Genetic Epidemiology 33:247–255 (2009)
Oral Facial Clefts and Gene Polymorphisms in Metabolism of Folate/
One-Carbon and Vitamin A: A Pathway-Wide Association Study
Abee L. Boyles,1?Allen J. Wilcox,1Jack A. Taylor,1Min Shi,2Clarice R. Weinberg,2Klaus Meyer3
A˚se Fredriksen,3Per Magne Ueland,3Anne Marte W. Johansen,4Christian A. Drevon,4Astanand Jugessur,5
Truc Nguyen Trung,6Ha ˚kon K. Gjessing,6Stein Emil Vollset,6Jeffrey C. Murray,7Kaare Christensen8
and Rolv T. Lie6
1Epidemiology Branch, NIEHS/NIH, Durham, North Carolina
2Biostatistics Branch, NIEHS/NIH, Durham, North Carolina
3Department of Pharmacology, University of Bergen, Bergen, Norway
4Department of Nutrition, Institute of Basic Medical Sciences, Faculty of Medicine, University of Oslo, Oslo, Norway
5Murdoch Children’s Research Institute, Royal Children’s Hospital, Parkville, Melbourne, Australia
6Department of Public Health and Primary Health Care, University of Bergen, Bergen, Norway
7Department of Pediatrics, University of Iowa, Iowa City, Iowa
8Center for Prevention of Congenital Malformation, Epidemiology Unit, University of Southern Denmark, Odense, Denmark
An increased risk of facial clefts has been observed among mothers with lower intake of folic acid or vitamin A around
conception. We hypothesized that the risk of clefts may be further moderated by genes involved in metabolizing folate or
vitamin A. We included 425 case-parent triads in which the child had either cleft lip with or without cleft palate (CL/P) or
cleft palate only (CPO), and no other major defects. We analyzed 108 SNPs and one insertion in 29 genes involved in folate/
one-carbon metabolism and 68 SNPs from 16 genes involved in vitamin A metabolism. Using the Triad Multi-Marker
(TRIMM) approach we performed SNP, gene, chromosomal region, and pathway-wide association tests of child or maternal
genetic effects for both CL/P and CPO. We stratified these analyses on maternal intake of folic acid or vitamin A during the
As expected with this high number of statistical tests, there were many associations with P-valueso0.05; although there
were fewer than predicted by chance alone. The strongest association in our data (between fetal FOLH1 and CPO,
P50.0008) is not in agreement with epidemiologic evidence that folic acid reduces the risk of CL/P in these data, not CPO.
Despite strong evidence for genetic causes of oral facial clefts and the protective effects of maternal vitamins, we found no
convincing indication that polymorphisms in these vitamin metabolism genes play an etiologic role. Genet. Epidemiol.
r 2008 Wiley Liss, Inc.
Key words: cleft lip; cleft palate; dietary supplements; folic acid; genetics; metabolism; vitamin A
Additional supporting information may be found in the online version of this article.
Contract grant sponsor: National Institutes of Health; Contract grant number: DE085592, RO1 DE-11948-04, N01-HG-65403, P50 DE-
16215, R37 DE-0559; Contract grant sponsor: National Institute of Environmental Health Sciences; Contract grant number: Z01 ES049027-
11, Z01 ES040007; Contract grant sponsor: the Research Council of Norway; Contract grant number: 166026/V50; Contract grant sponsor:
Foundation to promote research into functional vitamin B12-deficiency; Contract grant sponsor: the Freia Foundation; Contract grant
sponsor: the Throne-Holst Foundation; Contract grant sponsor: Faculty of Medicine, University of Oslo.
?Correspondence to: Abee L. Boyles, Epidemiology Branch, Mail Drop A3-05, P.O. Box 12233, National Institute of Environmental Health
Sciences/NIH, Durham, NC 27709. E-mail: email@example.com
Received 29 August 2008; Accepted 5 September 2008
Published online 1 December 2008 in Wiley InterScience (www.interscience.wiley.com).
Oral facial clefts, including cleft lip and palate, have a
strong genetic basis as determined by many studies of
recurrence risks in relatives and by segregation analyses in
diverse populations [Clementi et al., 1997; Murray et al.,
1997; Palomino et al., 1997; Scapoli et al., 1999; Sivertsen
et al., 2008; Vieira et al., 2003]. Supplemental intake of folic
acid and multivitamins around conception is suggested to
provide protection from these birth defects [Badovinac
et al., 2007; Bille et al., 2007; Chevrier et al., 2007; Krapels
et al., 2004; Mitchell et al., 2003; van Rooij et al., 2004]. In
the Norwegian population studied here, folic acid supple-
mentation provided a 39% reduction in the risk of cleft lip
with or without cleft palate (CL/P) [Wilcox et al., 2007],
whereas increased total vitamin A intake from food and
supplements reduced the risk of cleft palate only (CPO) by
53% [Johansen et al., 2008]. Excessive vitamin A can be
teratogenic, but not at the levels observed in this
population [Soprano and Soprano, 1995]. However, it is
r 2008 Wiley-Liss, Inc.
unclear to what extent the genetic risk of clefts is
intertwined with the risk from vitamin deficiencies despite
numerous smaller-scale association studies showing mod-
est effects, including our own [Boyles et al., 2008; Jugessur
et al., 2003a,b,c].
MATERIAL AND METHODS
Families of patients with oral facial clefts born in
Norway between 1996 and 2001 were enrolled in the
study. The overall study design has been described
previously [Wilcox et al., 2007]. Of approximately 300,000
live births during this time, 676 were referred for
corrective surgery on oral facial clefts. Twenty-four babies
were excluded due to death or mothers who did not speak
Norwegian. Five hundred and seventy-three of the
remaining 652 families (88%) agreed to participate in the
study. Of 1,022 randomly sampled live births, 763 (75%)
were enrolled using the same exclusion criteria. All
parents provided informed consent. For this study only
isolated cases of oral facial clefts were analyzed. Cases
were excluded if another birth defect was reported on the
mother’s questionnaire, in the Medical Birth Registry, or in
medical records from the time of surgery [Nguyen et al.,
2007]. We used controls only for assessing deviations from
Hardy-Weinberg equilibrium (HWE) and for calculations
of linkage disequilibrium (LD). Table I shows the number
of case families stratified by cleft type, the number of
families of cases with an isolated cleft on whom we had
information on both genotype and vitamin intake, and
who represent the analyses presented here [Wilcox et al.,
Most of the SNPs in this study comprise a subset of
those assayed as part of a larger project exploring
candidate genes and clefts in Norwegian and Danish
populations. Genes potentially implicated in oral facial
clefts were chosen from published association and linkage
studies, human cytogenetic rearrangements, Mendelian
forms of clefting identified in the OMIMTMdatabase
knockout experiments in mice, and gene expression
studies in human and mouse embryonic tissues [Brown
et al., 2003; Cai et al., 2005; Gong et al., 2005; Jugessur and
Murray, 2005; Lidral and Moreno, 2005; Mukhopadhyay
et al., 2004]. A list of 357 candidate genes was generated
which included functional categories likely related to oral
facial cleft etiology such as growth factors, detoxification
genes, genes for syndromes that include clefts, and
vitamin metabolism-related genes.
SNPs in these genes were selected primarily using
CEPH data from the International HapMap Consortium
(www.hapmap.org) to evaluate their haplotype-tagging
properties, minor allele frequency (MAF), and gene
coverage. Supplemental SNPs were chosen using dbSNP
(genome.ucsc.edu), CHIP SNPper tool (snpper.chip.org),
and SeattleSNPs (pga.mbt.washington.edu). The SNPs
were prioritized based on prior evidence of an association
with clefting, coding SNPs, and an MAF of at least 10%.
Additional selected SNPs were intragenic, in putative
regulatory regions in the UTRs, or had haplotype-tagging
properties. SNPs were evaluated using HAPLOVIEW
index.php) [Barrett et al., 2005], BEST (www.genomethods.
org/best/index.htm) [Sebastiani et al., 2003], and SNP
BrowserTM(Applied Biosystems, Foster City, CA) to
determine LD patterns and haplotype block structures
for the selection of haplotype tagging SNPs.
Some SNPs were removed due to properties detrimental
to assay design: nearby palindromic sequences, GC- and
AT-rich regions, repetitive sequences, and sequences that
are similar to other human sequences. SNP assays
designed by Illumina were evaluated by a ‘‘design score’’
that tests each SNP’s performance on the GoldenGateTM
(San Diego, CA; www.illumina.com) platform. After
thorough evaluation a custom panel of 1,536 SNPs was
designed for the 357 genes and genotyped by the US
Center for Inherited Disease Research (CIDR, www.cidr.
SNPs with call rates below 95% were eliminated, as were
those out of HWE (w2410) in the control population as
determined by an exact test [Wigginton et al., 2005]. In
addition, eight families were removed for excessive
Mendelian inconsistencies (65 and more, while those
included had no more than eight inconsistencies and
there were no families with between nine and 64
inconsistencies). Those with high numbers of Mendelian
inconsistencies were probably the result of sample
switches or misidentified paternity. Many of these
same family inconsistencies
reported [Boyles et al., 2008].) SNPs on the X and Y
chromosomes were used to confirm the sex of subjects and
The intent of the overall candidate gene study was to
evaluate a large number of SNPs in genes thought to be
related to oral facial clefts in the Norwegian and Danish
populations. These data also allow more specific explora-
tion of gene-environment interaction for exposures asso-
ciated in our data with the risk of clefting. The Norway
study included detailed questionnaires of maternal ex-
posures that were not available for the Danish samples.
Our previous work in the Norwegian population showed a
reduction in the risk of clefts from folic acid supplementa-
tion [Wilcox et al., 2007], vitamin A intake [Johansen et al.,
2008], and an SNP in CBS, a one-carbon metabolism gene
[Boyles et al., 2008].
have been previously
TABLE I. Samples sizes for study participants
subdivided by cleft type, those genotyped, and isolated
case families analyzed by each stratum of maternal
CL/P CPO All clefts
Isolated with folic acid information
Isolated with Vitamin A information
248Boyles et al.
From the CIDR genotyping set, 114 SNPs were chosen in
27 genes involved with folate/one-carbon metabolism.
Nine of these SNPs had poor call quality and three were
not in HWE, leaving 102 SNPs for analysis. An additional
seven polymorphisms in five folate/one-carbon metabo-
lizing genes had been previously genotyped in these
samples by matrix-assisted laser desorption/ionization
time-of-flight mass spectrometry (MALDI-TOF MS) [Boy-
les et al., 2008; Meyer et al., 2004], and are included in this
analysis. Figure 1 diagrams the relative genomic positions
of the 29 folate/one-carbon metabolism genes and
functional type of the 109 genotyped polymorphisms.
Further description of these genes and their functional
categories are included in Supplemental Table I. Sixteen
genes related to vitamin A metabolism are also included
in Figure 1. This set of genes includes transforming
growth factor signaling genes which are part of other
signaling pathways but have been linked to vitamin A
metabolism as well [Abbott et al., 2005; Baroni et al., 2006].
These genes are described further in Supplemental
Table II. Sixty-eight of 73 selected vitamin A-related SNPs
were analyzed (four had poor call rates and one was
not in HWE).
Mothers were mailed questionnaires approximately 3
months after delivery regarding vitamin intake during the
6 months prior to and the first 3 months of pregnancy
ncl/question.cfm). If mothers reported taking a vitamin
name. Mothers mailed empty pill bottles or product labels
to the study office for verification of the product and
The facial structures of the embryonic lip and palate fuse
during the first 2 months of gestation, so we considered
mothers to be supplemented if they took folic acid for at
least 1 month during the 3-month window starting a
month before the last menstrual period and going through
the first 2 months of pregnancy. Intake of 400mg or more of
folic acid supplements reduced the risk of CL/P, whereas
no protective effect was seen in CPO and there was no
protective effect at lower doses [Wilcox et al., 2007]. For
the folate/one-carbon metabolism analysis, we therefore
divided women into strata of o400mg/day and over 4001
mg/day of folic acid from dietary supplements.
Total vitamin A from food and supplements was shown
in this sample to reduce the risk of CPO in a dose-
dependent manner, but not CL/P [Johansen et al., 2008].
Recommended levels of vitamin A during pregnancy vary
among countries, and excessive vitamin A is teratogenic
[Finnell et al., 2004; Soprano and Soprano, 1995]. Vitamin
A level was determined from a food frequency ques-
tionnaire covering maternal diet and supplement use
during the first 3 months of pregnancy as described
previously [Johansen et al., 2008]. We divided the data set
at the mean level of vitamin A (1,257mg from both diet and
supplements calculated as retinol plus 1/12 beta-carotene).
The intake levels of both vitamins were used to stratify the
data, but no formal test of gene-vitamin interaction was
askedfor the product
The Triad Multi-Marker test (TRIMM) allows assess-
ment of associations with both the child’s and mother’s
genes using multiple markers from mother-father-child
triad families [Shi et al., 2007]. To assess a possible effect of
inherited haplotypes, the genotype vector of the offspring
is contrasted with that of a hypothetical ‘‘complement’’
child who would have inherited parental alleles not
transmitted to the observed offspring. The difference
between these two offspring genotype vectors has an
expected value of 0 at each locus under the null
hypothesis, similar to the pedigree disequilibrium test
[Martin et al., 2000]. The TRIMM method allows for
multiple linked SNPs, but does not assume that there is no
recombination from parent to child. Permutation of the
labels for case-versus-complement status (i.e. randomly
multiplying the difference vector by 11 or ?1) is used to
evaluate statistical significance. To optimize over scenarios
where risk depends on a single SNP and also over
scenarios where risk instead depends on a multi-SNP
susceptibility haplotype, TRIMM performs both maximum
and Hotelling’s T2tests, and generates a combined
P-value from the two tests (sum_logP). The program also
nominates risk-haplotype-tagging alleles, but does not
need to either calculate or impute phase from the observed
Fig. 1. Genomic location of genes in the folate/one-carbon (left
of the chromosome) and the vitamin A (right of the chromo-
some) pathways. SNPs indicated by a square with a star were
considered in the region of the gene of interest, but actually fell
within a neighboring gene. Those indicated with a 1 were
obtained from MALDI-TOF genotyping performed prior to the
CIDR genotyping, and previously reported [Boyles et al., 2008].
SNPs in the 5q and 11q LD regions are detailed in Figure 2.
172?214mm (300?300 DPI)
249 Oral Facial Clefts and Vitamin Metabolism Genes
The TRIMM test for a maternally mediated genetic
effect is similar except the difference vector used is based
genotypes. Under genetic mating symmetry the father
serves as a genetic control for the mother of the child with
cleft. When calculating the child genetic effect, the
calculations assumed parental symmetry unless there
was evidence of a maternal effect (Po0.05), in which
case families with missing genotypes are omitted from the
All genes were analyzed separately, generating a
sum_logP gene score as well as max Z2scores for
individual SNPs and a possible risk-haplotype was
nominated if the overall max Z2P-value was less than
0.1. LD between all pairs of polymorphisms was calculated
from the control population samples using Haploview
version 4.0 [Barrett et al., 2005]. Among the folate/one-
carbon metabolizing genes, we jointly analyzed three
genes on chromosome 5 and three more on chromosome
11 due to high LD (D040.5 and LOD42) between them.
The LOD score is a measure of confidence in D0. An overall
pathway analysis was also conducted for folate/one-
carbon and for vitamin A.
When genes are in close proximity, LD can create
dependency between results of individual genes. We
therefore calculated LD between SNPs in the same
pathway to identify regions in high LD for joint analysis.
For the folate/one-carbon metabolizing genes on 5q11-14
there was significant LD among DMGDH, BHMT2, and
BHMT1 but not with DHFR (Fig. 2). DMGDH, BHMT2,
and BHMT1 were included in the Chr5 LD region and
DHFR was analyzed separately. The folate/one-carbon
metabolizing genes on 11q (FOLR1, FOLR2, and FOLR3)
showed high LD with one another (Fig. 2), and so were
analyzed jointly as the Chr11 LD region. All other folate/
one-carbon and vitamin A metabolizing genes were not in
LD with one another (D0o0.5 or LODo2) and were
The TRIMM sum_logP value was calculated for each
gene or LD region, as were the max Z2scores for each
individual SNP. For both the folate/one-carbon and
vitamin A pathways, we found fewer tests with P-
valueso0.05 than we would expect by chance. In the
folate/one-carbon pathway we analyzed 25 genes or LD
regions, two genetic effects (analyzing either for the child’s
or the mother’s genotype), two types of facial clefts (CL/P
or CPO), and two strata of vitamin intake. If these tests had
all been independent then under the global null that all
variants on these pathways are unrelated to clefting we
would have expected ten of these 200 tests to produce
P-values less than 0.05, but we observed only eight.
Similarly with the 16 genes of the vitamin A pathway, we
expected six of the 128 tests to have P-values less than 0.05,
but we observed only four.
We graphically compared the observed P-values of the
resulting 328 independent tests to an expected uniform
distribution of P-values stratified by type of cleft and
pathway (Fig. 3). This quantile-quantile plot compares the
percentile of tests expected by chance to the observed P-
values. Specific SNPs or LD regions with P-valueso0.05
have been labeled. In three of the four panels, there is very
little evidence of P-values in excess of what would be
expected by chance (the dashed line). In the ‘‘CPO and
Folate’’ panel, there is a cluster of genes carried by the
child suggesting possible associations (in the high-folic
acid stratum FOLH1 P50.0008, SHMT2 P50.0089, and
CTH P50.040; in the low-folic acid stratum LD region
Chr11 P50.013 and SHMT1 P50.015). MTHFR, the most
widely studied folate metabolizing gene, has a maternal
genetic effect weakly associated with CPO in the high-folic
acid stratum (P50.044).
Tables II and III provide individual SNP P-values and
proposed risk haplotype alleles for the eight folate/one-
carbon genes or LD regions and the four vitamin A genes
with overall P-values less than 0.05. Complete results for
all SNPs in every gene or each LD region are provided in
Supplemental Tables III and IV.
Pathway-wide significance tests included all the poly-
morphisms in each pathway for all eight strata. In CPO
there was marginal evidence of a maternal effect when
mothers took o400mg of folic acid (P50.0075) and for a
child effect in the 4001mg mothers (P50.0325). In the
Fig. 2. 5q and 11q LD region Haploview LD plots. D0values are
indicated in the pair wise squares. Shading is according to the
alternative D0/LOD color scheme with darker shades indicating
higher D0values if the LOD score is Z2. Each SNP’s functional
status is indicated by the same symbols used in Figure 1.
168?253mm (300?300 DPI)
250Boyles et al.
vitamin A pathway, there was also a borderline maternal
effect for CPO in the o1,257mg group (P50.0242). The 13
other unadjusted pathway-wide P-values were40.05
(Supplemental Table V).
Numerous studies have attempted to associate vitamin
metabolism gene polymorphisms with oral facial clefts,
often with conflicting results [Boyles et al., 2008; Chevrier
et al., 2007; Jugessur et al., 2003a, b; Martinelli et al., 2006;
Mitchell et al., 2003; Mostowska et al., 2006; Rubini et al.,
2005; Scapoli et al., 2005; Shaw et al., 1998; van Rooij et al.,
2003; Zhu et al., 2005]. Here, we found no evidence of a
role for these gene variants in the risk of clefts, after
accounting for the large number of tests performed. The
strongest associations were between folate/one-carbon
pathway genes and CPO, which is unexpected given the
lack of epidemiologic evidence that folic acid supplemen-
tation prevents CPO in this population [Wilcox et al.,
2007]. Similarly, there was no evidence of an association of
genes related to metabolism of vitamin A with CL/P or
CPO, either independently or in conjunction with lower
vitamin A intake.
This article provides the first application of the TRIMM
approach for genetic data analysis of a metabolic pathway.
TRIMM is a versatile method for triad family data in that
there is no limit to the number of SNPs that can be
included, and no requirement of HWE. Prior specification
of haplotypes or phase inference is also not necessary.
TRIMM can evaluate maternal effects, which is especially
important in birth defects where the maternal genes can
contribute to the fetal in utero environment. Software for
implementing TRIMM is available for the R computing
TRIMM is a powerful method for this type of data, but it
does not estimate a meaningful risk parameter such as a
relative risk. Moreover, a formal test of interaction cannot
be performed. The nominated risk-haplotype-tagging
alleles may not represent a true single haplotype because
phase is not inferred from the data, but it may mark a
jointly relevant set of marker alleles. It is theoretically
possible that exactly complementary haplotypes that are
both protective (or both risk-conferring) could cancel each
other out, generating a null-value difference vector.
Considering the analysis more broadly, the unified
approach to pathway-wide analysis presented here may
be applicable to other large data sets with candidate genes
and strong prior expectations about gene-environment
interactions. A combined pathway-wide approach has the
possibility of identifying synergistic relationships between
genes that might be missed by single SNP analysis. Such
studies would require evidence of an environmental risk
factor associated with a group of biologically related genes
and evidence of a multigenic model of disease inheritance.
We found multiple SNPs that contributed to a gene or
LD region’s overall significance of association. As shown
in Table II several genes had multiple SNPs in the
nominated risk-haplotype contributing to their overall P-
value: FOLH1 (3 SNPs), Chr5 (4 SNPs in DMGDH), and
CBS (3 SNPs). In the case of FOLH1, the overall P-value
was more significant than all of the individual SNP P-
values. However, there were also cases where only one
SNP was individually significant and the addition of other
SNPs led to a less significant overall P-value: Chr11
(strongest SNP in FOLR2), CTH, and MTHFR.
Fig. 3. Quantile-quantile plot of the observed P-values (-log scale) versus an expected uniform distribution under the null (for 20
independent tests we expect one test to have a P-valueo0.05). CL/P and CPO are plotted separately for both folate-related genes and
vitamin A-related genes. Each plot contains the TRIMM sum_logP results for each gene or LD region for child (diamond) or maternal
(circle) genetic effects stratified by periconceptional vitamin supplementation7400lg of folic acid or 71,257lg of total vitamin A
intake (low-vitamin, open symbols; high-vitamin, filled symbols). The expected null distribution is indicated by the dashed diagonal
line (points above that line represent more significance than expected). For all results with observed P-values o0.05 (above the
horizontal line), the gene or LD region and the associated P-value are detailed in Tables II and III. Complete results for each pathway,
gene, LD region, and SNP are included in Supplemental Tables III–V. 147?239mm (300?300 DPI)
251 Oral Facial Clefts and Vitamin Metabolism Genes
The strongest association in this study was between
several SNPs in FOLH1 and risk of CPO in mothers who
took at least 400mg of folic acid. Even with the large
number of tests and little evidence that folic acid prevents
CPO, we cannot entirely dismiss the possibility that this
observed association is real. Formerly called GCP2
TABLE II. Folate/one-carbon associated genes
Gene (if in an
LD region)SNP Type
CPO Child 4001
CPO Child 4001
?Individual SNP max Z2P-value o0.05.
aAlthough selected for being in the region of BHMT2, this SNP actually lies in a DMGDH intron.
bSixty-eight base pair insertion at position 844 in CBS.
cThe SNP changes the protein to a thermolabile form (also referred to a C677T). It is the most widely studied SNP in folate metabolism.
Individual polymorphism max Z2P-values for all folate/one-carbon metabolizing genes or LD regions generating an overall sum_logP P-
valueo0.05. Risk haplotype alleles are coded 1 for major or 2 for minor allele. Functional type of polymorphism is indicated by the
abbreviations: near, near the assigned gene, but actually in another gene; ig, intergenic; Int, intronic; NS, coding, nonsynonymous; UTR, 30
untranslated region—there were no 50UTR SNPs; ins, insertion; and Syn, coding, synonymous.
252Boyles et al.
(glutamate carboxypepidase II), FOLH1 (folate hydrolase)
encodes an intestinal brush border membrane protein that
digests polyglutamylated folates into monoglutamyl fo-
lates [OMIM ? 600934]. A polymorphism in FOLH1 has
been associated with low folate and high homocysteine
levels possibly via decreased absorption of dietary folate
from the intestines [Devlin et al., 2000], however, a larger
study associated this polymorphism with high folate and
low total homocysteine [Halsted et al., 2007]. There are no
previously published genetic association studies of FOLH1
and oral facial clefts, and so we are unable to compare our
results with data from other similar studies.
We have provided detailed information on all our
results, which may be useful for future meta-analyses of
folate/one-carbon and vitamin A polymorphisms and
facial clefts (Supplemental Tables III, IV). Even so, we
suspect that further analyses are unlikely to yield strong
results, given the lack of evidence so far among this wide
array of vitamin metabolism-related genes. Ungenotyped
variants, other-related genes, or epigenetic effects may
also play a role, but perhaps the simplest explanation for
these results is that the genetic contribution to oral facial
clefts is independent of pathways by which vitamins
provide protection from oral facial clefts.
This research was supported by National Institutes of
Health Grants (DE085592, RO1 DE-11948-04, N01-HG-
65403, P50 DE-16215, R37 DE-0559), the Intramural
Research Program of the NIH, National Institute of
Environmental Health Sciences (Z01 ES049027-11, Z01
ES040007); the Research Council of Norway (166026/
V50); the Foundation to promote research into functional
vitamin B12-deficiency; the Freia Foundation; the Throne-
Holst Foundation; and the thematic area of perinatal
nutrition, Faculty of Medicine, University of Oslo, Oslo,
Norway. Genotyping services were provided by the Center
for Inherited Disease Research (CIDR), which is fully
funded through a federal contract from the National
Institutes of Health to The Johns Hopkins University (N01-
HG-65403). The authors would also like to acknowledge
the valuable contributions of Andrew Lidral, Bridget Riley,
Adela Mansilla, Kathy Frees, Corinne Boehm, Kim
Doheny, and Ivy McMullen to the overall cleft candidate
Abbott BD, Best DS, Narotsky MG. 2005. Teratogenic effects of retinoic
acid are modulated in mice lacking expression of epidermal
growth factor and transforming growth factor-alpha. Birth Defects
Res A Clin Mol Teratol 73:204–217.
Badovinac RL, Werler MM, Williams PL, Kelsey KT, Hayes C. 2007.
Folic acid-containing supplement consumption during pregnancy
and risk for oral clefts: a meta-analysis. Birth Defects Res A Clin
Mol Teratol 79:8–15.
Baroni T, Bellucci C, Lilli C, Pezzetti F, Carinci F, Becchetti E, Carinci P,
Stabellini G, Calvitti M, Lumare E, Bodo M. 2006. Retinoic acid,
GABA-ergic, and TGF-beta signaling systems are involved in
human cleft palate fibroblast phenotype. Mol Med 12:237–245.
Barrett JC, Fry B, Maller J, Daly MJ. 2005. Haploview: analysis and
visualization of LD and haplotype maps. Bioinformatics 21:
Bille C, Olsen J, Vach W, Knudsen VK, Olsen SF, Rasmussen K, Murray
JC, Andersen AM, Christensen K. 2007. Oral clefts and life style
factors—a case-cohort study based on prospective Danish data.
Eur J Epidemiol 22:173–181.
Boyles AL, Wilcox AJ, Taylor JA, Meyer K, Fredriksen A, Ueland PM,
Drevon CA, Vollset SE, Lie RT. 2008. Folate and one-carbon
metabolism gene polymorphisms and their associations with oral
facial clefts. Am J Med Genet A 146:440–449.
TABLE III. Vitamin A associated genes
Gene (Sum_logP)Cleft TypeEffect
P-value Risk haplotype
?Individual SNP max Z2P-value o0.05
aFamilies with missing genotypes were not used due to evidence of a maternal effect.
Individual SNP max Z2P-values for all vitamin A metabolizing genes generating an overall sum_logP P-valueo0.05. Risk haplotype alleles
are coded 1 for major or 2 for minor allele. All SNPs included are intronic.
253 Oral Facial Clefts and Vitamin Metabolism Genes
Brown NL, Knott L, Halligan E, Yarram SJ, Mansell JP, Sandy JR. 2003.
Microarray analysis of murine palatogenesis: temporal expression
of genes during normal palate development. Dev Growth Differ
Cai J, Ash D, Kotch LE, Jabs EW, Attie-Bitach T, Auge J, Mattei G,
Etchevers H, Vekemans M, Korshunova Y, Tidwell R, Messina DN,
Winston JB, Lovett M. 2005. Gene expression in pharyngeal arch 1
during human embryonic development. Hum Mol Genet 14:
Chevrier C, Perret C, Bahuau M, Zhu H, Nelva A, Herman C,
Francannet C, Robert-Gnansia E, Finnell RH, Cordier S. 2007. Fetal
and maternal MTHFR C677T genotype, maternal folate intake and
the risk of nonsyndromic oral clefts. Am J Med Genet A
Clementi M, Tenconi R, Forabosco P, Calzolari E, Milan M. 1997.
Inheritance of cleft palate in Italy. Evidence for a major autosomal
recessive locus. Hum Genet 100:204–209.
Devlin AM, Ling EH, Peerson JM, Fernando S, Clarke R, Smith AD,
Halsted CH. 2000. Glutamate carboxypeptidase II: a polymorph-
ism associated with lower levels of serum folate and hyperhomo-
cysteinemia. Hum Mol Genet 9:2837–2844.
Finnell RH, Shaw GM, Lammer EJ, Brandl KL, Carmichael SL,
Rosenquist TH. 2004. Gene-nutrient interactions: importance of
folates and retinoids during early embryogenesis. Toxicol Appl
Gong SG, Gong TW, Shum L. 2005. Identification of markers of the
midface. J Dent Res 84:69–72.
Halsted CH, Wong DH, Peerson JM, Warden CH, Refsum H, Smith
AD, Nygard OK, Ueland PM, Vollset SE, Tell GS. 2007. Relations of
glutamate carboxypeptidase II (GCPII) polymorphisms to folate
and homocysteine concentrations and to scores of cognition,
anxiety, and depression in a homogeneous Norwegian popula-
tion: the Hordaland Homocysteine Study. Am J Clin Nutr 86:
Johansen AM, Lie RT, Wilcox AJ, Andersen LF, Drevon CA. 2008.
Maternal dietary intake of vitamin A and risk of orofacial clefts: a
population-based case-control study in Norway. Am J Epidemiol
Jugessur A, Murray JC. 2005. Orofacial clefting: recent insights into a
complex trait. Curr Opin Genet Dev 15:270–278.
Jugessur A, Lie RT, Wilcox AJ, Murray JC, Taylor JA, Saugstad OD,
Vindenes HA, Abyholm F. 2003a. Variants of developmental
genes (TGFA, TGFB3, and MSX1) and their associations with
orofacial clefts: a case-parent triad analysis. Genet Epidemiol 24:
Jugessur A, Lie RT, Wilcox AJ, Murray JC, Taylor JA, Saugstad OD,
Vindenes HA, Abyholm FE. 2003b. Cleft palate, transforming
growth factor alpha gene variants, and maternal exposures:
assessing gene-environment interactions in case-parent triads.
Genet Epidemiol 25:367–374.
Jugessur A, Wilcox AJ, Lie RT, Murray JC, Taylor JA, Ulvik A,
Drevon CA, Vindenes HA, Abyholm FE. 2003c. Exploring
the effectsof methylenetetrahydrofolate
variants C677T and A1298C on the risk of orofacial clefts
in 261 Norwegian case-parent triads. Am J Epidemiol 157:
Krapels IP, van Rooij IA, Ocke MC, van Cleef BA, Kuijpers-Jagtman
AM, Steegers-Theunissen RP. 2004. Maternal dietary B vitamin
intake, other than folate, and the association with orofacial cleft in
the offspring. Eur J Nutr 43:7–14.
Lidral AC, Moreno LM. 2005. Progress toward discerning the genetics
of cleft lip. Curr Opin Pediatr 17:731–739.
Martin ER, Monks SA, Warren LL, Kaplan NL. 2000. A test for linkage
and association in general pedigrees: the pedigree disequilibrium
test. Am J Hum Genet 67:146–154.
Martinelli M, Scapoli L, Palmieri A, Pezzetti F, Baciliero U, Padula E,
Carinci P, Morselli PG, Carinci F. 2006. Study of four genes
belonging to the folate pathway: transcobalamin 2 is involved in
the onset of non-syndromic cleft lip with or without cleft palate.
Hum Mutat 27:294.
Meyer K, Fredriksen A, Ueland PM. 2004. High-level multiplex
genotyping of polymorphisms involved in folate or homocysteine
metabolism by matrix-assisted laser desorption/ionization mass
spectrometry. Clin Chem 50:391–402.
Mitchell LE, Murray JC, O’Brien S, Christensen K. 2003. Retinoic acid
receptor alpha gene variants, multivitamin use, and liver intake as
risk factors for oral clefts: a population-based case-control study in
Denmark, 1991–1994. Am J Epidemiol 158:69–76.
Mostowska A, Hozyasz KK, Jagodzinski PP. 2006. Maternal MTR
genotype contributes to the risk of non-syndromic cleft lip and
palate in the Polish population. Clin Genet 69:512–517.
Mukhopadhyay P, Greene RM, Zacharias W, Weinrich MC, Singh S,
Young Jr WW, Pisano MM. 2004. Developmental gene expression
profiling of mammalian, fetal orofacial tissue. Birth Defects Res A
Clin Mol Teratol 70:912–926.
Murray JC, Daack-Hirsch S, Buetow KH, Munger R, Espina L,
Paglinawan N, Villanueva E, Rary J, Magee K, Magee W. 1997.
Clinical and epidemiologic studies of cleft lip and palate in the
Philippines. Cleft Palate Craniofac J 34:7–10.
Nguyen RH, Wilcox AJ, Moen BE, McConnaughey DR, Lie RT. 2007.
Parent’s occupation and isolated orofacial clefts in Norway:
Palomino H, Cerda-Flores RM, Blanco R, Palomino HM, Barton SA, de
Andrade M, Chakraborty R. 1997. Complex segregation analysis of
facial clefting in Chile. J Craniofac Genet Dev Biol 17:57–64.
Rubini M, Brusati R, Garattini G, Magnani C, Liviero F, Bianchi F,
Tarantino E, Massei A, Pollastri S, Carturan S, Amadori A,
Bertagnin E, Cavallaro A, Fabiano A, Franchella A, Calzolari E.
2005. Cystathionine beta-synthase c.844ins68 gene variant and
non-syndromic cleft lip and palate. Am J Med Genet A 136:
Scapoli C, Collins A, Martinelli M, Pezzetti F, Scapoli L, Tognon M.
1999. Combined segregation and linkage analysis of non-
syndromic orofacial cleft in two candidate regions. Ann Hum
Scapoli L, Marchesini J, Martinelli M, Pezzetti F, Carinci F, Palmieri A,
Rullo R, Gombos F, Tognon M, Carinci P. 2005. Study of folate
receptor genes in nonsyndromic familial and sporadic cleft
lip with or without cleft palate cases. Am J Med Genet A 132:
Sebastiani P, Lazarus R, Weiss ST, Kunkel LM, Kohane IS, Ramoni MF.
2003. Minimal haplotype tagging. Proc Natl Acad Sci USA
Shaw GM, Wasserman CR, Murray JC, Lammer EJ. 1998. Infant TGF-
alpha genotype, orofacial clefts, and maternal periconceptional
multivitamin use. Cleft Palate Craniofac J 35:366–370.
Shi M, Umbach DM, Weinberg CR. 2007. Identification of risk-related
haplotypes with the use of multiple SNPs from nuclear families.
Am J Hum Genet 81:53–66.
Sivertsen A, Wilcox AJ, Skjaerven R, Vindenes HA, Abyholm F,
Harville E, Lie RT. 2008. Familial risk of oral clefts by morpholo-
gical type and severity: population based cohort study of first
degree relatives. Br Med J 336:432–434.
Soprano DR, Soprano KJ. 1995. Retinoids as teratogens. Annu Rev
van Rooij IA, Vermeij-Keers C, Kluijtmans LA, Ocke MC, Zielhuis GA,
Goorhuis-Brouwer SM, van der Biezen JJ, Kuijpers-Jagtman AM,
Steegers-Theunissen RP. 2003. Does the interaction between
maternal folate intake and the methylenetetrahydrofolate reduc-
tase polymorphisms affect the risk of cleft lip with or without cleft
palate? Am J Epidemiol 157:583–591.
van Rooij IA, Ocke MC, Straatman H, Zielhuis GA, Merkus HM,
Steegers-Theunissen RP. 2004. Periconceptional folate intake by
supplement and food reduces the risk of nonsyndromic cleft lip
with or without cleft palate. Prev Med 39:689–694.
254 Boyles et al.
Vieira AR, Romitti PA, Orioli IM, Castilla EE. 2003. Inheritance of cleft Download full-text
palate in South America: evidence for a major locus recessive.
Orthod Craniofac Res 6:83–87.
Wigginton JE, Cutler DJ, Abecasis GR. 2005. A note on exact tests of
Hardy–Weinberg equilibrium. Am J Hum Genet 76:887–893.
Wilcox AJ, Lie RT, Solvoll K, Taylor J, McConnaughey DR, Abyholm F,
Vindenes H, Vollset SE, Drevon CA. 2007. Folic acid supplements
and risk of facial clefts: national population based case-control
study. Br Med J 334:464–470.
Zhu H, Curry S, Wen S, Wicker NJ, Shaw GM, Lammer EJ, Yang W,
Jafarov T, Finnell RH. 2005. Are the betaine-homocysteine
methyltransferase (BHMT and BHMT2) genes risk factors for
spina bifida and orofacial clefts? Am J Med Genet A 135:
255 Oral Facial Clefts and Vitamin Metabolism Genes