Human Reproduction, Vol.30, No.8 pp. 1982 –1993, 2015
Advanced Access publication on June 3, 2015 doi:10.1093/humrep/dev126
ORIGINAL ARTICLE Reproductive genetics
Role of folate-homocysteine pathway
gene polymorphisms and nutritional
cofactors in Down syndrome:
A triad study
K.K. Sukla 1, S.K. Jaiswal 2, A.K. Rai 2, O.P. Mishra 3, V. Gupta 3,
A. Kumar 3, and R. Raman 1,*
1
Cytogenetics Laboratory, Department of Zoology, Banaras Hindu University, Varanasi, Uttar Pradesh, India 2Centre for Genetic Disorders,
Banaras Hindu University, Varanasi, Uttar Pradesh, India 3Department of Pediatrics, Institute of Medical Sciences, Banaras Hindu University,
Varanasi, Uttar Pradesh, India
*Correspondence address. E-mail: [email protected]
Submitted on July 1, 2014; resubmitted on March 24, 2015; accepted on April 24, 2015
study question: Do gene –gene and gene –environment interactions in folate-homocysteine (Hcy) pathway have a predisposing role for
Down syndrome (DS)?
summary answer: The study provides evidence that in addition to advanced age, maternal genotype, micronutrient deficiency and elevated Hcy levels, individually and in combination, are risk factors for Down syndrome.
what is known already: Polymorphisms in certain folate-Hcy-pathway genes (especially the T allele of MTHFR C677T), elevated Hcy
and poor folate levels in mothers during pregnancy have been shown to be risk factors for Down syndrome in certain Asian populations (including
the eastern region of India), while the same SNPs are not a risk factor in European populations. This conflicting situation alludes to differential
gene –environment (nutrition) interactions in different populations which needs to be explored.
study design, size, duration: Between 2008 and 2012, 151 Down syndrome triads and 200 age-matched controls (Control
mothers n ¼ 186) were included in the study. Seven polymorphisms in six genes of folate-Hcy metabolic pathway, along with Hcy, cysteine
(Cys), vitamin B12 (vit-B12) and folate levels, were analysed and compared among the case and control groups.
participants/materials, setting, methods: Genotyping was performed by the PCR-RFLP technique. Levels of homocysteine and cysteine were measured by HPLC while vitamin B12 and folate were estimated by chemiluminescence.
main results and the role of chance: We demonstrate that polymorphisms in the folate-Hcy pathway genes in mothers collectively constitute a genotypic risk for DS which is effectively modified by interactions among genes and by the environment affecting folate, Hcy
and vitamin B12 levels. The study also supports the idea that these maternal risk factors provide an adaptive advantage during pregnancy supporting live birth of the DS child.
limitations and reasons for caution: Our inability to obtain genotype and nutritional assessments of unaffected siblings of the
DS children was an important limitation of the study. Also, its confinement to a specific geographic region (the eastern part) of India, and relatively
small sample size is a limitation. A parallel investigation on another population could add greater authenticity to the data.
wider implications of the findings: For mothers genetically susceptible to deliver a DS child (particularly in South Asia), periconceptional nutritional supplementation and antenatal care could potentially reduce the risk of a DS child. Additionally, nutritional strategies
could possibly be used for better management of the symptoms of DS children.
study funding/competing interests: The work is funded through Programme support for Genetic disorders by Department
of Biotechnology, Government of India to R.R. The authors declare no conflict of interest.
Key words: Down syndrome / folate-homocysteine metabolic pathway / gene–gene and gene–environment interactions / MTHFR / vitamin B12
& The Author 2015. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.
For Permissions, please email: [email protected]
1983
Gene – nutrient interaction and Down syndrome
Introduction
Materials and Methods
In a remarkably prescient paper Penrose (1933) postulated that children with Down Syndrome (DS) were born to late age mothers
(≥35 years), although a large number of them were born to younger
mothers (,35 years). Penrose (1965) later produced evidence to
suggest that the young age DS mothers had actually been born to
older grandmothers. Thus maternal age was established as a risk
factor for Down syndrome. Further studies on high association of
adult DS with Alzheimer disease (AD) showed that DS mothers also
had a higher chance of developing AD, and that the young DS
mothers were five times more susceptible to AD than the late age
mothers (Schupf et al., 1994). These studies implied not only a
shared genetic susceptibility between DS and AD, but also a genetic
predisposition of the parents to DS child birth. However, despite this
indication, there is little consensus on genetic predisposition and candidate risk genes for Down syndrome.
James et al. (1999) were the first to report that the T allele of the SNP
C677T in MTHFR, a folate-homocysteine (Hcy) metabolic pathway gene,
predisposed mothers to have DS children. A similar investigation on a
cohort from the eastern part of India concurred with James et al.
(1999) in reporting that the mothers having the MTHFR 677T allele
(especially TT homozygotes) were susceptible to having DS children
(Rai et al., 2006). These authors, as well as Sherman et al. (2007), conjectured that this SNP could adversely affect global DNA methylation during
maternal meiosis leading to non-disjunction, and Down syndrome.
However, several comparable studies done on populations from different regions of the world have yielded conflicting results; while some,
mainly from the developing countries, have confirmed 677T of MTHFR
as a risk factor, others from Europe and the Americas have not
(Wu et al., 2013). Similar studies on other genes in the folate-Hcy
pathway, especially MTR, RCF1 (SLC19A1) and MTRR (see Coppedè
et al., 2013a,b, 2014), have shown, like MTHFR C677T, much heterogeneity in association with Down syndrome. Studies have therefore
been done to test if the combination of two or more SNPs in the Hcyfolate pathway genes could pose a risk in mothers for DS children
(Coppede 2009).
Since certain micronutrients, i.e. folates and vitamin B12 (vit-B12),
are important components of the Hcy-folate pathway, the possibility
remains that the population-specific difference in the risk susceptibility
of the genotypes could be influenced by the Hcy and micronutrient
levels in the mother. It is plausible therefore that the genetic risk
towards DS in a population could be rationally estimated when gene –
gene and gene –environment interactions are considered together in
parents as well as in the affected child. With this objective in view, the
present study has been undertaken to critically evaluate the role of
folate-Hcy metabolism in the aetiology of DS. We have studied 7 polymorphisms in 6 genes of folate-Hcy metabolic pathway along with Hcy,
Cysteine (Cys), vitamin B12 (vit-B12) and folate levels in 151 triads of
DS cases along with age-matched unrelated controls. We validate the
association of MTHFR 677T with DS susceptibility and also the strong
modulatory role of vit-B12, folate and Hcy, as risk factors for DS in the
case mothers in this cohort. The other variants individually do not
show an association but cumulatively have an additive role as genetic
risk factors.
Subjects
Cases were referred to the Centre for Genetic Disorders for karyotyping, following clinical examination in the Pediatrics Department of Institute of
Medical Sciences, Banaras Hindu University. The period of sample recruitment was from 2008 to 2012. The parents of the suspected proband were
asked to complete a questionnaire which provided information regarding
their age, previous history of any abortions, vitamin supplementation for
at least 2 months during the first trimester of pregnancy, feeding habits of
parents, ethnicity and medical history (incidence of congenital birth
defects, cardiovascular disorders, diabetes, and renal disorders) in both
parental lineages. Clinical details of the probands were recorded from the
clinical report and a consent from the parents was taken. By karyotyping,
free trisomy 21 was confirmed in 151 samples. In addition, there were
two cases of translocations (14q;21q) and one of mosaicism (46,XX/
47,XX+21). Cases of free trisomy 21 only were included in the study. The
median age of the probands was around 2 years, whereas the maternal
and paternal ages around the time of the DS child birth were 26 and 29, respectively and at the time of sample collection 29 and 32, respectively (see
Table I for Mean + SD). Out of the 151 mothers only 33 were ≥30 years
at the time of child birth, the rest (78%) being ,30 years. The frequency
of spontaneous abortion (SA), details of parity and pedigree of the affected
child were noted. The clinical features of the suspected DS children were
taken along with other medical history; a greater proportion of them suffered
from intellectual disability, hypotonia and pneumonia. The incidence of
congenital heart disease (CHD) in the studied cohort was 15%. The majority of the parents informed that the DS children showed reluctance to eating
solid food hinting at poor development of jaw muscles, as earlier reported
(Gisel et al., 1984). Age and gender matched controls were chosen randomly
from the general population of this region (Sukla and Raman, 2012), the inclusion criteria being age and parentage of at least two children. The exclusion
criteria for control subjects were previous history of having a child with any
congenital anomaly including DS. Since it was difficult to get age-matched
controls for children, the majority of samples were taken from newborns
(Sukla et al., 2013) and a few from healthy children of ,10 years (Sukla
and Raman, 2012) from families having no family history of congenital anomalies. The study was approved by Institutional ethical committee (Institute of
Medical Sciences), Banaras Hindu University.
Biochemical analysis
Homocysteine and cysteine were measured by reverse phase HPLC from
plasma samples (Shimadzu, 122 Kyoto, Japan) as described by Kumar et al.
(2005). Vitamin B12 and folate levels were measured from plasma by chemiluminescence (Immulite1000, Siemens-Diagnostic-Products, and Flanders,
NJ, USA) as per the manufacturer’s instructions.
Gene polymorphism analysis
Gene polymorphisms (7 in 6 genes) of the folate-Hcy metabolic pathway
were studied using gene-specific primers. Genomic DNA was subjected to
PCR-RFLP for SLC19A1 (RFC1) (G80A), MTHFR (C677T and A1298C),
MTR (A2756G), MTRR (A66G) and TCN2 (C776G), while CBS (844ins68bp)
was analysed by doing PCR with gene-specific primers and the presence or
absence of 68bp insert was determined by running the PCR product on 2%
agarose gel (see Naushad et al, 2008; Sukla and Raman, 2012; Sukla et al.,
2013). For 5% of the randomly selected samples from controls, cross-checks
were performed by DNA sequencing and matching the sequence with the
PCR-RFLP generated genotype.
1984
Sukla et al.
Table I Demographic profile of the study subjects.
Category
Age at conception
Mean + SD
Age at sample collection
Mean + SD
Vitamin
supplementation (%)
Vegetarian (%)
Spontaneous
abortion (%)
.............................................................................................................................................................................................
29.0 + 3.5
11.2
41
92
Control mother
28.0 + 4.5
14.0
32
33
P-value
0.13
0.152
,0.00012
DS mother
26.0 + 2.5
32.5 + 4.0
31
Control father
33.0 + 4.5
33
P-value
0.28
0.802
DS father
29.5 + 3.0
0.552
DS child
–
2.5 + 2.0
Control child
–
2.5 + 3.0
P-value
–
0.991
P-value ,0.05 is taken as significant and represented in bold.
1
Mann –Whitney test.
2
Chi-square test.
Statistical analysis
Genetic variability of folate-Hcy pathway in DS
Median and interquartile range (IQR) was calculated for Hcy, Cys, folate
and vit-B12. For comparison between two groups, the non-parametric
Mann– Whitney U-test was performed. Dunn’s and Bonferroni multiple
comparison tests were done as statistical correction. The allele frequencies
of the six studied SNPs were tested for Hardy – Weinberg equilibrium, and
to assess the genotype and allele distribution among groups, chi-square test
(df 1) was performed; P-values ,0.05 with Yates correction were taken to
be statistically significant. To ascertain any preferential transmission of alleles
from DS parents to the child, Transmission Disequilibrium Test (TDT) was
performed. Binary logistic regression analysis was done to find out potential
risk factors for the dependent variable DS in case of parents as well as the
affected child by using SPSS version 16.0 statistical package (IBM, Armonk,
NY, USA). Odds ratios (OR) were calculated and given with 95% confidence
interval, and P-values ,0.05 were taken as significant.
Seven polymorphisms were genotyped in 151 triads of DS, 186 control
mothers and 200 each of control fathers and children. The T allele frequency of MTHFR C677T in the case parents was significantly higher
than in the controls (Table II). Of the other genes, the frequency of
mutant alleles of 1298C MTHFR, 80G SLC19A1, 2756G MTR and 66G
of MTRR was marginally higher in DS mothers (not DS fathers) than
the control mothers but the differences were not significant. On the
other hand, T allele of MTHFR C677T, G allele of SLC19A1 G80A and
CC homozygotes of TCN2 C776G polymorphism were significantly
higher in DS children (Table II). The power of the study to detect association for the studied polymorphisms was evaluated under different
genetic models and the power was ≥0.80 for all the SNPs excluding
CBS 844ins68bp. For MTHFR C677T, the statistical power was ≥0.80
in the dominant and co dominant models whereas it was ,0.80 in the
recessive model (data not shown).
Table III shows the association of the genotypic and haplotypic combinations of the MTHFR variants with the case parents and DS. Briefly, the
combination of the mutant alleles, 677T & 1298C (risk combination),
was significantly higher in case mothers but there was not a single homozygote for the 677TT/1298CC combination, suggesting this to be a high
risk genotype. An earlier meta-analysis of the MTHFR mutants involving
nearly 10 000 subjects also failed to find more than one individual with
TT/CC genotype (Fredriksen et al., 2007). The referent wild type genotype (CC/AA genotype) was expectedly high in control mothers and
control children. T-C and T-A haplotypes were significantly higher in DS
mothers whereas the C-A haplotype was significantly higher in control
mothers. Among the paternal haplotypes, none, except the T-C haplotype, was significantly associated with DS. The T-C and T-A haplotypic
combinations were significantly higher in DS children, while the C-A combination (wild type) was significantly higher in control children (Table III).
When all the seven genetic variants were considered together, 56.1%
(85/151) of DS mothers harboured four or more genetic variants
compared with only 10.2% (19/186) in control mothers (Table IV).
However, a similar comparison between the DS fathers and their
controls did not reveal a significant difference between them. In DS
mothers, we also studied the effect of other studied gene polymorphisms
Results
A total of 151 samples with trisomy 21 were included for the study. The
median age of DS mothers around the time of conception was 26 years
(only 22% ≥ 30 years) with average parity rate of 3. Vegetarianism was
found to be 41 and 32% in case and control mothers, respectively, while
31 and 33% of case and control fathers were vegetarians, respectively.
Vitamin supplementation (iron and folic acid but not vitamin B12)
during conception was taken by 11.2 and 14.0% of case and control
mothers (Table I). The incidence of congenital birth defects was 1.3%
in case parents while it was nil in control parents. A history of diabetes
was recorded in 7.3 and 6.5% persons among the case parents and controls, respectively. There was no report of renal disorders in either of the
parental groups, while 3.5% of subjects in both the parental groups had a
history of cardiovascular disease (CVD). A high proportion, 92% (138),
of the DS mothers suffered at least one instance of spontaneous
abortion compared with 33% (62) in control mothers (Fig. 1). Of the
13 DS mothers who did not report a SA, in 12 the first pregnancy itself
resulted in the DS child, while in the other one, the DS child was the
second child. About 98% of the spontaneous abortions in DS mothers
occurred during the first trimester. The sex ratio of the DS children
was skewed towards males (2.8:1).
1985
Gene – nutrient interaction and Down syndrome
Figure 1 Incidence of spontaneous abortions (SA) in DS and control mothers.
in combination with MTHFR C677T polymorphism and found that
apparently ‘safe’ mutant alleles also added to the risk for DS. CT/AA,
CT/AC genotypic combination of MTHFR (C677T & A1298C); CT/
GA with SLC19A1 (G80A); CC/AG, CT/AA & CT/GG with MTR
(A2756G); CT/AG, CT/GG with MTRR (A66G) and CT/CG genotype
with TCN2 (C776G) were significant risk genotypic combinations in DS
mothers (Supplementary Table SI). A similar observation was also
reported by Scala et al. (2006). Interestingly among the DS children,
65% had ≥4 mutations compared with 13% in control children, a condition comparable to that seen in DS mothers.
TDT analysis
With respect to MTHFR C677T, the T allele in DS children was received
1.42 times more often than the C allele. Among the informative parents,
the T allele was preferentially transmitted both from the father (1.64)
and the mother (1.73 times) but this preferential transmission was statistically not significant. SLC19A1 was the only gene whose G (risk) allele
was preferentially transmitted to DS children (1.51 times) from DS
mothers (1.74 times), while the transmission from DS father was in equilibrium (Supplementary Table SII). However, since SLC19A is a chromosome 21 gene which is received in two copies from the mother, and its
major allele G is the risk allele, its preferential transmission to DS offspring
is expected.
Biochemical variability of folate-Hcy pathway
in DS
As seen from the data in Table V, in comparison with their respective
controls, the DS parents had higher levels of Hcy and lower levels of
vit-B12 and folate, while Cys levels were comparable between them.
In the DS children, Hcy levels were lower than in their parents as
well as in the control children, the Cys level was high but the levels of
vit-B12 and folate were low. All these differences were statistically significant (Table V). Among the DS parents, while 50% were hyperhomocyteinemic (hypHcy), 70% and 35% were deficient in vit-B12 and folates,
respectively.
Genetic and biochemical variability among
DS children with and without congenital
heart defect
Of the 151 DS patients, 23 (15%) suffered from congenital heart disorders (CHD) while 128 (85%) were devoid of it. In order to resolve
whether these two groups varied genetically or physiologically, we computed their genotypes and nutritional status separately. Genotypically,
the frequency of the risk alleles C of MTHFR A1298C (41 versus 33%)
and G of MTR A2756G (41 versus 29%) were higher in DS children
with CHD, but the difference was statistically not significant, possibly
due to low and vast numerical disparity between the samples. While
Vit-B12 levels and cysteine levels between them were comparable,
there were significantly raised levels of Hcy and significant deficiencies
of folic acid in DS children with CHD compared with those without
CHD (Supplementary Table SIII).
Regression analysis of associated risk factors
for DS
In order to define the role of individual factors amidst the variables analysed, the data were subjected to binary logistic regression analysis separately for mother, father and the DS child. Spontaneous abortion,
hyperhomocysteinemia (hypHcy), and deficiencies of vit-B12 and folic
acid as well as the T allele of MTHFR C677T individually turned out to
be risk factors in the case mothers (Table VI). In DS fathers, on the
other hand, no individual genetic marker was a risk factor but frequency
1986
Sukla et al.
Table II Comparative analysis of levels of different genotypes in case parents, DS children and their controls.
Genotype
Father
Mother
Child
.............................................................................................................................................................................................
MTHFR C677T
Genotype and Allele Frequency in DS
CC ¼ 91, CT ¼ 56, TT ¼ 4
C ¼ 0.79 and T ¼ 0.21
CC ¼ 86, CT ¼ 59, TT ¼ 6
C ¼ 0.76 and T ¼ 0.24
CC ¼ 83, CT ¼ 58, TT ¼ 10
C ¼ 0.74 and T ¼ 0.26
MTHFR C677T
Genotype and allele frequency in control
CC ¼ 149, CT ¼ 47, TT ¼ 4
C ¼ 0.86 and T ¼ 0.14
CC ¼ 141, CT ¼ 42, TT ¼ 3
C ¼ 0.87 and T ¼ 0.13
CC ¼ 154, CT ¼ 42, TT ¼ 4
C ¼ 0.87 and T ¼ 0.13
Odds ratio 95% CI
P-value for dominant model
1.93 (1.22–3.04)
0.006*
2.37 (1.49– 3.78)
0.0004*
2.74 (1.73–4.35)
<0.0001*
Odds ratio 95% CI
P-value for recessive model
1.34 (0.33– 5.42)
0.96
2.52 (0.62–10.27)
0.32
3.47 (1.07–11.31)
0.05
Odds ratio 95% CI
P-value for co-dominant model
1.95 (1.23–3.12)
0.007*
2.30(1.43 –3.72)
0.0009*
2.62 (1.62–4.24)
0.0001*
MTHFR A1298C
Genotype and Allele Frequency in DS
AA ¼ 71, AC ¼ 68, CC ¼ 12
A ¼ 0.70 and C ¼ 0.30
AA ¼ 69, AC ¼ 68, CC ¼ 14
A ¼ 0.68 and C ¼ 0.32
AA ¼ 67, AC ¼ 65, CC ¼ 19
A ¼ 0.66 and C ¼ 0.34
MTHFR A1298C
Genotype and allele frequency in control
AA ¼ 94, AC ¼ 87, CC ¼ 19
A ¼ 0.69 and C ¼ 0.31
AA ¼ 104, AC ¼ 65. CC ¼ 17
A ¼ 0.73 and C ¼ 0.27
AA ¼ 98, AC ¼ 83, CC ¼ 19
A ¼ 0.70 and C ¼ 0.30
Odds ratio 95% CI
P-value for dominant model
0.99 (0.65– 1.53)
0.99
1.51 (0.98–2.32)
0.08
1.20 (0.78– 1.84)
0.45
Odds ratio 95% CI
P-value for recessive model
1.15 (0.51– 2.56)
0.90
1.01 (0.48–2.13)
0.97
1.37 (0.70– 2.70)
0.45
Odds ratio 95% CI
P-value for co-dominant model
1.03 (0.67– 1.61)
0.97
1.57 (0.99–2.49)
0.07
1.14 (0.73– 1.80)
0.63
CBS 844ins68bp
Genotype and Allele Frequency in DS
WW ¼ 137, WI ¼ 14
W ¼ 0.95 and I ¼ 0.05
WW ¼ 140, WI ¼ 11
W ¼ 0.96 and I ¼ 0.04
WW ¼ 138, WI ¼ 13
W ¼ 0.96 and I ¼ 0.04
CBS 844ins68bp
Genotype and allele frequency in control
WW ¼ 187, WI ¼ 13
W ¼ 0.97 and I ¼ 0.03
WW ¼ 172, WI ¼ 14,
W ¼ 0.96 and I ¼ 0.04
WW ¼ 185, WI ¼ 15
W ¼ 0.96 and I ¼ 0.04
Odds ratio 95% CI
P-value co-dominant model
1.47 (0.67– 3.3)
0.45
0.96 (0.42–2.19)
0.93
1.16 (0.53– 2.52)
0.86
SLC19A1 G80A
Genotype and Allele Frequency in DS
GG ¼ 57, GA ¼ 70, AA ¼ 24
G ¼ 0.61 and A ¼ 0.39
GG ¼ 54, GA ¼ 75, AA ¼ 22
G ¼ 0.61 and A ¼ 0.39
GG ¼ 65, GA ¼ 67, AA ¼ 19
G ¼ 0.65 and A ¼ 0.35
SLC19A1 G80A
Genotype and allele frequency in control
GG ¼ 73, GA ¼ 90, AA ¼ 37
G ¼ 0.59 and A ¼ 0.41
GG ¼ 64, GA ¼ 80, AA ¼ 42
G ¼ 0.56 and A ¼ 0.44
GG ¼ 60, GA ¼ 95, AA ¼ 45
G ¼ 0.55 and A ¼ 0.45
Odds ratio 95% CI
P-value for dominant model
0.95 (0.61– 1.46)
0.89
0.94 (0.61–1.48)
0.88
0.56 (0.36–0.88)
0.02*
Odds ratio 95% CI
P-value for recessive model
0.83 (0.47– 1.46)
0.63
0.58 (0.33–1.03)
0.08
0.50 (0.27–0.89)
0.02*
Odds ratio 95% CI
P-value for co-dominant model
0.99 (0.62– 1.59)
0.98
1.11 (0.68–1.80)
0.76
0.65 (0.41– 1.04)
0.09
MTR A2756G
Genotype and Allele Frequency in DS
AA ¼ 70, AG ¼ 61, GG ¼ 20
A ¼ 0.67 and G ¼ 0.33
AA ¼ 73, AG ¼ 59, GG ¼ 19
A ¼ 0.68 and G ¼ 0.32
AA ¼ 72, AG ¼ 65, GG ¼ 14
A ¼ 0.69 and G ¼ 0.31
MTR A2756G
Genotype and allele frequency in control
AA ¼ 99, AG ¼ 82, GG ¼ 19
A ¼ 0.70 and G ¼ 0.30
AA ¼ 95, AG ¼ 79, GG ¼ 12
A ¼ 0.72 and G ¼ 0.28
AA ¼ 100, AG ¼ 86, GG ¼ 14
A ¼ 0. 71 and G ¼ 0.29
Odds ratio 95% CI
P-value for dominant model
1.13 (0.74– 1.73)
0.64
1.11 (0.73–1.70)
0.70
1.10 (0.72– 1.68)
0.74
Odds ratio 95% CI
P-value for recessive model
1.45 (0.75– 2.83)
0.35
2.07 (0.98–4.34)
0.07
1.36 (0.63– 2.94)
0.56
Odds ratio 95% CI
P-value for co-dominant model
1.05 (0.67– 1.65)
0.91
0.97 (0.62–1.52)
0.98
1.05 (0.67– 1.63)
0.92
MTRR A66G
Genotype and Allele Frequency in DS
AA ¼ 24, AG ¼ 78, GG ¼ 49
A ¼ 0.42 and G ¼ 0.58
AA ¼ 19, AG ¼ 75, GG ¼ 57
A ¼ 0.37 and G ¼ 0.63
AA ¼ 20, AG ¼ 73, GG ¼ 58
A ¼ 0.37 and G ¼ 0.63
MTRR A66G
Genotype and allele frequency in control
AA ¼ 43, AG ¼ 91, GG ¼ 66
A ¼ 0.44 and G ¼ 0.56
AA ¼ 37, AG ¼ 87, GG ¼ 62
A ¼ 0.43, G ¼ 0.57
AA ¼ 39, AG ¼ 89, GG ¼ 72
A ¼ 0.42 and G ¼ 0.58
Odds ratio 95% CI
P-value for dominant model
1.02 (0.65– 1.61)
0.91
0.81 (0.52–1.26)
0.42
0.91 (0.58– 1.40)
0.73
Continued
1987
Gene – nutrient interaction and Down syndrome
Table II Continued
Genotype
Father
Mother
Child
.............................................................................................................................................................................................
Odds ratio 95% CI
P-value for recessive model
0.69 (0.40 –1.20)
0.24
0.56 (0.31–1.01)
0.07
0.63 (0.35– 1.13)
0.16
Odds ratio 95% CI
P-value for co-dominant model
1.15 (0.72 –1.86)
0.64
0.94 (0.59–1.49)
0.87
1.02 (0.64– 1.62)
0.94
TCN2 C776G
Genotype and Allele Frequency in DS
CC ¼ 19, CG ¼ 71, GG ¼ 61
G ¼ 0.64 and C ¼ 0.36
CC ¼ 27, CG ¼ 69, GG ¼ 55
G ¼ 0.59 and C ¼ 0.41
CC ¼ 35, CG ¼ 61, GG ¼ 55
G ¼ 0.57 and C ¼ 0.43
TCN2 C776G
Genotype and allele frequency in control
CC ¼ 24, CG ¼ 97, GG ¼ 79
G ¼ 0.64 and C ¼ 0.36
CC ¼ 24, CG ¼ 86, GG ¼ 76
G ¼ 0.64 and C ¼ 0.36
CC ¼ 24, CG ¼ 85, GG ¼ 91
G ¼ 0.67 and C ¼ 0.33
Odds ratio 95% CI
P-value for dominant model
0.96 (0.63 –1.48)
0.95
1.21 (0.79–1.87)
0.45
1.45 (0.94– 2.25)
0.11
Odds ratio 95% CI
P-value for recessive model
1.05 (0.56 –2.01)
0.99
1.52 (0.85–2.75)
0.21
2.21 (1.25–3.91)
0.010*
Odds ratio 95% CI
P-value for co-dominant model
0.95 (0.60 –1.50)
0.91
1.10 (0.69–1.76)
0.75
1.18 (0.74– 1.90)
0.55
P-value ,0.05 is taken as significant and represented in bold.
*Significant after Yates Correction.
Table III MTHFR genotypic combination and haplotypic analysis in case parents, DS children and their controls.
Genotypic combination
DS mother
Control
mother
DS father
Control father
Case child
Control child
.............................................................................................................................................................................................
Referent combination MTHFR
(C677T & A1298C)
32
73
47
60
32
72
Risk Combination
MTHFR (C677T & A1298C)
119
113
104
140
119
128
X 2-value (Yates correction)
OD (95% CI)
P-value
11.84
2.40 (1.47–3.92)
0.0006*
Haplotype
DS mother
frequency
0.01
0.95 (0.60– 1.5)
0.91
Control mother
frequency
DS father
frequency
8.35
2.1 (1.30–3.40)
0.004*
Control father
frequency
Case child
frequency
Control child
frequency
.............................................................................................................................................................................................
T-C
2
0.038
0.014
0.090
0.025
0.065
x test
4.54
7.25
4.96
P-value
0.0316
0.0007*
0.0261
T-A
0.197
x 2 test
10.11
0.106
0.121
0.112
1.51
0.193
P-value
0.0012*
0.280
0.2196
x 2 test
0.68
2.38
0.004
P-value
0.3854
0.1226
0.9488
0.631
0.214
0.574
0.091
13.92
C-C
0.252
0.034
0.0002*
0.287
0.575
0.276
C-A
0.485
x 2 test
13.42
0.362
12.74
0.465
P-value
0.0003*
0.5472
0.0004*
0.269
0.606
Referent haplotype MTHFR 677CC/1298AA while the rest of the combinations were taken as Risk haplotype. P-value ,0.05 is taken as significant and represented in bold.
*P-value significant after Bonferroni correction.
of hypHcy and vit-B12 and folate deficiencies was significantly higher
(Supplementary Table SIV). In the case of DS child, male gender, elevated
cysteine and deficiencies of vit-B12 and folate were significant risk factors.
Genotypically, frequencies of the T allele of MTHFR C677T and G
allele of SLC19A1 G80A were significantly higher in DS children (Supplementary Table SV).
1988
Sukla et al.
Table IV Incidence of risk genotypic presence among the case parents, their DS children and the controls.
Risk allele variants
DS mother (n 5 151)
Control mother (n 5 186)
Odds (95% CI)
X 2 (Yates correction)
P-value
.............................................................................................................................................................................................
One
9 (6%)
41 (22%)
0.88 (0.20 –3.77)
0.03
0.86
Two
23 (15.2%)
63 (33.9%)
1.46 (0.38 –5.65)
0.053
0.82
Three
31 (20.5%)
51 (27.4%)
2.43 (0.64 –9.30)
1.07
0.31
Four
41 (27.1%)
11 (5.9%)
14.91 (3.57 –62.30)
15.37
,0.0001
Five
30 (19.8%)
6 (3.2%)
20.0 (4.30 –93.25)
15.93
,0.0001
Six and more
14 (9.2%)
2 (1.1%)
28.0 (3.99 –196.56)
11.65
0.0006
3 (2%)
12 (6.5%)
Referent
–
Control father (n 5 200)
Odds (95% CI)
X 2 (Yates correction)
P-value
No variant
Risk allele variants
DS father (n 5 151)
.............................................................................................................................................................................................
One
16 (10.6%)
36 (18%)
0.56 (0.17 –1.75)
0.50
0.48
Two
43 (28.5%)
57 (28.5%)
0.95 (0.33 –2.76)
0.007
0.93
Three
56 (37.1%)
74 (37%)
0.96 (0.33 –2.73)
0.005
0.94
Four
15 (9.9%)
17 (8.5%)
1.13 (0.34 –3.80)
0.04
0.72
Five
8 (5.3%)
5 (2.5%)
2.06 (0.46 –9.14)
0.34
0.56
Six and more
6 (4%)
2 (1%)
3.86 (0.60 –25.3)
1.03
0.31
No variant
Risk allele variants
7 (4.6%)
DS child (n 5 151)
9 (4.5%)
Control child (n 5 200)
Referent
–
Odds (95% CI)
X 2 (Yates correction)
P-value
.............................................................................................................................................................................................
One
7 (4.6%)
45 (22.5%)
0.54 (0.14 –2.13)
0.25
0.61
Two
18 (11.9%)
64 (32%)
0.98 (0.29 –3.36)
0.0006
0.98
Three
24 (15.9%)
51 (25.5%)
1.65 (0.49 –5.54)
0.27
0.59
Four
46 (30.4%)
18 (9%)
8.94 (2.60 –30.85)
12.55
0.004
Five
37 (24.5%)
6 (3%)
21.60 (5.29 –88.15)
20.65
,0.0001
Six and more
15 (9.9%)
2 (1%)
26.25 (4.14 –166.53)
12.81
0.0003
4 (2.6%)
14 (7%)
Referent
–
No variant
Discussion
Parental genotype, Hcy, and micronutrients
status as risk factors for Down syndrome
In an earlier report from our lab, MTHFR 677T as well as 1298C were
reported to be risk factors in DS mothers, 677T being a greater risk
than 1298C (Rai et al., 2006). In the present study also, MTHFR 677T
emerged a clear risk factor in DS mothers, with the mutant homozygote
(TT) presenting much more risk than the CT heterozygote. MTHFR
1298C, also occurred more frequently in the DS mothers but the difference from the controls was not statistically significant. In the majority of
earlier studies done on individual gene polymorphisms, a larger body of
data supports MTHFR 677T as a risk factor in DS mothers, while MTHFR
1989
Gene – nutrient interaction and Down syndrome
Table V Comparative analysis of levels of different biochemical parameters (Hcy, Cys, folate and vit-B12) in case parents,
their DS children and the controls.
Biochemical parameter
Father
Mother
Child
.............................................................................................................................................................................................
DS (n ¼ 151)
Hcy in mmol/l, median and IQR
(% .15 mmol/l)
15.1 (12.4– 17.2)
(52%)
15.9 (13.6 –18.9)
(54%)
11.2 (8.9–14.2)
(18%)
Control (n ¼ 200)
Hcy in mmol/lit, median and IQR
(% .15 mmol/l)
12.6 (11.7– 15.6)
(29%)
11.7 (10.2– 14.5)
(23%)
11.7 (9.4–13.7)
(20%)
P-value
(Mann–Whitney test)
<0.0001*
<0.0001*
0.0945
DS (n ¼ 151)
Cys in mmol/l, median and IQR
204 (173–215)
206 (181–219)
217 (198–236)
Control (n ¼ 200)
Cys in mmol/l, median and IQR
202 (176–218)
201 (174–216)
198 (185–215)
P-value
(Mann–Whitney test)
0.865
0.379
<0.0001*
DS (n ¼ 151)
B12 in pg/ml, median and IQR
(% ,220 pg/ml)
193 (165–236)
(69%)
190 (163– 232)
(71%)
188 (158–226)
(74%)
Control (n ¼ 200)
B12 in pg/ml, median and IQR
(% ,220 pg/ml)
209(177 –261)
(54%)
228 (171– 262)
(45%)
216 (177–257)
(57%)
P-value (Mann–Whitney test)
<0.0001*
<0.0001*
<0.0001*
DS (n ¼ 151)
Folic acid in ng/ml, median and IQR
(% ,3 ng/ml)
3.3 (2.9 –3.7)
(33%)
3.1 (2.7– 3.7)
(37%)
3.2 (2.7 –3.8)
(36%)
Control (n ¼ 200)
Folic acid in ng/ml, median and IQR
(% ,3 ng/ml)
4.5 (3.6 –5.6)
(12%)
4.8 (3.5– 6.0)
(∼11%)
3.9 (3.2 –5.6)
(19%)
P-value (Mann–Whitney test)
<0.0001*
<0.0001*
<0.0001*
P-value ,0.05 is taken as significant and represented in bold.
*Significant after Bonferroni correction. For Control mother n ¼ 186.
1298C, SLC19A1 80G and MTRR 66G have been identified as risk factors
at least in certain populations (e.g. SLC19A1 80G in Brazilians and MTRR
66G in Caucasian whites) (see Coppedè et al., 2013b, 2014). CBS
844in/del 68bp, almost totally, and MTR, in the majority of studies, have
not been reported to have an association with DS (Scala et al., 2006;
Coppedè et al., 2013a; Yang et al., 2013). Our choice of studying TCN2
C776G was to assess whether this polymorphism influenced the vit-B12
level, one of the important nutritional risk factor for DS in this cohort,
but it did not show any association. There are also a few studies where
more than one folate-Hcy pathway gene polymorphisms have been examined within the same cohort in order to assess the effect of their interaction
in the development of DS (Martinez-Frias et al., 2006; Scala et al., 2006;
Coppedè et al., 2009). In an Italian cohort, Scala et al. (2006) showed an
additive risk effect of MTHFR 677T with 1298C and SLC19A1 but not
with MTRR A66G and MTR A2756G. Coppedè et al. (2006) showed the
MTHFR 677TT and SLC19A1 80GG genotypic combination to be more
frequent in DS mothers whereas the MTHFR 1298AA genotype with
SLC19A1 80GA/GG genotypic combination was protective. In our
study, both T allele of MTHFR C677T and G allele of SLC19A1 G80A
were found to be associated with risk in DS mothers (Supplementary
Table SI). Martinez-Frias et al., (2006) on the other hand, found that in
Spanish samples, MTHFR 1298C and MTRR 66G were associated with
added risk by elevating the level of homocysteine significantly but MTHFR
677T was not a risk factor. There are more studies which compare three
or more mutants together and their combined risk on DS (da Silva et al.,
2005; Biselli et al., 2008; Brandalize et al., 2010; Yang et al., 2013). In the
present study even though none of the alleles, except MTHFR 677T, individually were risk factors, almost each of them together with others added
to the risk potential seen in DS mothers, especially in association with
MTHFR 677T. The additive effect of these mutations was best illustrated
by the fact that 56% of DS mothers carried four or more of the folate-Hcy
pathway gene variants compared with 11% of the control mothers
(Table IV). That mutants other than MTHFR 677T do have a subtle and
additive role as risk factor was confirmed because even after excluding
MTHFR 677T from the consideration, 35% of the case mothers had
more than 4 mutations (compared with 11% of the controls). Taken together, there is substantial evidence that MTHFR 677T individually and
other Hcy-pathway genes cumulatively are risk factors for DS in this population, which is in conformity with the above-cited evidence from Italian and
Spanish cohorts in Europe.
In addition to the Hcy-pathway gene polymorphism, a mass of data
supports low folate and vit-B12 and high maternal Hcy levels as risk
1990
Sukla et al.
Table VI Binary logistic regression analysis for risk factors among DS and control mothers.
Factors associated with DS
predisposition in mothers
DF
P-value
Odds ratio
95% CI for Odds
........................................
Lower
Upper
.............................................................................................................................................................................................
Age
1
0.756
0.82
0.56
2.18
Veg/Nonveg
1
0.074
1.72
0.86
3.34
Vit- supplementation
1
0.829
0.87
0.41
5.61
Birth defects history
1
0.612
1.23
0.69
4.67
CVD history
1
0.991
1.05
0.61
4.15
Diabetic history
1
0.847
0.91
0.57
4.08
Spontaneous abortion
1
<0.0001
26.25
12.54
50.93
Hyperhomocysteinemia
1
0.01
2.76
1.49
4.82
Elevated cysteine
1
0.731
0.92
0.39
5.51
Vit-B12 deficiency
1
0.016
2.63
1.32
5.23
Folate deficiency
1
0.011
3.01
1.37
6.04
MTHFR 677T
1
0.018
2.98
1.32
6.73
MTHFR 1298C
1
0.186
1.72
0.65
3.86
CBS 844ins68bp
1
0.458
1.02
0.45
3.07
SLC19A1 80G
1
0.143
1.38
0.79
6.25
MTR 2756G
1
0.082
1.65
0.78
5.31
MTRR 66G
1
0.127
1.43
0.85
6.4
TCN2 776C
1
0.326
1.36
0.68
5.85
P-value ,0.05 is taken as significant and represented in bold. Cox and Snell R square value ¼ 0.28, Nagelkerke R square value ¼ 0.37.
factors in DS mothers (James et al., 1999; Bosco et al., 2003; Takamura
et al., 2004; da Silva et al., 2005; Eskes 2006; Martinez et al., 2006; Wang
et al., 2007; Biselli et al., 2008; Meguid et al., 2008; Santos-Reboucas et al.,
2008). Considering that in our samples, 71% of the DS mothers were deficient in vit-B12 and 37% were deficient in folic acid, while 57% were
hyperhomocysteinemic (hypHcy), and that those with MTHFR 677T
have higher levels of Hcy, there is little doubt that genetic susceptibility
(MTHFR 677T) and nutritional deficiency synergistically enhance the
risk in DS mothers through impairment of the folate-Hcy pathway.
Unlike the maternal contribution, paternal gametogenesis-induced
aneuploidy is responsible for only 5–10% of DS. Paternal age is not considered a risk factor. However, an earlier TDT study by Hobbs et al.
(2002) had shown a skewed transmission of the paternal T allele of
MTHFR 677 to DS children, and comparable results were obtained
by us in an earlier study (Rai et al., 2006), suggesting preferential transmission of a mutant allele from the father. However, in the present study, the
DS fathers did show high frequency of MTHFR 677T in univariate analysis,
but upon multivariate analysis the association was found to be not significant. This allele also transmitted preferentially from DS father to the child
but here too, it was statistically not significant. Though risk alleles of other
studied genes also showed no significant preferential transmission of any
of the paternal allele, the haplotypic combination of MTHFR 677T/
1298C did indicate preferential transmission (Table III). Obviously,
more studies on larger sample sizes may clarify the contribution of the
paternal genome.
In contrast to the Hcy-pathway genes, high Hcy, and low vit-B12 and
folate in fathers did appear as independent risk factors just as in DS
mothers. Young et al. (2008), studied the association of diverse micronutrients with aneuploidy in sperm by employing FISH, and showed an
association of folate level with sperm aneuploidy. There is indeed sufficient indication that low folates and/or polymorphism in Hcy-folate
pathway genes impair spermatogenesis due to hypomethylation,
errors in DNA replication or DNA damage due to high level of free radicals (Loscalzo 1996; Singh et al., 2005). The scale of vit-B12 and folic acid
deficiency was comparable between DS fathers and mothers, which
could be possibly due to their similar dietary pattern within the
common familial environment. Nevertheless, it is reasonable to
assume that the paternal MTHFR haplotype (677T/1298C) together
with folate and vit-B12 deficiency could affect chromosomal disjunction
in meiosis by augmenting pericentromeric hypomethylation, as suggested by Hobbs et al. (2000) for female meiosis. Ideally, FISH-based detection of aneuploidy in sperm would have offered a clearer
understanding of the situation but at present it remains a limitation of
this work due to logistical problems.
In the DS mothers, beside the hypomethylation of pericentromeric
region generating chromosomal mal-segregation and aneuploidy, an alternative explanation is that elevated Hcy levels cause endoplasmic stress and
generation of free radicals leading to uneven cellular activity and DNA
damage (Perluigi and Butterfield, 2012). In an Egyptian study, low levels
of antioxidants and trace elements, along with elevated levels of Hcy,
have been associated with oxidative stress in DS mothers (Meguid et al.,
2008) which could, among other things, adversely affect development
of fetal organs, including the brain (Boldyrev et al., 1999; Perrone et al.,
2007). A rather novel explanation has recently been put forth by
Costa-Lima et al. (2013) whose meta-analysed data show that MTHFR
677T is a distinct risk for Down syndrome in populations belonging to subtropical regions compared with northern and tropical regions where little
association was seen. These authors attributed this zonal association to
Gene – nutrient interaction and Down syndrome
photolysis of folates due to emission of specific wavelength UV radiation in
subtropical regions. Incidentally, the eastern Indian region, where the
present study was done, falls within the subtropical region.
Spontaneous abortions and Down syndrome
One of the most intriguing observations in the present study is that 92%
of case mothers have suffered at least one spontaneous abortion compared with 32% in control mothers. Earlier studies have also indicated
a higher incidence of SA in DS mothers (Lippman and Aymé, 1984;
Hassold and Hunt 2001). Considering that more than 50% of early
pregnancy terminations are due to chromosomal aneuploidy and more
than 80% of DS conceptuses abort, and that MTHFR 677T and 1298C
SNPs are risk factors in mothers with recurrent pregnancy failure (Nair
et al., 2012, 2013), it would seem natural that DS mothers have a
higher propensity towards spontaneous abortions. Since aneuploidy is
one of the major genetic causes of abortions, this evidence strengthens
the genetic predisposition of DS mothers to aneuploidy. Conceding that
SA could be a genetic attribute of DS mothers, it could be argued that
the occurrence of SA in 32% of controls could impair the comparison
between the case and control mothers. In view of this possibility, we
have also made the comparison with DS mothers after excluding the 62
control mothers who had SAs, and the results do not deviate in any significant way (Supplementary Tables SVI and SVII). Hence, it is reasonable to
conclude that DS mothers are genetically predisposed to spontaneous
abortion. But the scale at which SA occurs in the present report is much
higher than in any previous report which perhaps is a reflection of the
general health scenario in the population in question, that suffers from
high maternal and infant mortality rates due to poor nutritional status of
the mothers (NFHS-3, 2007; James 2011).
Congenital heart defects in Down syndrome
children
Globally, nearly 50% of DS patients suffer from congenital heart defects
(CHD). Several authors have explored the possibility of Hcy-pathway
gene polymorphism contributing to it, but the results are rather heterogeneous. While Brandalize et al. (2009) found a marginal association of
MTHFR 677T with CHD in DS, Hobbs et al. (2006) found no correlation
with this SNP but reported MTHFR 1298C to be protective. Locke et al.
(2010) noticed a higher frequency of SLC19A1 80G in DS children with
atrial valve septal defect while Božović et al. (2011) found no association
with this phenotypic difference. In our study group, only 23 DS children
(15%) were recorded to have CHD. Though the diagnosis of CHD was
largely based on clinical symptoms rather than on cardiac imageries,
there is little chance of large scale misdiagnosis or missing cases. We
have no obvious clue to this disparity. Although we are unaware what
proportion of abortuses of the DS mothers might have had DS, and
carried CHD, considering that more than 90% DS mothers experienced
abortions, there may be a possibility that a major fraction of DS fetuses
with the CHD phenotype, compounded by gestational nutritional deficiency, were aborted. That apart, a comparison of genotypes and nutritional status of the two groups of DS children revealed a distinctly higher
frequency of MTHFR 1298C and MTR 2756G in DS children with CHD,
though the difference was statistically not significant. The difference in the
nutritional status was more acute with respect to Hcy, which was
2.5 mmol/l higher, and folates, which were severely low (67% had
folate deficiency) in the DS children with CHD, as compared to
1991
without CHD, and the differences were significant. We infer that there
is a genuine genotypic and physiological difference in the DS children
with CHD and without CHD. It is possible that the vast numerical disparity of sample size (n ¼ 23 of DS children with CHD versus n ¼ 128 of DS
children without CHD) may have affected the statistical estimates with
respect to their genotypic difference, and a larger sample size may
further clarify it. However, the nutritional disparity does indicate a
larger developmental impediment for DS children with CHD and a possible cause of loss during pregnancy.
Down syndrome and impaired folate-Hcy
metabolism pathway: a selective advantage?
Considering that nearly 80% of DS conceptuses abort, do those who are
born alive harbour a selective advantage? Hobbs et al. (2002) have suggested that mutations in the Hcy-pathway genes could impart an advantage to the DS children for live births, and our results tend to support this
view. Pogribna et al. (2001) in a wide metabolic screen showed that DS
children had low level of Hcy and low activity of the folate-methionine
pathway substrates and products (methionine, S-adenosyl methionine)
and elevated levels of the trans-sulfuration products, cystathionine and
cysteine. Three copies of the trans-sulfuration pathway gene, CBS, in
DS children due to its location on chromosome 21, are likely to tilt the
metabolism of Hcy towards trans-sulfuration at the cost of the methylation pathway, leading to low homocysteine and hypomethylation. In the
present report also, the fact that in DS children, cysteine levels (217 mmol/
l) were higher than all the other studied case and control groups, and Hcy
levels were the lowest (median: 11.2 mmol) despite highest frequency of
vit-B12 and folic acid deficiency and MTHFR 677T allele, demonstrates that
Hcy metabolism in DS children is clearly driven towards the transsulfuration pathway, causing irretrievable loss of Hcy which would necessarily lead to low S-Adenosylmethionine and low methylation, as shown by
Pogribna et al. (2001). Elevation of cysteine, on the other hand, would generate free radicals inducing oxidative stress, which could be detrimental to
various organs including brain (Boldyrev et al., 1999).
Under this condition, the mutations in the 1-Carbon pathway genes
and lowered micronutrients may provide a survival advantage to DS.
As shown in the present report and earlier by Ueland et al. (2001) and
Hobbs et al. (2002), MTHFR 677CC DS children have much lower Hcy
(11.3 + 2.2) than MTHFR 677CT/TT mutants (13.4 + 3.1), raising
the possibility that in DS children with the wild type MTHFR (CC677)
and micronutrient deficiency, the Hcy level may be too low for the synthesis of the essential amino acid, methionine and the methyl donor
S-adenosylmethionine, leading to hypomethylation, a condition that
could prove fatal. We contend that under the condition of the mutation
as well as lowered micronutrients, the 1-Carbon pathway will draw less
Hcy than under the wild-type gene(s) or adequate micronutrient, offering better survivability to the former. A similar condition of the mothers
at risk of a DS child during pregnancy (hyperHcy and vit-B12 and folate
deficiency), we suggest is of mutual advantage to the fetus and the
mother, as the latter would reduce its Hcy load through its transplacental
supply to the overdrawing DS fetus. Better experimentation and measurements should provide a more reliable assessment.
This study thus adds to the evidence that supports the association of
the Hcy-pathway genes and micronutrient deficiency as risk factors with
DS mothers. We also suggest that DS mothers have a higher risk of spontaneous abortions and the association of the folate-Hcy pathway in DS
1992
children with CHD appears to be greater than in DS children without
CHD. Our results also provide indirect evidence that adds support to
the idea that these risk factors for the mother could well be an adaptive
advantage to the live birth of the DS child. The idea that nutritional intervention during an early stage of life could ameliorate the severity of
various associated disorders in DS children is an encouraging prospect
to work on.
Supplementary data
Supplementary data are available at http://humrep.oxfordjournals.org/.
Acknowledgements
Authors take pleasure in dedicating this paper to Prof.
Stanley M. Gartler, Department of Genetics, University of
Washington, Seattle, USA on his completing 90 eventful
years. The authors record their appreciation of the anonymous
reviewers (especially reviewer 2) for extensive and constructive criticism
of the paper. We believe this has enabled us to improve the paper considerably. The authors are thankful to Prof. S.K. Singh, Head, and Mr R.P.
Bhat, Senior Technical Officer, Department of Endocrinology & Metabolism, Institute of Medical Sciences (IMS), BHU, for help in performing
the chemiluminescence assay of vit-B12 and folate. The authors also
thank Prof. K.K. Singh, Statistics Department, Faculty of Science, BHU,
for his valuable suggestions in statistical analysis. The authors thank
Dr Amit Kumar Chaurasia, Application Scientist, NGS facility, BHU,
for his suggestions and help in analysis of data. This work forms part of
the contribution of the disease biology thrust area under DBTsponsored Interdisciplinary School of Life Sciences, BHU.
Authors’ roles
K.K.S. and R.R. conceived and designed the experiments. K.K.S., S.K.J.
and A.K.R. performed the experiments. K.K.S. and R.R. analysed the
data. K.K.S. and R.R. wrote the manuscript. R.R. provided the grants
for experiments. A.K., O.P.M. and V.G. evaluated the clinical profile of
the newborns. K.K.S. and S.K.J. collected the samples. A.K.R., A.K.,
O.P.M. and V.G. gave critical comments on the manuscript.
Funding
The work is funded through programme support for genetic disorders
by the Department of Biotechnology, Government of India, to R.R.
Conflict of interest
None declared.
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