Margaret DiPietro
The Role of Gene-Environment and Gene-Gene interaction in relation to Neural Tube
Defects.
Neural tube defects (NTD) is a general term for a congenital malformation of the central
nervous system CNS) occurring secondary to lack of closure of the neural tube. Neural tube
defects are the second most common form of birth defects and affect approximately 300,000
newborns worldwide each year (CDC, 2005) The two most common NTDs are spina bifida (17.9
per 100,000 live births in the United States) and anencephaly, (11.11 per 100,000 live births in
the United States; CDC, 2007). Spina bifida, which affects approximately 1,500 – 2,000
newborns every year in the United States, results from a failure of the neural tube to close
properly, a process that is normally completed at twenty-eight days gestation in humans.
Anencephaly, is the absence of a large part of the brain and the skull (Shookhoff et al. 2010)
Spina bifida can occur from either failures of the posterior neural pore to close (primary
neurulation) or when the surface ectoderm fails to separate from the neural tube (secondary
neurulation). Primary neurulation, which forms the brain and the majority of the spinal cord,
results in the formation of the neural tube and midline fusion of the neural plate. Secondary
neurulation is continuation of the neural tube folding. Open NTDs are due to the improper
closure during primary neurulation (meningocele and myelomeningocele), whereas closed NTDs
are due to improper closure during secondary neurulation (spina bifida occulta (group of
conditions involving the spinal column)). This improper closure may be the result of an
abnormally reduced rate of cell proliferation in the notochord but not the neuroepithelium (Sing
Au et al. 2010). (See Figure 1)
Fig. 1. Schematic diagram of neural plate bending. Neural
folds form at the lateral extremes of the neural plate (a,
arrows), elevate (b, arrows) and converge toward the dorsal
midline (c). Bending or hinge points form at two sites: the
median hinge point (MHP) overlying the notochord and
the paired dorsolateral hinge points (DLHP) at the lateral
sides of the folds (c). nc, notochord; np, neural plate,
pe, presumptive epidermis.
Supplementation of folic acid before and during pregnancy has been associated with
decreased risk of neural tube defects (Berry et al. 1999). The benefit of daily folic acid (FA)
supplementation during periconception resulted in the decreased risk of neural tube defects, a
fact that led to recommendations that all women at risk, for pregnancy, take 400 ug of FA per
day (ug/d) from fortified supplements. In the United States cereal grains have been fortified with
folic acid to reduce the incidence of neural tube defects because the unintended pregnancy rate in
is approximately 49 % (Berry et al. 1999).
A study was conducted to analyze the effect of folic acid fortified foods on the incidence
of neural tube defects in live newborns at Prince Badea Teaching Hospital, in northern Jordan
(Amarin et al. 2010). The researchers retroactively studied a seven year period which included
times before the fortification of cereal was implemented and after folic acid fortification of cereal
was implemented. The differences between the incidence of neural tube defects in the periods
before and after fortification of folic acid were statistically significant; they saw a reduction of
49%. (See Table 1) This reduction is consistent with reductions observed in other countries that
have fortified their food supplies with folic acid (Amarin et al. 2010).
Table 1
Period
Years
Live births
NTDs
Rate per 1000
(95% CI)
2000-01
18392
34
1.85
(1.2, 2.4)
Introduction period
2002-04
26286
28
1.07
(0.7, 1.5)
After fortification
2005-06
16769
16
0.95
(0.5, 1.5)
Before fortification
The risk of neural tube defects is determined by both genetic and environmental factors,
among which folate status appears to have a key role (Burren et al. 2008). However the precise
nature of the link between folate status and NTDs is poorly understood and it remains unclear
how folic acid prevents NTDs. We know that folate one-carbon metabolism plays several key
cellular roles including provision of nucleotides for DNA synthesis and generation of sadenosylmethionine (SAM), which is a key methylation cycle intermediate (methyl donor).
Burren et al, (2008) showed that lack of folates can cause a profound retardation of embryonic
growth and development progression, suggesting that cell proliferation is compromised. Thus, at
a given gestational age, folate-deficient embryos contain less protein, and have smaller crownrump length and fewer somites, than embryos developing under folate-replete conditions.
Population and family-based genetic studies indicate a complex multigenic cause of
neural tube defects (Kibar et al. 2007). Although no causative gene has been identified in
humans, several genes, such as Pax3, Vangl1, and Vangl2 have been associated with neural tube
defects (Doudney et al. 2005). Researchers have investigated the effect of folate level on the risk
of NTDs in splotch (Sp2h) mice, which carry a mutation in Pax3. (Pax 3 is a family of tissue
specific transcription factors, who have been identified with ear, eye and facial development)
Folate deficiency does not cause NTDs in wild-type mice, but causes significant increase in
cranial NTDs among Sp2H embryos, demonstrating a gene-environmental interaction (Burren et
al. 2008). (See Table 2)
Table 2
Development, growth
and incidence of
NTDs among splotch
embryos cultured in
the presence of 250
µM homocysteine
(Hcy)
Treatment/genotype
Control
+/+
Sp2H/+
Sp2H/Sp2H
Hcy
+/+
Sp2H/+
Sp2H/Sp2H
No.
Cranial
NTDs
PNP length
(mm)
Somites
Crown-rump length
(mm)
Yolk sac score
2
10
5
0
0
2 (40%)
0.49 ± 0.09
0.42 ± 0.12
1.54 ± 0.15*
25.0 ± 0.0
25.7 ± 0.78
24.6 ± 0.51
3.33 ± 0.17
3.37 ± 0.13
3.47 ± 0.14
2.5 ± 0.5
2.4 ± 0.3
1.6 ± 0.4
4
12
8
0
0
2 (25%)
0.18 ± 0.09
0.19 ± 0.05
1.32 ± 0.19*
23.0 ± 1.9
25.4 ± 0.6
25.0 ± 0.7
3.19 ± 0.30
3.44 ± 0.10
3.6 ± 0.20
1.3 ± 0.3
2.1 ± 0.2
2.0 ± 0.3
Embryos were cultured for 40 h from E8.5, and no difference was observed between treatment groups in
number of somites, crown-rump length (indicators of developmental progression and growth respectively) or
yolk sac circulation score (indicator of viability). As expected the PNP is significantly enlarged in Sp2H/Sp2H
embryos compared with other genotypes (*P < 0.05; one-way ANOVA), but no difference was observed
between treatments for any genotype. The frequency of cranial NTDs among Sp2H/Sp2H embryos was not
affected by homocysteine treatment (P > 0.05; Fisher exact test).
Although no causative gene for NTDs has been identified in humans, mutated Vangl2 is
present in the loop-tail (Lp) mouse mutant with a severe defect known as craniorachischisis, a
congenital fissure of the skull and spine (Stein et al. 1953). The loop-tail mouse mutant causes
affected heterozygote and homozygote mice to have a short curled tails and some
affected heterozygotes mice may also display spina bifida at birth. No homozygotes with Spina
bifida have been observed. Wild type siblings are phenotypically normal. The loop tail-like
mutation is mapped to Chromosome 1, near the location of the gene Vangl2. Vangl2 is the
mammalian homologue of the drosophila gene Stbm/Vang, which is required for establishing
cell polarity in the developing eye, wing, and leg tissues. The study by Kibar et al. (2007) in the
New England Journal of Medicine looked at 144 patients with neural tube-defects, and identified
three mutations in the VANGL1 gene in patients with familiar types (V2391 and R274Q) and a
sporadic type (M328T) of the disease, including a spontaneous mutation (V2391) appearing in a
familiar setting (Kibar et al. 2007). (See Figure2)
In order to understand the role of Vangl1 in normal development additional research has
been conducted on the Vangl1 and Vangl2 gene mutation association with NTDs. Torban’s
group (Torban et al. 2008) studied the Looptail mouse, where homozygosity for loss-of-function
alleles at Vangl2 caused severe NTD craniorachischisis. In sporadic cases of neural tube defects
found in humans there is an association between Vangl1 heterozygosity and NTDs. The Vangl1
gene shows a dynamic pattern of expression in the developing neural tube and notochord at the
time of neural tube closure. Based on these results, Torban et al. (2008) proposed that the Vangl
pathway is exquisitely sensitive to gene dosage during NT formation, with a minimum threshold
level of combined Vangl activity required for neural tube closure. Furthermore, they proposed
that Vangl1 and Vangl 2 have a redundant function so that, in cells that have high levels of
Vangl2, loss of Vangl1 may be completely masked. In conclusion the Vang1 functions in the
mammalian planar cell polarity (PCP) pathway (The planar cell polarity Wnt signaling pathway
(PCP) plays a major role in embryonic tissue patterning, cell polarization, migration and
morphogenesis), acting in concert with Vangl2 to control neural tube formation. Their results
show that genetic interactions between Vangl1 and Vangl2 can cause NTDs and raise the
possibility that interactions between Vangl genes and other genetic loci and/or environmental
factors may contribute to the etiology of NTDs (Torban et al. 2008).
Fig. 2.
Genetic interaction between Vangl1 and Vangl2 during neural tube
closure. The presence of craniorachischisis in Vangl2lp/lp (E) and in Vangl1
;Vangl2
gt/+
lp/+
double heterozygote embryos (arrows in C and D) is
compared with phenotypically normal Vangl1
;Vangl2
gt/+
+/+
controls (A
and B). E13.5 embryos are shown in A–D. E18.5 embryos are shown in E
and F. B and D are dorsal views of embryos in A and C. Closed vs. open
eyelid is identified by white arrows in E and F.
Perhaps the most compelling case for gene-environment interactions is the association of
NTDs with vitamin deficiency and the capacity for maternal folic acid supplementation to reduce
the occurrence rate of anencephaly or spina bifida in some populations. Yet, after more than two
decades of research, the mechanism(s) by which folic acid can intervene to prevent NTDs are not
completely understood. A significant challenge has been the illumination of which gene
mutation(s) confer NTD risk and how/what part of the folic acid metabolic pathway compensates
for a given deficit (Marean et al. 2011).
The folic acid fortification of milled grains was instituted in 1998 as a result of the data
which showed its role in the reduction of NTDs. Currently, a little over 50% of pregnant women
in the United States report daily supplementation of folic acid in the periconceptional period and,
another report starting intake in the prenatal period (Carmichael et al. 2006). Reports following
this increase in folic acid intake, showed a link of a lower risk of involuntary abortion (George et
al. 2006), pre-clampsia (Wen et al. 2006), gestational hypertension (Hernandez-Diaz et al. 2002),
and congenital abnormalities (Godwin et al. 2008) in relation to folic acid intake (Hoyo et al.
2011).
However, more recently, periconceptional folic acid intake has also been associated with
an increased risk of wheezing and asthma in both mice and humans (Haberg et al. 2009). (See
Table 3) The study by Marean et al. (2011) showed that folic acid supplementation adversely
affect murine neural tube closure and embryonic survival. As a result of having folic acid
fortification in our grain supply Marean’s group looked at folic acid exposure over a long time
period and reported results that showed three genetic mutations which respond adversely to folic
acid supplementation by displaying an increased incidence of NTDs in homologous mutants
(Marean et al. 2011).
Table 3 Incidence proportions (%) and adjusted* relative risks with 95% CI for wheeze, lower respiratory tract
infections (LRTIs) and hospitalisations for LRTIs up to 18 months of age according to prenatal exposure to folic acid
supplements for children born in 2000–2005 (Haberg et al. 2009)
Folic acid
supplements in
pregnancy
Wheeze, 6–18 months
LRTI, 0–18 months
LRTI hospitalised, 0–
18 months
After
Before week
week n
12
12
%
No
No
6835
38.2 1.00
No
Yes
4431
39.5
1.01 (0.96
0.97 (0.88 to
0.92 (0.73 to
16.0%
3.8%
to 1.07)
1.08)
1.15)
Yes
No
7145
41.0
1.07 (1.03
1.10 (1.01 to
1.28 (1.07 to
17.3%
5.0%
to 1.12)
1.20)
1.53)
Yes
Yes
13 666 41.2 1.07 (1.02 16.8% 1.07 (0.98 to 4.2% 1.08 (0.90 to
aRR (95%
%
CI)
aRR
(95%CI)
16.7% 1.00
%
aRR (95%CI)
4.3% 1.00
Folic acid
supplements in
pregnancy
Wheeze, 6–18 months
After
Before week
week n
12
12
%
LRTI, 0–18 months
aRR (95%
%
CI)
aRR
(95%CI)
to 1.12)
1.16)
LRTI hospitalised, 0–
18 months
%
aRR (95%CI)
1.29)
*Adjusted for other vitamin supplements and cod liver oil in pregnancy, vitamin supplements and cod liver oil
at 6 months of age, and for maternal age, maternal atopy, maternal smoking in pregnancy, maternal
educational level, postnatal parental smoking, sex, parity, birth weight, season born, breast feeding and
type of day care.
In conclusion, decades of investigation have yielded a diverse collection of genetic
associations with NTDs in the mouse, epidemiological evidence for a few gene associations
in human populations, and identification of environmental exposures and health conditions
that increase NTD risk. Accumulating evidence indicates that no single factor ensures that
neurulation will fail, but that polymorphisms in several genes, and/or coupled with the in
utero environment, must collaborate to result in an NTD. It is now becoming feasible to take
a systems approach to integrating these influences into profiles that may permit assessment
of an individual couple’s risk for having a child with an NTD, and to predict the preventative
measure most likely to promote a healthy birth outcome. It has been predicted that because
the overt NTD phenotypes are readily recognized in humans and experimental animals,
NTDs may well be the first complex genetic disorder for which gene-gene and geneenvironment interactions may be understood in depth. Progress made for this disorder can
provide useful analytical tools for identifying molecular network interactions relevant to
later-onset complex genetic disorders, like schizophrenia and autism (Ross 2010).
Work cited:
Kibar, Zoha, E. Torban, J. McDearmid, A. Reynolds, J. Berghout, M. Mathieu, I. Kirillova, P.
De Marco, E. Merello, J. Hayes, J. Wallingford, P. Drapeau, V. Capra, and P. Gros.
"Mutations in VANGL1 Associated with Neural-tube Defects. 2007." The New England
Journal of Medicine. 356 [14] : 1432-1437.
Burren, Katie, D. Savery, V. Massa, R. Kok, J. Scott, H. Blom, A. Copp, and N. Greene. 2008.
“Gene-environmental interactions in the causation of neural tube defects: folate defiency
increases susceptibility conferred by loss of Pax3 function.” Human Molecular Genetics 17
[23] (2008): 3675-3685.
Torban, Elena, A. Patenaude, S. Leclerc, S. Rakowiecki, S. Gauthier, G. Andelfinger, D. Epstein,
and P. Gros. 2008. 3 "Genetic interaction between members of the Vangl." PNAS . 105 [ 9] :
3449-3454.
Marean, Amber, A. Graf, Y. Zhang, and L. Niswander. 2011. 6 "Folic acid supplementation can
adversely affect murine neural tube closure and embryonic survival." Human Molecular
Genetics . 20 [18]: 3678-3683.
Hoyo, Cathrine, A. Murtha, J. Schildkraut, R. Jirtle, W. Demark-Wahnefried, M. Forman, E.
Iversen, J. Kurtzberg, F. Overcash, Z. Huang, and S. Murphy. 2011. 7 "Methylation variation
at igf2 differentially methylated regions and maternal folic acid use before and during
pregnancy." Epigenetics . 6 [7]: 928-936.
Ross, M. Elizabeth. 2010. "Gene-environment interactions, folate metabolism and the
embryonic nervous system." NIH-PA . 2 [ 4]: 471-480.
Sing au, Kit, A. Ashley-Koch, and H. Northrup. 2010 "Epidemiologic and genetic aspects of
spina bifida and other neural tube defects." NIH Public Access. 16 [1] 6-15.
Shookhoff, J. M. and G. Gallicano. 2010 "A new perspective on neural tube defects: folic acid
and microrna misexpression." Genesis. 48. 282-294.
Berry RJ, Li Z, Erickson JD, Li S, Moore CA, Wang H, et al.1999 ” Prevention of neural-tube
defects with folic acid in China. China-U.S. Collaborative Project for Neural Tube Defect
Prevention.” N Engl J Med. 34 [1] 1485–1490.
Doudney K, Ybot-Gonzalez P, Paternotte C, et al. 2005 “Analysis of the planar cell polarity gene
Vangl2 and its co-expressed paralogue Vangl1 in neural tube defect patients.” Am J Med Genet
A 136 [415] 90-92.
Stein KF, Rudin IA. 1953 “Development of mice homozygous for the gene for Looptail.” J
Hered 44:59-69.
George L, Mills JL, Johansson AL, Nordmark A, Olander B, Granath F, et al. 2002 “Plasma
folate levels and risk of spontaneous abortion.” JAMA; 288:1867-73.
Wen SW, Chen XK, Rodger M, White RR, Yang Q, Smith GN, et al. 2008 “Folic acid
supplementation in early second trimester and the risk of preeclampsia.” Am J Obstet Gynecol
198:45 e1-7.
Hernandez-Diaz S, Werler MM, Louik C, Mitchell AA. 2002 “Risk of gestational hypertension
in relation to folic acid supplementation during pregnancy.” Am J Epidemiol 156:806-12.
Godwin KA, Sibbald B, Bedard T, Kuzeljevic B, Lowry RB, Arbour L. 2008 “Changes in
frequencies of select congenital anomalies since the onset of folic acid fortification in a Canadian
birth defect registry.” Can J Public Health 99:271-5.
Carmichael SL, Shaw GM, Yang W, Laurent C, Herring A, Royle MH, et al. 2006 “Correlates
of intake of folic acid-containing supplements among pregnant women.” Am J Obstet Gynecol
194:203-10.
Haberg, S e. 2009 "Folic acid supplements in pregnancy and early childhood respiratory health."
Arch Dis Child . 94 [3] 180-184.Print.