Genes encoding critical transcriptional activators for murine neural tube development and human spina bifida: a case-control study.
ABSTRACT Spina bifida is a malformation of the neural tube and is the most common of neural tube defects (NTDs). The etiology of spina bifida is largely unknown, although it is thought to be multi-factorial, involving multiple interacting genes and environmental factors. Mutations in transcriptional co-activator genes-Cited2, p300, Cbp, Tfap2α, Carm1 and Cart1 result in NTDs in murine models, thus prompt us to investigate whether homologues of these genes are associated with NTDs in humans.
Data and biological samples from 297 spina bifida cases and 300 controls were derived from a population-based case-control study conducted in California. 37 SNPs within CITED2, EP300, CREBBP, TFAP2A, CARM1 and ALX1 were genotyped using an ABI SNPlex assay. Odds ratios and 95% confidence intervals were calculated for alleles, genotypes and haplotypes to evaluate the risk for spina bifida.
Several SNPs showed increased or decreased risk, including CITED2 rs1131431 (OR = 5.32, 1.04~27.30), EP300 rs4820428 (OR = 1.30, 1.01~1.67), EP300 rs4820429 (OR = 0.50, 0.26~0.50, in whites, OR = 0.7, 0.49~0.99 in all subjects), EP300 rs17002284 (OR = 0.43, 0.22~0.84), TFAP2A rs3798691 (OR = 1.78, 1.13~2.87 in Hispanics), CREBBP rs129986 (OR = 0.27, 0.11~0.69), CARM1 rs17616105 (OR = 0.41, 0.22~0.72 in whites). In addition, one haplotype block in EP300 and one in TFAP2A appeared to be associated with increased risk.
Modest associations were observed in CITED2, EP300, CREBBP, TFAP2A and CARM1 but not ALX1. However, these modest associations were not statistically significant after correction for multiple comparisons. Searching for potential functional variants and rare causal mutations is warranted in these genes.
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ABSTRACT: The California Birth Defects Monitoring Program maintains a population-based birth defects registry of structural congenital malformations, monitoring over 600000 resident births annually. Cases are actively ascertained from hospitals and genetic centres throughout California and from selected facilities in adjacent states. Field staff identify presumptive cases from careful review of medical records. Diagnostic and demographic information is collected from in-patient and genetic centre medical charts for children diagnosed with major structural malformations between conception and 1 year of age. The application of these data to epidemiological investigations of birth defects is described in the context of prevalence studies, aetiological studies and evaluative studies, and the strengths and limitations of the registry data are discussed.Paediatric and Perinatal Epidemiology 09/1991; 5(4):423 - 427. · 2.16 Impact Factor
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ABSTRACT: The protein EP300 and its paralog CREBBP (CREB-binding protein) are ubiquitously expressed transcriptional co-activators and histone acetyl transferases1. The gene EP300 is essential for normal cardiac and neural development, whereas CREBBP is essential for neurulation, hematopoietic differentiation, angiogenesis and skeletal and cardiac development2, 3, 4, 5. Mutations in CREBBP cause Rubinstein-Taybi syndrome, which is characterized by mental retardation, skeletal abnormalities and congenital cardiac defects6, 7. The CBP/p300-interacting transactivator with ED-rich tail 2 (CITED2) binds EP300 and CREBBP with high affinity8 and regulates gene transcription8, 9, 10. Here we show that Cited2 -/- embryos die with cardiac malformations, adrenal agenesis, abnormal cranial ganglia and exencephaly. The cardiac defects include atrial and ventricular septal defects, overriding aorta, double-outlet right ventricle, persistent truncus arteriosus and right-sided aortic arches. We find increased apoptosis in the midbrain region and a marked reduction in ErbB3-expressing neural crest cells in mid-embryogenesis. We show that CITED2 interacts with and co-activates all isoforms of transcription factor AP-2 (TFAP2). Transactivation by TFAP2 isoforms is defective in Cited2 -/- embryonic fibroblasts and is rescued by ectopically expressed CITED2. As certain Tfap2 isoforms are essential in neural crest, neural tube and cardiac development11, 12, 13, we propose that abnormal embryogenesis in mice lacking Cited2 results, at least in part, from its role as a Tfap2 co-activator.Nature Genetics 11/2001; 29(4):469-474. · 35.21 Impact Factor
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ABSTRACT: During closure of the neural tube in the mouse, transcription factor AP-2 is expressed in ectoderm and in neural-crest cells migrating from the cranial neural folds. Cranial neural crest cells provide patterning information for craniofacial morphogenesis, generate most of the skull bones, and together with placodal ectoderm, form the cranial ganglia. To study the role of AP-2 during embryogenesis, we undertook a targeted mutagenesis of the AP-2 gene in the mouse. Here we report that AP-2(-/-) mice died perinatally with cranio-abdominoschisis and severe dismorphogenesis of the face, skull, sensory organs and cranial ganglia. Failure of cranial closure between 9 and 9.5 days postcoitum coincided with increased apoptosis in the midbrain, anterior hindbrain and proximal mesenchyme of the first branchial arch, but did not involve loss of expression of twist or Pax-3, two other regulatory genes known to be required for cranial closure.Nature 06/1996; 381(6579):235-8. · 38.60 Impact Factor
RESEARCH ARTICLEOpen Access
Genes encoding critical transcriptional activators
for murine neural tube development and human
spina bifida: a case-control study
Wei Lu1, Adrian R Guzman1, Wei Yang2, Claudia J Chapa5, Gary M Shaw2, Robert M Greene3, M Michele Pisano3,
Edward J Lammer4, Richard H Finnell5, Huiping Zhu5*
Background: Spina bifida is a malformation of the neural tube and is the most common of neural tube defects
(NTDs). The etiology of spina bifida is largely unknown, although it is thought to be multi-factorial, involving
multiple interacting genes and environmental factors. Mutations in transcriptional co-activator genes-Cited2, p300,
Cbp, Tfap2a, Carm1 and Cart1 result in NTDs in murine models, thus prompt us to investigate whether
homologues of these genes are associated with NTDs in humans.
Methods: Data and biological samples from 297 spina bifida cases and 300 controls were derived from a
population-based case-control study conducted in California. 37 SNPs within CITED2, EP300, CREBBP, TFAP2A, CARM1
and ALX1 were genotyped using an ABI SNPlex assay. Odds ratios and 95% confidence intervals were calculated for
alleles, genotypes and haplotypes to evaluate the risk for spina bifida.
Results: Several SNPs showed increased or decreased risk, including CITED2 rs1131431 (OR = 5.32, 1.04~27.30),
EP300 rs4820428 (OR = 1.30, 1.01~1.67), EP300 rs4820429 (OR = 0.50, 0.26~0.50, in whites, OR = 0.7, 0.49~0.99 in all
subjects), EP300 rs17002284 (OR = 0.43, 0.22~0.84), TFAP2A rs3798691 (OR = 1.78, 1.13~2.87 in Hispanics), CREBBP
rs129986 (OR = 0.27, 0.11~0.69), CARM1 rs17616105 (OR = 0.41, 0.22~0.72 in whites). In addition, one haplotype
block in EP300 and one in TFAP2A appeared to be associated with increased risk.
Conclusions: Modest associations were observed in CITED2, EP300, CREBBP, TFAP2A and CARM1 but not ALX1.
However, these modest associations were not statistically significant after correction for multiple comparisons.
Searching for potential functional variants and rare causal mutations is warranted in these genes.
Studies using genetically modified mouse models have
contributed a great deal to our understanding of molecu-
lar mechanisms that govern critical embryonic develop-
ment processes, such as neural tube closure. Among the
over 200 existing mouse models for neural tube defects
(NTDs), several harbor mutations in genes encoding
transcriptional activators or co-activators, such as Cited2,
Crebbp, p300, Tfap2a, Carm1 and Cart1 [1-9].
CREB binding protein (CREBBP; CBP) was originally
identified as a protein that binds to the phosphorylated
form of the CREB transcription factor and increases the
expression of genes containing CRE elements. It is clo-
sely related to the adenovirus E1A-associated protein
p300. CBP and p300 are functionally redundant histone
deacetylases essential for proliferation and embryonic
development [5,8,10]. Both Crebbp and p300 genes are
expressed at high level in the neural folds at embryonic
day E8.5 in mice . Homozygous deletions of either
the Crebbp or p300 genes in mice results in lethality on
E9~11, with subsequent defects in neurulation, cell pro-
liferation and cardiac development [5,8].
The methylation of the Crebbp/p300 complex is cata-
lyzed by co-activator-associated arginine methyltransferase
(CARM1). This process disables the recruitment of cAMP
response element binding protein (CREB). Thus, CARM1
functions as a co-repressor in the cAMP signaling
* Correspondence: email@example.com
5Dell Pediatric Research Institute, UT Austin, Austin, TX, USA
Full list of author information is available at the end of the article
Lu et al. BMC Medical Genetics 2010, 11:141
© 2010 Lu et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.
pathway, via its methyltransferase activity . In addition
to its enzymatic function, CARM1 is also required for NF-
kappaB-dependent gene expression as a transcriptional
cofactor . Carm1 gene expression was observed in the
fore- and hindbrain, neural folds, somites and posterior
lateral plates of E8.25 mouse embryos, and later on in
development (E8.75) expression became prominent in the
neural tube and somites .
CITED2 is a nuclear co-activator that binds to CBP/
p300. Disruption of the Cited2 gene in mice is embryonic
lethal, with the mice dying secondary to significant
defects in the development of the neural tube and the
heart [2,6,14,15]. The interaction of CBP and p300 with
Cited2 is required for activation of important transcrip-
tional factors such as AP-2a [1,16]. Mice carrying homo-
zygous disruptions of the gene coding for the protein
AP-2 a (Tfap2 a) die at term as a result of severe devel-
opmental abnormalities. The earliest defect observed was
a complete failure of neural tube closure in the cranial
region, and it has been suggested in a chimera model
study that there is an independent requirement for AP-2
a in the formation of the neural tube [17-19].
CART1 is a paired-class homeobox-containing tran-
scription factor regulated by CBP/p300 acetylation of a
highly conserved lysine residue that increases the interac-
tion between CBP/p300 and CART1, ultimately enhancing
its transcriptional activation . The Cart1 gene is
expressed in chondrocytes [21,22], and appears to be
required for embryonic forebrain mesenchymal survival.
The absence of CART1 disrupts cranial neural tube
morphogenesis by blocking the initiation of closure in the
midbrain region. Homozygous Cart1 mutant mice are
born alive with neural tube closure defects including
acrania and meroanencephaly, but die soon after birth .
The Cart1 knockout mouse is among several NTD mouse
models that can be rescued by maternal supplementation
with folic acid . The gene encoding human CART1
is ALX1 (ALX homobox 1). In summary, CBP, p300,
CARM1, TFAP2a, CITED2 and CART1 are transcrip-
tional activator/co-activators that contributes to the onto-
geny of the neural tube in mammalian systems. Whether
gene homologues are associated with human NTDs
remains unknown. Three single nucleotide polymorphisms
(SNP) in the un-translated region of CITED2 gene were
studied in a group of patients with spina bifida and con-
trols, modest associations were observed but results were
imprecise owing to small sample size . Mutations in
the human CBP gene have been identified as being respon-
sible for the Rubinstein-Taybi syndrome (RTS), which has
a defined craniofacial phenotype . The purpose of this
study is to evaluate the potential association between var-
iations among human CITED2, CREBBP, EP300, TFAP2A,
CARM1 and ALX1 genes and the risk for spina bifida.
Data and biological samples were obtained from a case-
control study conducted by the California Birth Defects
Monitoring Program (CBDMP). The CBDMP is an active,
population-based surveillance system for collecting infor-
mation on infants and fetuses with structural congenital
malformations, which has been described elsewhere .
Included were 297 singleton infants with spina bifida
(cases) and 300 non-malformed infants (controls). Both
cases and controls were randomly selected from the
samples with bloodspots available. Cases were selected
from California Birth Defect Registry 1990-1999; non-
malformed controls were selected from Vital Statistics
matching the birth years with the selected cases. Case and
control infants were linked to their newborn screening
bloodspots. All samples were obtained with approval from
the State of California Health and Welfare Agency
Committee for the Protection of Human Subjects.
Candidate genes and SNPs
The 6 candidate genes and SNPs genotyped are listed in
Table 1. Tagging SNPs were selected for each candidate
gene using the SNPbrowser™ Software (ABI, Foster City,
CA) based on data from the four HapMap populations
(CEU, YRI, JPT and CHB) . All validated tagging
SNPs with pairwise r2> = 0.8 with a minor allele fre-
quency (MAF) no less than 5% were included for geno-
typing. Also included for study were all non-synonymous
coding SNPs in these genes. Based on these criteria, 37
SNPs were genotyped.
DNA was extracted from blood spots using the Gentra
Puregene DNA Extraction Kit (Qiagen, Valencia, CA)
following the standard protocol. Prior to genotyping,
genomic DNA was amplified using a commercially avail-
able multiple displacement amplification (MDA) kit,
GenomiPhi DNA Amplification kit (GE Healthcare, Pis-
cataway, NJ). The whole genome amplification (WGA)
product was then quantified using RNase P assay
(AppliedBiosystems, Foster City, CA). 200 ng of WGA
product was used for the SNPlex assay containing the
37 SNPs. All genotyping assays were performed blinded
to subjects’ case or control status.
The SNPs selected for analyses were submitted to
Applied Biosystems to create a unique SNPlex assay.
The SNPlex™ genotyping system (Applied Biosystems,
Foster City, CA) is based on a universal multiplexed oli-
gonucleotide ligation assay (OLA)/PCR and drag chute
mobility modifier technology. The resulting genotype
data were analyzed using GeneMapper™ v4.0 (Applied
Biosystems, Foster City, CA).
Lu et al. BMC Medical Genetics 2010, 11:141
Page 2 of 7
Analyses were performed for the overall study group as
well as for specific ethnic/race groups. Hispanic infants
were further subdivided into those whose mothers were
born in the United States, or native-born (Hisp-NB) and
those whose mothers were not, or foreign-born (Hisp-FB),
as it has been shown that these two groups are different
with respect to ethnic composition and social economic
Table 1 Characteristics of all genotyped SNPs
rs116087731284229006 A/G100.00.229 0.4350.274 0.4780.217 0.391
rs4761130 1284226396C/T 99.8 0.4210.286 0.3580.1540.4010.465
rs72952421284222948A/G100.0 0.161 0.7810.074 0.5570.1590.358
rs11667234 1910880581 A/C99.8 0.0210.010 0.039 0.071 0.0160.934
rs1167036519 10865833C/T100.0 0.0560.9180.0840.754 0.0450.702
rs1529711 1910884434 A/G 99.70.0970.492 0.1610.9320.0810.519
rs154159619 10848013 A/G99.50.475 0.3260.4970.9690.408 0.957
rs17616105 1910883159C/T 100.00.1890.732 0.2350.3650.1520.072
rs725470819 10863579A/G100.0 0.071 0.0430.123 0.1160.0590.441
rs11076786 163751597 C/T100.0
rs129986 163744242 C/T 100.00.0230.530 0.0320.5900.020 0.735
rs130003 163768173 A/G99.80.015 0.7920.032 0.7110.0100.967
rs13000816 3721953 C/G99.8 0.0110.8380.0320.752 0.001 1.000
rs17136507163740546 C/T100.0 0.178 0.5360.1100.229 0.2270.449
rs886528163751557 C/T99.70.383 0.5900.3900.986 0.3680.839
rs9392163715170 A/G99.8 0.1890.566 0.2230.625 0.1380.700
rs1700228422 39826792A/G 100.0 0.265 0.2200.313 0.745 0.2170.522
rs17433014 2239829768A/G 99.8 0.0180.7690.029 0.793 0.0190.834
rs22301112239851949A/G99.8 0.0020.9770.006 0.9591.000 1.000
rs48204292239868057 C/G100.00.1590.3820.258 0.0400.1240.471
rs48220062239849308C/T 100.00.247 0.8440.355 0.5870.185 0.740
rs5758246 22 39871792A/G100.0 0.0300.635 1.000 1.0000.012 0.934
rs96115022239861182C/T 100.0 0.014 0.8380.019 0.9180.013 0.867
rs1621700610528084 A/G 99.70.2880.6710.252 0.0810.3570.773
rs3030506 10511169 C/T 100.0 0.2570.061 0.1650.259 0.2620.267
rs3030556 10527462C/T100.00.353 0.150 0.435 0.5690.3350.025
rs303056610528467 C/G 100.00.1590.2290.0940.477 0.1261.000
rs3798691610519381 C/G100.00.112 0.0210.042 0.0110.134 0.111
rs3798694610516743 A/G100.0 0.0620.1220.0870.2250.059 0.293
rs533558 6 10503558A/G 100.00.4410.8480.361 0.9550.4810.858
rs537112610503192 A/G 100.00.180 0.5710.1030.7540.1950.255
*MAF: minor allele frequency; **HWE: Hardy-Weinberg Disequilibrium.
Lu et al. BMC Medical Genetics 2010, 11:141
Page 3 of 7
status [26,27]. Departure from Hardy-Weinberg Equili-
brium (HWE) was assessed by Chi-square analysis in each
subgroup using data from controls only. Odds ratios (OR)
for SNP genotypes were calculated using logistic regres-
sion analyses. Since the mode of inheritance is unknown,
potential associations with spina bifida risk were evalua-
ted assuming dominant, recessive and additive models
for each SNP. Chi-square tests were used to assess the sig-
nificance of dominant and recessive models. Cochran-
Armitage trend tests were used to assess the significance
of additive models. If numbers were less than 5, Fisher’s
exact test and Exact Armitage trend test were applied.
Analyses were performed using SVS v7.1.1 (GoldenHelix,
Bozeman, MT) software. The false discovery rate (FDR)
 was used to account for multiple testing (data
not shown). Haplotype blocks were constructed using
HaploView 4.2 http://www.broadinstitute.org/haploview/
haploview in each race/ethnicity group with pair-wise
D’≥0.85. Haplotype frequencies were estimated based on
the Maximum Expectation (EM) algorithm. Association of
each haplotype was evaluated by calculating ORs and 95%
confidence intervals against all other haplotypes. Estimated
risks were considered statistically precise if 95% confiden-
tial interval of OR does not include 1.00.
Results and Discussion
Demographic characteristics of the cases and controls
are listed in Table 2. Cases and controls were similar
with respect to maternal age, parity, and plurality but
expectedly differed with respect to race/ethnicity and
maternal educational attainment. That is, the data
revealed greater frequencies of Hispanics (primarily for-
eign born) and women with low educational level (less
than high school) in the spina bifida case group.
Genotyping of 37 SNPs was performed using a custom-
made Applied Biosystems SNPlex panel. Due to low call
rates, two SNPs, rs7775752 (45.1%) and rs11076785
(46.6%), were excluded from analysis. Departure
from HWE in the controls (p < 0.01) was observed in
rs11076786 and rs4820428 when analyzing the whole
dataset. This deviation was eliminated when stratified by
race/ethnicity (Table 1). Several SNPs and haplotypes con-
taining these SNPs showed modest associations with spina
bifida risk (p ≤ 0.05, Table 3 and 4). These associations are
summarized below for each gene. The results, however,
are largely indicative of no or very limited associations,
particularly after correction for multiple comparisons.
Only one of the two SNPs genotyped was fully analyzed.
SNP rs1131431 showed a genotypic association (OR =
5.32, 95% CI: 1.04~27.30) based on a recessive mode of
inheritance, specifically in the White population. It is
noteworthy that the confidence interval had a wide
range, suggesting that the estimate may be imprecise
due to the small number of White cases and controls
with the variant. This SNP, however, was previously
associated with a modest increase in risk for spina bifida
and congenital heart defects .
Among the 8 SNPs analyzed, the G allele of SNP
rs4820428 was over-represented in spina bifida cases
(OR = 1.30, 95% CI: 1.01~1.67) in all subjects. Analyses
showed that rs4820428 was associated with spina bifida
risk under a dominant model (OR = 1.54 95%CI:
1.10~2.17). The minor allele of rs4820429 (C) showed a
reduced risk in the Whites (OR = 0.50, 95% CI:
0.26~0.50) and in the whole dataset (OR = 0.70, 95% CI:
0.49~0.99) under a dominant model. Minor allele of
rs17002284 (G) also showed a reduced risk (OR = 0.43,
95%CI: 0.22~0.84), but under a recessive model. These
three SNPs were in linkage disequilibrium. Subsequent
analysis showed that haplotype ACGG (rs17002284-
rs4822006-rs4820428-rs4820429) accounted for 50.3% of
Table 2 Demographic distribution of malformed cases
and non-malformed controls, California 1990-1999
n = 297 (%1)
n = 300 (%1)
Hispanic, Native Born
Hispanic, Foreign Born
Maternal age at delivery (years)
Less than high school
Greater than High school
1Percentages may not equal 100 owing to missing data or rounding.
Lu et al. BMC Medical Genetics 2010, 11:141
Page 4 of 7
all haplotypes in the Whites and was over-represented
in the white cases (OR = 1.30, 95%CI: 1.01~1.67). It is
noteworthy that this high risk haplotype contains the
high risk alleles for rs17002284 (A), rs4820428 (G) as
well as rs4820429 (G) (Table 5).
Among the 9 SNPs analyzed, rs303056 (C), rs3798691
(C), rs533558 (G) showed allelic association with spina
bifida risk in Hispanics. Genotypic association analysis
showed that rs3798691 was associated with spina bifida
risk under a dominant model in Hispanics (OR = 1.78,
95% CI: 1.13~2.87). SNP rs533558 also showed an asso-
ciation under a dominant model, but only in native-born
Hispanics (OR = 3.32, 95%CI: 1.08~10.18). Subsequent
haplotype analysis predicted two haplotype blocks in
Hispanics. Haplotype AGCGC of block1 (rs537112-
rs533558-rs303050-rs3798694-rs3798691) had an average
Table 3 Allele associations (P < 0.05) among subgroups
SNP Gene EthnicityrefSNP alleles Minor AlleleRatio Counts
P value Odds Ratio95% CI
white C/TT118:0, 182:10 0.008* n/a n/a
whiteC/TC101:17, 136:56 0.0032.451.34-4.46
whiteC/GC16:102, 13:179 0.0462.16 1.00-2.15
HispanicC/GC65:333, 29:2670.013 1.801.13-2.87
Hispanic-FB C/GC 52:280, 22:2080.0361.761.03-2.98
Hispanic-NB A/GG36:30, 24:42 0.0362.10 1.05-4.22
All subjectsA/GG276:240, 229:2590.0381.301.01-1.67
Table 4 Genotype associations (P < 0.05) among subgroups under different modes of inheritance
P valueMode of Inheritance Odds Ratio
[(ratio count (case, control))
All subjects 0.012Recessive
0.43 (13, 29; 284, 271)0.22 - 0.84
All subjects0.011Additive [(Dd) vs (dd)]
1.46 (132, 122; 87, 117)1.00 - 2.11
[(DD) vs (Dd)] 1.18 (78, 61; 132, 122)0.78 - 1.79
All subjects0.012 Dominant
1.54 (210, 183; 87, 117) 1.10 - 2.17
0.70 (75, 98; 222, 202)0.49 - 0.99
0.50 (22, 52; 37, 44)0.26 - 0.50
All subjects 0.041Additive [(Dd) vs (dd)]1.30 (131, 118; 138, 161)0.92 - 1.81
[(DD) vs (Dd)]1.33 (28, 19; 131, 118) 0.70 - 2.50
Hispanic0.015Additive [(Dd) vs (dd)]
1.78 (48, 23; 143, 122)1.04 - 3.19
[(DD) vs (Dd)]1.40 (9, 3; 47, 22) 0.35 - 5.70
1.84 (56, 26; 143, 122)1.13 - 3.25
1.82 (44, 19; 122, 92)1.00 - 3.40
Hispanic-NB0.038 Additive [(Dd) vs (dd)]3.00 (18, 14; 6, 14)0.92 - 9.80
[(DD) vs (Dd)] 1.40 (9, 5; 18, 14)0.38 - 5.12
Hispanic-NB 0.032 Dominant
3.32 (27, 19; 6, 14)1.08 - 10.18
Hispanic0.0Recessive n/a (6, 0; 193, 148)n/a
White0.004Additive [(Dd) vs (dd)]0.48 (15, 36; 43,50) 0.23 - 1.00
[(DD) vs (Dd)]0.24 (1,10; 15,36) 0.03 - 2.04
0.40 (16, 46; 43, 50)0.40 - 0.20
5.32 (6, 2; 53, 94) 1.04 - 27.30
All subjects 0.005Additive [(Dd) vs (dd)]
0.27 (6, 21; 291, 279)0.11 - 0.69
[(DD) vs (Dd)] n/a (0, 0; 6, 21)n/a
0.27 (6, 21; 291, 279)0.11 - 0.69
White0.014 Additive [(Dd) vs (dd)] n/a (0, 10; 59, 86)n/a
[(DD) vs (Dd)]n/a (0, 0; 0, 10) n/a
White 0.010Dominant n/a (0, 10; 59, 86)n/a
Lu et al. BMC Medical Genetics 2010, 11:141
Page 5 of 7
frequency of 0.133 and was associated with an increased
risk, OR = 1.73 (1.08-2.77) compared to all other haplo-
types. Since no high risk SNP has been discovered, these
high risk blocks will be thoroughly sequenced to search
for other variants (Table 5).
Among the 8 SNPs analyzed, the heterozygous status of
SNP rs129986 (TC) was associated with a reduced risk
in all subjects (OR = 0.27, 95% CI: 0.11~0.69). The
homozygous status of the minor allele, CC, was not
observed. The TC genotype was observed in only 6
cases and they were all Hispanics.
6 SNPs were genotyped. In Whites, the minor allele (C) of
rs17616105 showed a reduced risk with OR = 0.41 (95%
CI: 0.22~0.75). Genotype analysis showed that this effect
followed a dominant inheritance model (ORCC+CT= 0.40,
95% CI: 0.20~0.40), and was not observed in the
3 SNPs were genotyped. No association was observed
between any SNP and spina bifida risk. It is known that
the Cart1 knockout mouse responses to maternal supple-
mentation with folic acid . The majority of the DNA
samples for this study were collected after folic acid forti-
fication is implied in the USA, which might interact with
the genotyping effect; however, our study lacks the statis-
tic power to analyze the gene-environment interaction.
To our knowledge, this is the first study evaluating
human CREBBP, EP300, CARM1, TFAP2A, and ALX1
genes as possible risk factors for spina bifida. Associa-
tion between CITED2 and spina bifida has been studied
before, and results from our current study are consistent
with the previous findings . Modest associations
were observed between spina bifida risk and TFAP2A,
EP300, CITED2, CREBBP1 and CARM1. No association
was observed in ALX1 gene. However, given the number
of comparisons made, relatively few associations
emerged. Although NTDs are multifactorial diseases, it
cannot be excluded that the studied genes may contain
rare mutations that cause some of the NTDs. With the
most advanced next-generation sequencing technique
being available, we may be able to discover such rare
variants in our study population.
The strengths of this study include its population-
based ascertainment of cases and controls and its eva-
luation of race/ethnicity as potential modifiers of risk in
the presence of variant genotypes and haplotypes. A
major limitation of our study was the small sample size,
which reduced the statistical power of analyses. Replica-
tion of some of the modest findings in a larger sample
set may be warranted.
We have observed modest associations with risk of spina
bifida in CITED2, EP300, CREBBP, TFAP2A and CARM1
genes but not ALX1 gene. These modest associations
were not statistically significant after correction for mul-
tiple comparisons. Future effort will focus on searching
for potential functional variants and rare causal muta-
tions in these potential candidate genes.
CREB: cAMP response element binding protein; CRE: cyclic AMP-responsive;
CITED2: Cbp/p300-interacting transactivator, with Glu/Asp-rich carboxy-
terminal domain, 2; EP300: E1A binding protein p300 (Alias: p300); CREBBP:
CREB binding protein (Alias: CBP); TFAP2A: transcription factor AP-2 alpha
(activating enhancer binding protein 2 alpha) (Alias: AP-2); CARM1:
coactivator-associated arginine methyltransferase 1; ALX1: ALX homeobox 1
Table 5 Haplotype associations (only significant associations are shown)
Haplotype BlockFrequency P Value Odds Ratio (case, control) 95% CI
TFAP2A-rs537112, rs533558, rs303050, rs3798694, and rs3798691 in Hispanic
0.0205 1.73 (63.0 : 335.0, 29.0 : 267.0)
0.2019 0.82 (197.0 : 201.0, 161.0 : 135.0)
0.8135 0.96 (78.9 : 319.1, 60.8 : 235.2)
0.4974 1.26 (25.0 : 373.0, 15.0 : 281.0)
0.3207 0.69 (13.9 : 384.1, 14.8 : 281.2)
0.6568 0.81 (10.1 : 387.9, 9.2 : 286.8)
0.9639 0.97 (8.1 : 389.9, 6.2 : 289.8)
EP300- rs17002284, rs4822006, rs4820428 and rs4820429 in non-Hispanic white
0.0377 1.30 (276.0 : 240.0, 229.0 : 259.0)
0.1665 0.82 (117.0 : 399.0, 129.0 : 359.0)
0.054 0.72 (73.0 : 443.0, 91.0 : 397.0)
0.8074 1.06 (38.0 : 478.0, 34.0 : 454.0)
0.2348 1.91 (10.0 : 506.0, 5.0 : 483.0)
Lu et al. BMC Medical Genetics 2010, 11:141
Page 6 of 7
This research was supported in part by funds from NIH grants R01 NS050249
(GMS), R01 HD053509 (RMG), P20 RR017702 from the COBRE program of the
National Center for Research Resources (RMG), as well as a contract from the
Centers for Disease Control and Prevention, Center of Excellence Award UO1/
DD000491. We thank the California Department of Public Health Maternal Child
and Adolescent Health Division for providing biological samples and data for
these analyses. The findings and conclusions in this report are those of the
authors and do not necessarily represent the views of the California Department
of Public Health. The authors thank Ms. Consuelo Vega for her technical
1Institute of Biosciences and Technology, Texas A&M University System
Health Science Center, Houston, TX, USA.2Stanford University, Stanford, CA,
USA.3University of Louisville Birth Defects Center, Louisville, KY, USA.
4Children’s Hospital Oakland Research Institute, Oakland, CA, USA.5Dell
Pediatric Research Institute, UT Austin, Austin, TX, USA.
All authors have read and approved the final manuscript. WL performed and
supervised most of the experiments, and also performed part of the
statistical analysis. ARG and CJC performed most of the experiments and
helped with manuscript writing. WY performed part of the statistical analysis
and helped with the manuscript writing. GMS contributed to the study
design and authorized the use of CBDMP samples. RMG and MMP
contributed to candidate gene selection and study. RHF contributed to the
study design and laboratory instruction of the molecular biology
experiments. EJL reviewed and confirmed the clinical diagnosis of study
subjects. HZ contributed to the overall study design, quality assurance of
genotyping experiments, statistical analysis and most of the manuscript
The authors declare that they have no competing interests.
Received: 1 June 2010 Accepted: 8 October 2010
Published: 8 October 2010
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The pre-publication history for this paper can be accessed here:
Cite this article as: Lu et al.: Genes encoding critical transcriptional
activators for murine neural tube development and human spina bifida:
a case-control study. BMC Medical Genetics 2010 11:141.
Lu et al. BMC Medical Genetics 2010, 11:141
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