have been reported for Vitamin B6 (22). In addition, this region
also harbors GWAS associations with traits ranging from TNFα
response in patients with rheumatoid arthritis (45) to hemato-
logic traits (46). While the physiological function of ALPLs are
unknown, and no direct correlations have been made between
ALPL variants and cognitive function, tissue non-specific ALPL
is increased in Alzheimer’s disease patients (47). Furthermore,
Alzheimer’s disease patients have an increased risk of suffering
a stroke (48).
The data were collected as part of a randomized clinical trial is
a systematic and standardized fashion, which is a major strength
of the study. VISP used centralized laboratory analysis on all
samples that complied with strict quality standards. The study
population all had ischemic stroke and had elevated measures of
serum homocysteine, which might limit generalizability. However,
we replicated a substantial proportion of the previously identified
genetic variants from studies using a more “general population.”
All participants in the VISP clinical trial were 35 years of age or
older and suffered a stroke within 120 days of enrollment. This
study population also represents an older group of individuals
(mean age 67.2 years) that is most prone to vitamin deficiency and
subsequent public health concerns including dementia and stroke.
We are unable to make any comparisons in normal, healthy indi-
viduals, or assess the relation of such associations on stroke risk
and other vascular disorders; however, collectively,our finding may
provide some insight into the genetic factors influencing measures
of B vitamins, in a vulnerable population. Although some dietary
measures were collected as part of the VISP trial, we were not able
to incorporate dietary “exposure” as a covariate in our analyses.
Thus, we cannot identify gene by environmental interactions.
In summary, we performed a GWAS for measures of Vitamin
B6, B12, and folate observing six genome-wide significant associ-
ations, nine suggestive associations, and successfully replicating 5
of 16 SNPs previously reported in the literature. Our study is the
first of its kind evaluating genetic contributors for measurements
of B vitamins in a stroke population. Additionally, this knowledge
could lead to genetic screening approaches, which could identify
pre-symptomatic individuals that could benefit from interven-
tions such as enhanced vitamin supplementation prior to clinical
manifestations.
AUTHORS CONTRIBUTION
Keith L. Keene – performed locus specific analyses, drafted man-
uscript, and constructed primary tables and figures. Wei-Min
Chen – lead VISP statistical analyst, reviewed and edited man-
uscript. Fang Chen – performed initial GWAS analyses under the
supervision of Wei-Min Chen. Stephen R. Williams – assisted with
figures and summary statistics, reviewed and edited manuscript.
Stacey D. Elkhatib – conducted initial review of literature for
GWAS of B vitamin phenotypes, ran analyses of several candidates
prior to GWAS data, reviewed and edited manuscript. Fang-Chi
Hsu – contributed to the overall GWAS design and the writing
of the manuscript. Josyf C. Mychaleckyj – assisted with statistical
analyses, reviewed and edited manuscript. Kimberly F. Doheny –
generation of GWAS data and QC of GWAS data, reviewed and
edited manuscript. Elizabeth W. Pugh – generation of GWAS data
and QC of GWAS data, reviewed and edited manuscript. Hua
Frontiers in Public Health | Epidemiology August 2014 | Volume 2 | Article 112 | 6
Keene et al. GWAS of plasma B12, B6, and folate in VISP
Ling – generation of GWAS data and QC of GWAS data, reviewed
and edited manuscript. Cathy C. Laurie – quality control of the
VISP dataset, assisted with statistical analysis, reviewed and edited
manuscript. Stephanie M. Gogarten – quality control of the VISP
dataset, reviewed and edited manuscript. Ebony B. Madden –
Program Director for the project and made contributions to the
writing of the manuscript. Bradford B. Worrall – Co-Principal
investigator on GARNET, contributed to the design and analysis
plan for paper, and made contributions to the writing of the man-
uscript. Michele M. Sale – Co-Principal investigator on GARNET,
contributed to the design and analysis plan for paper, and made
contributions to the writing of the manuscript.
ACKNOWLEDGMENTS
Study recruitment and collection of datasets for the VISP clini-
cal trial were supported by an investigator-initiated research grant
(R01 NS34447; PI James Toole) from the National Institute of Neu-
rological Disorders and Stroke. GWAS genotyping was provided
by the Center for Inherited Disease Research (U01 HG004438l; PI
David Valle). Assistance with genetic data cleaning was provided
by the GARNET Coordinating Center (U01 HG005157; PI Bruce
S. Weir).
SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found
online at http://www.frontiersin.org/Journal/10.3389/fpubh.2014.
00112/abstract
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Conflict of Interest Statement: The authors declare that the research was conducted
in the absence of any commercial or financial relationships that could be construed
as a potential conflict of interest.
Received: 12 May 2014; accepted: 21 July 2014; published online: 06 August 2014.
Citation: Keene KL, Chen W-M, Chen F, Williams SR, Elkhatib SD, Hsu F-C,
Mychaleckyj JC, Doheny KF, Pugh EW, Ling H, Laurie CC, Gogarten SM, Mad-
den EB, Worrall BB and Sale MM on behalf of the GARNET Collaborative Research
Group (2014) Genetic associations with plasma B12, B6, and folate levels in an ischemic
stroke population from the Vitamin Intervention for Stroke Prevention (VISP) trial.
Front. Public Health 2:112. doi: 10.3389/fpubh.2014.00112
This article was submitted to Epidemiology, a section of the journal Frontiers in Public
Health.
Copyright © 2014 Keene, Chen, Chen,Williams, Elkhatib, Hsu,Mychaleckyj, Doheny,
Pugh, Ling , Laurie, Gogarten, Madden, Worrall and Sale on behalf of the GARNET
Collaborative ResearchGroup. This is an open-access article distributed under the terms
of the Creative Commons Attribution License (CC BY). The use, distribution or repro-
duction in other forums is permitted, provided the original author(s) or licensor are
credited and that the original publication in this journal is cited, in accordance with
accepted academic practice. No use, distribution or reproduction is permitted which
does not comply with these terms.
Frontiers in Public Health | Epidemiology August 2014 | Volume 2 | Article 112 | 8