Transcobalamin II Receptor Polymorphisms Are Associated with Increased Risk for Neural Tube Defects

Abstract

Women who have low cobalamin (vitamin B(12)) levels are at increased risk for having children with neural tube defects (NTDs). The transcobalamin II receptor (TCblR) mediates uptake of cobalamin into cells. Inherited variants in the TCblR gene as NTD risk factors were evaluated.
Case-control and family-based tests of association were used to screen common variation in TCblR as genetic risk factors for NTDs in a large Irish group. A confirmatory group of NTD triads was used to test positive findings.
2 tightly linked variants associated with NTDs in a recessive model were found: TCblR rs2336573 (G220R; p(corr)=0.0080, corrected for multiple hypothesis testing) and TCblR rs9426 (p(corr)=0.0279). These variants were also associated with NTDs in a family-based test before multiple test correction (log-linear analysis of a recessive model: rs2336573 (G220R; RR=6.59, p=0.0037) and rs9426 (RR=6.71, p=0.0035)). A copy number variant distal to TCblR and two previously unreported exonic insertion-deletion polymorphisms were described.
TCblR rs2336573 (G220R) and TCblR rs9426 represent a significant risk factor in NTD cases in the Irish population. The homozygous risk genotype was not detected in nearly 1000 controls, indicating that this NTD risk factor may be of low frequency and high penetrance. 9 other variants are in perfect linkage disequilibrium with the associated single nucleotide polymorphisms. Additional work is required to identify the disease-causing variant. Our data suggest that variation in TCblR plays a role in NTD risk and that these variants may modulate cobalamin metabolism.

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doi: 10.1136/jmg.2009.073775
published online June 24, 2010J Med Genet
F Pangilinan, A Mitchell, J VanderMeer, et al.
defects
associated with increased risk for neural tube
Transcobalamin II receptor polymorphisms are
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Transcobalamin II receptor polymorphisms are
associated with increased risk for neural tube defects
F Pangilinan,
1
A Mitchell,
1
J VanderMeer,
1
A M Molloy,
2
J Troendle,
3
M Conley,
3
P N Kirke,
4
M Sutton,
4
J M Sequeira,
5
E V Quadros,
5
J M Scott,
6
J L Mills,
3
L C Brody
1,7
ABSTRACT
Objective Women who have low cobalamin (vitamin B
12
)
levels are at increased risk for having children with neural
tube defects (NTDs). The transcobalamin II receptor(TCblR)
mediates uptake of cobalamin into cells. Inherited variants
in the TCblR gene as NTD risk factors were evaluated.
Methods Caseecontrol and family-based tests of
association were used to screen common variation in
TCblR as genetic risk factors for NTDs in a large Irish
group. A confirmatory group of NTD triads was used to
test positive findings.
Results 2 tightly linked variants associated with NTDs in
a recessive model were found: TCblR rs2336573 (G220R;
p
corr
¼0.0080, corrected for multiple hypothesis testing)
and TCblR rs9426 (p
corr
¼0.0279). These variants were
also associated with NTDs in a family-based test before
multiple test correction (log-linear analysis of a recessive
model: rs2336573 (G220R; RR¼6.59, p¼0.0037) and
rs9426 (RR¼6.71, p¼0.0035)). A copy number variant
distal to TCblR and two previously unreported exonic
insertionedeletion polymorphisms were described.
Conclusions TCblR rs2336573 (G220R) and TCblR
rs9426 represent a significant risk factor in NTD cases in
the Irish population. The homozygous risk genotype was
not detected in nearly 1000 controls, indicating that this
NTD risk factor may be of low frequency and high
penetrance. 9 other variants are in perfect linkage
disequilibrium with the associated single nucleotide
polymorphisms. Additional work is required to identify the
disease-causing variant. Our data suggest that variation in
TCblR plays a role in NTD risk and that these variants may
modulate cobalamin metabolism.
Neural tube defects (NTDs) are common birth
defects, affecting approximately 1 in 1000 preg-
nancies.
1 2
The neural tube closes during the
4 weeks of embryogenesis and gives rise to the
spinal cord and brain. Incomplete closure of
the neural tube causes a range of birth defects
including spina bifida and anencephaly.
NTD aetiology is multifactorial and includes
environmental and genetic factors.
3 4
Periconcep-
tional folic acid supplementation in mothers can
reduce the risk of an NTD-affected pregnancy by
up to 70%.
56
Genetic variants relating to folate
metabolism have also been implicated in contrib-
uting to NTD risk. The most well studied of these
is the 677C/T variant in the methylene tetrahy-
drofolate reductase (MTHFR) gene (reviewed by
Botto and Yang
7
). In the European population, this
variant may account for 26% of NTDs.
8
Cobalamin (vitamin B
12
) plays an important role
in folate metabolism as a cofactor for methionine
synthase (MTR), the enzyme that catalyses the
transfer of the methyl group from 5-methyl tetra-
hydrofolate to homocysteine to produce methio-
nine. Compromised methionine synthase function
results in functional intracellular folate deficiency
and a deficit of methyl groups for methylation
reactions. Low maternal blood
9e17
or
amniotic fluid
18e22
cobalamin levels have been
associated with NTD risk. Levels of the cobala-
minetranscobalamin II complex (holoTC) are also
reduced in mothers during
23
or after
24
an NTD
pregnancy.
Polymorphisms in cobalamin-related genes have
been investigated for NTD risk. Several transporter
proteins, receptors and converting enzymes are
required to ensure that dietary cobalamin is deliv-
ered in an active form to methionine synthase and
methylmalonyl coenzyme A mutase (MUT).
Transcobalamin II (TCN2) transports cobalamin
from the intestinal circulation into the bloodstream
and ultimately to target tissues.
25
TCN2 P259R
(rs1801198) was reported to be a maternal NTD
risk factor,
26
although other studies examined
multiple TCN2 variants without detecting an
effect.
24 27
Methionine synthase reductase (MTRR)
rereduces oxidised cobalamin, thereby maintaining
methionine synthase activity. Although some
studies found no association between NTDs and
variants in MTR or MTRR,
28e32
others observed
independent and/or joint effects.
16 33e37
Cubilin
(CUBN) is a receptor found on the luminal
epithelium of the intestine and the kidney that
binds the cobalamineintrinsic factor complex. An
intronic single nucleotide polymorphism (SNP,
rs1907362) in CUBN has been associated with
NTDs.
38
Uptake of the cobalaminetranscobalamin II
complex into cells and tissues occurs via binding to
the recently identified transcobalamin II receptor
(TCblR, also known as CD320 and 8D6).
39
Because
of TCblR’sessential role in cobalamin bioavail-
ability, we evaluated its genetic variation for asso-
ciation with NTDs in a large Irish cohort. We report
a significantly associated genetic risk factor for
NTDs in TCblR.
MATERIALS AND METHODS
Study populations
Recruitment of our Irish case and control popula-
tions has been previously described.
40e42
Our
complete Irish case population consists of 586
families with an NTD-affected child. This includes
442 full family triads (case, mother and father), 57
1
Molecular Pathogenesis
Section, Genome Technology
Branch, National Human
Genome Research Institute,
Bethesda, Maryland, USA
2
Department of Clinical
Medicine, Trinity College Dublin,
Dublin, Ireland
3
Division of Epidemiology,
Statistics and Prevention
Research, The Eunice Kennedy
Shriver National Institute of
Child Health and Human
Development, Department of
Health and Human Services,
National Institutes of Health,
Bethesda, Maryland, USA
4
Child Health Epidemiology Unit,
Health Research Board, Dublin,
Ireland
5
Departments of Medicine and
Cell Biology, The State
University of New York
Downstate Medical Center,
Brooklyn, New York, USA
6
Department of Biochemistry,
Trinity College Dublin, Dublin,
Ireland
7
Molecular Pathogenesis
Section, Genome Technology
Branch, National Human
Genome Research Institute,
Bethesda, Maryland, USA
Correspondence to
Lawrence C Brody, Molecular
Pathogenesis Section, Genome
Technology Branch, National
Human Genome Research
Institute, Room 5306, Building
50, 50 South Drive, MSC 8004,
Bethesda, MD 20892-8004,
USA; lbrody@helix.nih.gov
Received 6 October 2009
Revised 16 December 2009
Accepted 1 February 2010
Pangilinan F, Mitchell A, VanderMeer J, et al.J Med Genet (2010). doi:10.1136/jmg.2009.073775 1 of 9
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caseemother pairs, 4 caseefather pairs, 44 cases only and 39
families with parents or single parent only. In total, there are 530
spina bifida and 21 encephalocoele cases. Our control samples
(n¼999) were randomly selected from a bank of 56 049 blood
samples taken from women during their first prenatal visit at
the three major maternity hospitals in Dublin between 1986 and
1990, after excluding samples from women whose current or
past pregnancies involved an NTD.
An additional 367 case families were recruited in the UK
between 2001 and 2003, with the assistance of the UK Associ-
ation for Spina Bifida and Hydrocephalus (ASBAH). This
cohort includes 258 full family triads, 66 caseemother pairs, 2
caseefather pairs, 38 caseeonly samples and 3 families with
parents only. There are 354 spina bifida cases, 8 encephalocoele
cases and 2 cases with spina bifida and encephalocoele. All the
UK families gave buccal swab samples collected using the
protocol of Meulenbelt et al
43
and completed questionnaires
detailing the case’s NTD type, maternal pregnancy history and
family history of birth defects.
These cohorts are comparable in folic acid supplementation.
Of the Irish NTD mothers, 91 definitively answered whether
they were taking any folic acid or multivitamins in the month
before their last monthly period; 23 (25.3%) were on supple-
ments. Of the UK NTD mothers, 73 definitively answered the
same question; 23 (31.5%) were on supplements. Additionally,
the distributions of case birth years in the two cohorts were
similar and largely predated the recommendation of folate
supplementation by the United States Public Health Service in
1992. Case birth years in the Irish NTD cohort ranged from 1938
to 2003 with a median of 1981 and an SD of 11 years. Case birth
years in the UK NTD cohort ranged from 1926 to 2001 with
a median of 1978 and an SD of 15 years.
The sample collections were approved by the Health Research
Board Research Ethics Committee (Dublin, Ireland), the UK
Multi-Centre Research Ethics Committee (University of
Newcastle, UK) in collaboration with UK ASBAH and the
Institutional Review Board at the National Human Genome
Research Institute (Bethesda, Maryland, USA). Written consent
was obtained from all the participants.
Genomic DNA was extracted from all the blood samples and
buccal swabs using the QIAamp DNA Blood Mini Kit (Qiagen,
Valencia, CA, USA).
African American control DNA (HD100AA) and Caucasian
control DNA (HD200CAU) samples were purchased from the
Coriell Cell Repositories, Camden, NJ, USA.
SNP selection and discovery
To select a set of SNPs to capture common genetic variation in
TCblR, we evaluated SNPs genotyped by HapMap.
44
SNPs
within and up to 10 kilobases (kb) from the gene were consid-
ered. A minimum SNP set representing all such HapMap SNPs
was manually selected, while allowing exclusion of redundant
SNPs (r
2
>0.8).
Up to 18 Irish individuals, most of which were drawn from
NTD families, were screened for unreported polymorphisms.
Primers were designed to polymerase chain reaction
(PCR)eamplify exons 1 through 5; the resulting amplicons were
analysed via fluorescent automated dideoxy DNA sequencing.
Genotyping
Multiplex ligation-dependent probe amplification (MLPA) was
used to estimate the relative copy number for the 14.8 kb copy
number variant (CNV) immediately downstream from TCblR.
Briefly, probe pairs were designed to hybridise to test and control
loci (primer sequences available upon request). The multiplex
reaction contains probe pairs to detect five test loci within the
CNV. The control loci included four single copy regions in
TCblR, and one single copy probe was placed in TCN2 for rela-
tive quantification. A locus in the androgen receptor was
included as an additional control to ensure that the method
detects copy number differences of the X chromosome.
The MLPA reaction was performed as directed with the
SALSA kit reagents obtained from MRC-Holland (Amsterdam,
The Netherlands) using at least 50 ng genomic DNA for each
sample. After hybridisation and ligation, products were PCR
amplified and analysed on an ABI 3100 Genetic Analyser
(Applied Biosystems, Foster City, CA, USA). Comparisons of
amplicon peak areas were used to estimate relative DNA quan-
tity scores for each locus. A real-time PCR assay using the
Applied Biosystems 7900HT Fast Real-Time PCR System
(Applied Biosystems) was used to independently estimate copy
number in a subset of samples with good correlation.
All other variants were genotyped by detection of allele-
specific extension products via matrix-assisted laser desorption/
ionisationetime of flight (MALDI-TOF) mass spectrometry
(Sequenom, San Diego, CA, USA). Primer sequences and assay
conditions are available upon request.
Genotyping quality was assessed in each sample population.
More than 10% of the Irish samples were repeated with $99%
concordance for all the variants. More than 8% of the UK
samples were repeated with $98% concordance for all the
variants. More than 7% of the Caucasian (Coriell) samples were
repeated with 100% concordance for all the variants. More than
10% of the African American (Coriell) samples were repeated
with 100% concordance for all but two variants (E88del and
rs9426, one discrepant sample each).
The mean genotyping call rates (ie, success rates) were 97%
for the Irish and UK samples. Call rates were at least 95% for all
the variants in all the groups with the exception of rs4147651 in
the Irish NTD cases (94%) and rs2232775 (Q8R) in the Irish
controls (93%). The mean genotyping call rates were 98% in the
Caucasian Coriell samples and 92% in the African American
Coriell samples. Call rates were at least 93% for all the variants
in both Coriell groups with the exception of rs250503 (87%) in
African Americans.
Only the NTD cases from Ireland were observed to deviate
from the HardyeWeinberg equilibrium (HWE, p<0.01): the
rs2232775 (Q8R) NTD cases (
c
2
¼15.4, p¼8.9310
05
), the
rs2336573 (G220R) NTD cases (
c
2
¼27.8, p¼1.3E
07
), the rs9426
NTD cases (
c
2
¼21.2, p¼7.2E
06
), and the E88del NTD cases
(
c
2
¼51.1, p¼8.7E
13
). These four SNPs were in HWE in all the
other genotyped groups. Additionally, the first three SNPs are in
strong r
2
linkage disequilibrium (LD; r
2
$0.94), and similar
genotyping results were obtained with these independent
genotype assays. The E88del polymorphism has a low minor
allele frequency (MAF; p=0.02), which may account for its
failure to adhere to HWE in the NTD cases.
Discordant genotypes and triads exhibiting noneMendelian
inheritance for any single marker were excluded for that marker.
Twenty-four samples with discrepancies for >1 marker were
excluded from all the analyses.
Haplotype analysis
LD in the region was estimated using Haploview (http://www.
broad.mit.edu/mpg/haploview/).
45
Haplotype blocks were
defined based on D9values using the solid spine of LD option in
Haploview. Haplotype frequency estimates based on these block
definitions were then generated for Irish NTD cases, NTD
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mothers and controls using PHASE V.2.1.
46 47
A permutation
test within PHASE V.2.1 was used to test whether haplotype
frequency distributions differed between the controls and the
NTD cases or the NTD mothers in the Irish cohort.
Statistical analyses
A logistic regression model, with the number of risk alleles as the
independent variable, was used as a primary test to evaluate
each polymorphism for NTD association. In this multiplicative
model, the odds ratio (OR) of two risk alleles is the square of the
OR of one risk allele. Additionally, one degree of freedom (DOF)
models of dominant and recessive genetic risk were applied via
logistic regression. These tests are not valid when any of the
genotypes cells are empty; in these cases, the Fisher exact test
was used in place of the logistic regression to test for genotypic
differences. These tests were used to perform NTD caseecontrol
comparisons and NTD motherecontrol comparisons in the Irish
NTD cases, the NTD mothers and the controls.
The (mother, father and case) triads were analysed by fitting
multiplicative, dominant and recessive log-linear models with
one DOF to test for case effects and one DOF to test for direct
maternal effects.
48
The case term in the multiplicative log-linear
model provides comparable information to the transmission
disequilibrium test.
49
These analyses were modified to incorpo-
rate data from incomplete triads by using the expectation
maximisation algorithm.
50
These family-based tests were
applied to the Irish and UK NTD triads to test TCblR variants
for association with NTDs.
Correction for multiple tests was by permutation (99 999
random permutations). This method accounts for any LD
(non-independence) between evaluated SNPs. Multivariate
permuting of triads for log-linear analysis involved treating the
test as a one-sample test and permuting the hypothetical risk
allele. Permutations of the cases and the controls were inde-
pendent of permutations of triads, and the results were
combined by Bonferroni adjustment so that the resulting
adjusted p values accounted for all tests and all SNPs
while controlling the chance of any false positive (familywise
error) at 5%.
RESULTS
Genomic analysis
The TCblR gene spans approximately 6 kb on chromosome 19
(figure 1a). It is flanked by a gene encoding a subunit of
ubiquinone, NDUAF7, and the LAG1 longevity assurance
homologue 4 (LASS4) gene, which encodes a ceramide synthase.
TCblR and NDUFA7 are in close proximity (approximately 3 kb)
and transcribed from the same strand. In contrast, LASS4 is
transcribed from the opposite strand and its 39terminus is
mapped approximately 40 kb away from the last exon of TCblR.
The reference human genomic sequence appears contiguous
through this region. Close examination of the genomic
sequencing used to build this region of the genome revealed that
the sequence in this interval had not been completed. The gap in
the sequence was annotated in GenBank (contig, NT_077812) as
including an undetermined number of an approximately 15 kb
repetitive element. During the course of this work, genome
annotation was expanded to include areas containing potential
segmental duplications. To ensure that we were capturing all
regional variability related to the TCblR gene, we first asked if
this segmental duplication varied in copy number between
individuals. The CNVs have been implicated in disease and have
been shown to affect expression for nearby genes. We developed
an MLPA assay for this region and screened Caucasian and
African American samples available from the Coriell Cell
Repositories. We found that this region was polymorphic (ie,
this a bona fide CNV) in the African American (MAF¼0.054)
and Caucasian (MAF¼0.021) samples tested. MLPA alone does
not definitively allow determination of copy number; however,
the most parsimonious model consistent with the MLPA-derived
data is that the common allele has two repeats, whereas the
minor allele has three repeats. The CNV was not found to be in
strong LD with any other marker (figure 1b). Although it could
serve as a candidate variant, we excluded it from further
consideration because of its low MAF and the relatively large
quantity of DNA consumed by the MLPA assay.
SNP analysis and LD in the TCblR region
We selected 10 HapMap
44
polymorphisms to cover the region
within and flanking TCblR. Additionally, while developing the
MLPA assay, we discovered a C/GA insertionedeletion (indel)
polymorphism in the 39UTR of exon 5 located 83 bases down-
stream from the stop codon. Lastly, to search for unreported
common variation, limited screening of TCblR coding regions
was performed by directly sequencing exons in at least 12 Irish
individuals. This revealed a three nucleotide insertionedeletion
(indel) polymorphism in exon 2, resulting in the presence or
absence of one of the three tandem glutamic acid (E) residues
starting at codon 86 (TCblR E88del).
These 12 polymorphisms and the CNV were typed in
Caucasians and African Americans to determine the LD struc-
ture in the region (figure 1b). Blocks defined by measures of D9
revealed that the upstream region and most TCblRs are in
a single block of LD (figure 1). In the Irish population, this block
encompassed all markers within TCblR. Most of the markers
examined are independently informative (r
2
<0.8). The excep-
tions consist of two sets of markers that are in high r
2
LD: (1)
rs2232775 (Q8R), rs2336573 (G220R) and rs9426 (r
2
$0.94) and
(2) rs250511 and rs173665 (r
2
¼0.99). A total of 12 markers were
genotyped in the Irish NTD cohort (table 1). The exon 5 indel
had the lowest MAF (0.001) and was excluded from further
study.
Evaluation of individual markers for association with NTDs
Each marker (n¼11) was tested for caseecontrol associations
with NTDs in the Irish population using three models of logistic
regression: multiplicative, dominant and recessive models.
Significant associations are shown in table 2. Essentially, three
positive associations were found. First, the three highly linked
SNPs were found to be significantly associated in a recessive
model of case effect (rs2232775 (Q8R), OR¼10.6 (95% confi-
dence interval (CI) 1.28 to 88.56), p¼0.029; rs2336573 (G220R),
Fisher exact test, p¼0.0002; rs9426, Fisher exact test, p¼0.0006).
These SNPs share very high r
2
values and, as predicted, yield
similar association results. Second, TCblR E88del is also signifi-
cantly associated with NTDs in a recessive model of case effect
(OR¼9.2 (95% CI 1.3 to 88.9), p¼0.04). Third, the highly linked
SNP pair (rs250511, OR¼0.7 (95% CI 0.56 to 0.99), p¼0.04;
rs173665, OR¼0.7 (95% CI 0.51 to 0.94), p¼0.017) is signifi-
cantly associated with NTDs in a dominant model of case effect.
Additionally, rs173665 was associated when a multiplicative
model of case effect was applied (OR¼0.7 (95% CI 0.55 to 0.96),
p¼0.02). Upon correcting for multiple tests, two markers from
the highly linked trio remained significant: rs2336573 (G220R;
p¼0.008) and rs9426 (p¼0.03), by Fisher exact test of a recessive
model in the cases versus the controls. No other comparisons
were significant after adjusting for multiple tests.
Pangilinan F, Mitchell A, VanderMeer J, et al.J Med Genet (2010). doi:10.1136/jmg.2009.073775 3 of 9
Original article
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Examining the same markers in the NTD mothers and the
controls revealed no significant associations (data not shown).
A family-based test of association was also used to evaluate
these 11 markers for case and maternal NTD risk (table 3).
Multiplicative (data not shown), dominant and recessive models
of log-linear analysis were applied. Depending on the applied
model and the corresponding genotype frequencies, the test
failed to converge (no result obtained) for some SNPs. One
positive result was observed among the successfully tested SNPs.
All the three highly linked SNPs were significantly associated
with a strong RR in log-linear analysis of the recessive model in
the cases (rs2232775 (Q8R), RR¼6.0, p¼0.0086; rs2336573
Figure 1 Genomic structure and LD in
the TCblR region. (A) Genomic structure
of the region including TCblR.
Orientation, relative size and distance of
LASS4 (53 kb), the 14.8 kb CNV, TCblR
(6 kb), NDUFA7 (10 kb) and RPS28
(0.9 kb) are shown. The 14.8 kb CNV is
shown in single copy, although two
copies are predicted to be present on
most chromosomes. (B) LD plots of
polymorphisms within and surrounding
TCblR. Genotyping data from the Irish
controls (n¼993), the Caucasian
Americans (n¼79) and the African
Americans (n¼83) were used to
construct D9and r
2
LD plots. Labels for
variants within TCblR are green.
Relative distances between markers are
shown with two exceptions: the CNV is
approximately 450 bases downstream
from TCblR, and rs250508 is
approximately 32 kb downstream from
TCblR.
4 of 9 Pangilinan F, Mitchell A, VanderMeer J, et al.J Med Genet (2010). doi:10.1136/jmg.2009.073775
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(G220R), RR¼6.6, p¼0.0037; and rs9426, RR¼6.7, p¼0.0035).
However, these results did not withstand correction for multiple
tests (data not shown).
Further evidence was sought by evaluating TCblR variation for
NTD risk in an independent population. Seven SNPs were tested
in a UK cohort of NTD triads (table 3). Again, some tests failed
to converge, including the log-linear analysis of the recessive
model as applied to two of the associated SNPs (rs2232775
(Q8R) and rs2336573 (G220R)). The remaining models of SNPs
that were successfully tested were not found to be associated
with the case or maternal NTD risk in the UK population.
Haplotype analysis of TCblR variants for NTD risk
To test for TCblR haplotype association, a haplotype was
constructed based on the single block of LD in the Irish popu-
lation (figure 1b). rs2336573 (G220R) was retained to tag
rs2232775 (Q8R) and rs9426, which were not included in these
analyses. Similarly, rs173665 was retained as a tag for rs250511.
The resulting haplotype block is defined by the remaining seven
markers: rs4147651, rs7408841, rs2927707, E88del, rs2336573
(G220R), rs2227288 and rs173665. Haplotype frequencies were
estimated in the Irish controls, the NTD cases and the mothers
(table 4). Five haplotypes account for >90% of the variation
present in this haplotype block in the Irish population.
A permutation test did not detect differences in haplotype
frequencies between the NTD cases and the controls (p¼0.3) or
the NTD mothers and the controls (p¼1.0).
DISCUSSION
We evaluated the association between genetic variation in the
transcobalamin II receptor gene, TCblR, and NTD risk. When
considered singly, several independent (r
2
<0.8) TCblR
Table 1 Genotype distributions and allele frequencies in Irish controls
and NTD triads
Marker Controls NTD children NTD mothers NTD fathers
NDUFA7 rs4147651
GG 636 (0.66) 314 (0.63) 310 (0.62) 289 (0.64)
GA 290 (0.30) 167 (0.33) 175 (0.35) 140 (0.31)
AA 31 (0.03) 21 (0.04) 16 (0.03) 24 (0.05)
G 0.82 0.79 0.79 0.79
A 0.18 0.21 0.21 0.21
rs7408841
GG 377 (0.39) 210 (0.41) 223 (0.44) 193 (0.42)
GC 459 (0.48) 218 (0.43) 218 (0.43) 201 (0.44)
CC 129 (0.13) 81 (0.16) 63 (0.13) 61 (0.13)
G 0.63 0.63 0.66 0.65
C 0.37 0.37 0.34 0.35
TCblR rs2232775 Q8R
TT 846 (0.91) 479 (0.91) 479 (0.93) 423 (0.91)
TC 82 (0.09) 44 (0.08) 33 (0.06) 39 (0.08)
CC 0 (0.00) 6 (0.01) 2 (0.00) 3 (0.01)
T 0.95 0.95 0.96 0.95
C 0.05 0.05 0.04 0.05
TCblR rs2927707
TT 481 (0.50) 259 (0.51) 243 (0.48) 227 (0.50)
TC 409 (0.43) 212 (0.41) 212 (0.42) 189 (0.42)
CC 71 (0.07) 41 (0.08) 51 (0.10) 38 (0.08)
T 0.71 0.71 0.69 0.71
C 0.29 0.29 0.31 0.29
TCblR E88del
E/E 915 (0.96) 495 (0.95) 493 (0.96) 436 (0.56)
E/del 35 (0.04) 20 (0.04) 35 (0.04) 18 (0.38)
del/del 1 (0.00) 5 (0.01) 1 (0.00) 0 (0.06)
E 0.98 0.97 0.98 0.96
del 0.02 0.03 0.02 0.04
TCblR rs2336573 G220R
CC 889 (0.92) 464 (0.90) 468 (0.93) 417 (0.91)
CT 80 (0.08) 42 (0.08) 35 (0.07) 39 (0.08)
TT 0 (0.00) 8(0.02) 1 (0.00) 3 (0.01)
C 0.96 0.94 0.96 0.95
T 0.04 0.06 0.04 0.05
TCblR rs2227288
GG 780 (0.81) 417 (0.82) 404 (0.80) 364 (0.80)
GC 179 (0.19) 90 (0.18) 97 (0.19) 89 (0.20)
CC 6 (0.01) 4 (0.01) 6 (0.01) 2 (0.00)
G 0.90 0.91 0.89 0.90
C 0.10 0.10 0.11 0.10
TCblR rs250511
TT 802 (0.82) 453 (0.86) 428 (0.83) 392 (0.84)
TA 175 (0.18) 72 (0.14) 83 (0.16) 76 (0.16)
AA 7 (0.01) 4 (0.01) 6 (0.01) 0 (0.00)
T 0.90 0.92 0.91 0.84
A 0.10 0.08 0.09 0.16
TCblR exon 5 indel
del/del 982 (1.00) 508 (1.00) 504 (1.00) 456 (1.00)
del/ins 1 (0.00) 1 (0.00) 1 (0.00) 1 (0.00)
Continued
Table 1 Continued
Marker Controls NTD children NTD mothers NTD fathers
ins/ins 0 (0.00) 0 (0.00) 0 (0.00) 0 (0.00)
del 1.00 1.00 1.00 1.00
ins 0.00 0.00 0.00 0.00
TCblR rs9426
CC 882 (0.91) 470 (0.90) 473 (0.93) 425 (0.91)
CT 87 (0.09) 44 (0.08) 35 (0.07) 39 (0.08)
TT 0 (0.00) 7 (0.01) 1 (0.00) 3 (0.01)
C 0.95 0.94 0.96 0.95
T 0.05 0.06 0.04 0.05
rs173665
GG 790 (0.82) 445 (0.86) 421 (0.83) 389 (0.84)
GA 171 (0.18) 66 (0.13) 79 (0.16) 74 (0.16)
AA 8 (0.01) 4 (0.01) 6 (0.01) 1 (0.00)
G 0.90 0.93 0.91 0.92
A 0.10 0.07 0.09 0.08
rs250508
CC 304 (0.32) 176 (0.35) 166 (0.34) 140 (0.30)
CT 482 (0.50) 222 (0.44) 235 (0.48) 222 (0.47)
TT 169 (0.18) 107 (0.21) 87 (0.18) 107 (0.23)
C 0.57 0.57 0.58 0.54
T 0.43 0.43 0.42 0.46
Because of rounding, group frequencies may not sum to 1. Genotypes are expressed in n
(frequency); and alleles, in frequency.
Pangilinan F, Mitchell A, VanderMeer J, et al.J Med Genet (2010). doi:10.1136/jmg.2009.073775 5 of 9
Original article
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polymorphisms were implicated as NTD risk factors in the
Irish population. One pair of redundant SNPs (rs2336573
(G220R) and rs9426, r
2
¼1) remained significantly associated
with NTDs even after rigorous correction for multiple tests. One
possible concern about the validity of this result is that the NTD
cases are not in HWE for these SNPs (see Materials and
methods), which can result from genotyping error. We consider
this unlikely because these two highly linked SNPs were geno-
typed with two independent assays, producing concordant
results. Moreover, these variants were also significantly associ-
ated in an unadjusted recessive model of log-linear analysis that
does not use control data, eliminating the possible problem of
population stratification. Furthermore, this finding may not
have withstood correction because power is reduced when
examining a variant with a low MAF. Thus, we conclude
that the signal seen with the rs2336573 (G220R) and rs9426
variants represents a true genetic risk factor in TCblR in the Irish
population.
We sought to replicate this association in a sample of triads
from the UK. As this sample did not include population
controls, we were restricted to family-based tests of association.
The lack of individuals homozygous for the minor allele of
rs2336573 (G220R) precluded the use of the log-linear test for
a recessive case effect. Because testing of the Irish cohort
revealed a case effect, we also performed the transmission
disequilibrium test
49
but did not detect a case effect for either
rs2336573 G220R or rs2232775 Q8R in the UK cohort (data not
shown). The rs9426 SNP was not directly tested, but because of
its high LD with the other two markers, we would predict the
same result in the UK NTD cohort. This lack of replication may
be due to decreased power to detect an effect (approximately
525 Irish NTD triads vs approximately 345 UK NTD triads).
Alternatively, because of differences in dietary factors, prenatal
screening and/or greater ethnic heterogeneity, these variants
may not contribute to NTDs in the UK population.
Haplotype analysis of all the TCblR variants did not yield
further evidence of association; therefore, the identified SNP pair
exhibits the strongest signal for NTD risk. It is possible that
these variants do not alter gene function, and a yet unidentified
causal risk SNP may be linked to these two and reside on
a haplotype we have yet to test. In addition, these SNPs
(rs2336573 G220R and rs9426) have been typed in the HapMap
CEU (Caucasian) population and are in very high LD (r
2
¼1)
with five additional TCblR SNPs: rs17160390 (intron 1),
rs2232783 (T149T), rs2232784 (S161S), rs2232785 (intron 3)
and rs2227289 (T279T). Additionally, there are four such
SNPs in introns of upstream genes: rs7249111, rs7250792,
rs2288414 in NDUFA7 and rs4147645 in ribosomal protein S28
(RPS28).
We aligned the TCblR protein sequences from multiple species
to determine whether any of these variants are in highly
conserved regions. The conservation track of the University of
CaliforniadSanta Cruz Genome Browser (http://genome.ucsc.
edu/)
51
displays syntenic regions of up to 17 species, and full
alignments for TCblR were obtained only for chimp, rhesus, dog,
cow, mouse, rat, tenrec and elephant. The only intronic SNP
with a non-zero phastCons conservation score is rs17160390
(TCblR intron 1); its signal is relatively low. In contrast,
rs2232783 (T149T) and rs2232784 (S161S) are the only other
SNPs in this set with phastCons conservation scores (0.742 and
0.677, respectively). These SNPs are part of the coding region for
the second low-density lipoprotein receptor class A (LDLRa)
domain in TCblR. As the name implies, these domains are shared
by proteins in the LDL receptor superfamily, which includes the
LDL receptor (LDLR), the LDL receptorerelated protein (LRP1),
megalin/LRP2, the apolipoprotein 2 receptor (LRP8) and the
very-lowedensity lipoprotein receptor (reviewed by May et al).
52
The LDLRa domains shared by these proteins contain cysteine-
rich regions of approximately 40 amino acids and are involved in
ligand binding. While rs2232783 (T149T) and rs2232784 (S161S)
fall into this well-conserved region, they are synonymous
coding SNPs and appear to be weak candidates for affecting
functionality.
Perhaps, the most likely causal candidate SNP is rs2336573
(G220R), the nonsynonymous coding SNP directly tested in this
study. This amino acid substitution replaces a glycine with
arginine and is predicted to reside 10 residues from the trans-
membrane and cytoplasmic domains found at the carboxyl
terminus of the protein. We aligned and examined the TCblR
orthologs from 23 mammalian species. This position contains
glycine in 14 species. The other species contain glutamic acid,
arginine, alanine or tryptophan at this residue. The moderate
conservation of this amino acid implies functional tolerance of
the rs2336573 (G220R) variant in TCblR. While such speculation
is intriguing, functional analyses of these individual variants will
be required to determine which of these NTD-associated SNPs
may be the direct contributor to NTD risk.
Strengths of the current study include a large, homogenous
Irish population and a large UK population for replication. One
limitation was the lack of UK populationebased controls, which
prevented replication of the same caseecontrol analyses that
identified rs2336573 (G220R) and rs9426 as NTD risk SNPs in
the Irish group. Thus, replication studies in independent cohorts
are required to determine whether these TCblR polymorphisms
contribute to NTDs in other populations. Additionally, the lack
of metabolite measurements in either population prevented
testing whether the risk genotype influences circulating cobal-
amin levels.
Table 2 Caseecontrol logistic regression of significantly associated TCblR variants in the Irish population
Model
Logistic regression Fisher exact test
ORy(95% CI) p Value corrected p Value p Value Corrected p value
rs2232775 (Q8R) Recessive 10.6 (1.28 to 88.56) 0.0288 0.3747
E88del Recessive 9.2 (1.08 to 79.16) 0.0428 0.6887
rs2336573 (G220R) Recessive FTC 0.0002 0.0080
rs250511 Dominant 0.7 (0.55 to 0.99) 0.0422 1.0000
rs9426 Recessive FTC 0.0006 0.0279
rs173665 Dominant 0.7 (0.51 to 0.94) 0.0170 0.7217
rs173665 Multiplicative 0.7 (0.55 to 0.96) 0.0249 1.0000
FTC, failed to converge.
yOR for the recessive model is the odds of disease with two copies divided by the odds of disease with zero or one copy; OR for the dominant model is the odds of disease with one or two
copies divided by odds of the disease with zero copies.
OR for the multiplicative model is the OR for disease for each copy of the allele.
6 of 9 Pangilinan F, Mitchell A, VanderMeer J, et al.J Med Genet (2010). doi:10.1136/jmg.2009.073775
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In summary, we identified a highly linked SNP pair (rs2336573
(G220R) and rs9426) in TCblR that is significantly associated in
a recessive model of NTD case risk. The risk genotype (minor
allele homozygote) was undetected in the large control group
(n¼979), contributing to the large RR (RRz6) observed in NTD
cases. As a complex disease, multiple genetic risk factors are
expected to contribute to the development of NTDs. Because of
the relative rarity of the TCblR 2336573 (G220R) and TCblR
rs9426 risk genotypes (approximately 1% in the NTD cases), we
lack the power to perform meaningful interaction analyses of
these genotypes with other established NTD risk factors such as
MTHFR 677C/T. Its low frequency also means that the risk
genotype does not contribute greatly to the total genetic risk
for NTDs in the general population. However, for those few
individuals carrying the risk genotype, risk is increased approx-
imately sixfold.
Finding a genetic risk factor in the receptor required for
cobalamin bioavailability further establishes the role of cobal-
amin in the development of NTDs. This raises the possibility
that women with the risk variant may need additional dietary
cobalamin to provide sufficient intracellular cobalamin for the
developing embryo at the time of neural tube closure. There may
also be individuals with these polymorphisms in the general
population who need dietary supplements to maintain normal
Table 3 Dominant and recessive models of log-linear analysis of TCblR variants
Variant
Republic of Ireland UK
Dominant
RRy
Dominant
p value
Recessive
RR
Recessive
p value
Dominant
RR
Dominant p
value
Recessive
RR
Recessive
p value
rs4147651
Case 1.1048 0.4329 0.7598 0.3496 FTC FTC 0.6830 0.3386
Mother 1.0342 0.5795 0.5868 0.1055 0.9765 0.5303 1.0637 0.8976
rs7408841
Case FTC FTC 1.3588 0.0824 1.2350 0.2127 1.0259 0.9091
Mother FTC FTC 0.8482 0.4135 1.1464 0.3378 0.8033 0.3594
rs2232775
Case FTC FTC 6.000 0.0086 0.7965 0.4081 FTC FTC
Mother FTC FTC 0.4567 0.3357 0.8394 0.1738 FTC FTC
rs2927707
Case 1.0561 0.6148 0.7133 0.1091 0.9483 0.7146 0.9659 0.9014
Mother FTC FTC 1.2461 0.3263 0.9582 0.6013 0.8331 0.4988
E88del
Case 1.3405 0.7462 FTC FTC ND ND ND ND
Mother FTC FTC FTC FTC ND ND ND ND
rs2336573
Case FTC FTC 6.5871 0.0037 0.7064 0.2550 FTC FTC
Mother FTC FTC 0.2469 0.1858 0.7518 0.1386 FTC FTC
rs2227288
Case 1.0210 0.8971 0.6856 0.4932 0.8320 0.3962 0.4258 0.2315
Mother FTC FTC 2.6990 0.2006 0.8055 0.5434 0.4428 0.3369
rs250511
Case 0.7980 0.2625 FTC FTC ND ND ND ND
Mother FTC FTC FTC FTC ND ND ND ND
rs9426
Case FTC FTC 6.7127 0.0035 ND ND ND ND
Mother FTC FTC 0.2471 0.1656 ND ND ND ND
rs173665
Case 0.7253 0.1313 FTC FTC 0.9480 0.8021 0.7332 0.7084
Mother FTC FTC FTC FTC FTC FTC 2.4150 0.4509
rs250508
Case 0.8428 0.1861 1.2416 0.1621 ND ND ND ND
Mother 0.9832 0.6401 0.7748 0.1239 ND ND ND ND
ND, not done.
None of the reported p values are <0.05 upon correction for multiple tests (permutation).
yRR for the recessive model is the risk of disease with 2 copies divided by risk of disease with 0 or 1 copy; RR for the Dominant Model is the risk of disease with 1 or 2 copies divided by risk of
disease with 0 copies.
Pangilinan F, Mitchell A, VanderMeer J, et al.J Med Genet (2010). doi:10.1136/jmg.2009.073775 7 of 9
Original article
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cobalamin homeostasis. The reported NTD risk SNPs are prime
candidates to investigate in any disease state influenced by
cobalamin metabolism.
Acknowledgements These studies would not be possible without the participation
of the affected families and their recruitment by the Irish Association of Spina Bifida
and Hydrocephalus and the Irish Public Health Nurses in Ireland, and by the UK
ASBAH. The authors would like to thank Amanda Samuels and David Bernard for the
technical assistance in screening TCblR coding regions for polymorphisms.
Funding The authors acknowledge the research support from the intramural research
programmes of the National Human Genome Research Institute, the Eunice Kennedy
Shriver National Institute of Child Health and Human Development, the National
Institutes of Health, the Department of Health and Human Services and the Health
Research Board, Ireland. EVQ and JMS are supported by the National Institutes of
Health grant DK064732.
Competing interests None.
Patient consent Obtained.
Ethics approval This study was conducted with the approval of the National
Institutes of Health, Bethesda, Maryland, USA; the Health Research Board, Dublin,
Ireland, and the Multi-Center Research Ethics Committee, University of Newcastle,
UK.
Provenance and peer review Not commissioned; externally peer reviewed.
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Table 4 Haplotype frequency estimates of the TCblR D9block
Haplotype Controls NTD cases NTD mothers
GCTdelCGG 0.33 0.32 0.30
GGTdelCGG 0.24 0.24 0.24
AGCdelCGG 0.19 0.21 0.21
GGTdelCCG 0.10 0.10 0.11
GGCdelCGA 0.10 0.07 0.09
GCTdelTGG 0.02 0.03 0.02
GCTinsTGG 0.02 0.03 0.01
GGCdelCGG 0.01 0.01 0.01
p Value 0.3y1.0y
The haplotype block consists of the following markers: rs4147651, rs7408841, rs2927707,
E88del, rs2336573 (G220R), rs2227288 and rs173665.
yResult of the permutation test for significant differences in haplotype frequencies in the
NTD cases versus the controls or the NTD mothers versus the controls.
8 of 9 Pangilinan F, Mitchell A, VanderMeer J, et al.J Med Genet (2010). doi:10.1136/jmg.2009.073775
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