Neuroscience Letters 449 (2009) 1519

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Neuroscience Letters
journal homepage: www.elsevier.com/locate/neulet

Association of maternal polymorphisms in folate metabolizing genes

with chromosome damage and risk of Down syndrome offspring
Fabio Copped a, , Francesca Migheli b , Stefania Bargagna c , Gabriele Siciliano a ,
Ivana Antonucci d , Liborio Stuppia d,e , Giandomenico Palka d,f , Lucia Migliore b,
a

Department of Neuroscience, University of Pisa, Pisa, Italy

Department of Human and Environmental Sciences, University of Pisa, Via S. Giuseppe 22, 56126 Pisa, Italy
Scientic Institute Stella Maris, Calambrone, Pisa, Italy
d
Department of Biomedical Sciences, G. DAnnunzio University Foundation, Chieti, Pescara, Italy
e
I.G.M. CNR, c/o IOR, Bologna, Italy
f
Human Genetic Division, Pescara Hospital, Pescara, Italy
b
c

a r t i c l e

i n f o

Article history:
Received 30 September 2008
Received in revised form 20 October 2008
Accepted 21 October 2008
Keywords:
Down syndrome (DS)
Folic acid
Gene polymorphisms
MTHFR
MTR
MTRR
TYMS
Chromosome damage

a b s t r a c t
We analyzed the role of six common polymorphisms in folate metabolizing genes as possible risk factors for having a child with Down syndrome (DS) in 94 Italian mothers of a DS child (MDS) and 113
matched control mothers, both aged less than 35 years at conception. Investigated polymorphisms
include methylenetetrahydrofolate reductase (MTHFR) 677C > T and 1298A > C, methionine synthase (MTR)
2756A > G, methionine synthase reductase (MTRR) 66A > G, and thymidylate synthase (TYMS) 28 bp repeat
and 1494del6. We also measured the amount of chromosome damage in peripheral blood lymphocytes
of 42 MDS and 41 matched controls, by means of the micronucleus assay, and searched for association
between this cytogenetic endpoint and any of the studied polymorphisms. Micronuclei in peripheral blood
lymphocytes have been analyzed several years after conception: the mean age at sampling was 45.6 11.4
years for MDS and 47.95 6.9 years for controls. The combined MTHFR 677TT/MTR 2756AA genotype was
associated with increased DS risk (P = 0.034), and the combined MTHFR 1298AC/TYMS 2R/2R genotype
with reduced risk (P = 0.003). Moreover, we observed a signicant increased frequency of micronucleated lymphocytes in MDS as compared to controls (P < 0.0001) and, in the total population, a signicant
correlation between micronucleated cells and both MTHFR 677C > T (P = 0.031) and 1298A > C (P = 0.047)
polymorphisms.
2008 Elsevier Ireland Ltd. All rights reserved.

Primary trisomy 21 leading to Down syndrome (DS) is caused by

the failure of normal chromosome 21 segregation during meiosis.
In 95% of the cases the nondisjunction event is of maternal origin, occurring primarily during meiosis I in the maturing oocyte.
Advanced maternal age at conception represents the major risk
factor for trisomy 21, and after age 35 the risk for a DS pregnancy increases proportionally to increasing maternal age [2].
However, several women aging less than 35 years have a DS child,
suggesting that their cell division machineries could be physiolog-

Corresponding author at: Department of Neuroscience, Neurological Clinic, University of Pisa, Via Roma 67, 56126 Pisa, Italy. Tel.: +39 050993347;
fax: +39 050992748.
Corresponding author at: Department of Human and Environmental Sciences,
University of Pisa, Via S. Giuseppe 22, 56126 Pisa, Italy.
E-mail addresses: f.coppede@geog.unipi.it (F. Copped), l.migliore@geog.unipi.it
(L. Migliore).
0304-3940/$ see front matter 2008 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.neulet.2008.10.074

ically aged and/or genetically predisposed to early chromosome

malsegregation [21]. To test this hypothesis, we recently measured chromosome damage and malsegregation events by means
of the micronucleus assay coupled by FISH technique in peripheral lymphocytes of mothers of DS infants (MDS) aged less than 35
years at conception, observing a signicant increased frequency of
both binucleated micronucleated (BNMN) cells and chromosome
13 and 21 malsegregation events as compared to a control group.
This suggests that MDS have a tendency to chromosome damage
and malsegregation not limited to gametes, but occurring even in
somatic cells [17].
A deciency in cellular folates results in aberrant DNA methylation, chromosome breakage, increased frequency of micronuclei
(MN), defective chromosome recombination and aneuploidy [9].
Therefore, it was postulated that impairments in folate metabolism
due to genetic polymorphisms of metabolic enzymes could increase
the risk for having an infant with DS [12]. There is also increasing evidence of association between polymorphisms in folate

16

F. Copped et al. / Neuroscience Letters 449 (2009) 1519

Fig. 1. Simplied overview of the human folate metabolic pathway. Enzymes: MTHFR: methylenetetrahydrofolate reductase; MTR: methionine synthase; MTRR: methionine
synthase reductase; TYMS: thymidilate synthase. Metabolites: DHF: dihydrofolate; THF: tetrahydrofolate; dTMP: deoxythymidine monophosphate; dUMP: deoxyuridine
monophosphate; Met: methionine; Hcy: homocysteine; SAM: S-adenosylmethionine.

metabolizing genes and the amount of chromosome damage and

malsegregation measured in human blood cells [1,6,14].
Folate is obtained from the diet and its metabolism requires
intestinal uptake, reduction and methylation into the liver to form
5-methyltetrahydrofolate (5-methylTHF), release into the blood
and cellular uptake; then it can be used for the synthesis of DNA
precursors or for the conversion of homocysteine (Hcy) to methionine, which is then used to form the main DNA methylating agent
S-adenosylmethionine (SAM) (Fig. 1). Methylenetetrahydrofolate
reductase (MTHFR) plays a pivotal role in regulating DNA methylation, through the reduction of 5,10-methylentetrahydrofolate
(5,10-MTHF) to 5-methylTHF, the one carbon donor for the
remethylation of Hcy to methionine mediated by the activity of
methionine synthase (MTR), which is maintained in its active state
by the methionine synthase reductase (MTRR) protein [3]. This
reaction is important for the synthesis of SAM, the major DNA
methyl donor. Two common polymorphisms are known to reduce
MTHFR activity: a T variant at nucleotide 677 (MTHFR 677C > T),
and a C variant at nucleotide 1298 (MTHFR 1298A > C), this latter
to a lesser extent [26]. A G variant at nucleotide 2756 of the MTR
gene (MTR 2756A > G) and a G variant at nucleotide 66 of the MTRR
gene (MTRR 66A > G) are known, the former being associated with
aberrant methylation [19]. 5,10-MTHF is also used by thymidylate
synthase (TYMS) for the conversion of deoxyuridine monophosphate (dUMP) to deoxythymine monophospate (dTMP) (Fig. 1).
Therefore, polymorphisms affecting TYMS activity might shift the
pools of 5,10-MTHF from DNA synthesis toward DNA methylation
[22]. Two common polymorphisms are known in the TYMS gene, a

double (2R) or triple (3R) 28 bp tandem repeat sequence in the promoter enhancer region and a 6 bp deletion/insertion (6bp/6bp+)
at position 1494 (1494del6) in the 3 -untranslated region (3 -UTR);
the rst inuences TYMS mRNA expression, the latter is thought to
inuence mRNA expression and/or stability [11,25].
Several studies support a role for polymorphisms of MTHFR, MTR
and MTRR genes, alone or combined, in the risk of having a DS child
[5,7,8,10,12,16,20]. On the contrary, at best of our knowledge, TYMS
variants have not been investigated as risk factors for a DS offspring,
yet.
Here, we evaluated the relationship between MTHFR, MTR, MTRR
and TYMS gene polymorphisms, alone or combined, and the risk of
having a DS child. Moreover, we measured the amount of chromosome damage and malsegregation events, by means of the micronucleus assay, in peripheral lymphocytes of a subgroup of MDS and
control mothers, and searched for association between any of the
studied polymorphisms and the frequency of BNMN lymphocytes.
Peripheral blood samples were collected from 94 women who
had a DS child with kariotipically conrmed full trisomy 21 at an age
below 35 years. Most MDS had only the DS child, but some of them
(30%) had also healthy children, mainly before the birth of the DS
one. The control group was composed by 113 healthy women who
had at least one healthy child before age 35 years, and no experience of miscarriages, abnormal pregnancies or children affected by
genetic disorders in their life. All subjects were white Caucasians
of Italian origin and gave informed consent for participation to the
study, that was approved by the Scientic Institute Stella Maris
Ethics Committee.

F. Copped et al. / Neuroscience Letters 449 (2009) 1519

Table 1
Study population, CBMN assay results and sets of primers used for genotyping.
Age, smoking habits and CBMN assay results

MDS (n = 42)

Controls (n = 41)

Age (years SD)

45.6 11.4

47.95 6.9

Number of smokers and ex-smokers

Ex-smokers
Light smokers (110 cigarette/day)
Heavy smokers (>10 cigarette/day)

9
4
2
3

BNMN a (mean SD)

15.99 8.36*

Primers


11
3
1
7

F: 5 -TGAAGGAGAAGGTGTCTGCGGGA-3
R: 5 -AGGACGGTGCGGTGAGAGTG-3
F: 5 -CTTTGGGGAGCTGAAGGACTACTA-3
R: 5 -CACTTTGTGACCATTCCGGTTTG-3
F: 5 -TGTTCCCAGCTGTTAGATGAAAATC-3
R: 5 -GATCCAAAGCCTTTTTACACTCCTC-3
F: 5 -GCAAAGGCCATCGCAGAAGACAT-3
R: 5 -TGGTGGTATTAGTGTCCTTTTG-3
F: 5 -GTGGCTCCTGCGTTTCCCCC-3
R: 5 -GGCTCCGAGCCGGCCACAGGCATGGCGCGG-3
F: 5 -CAAATCTGAGGGAGCTGAGT-3
R: 5 -CAGATAAGTGGCAGTACAGA-3

10.29 4.33

Polymorphism

References

MTHFR
677C > T
MTHFR
1298A > C
MTR
2756A > G
MTRR
66A > G
TYMS
28 bp repeat
TYMS
1494del6

[7]
[7]

Table 2
Genotype and allele frequencies of the six polymorphisms in MDS and control
mothers.
Polymorphism

Genotype

MDS (%)

Controls (%)

MTHFR 677C > T

94 MDS
113 Controls

CC
CT
TT
Allele T

25 (26.6)
52 (55.3)
17 (18.1)
86 (45.7)

40 (35.4)
55 (48.7)
18 (15.9)
91 (40.3)

1.0 (Reference)
1.51 (0.812.83)
1.51 (0.663.47)
1.25 (0.851.85)

MTHFR 1298A > C

88 MDS
107 Controls

AA
AC
CC
Allele C

44 (50.0)
38 (43.2)
6 (6.8)
50 (28.4)

47 (43.9)
53 (49.5)
7 (6.6)
67 (31.3)

1.0 (Reference)
0.77 (0.431.38)
0.92 (0.292.94)
0.87 (0.561.35)

MTR 2756A > G

90 MDS
111 Controls

AA
AG
GG
Allele G

66 (73.3)
22 (24.5)
2 (2.2)
26 (14.5)

78 (70.3)
30 (27.0)
3 (2.7)
36 (16.2)

1.0 (Reference)
0.87 (0.461.64)
0.79 (0.134.86)
0.87 (0.501.51)

MTRR 66A > G

81 MDS
111 Controls

AA
AG
GG
Allele G

20 (24.7)
39 (48.1)
22 (27.2)
83 (51.2)

29 (26.1)
62 (55.8)
20 (18.1)
102 (45.9)

1.0 (Reference)
0.91 (0.451.83)
1.60 (0.69366)
1.24 (0.821.85)

TYMS 28-bp repeat

82 MDS
107 Controls

2R/2R
2R/3R
3R/3R
Allele 3R

19 (23.1)
40 (48.8)
23 (28.1)
86 (52.4)

26 (24.3)
45 (42.1)
36 (33.6)
117 (55.7)

1.0 (Reference)
1.22 (0.592.52)
0.87 (0.401.93)
0.88 (0.581.32)

TYMS 1494del6
89 MDS
111 Controls

6bp+/6bp+
6bp+/6bp
6bp/6bp
Allele 6bp

29 (32.6)
44 (49.4)
16 (18.0)
76 (42.7)

39 (35.1)
53 (47.8)
19 (17.1)
91 (41.0)

1.0 (Reference)
1.12 (0.602.09)
1.13 (0.502.57)
1.07 (0.721.60)

[6]
[28]
[27]
[27]

a
Frequency of micronucleated binucleated lymphocytes calculated analyzing
2000 cells.
*
MANOVA: P < 0.0001, MDS versus controls.

Genomic DNA was isolated from whole blood by means of the

QIAamp Blood Mini Kit (Qiagen, Milan, Italy). All genotype analyses were performed by PCR-RFLP technique by means of standard
validated protocols [6,7,27,28]. Internal control samples, whose
genotypes had been previously assessed, were always included and
analyzed on each gel. Not all loci were amplied from every subject
due to a limited DNA quantity.
We selected 42 MDS and 41 control mothers for the analysis of chromosome damage in peripheral lymphocytes (Table 1).
Recent exposure to X-rays or nuclear magnetic resonance, viral
infections and inammatory disorders experienced during the last
three months preceding blood drawing, or the current use of
pharmacological products known to interfere with the micronucleus frequency were used as criteria to exclude subjects from the
analysis. The cytokinesis-block micronucleus assay (CBMN) was
performed according to the procedure previously described [18].
The statistical analysis was performed using the SPSS 11.0
statistical package for Windows. To assess differences in allele distributions, and to verify HardyWeinberg equilibrium, we used the
Chi-square (2 ) analysis. The differences in genotype frequencies
and in the combined genotype frequencies were analyzed between
the case and control groups by 2 2 contingency tables using 2
analysis or Fishers exact test if one or more variables in 2 2 tables
were <5. Odds ratios (ORs) have been calculated and given with 95%
condence intervals (CIs) using unconditional logistic regression
analysis with the low risk combination as a reference category. To
correct for inated alfa error resulting from multiple group comparisons within sets, we used the modied Bonferroni test provided by
Keppel [13].
Data obtained from the micronucleus test have been normalised,
and the natural logarithmic-transformed data have been elaborated through the multifactorial analysis of variance (MANOVA)
after including age and smoking habits in the model as confounding
factors. MANOVA was also used to test for associations between the
BML frequency and the particular genotype for each of the six polymorphisms under study. The relationship between the age and the
BML frequency was tested by regression analysis. Data were analyzed using the STATGRAPHICS Plus software package for Windows
(SWGIN, version 5.1), and the level of signicance was P < 0.05.

17

OR (95% CI)

The frequencies of MTHFR 677C > T and 1298A > C, MTR

2756A > G, MTRR 66A > G, TYMS 28 bp repeat and 1494del6 alleles and genotypes are listed in Table 2. No statistically signicant
differences in allele and genotype frequencies have been observed
between MDS and control mothers (Table 2). Genotype frequencies
were consistent with HardyWeinberg equilibrium expectations.
The combined MTHFR 677TT/MTR 2756AA genotype resulted in
a signicant increased DS risk (P = 0.034), and the combined MTHFR
1298AC/TYMS 2R/2R genotype resulted in a signicant decreased
DS risk (P = 0.003) (Table 3). None of the other combined genotypes
was associated with DS risk (data not shown).
The mean frequency of BNMN lymphocytes in MDS
(15.99 8.36) was signicantly higher (P < 0.0001) if compared
to the control group (10.29 4.33) (Table 1). MANOVA analysis
revealed also signicant correlations between both MTHFR 677C > T
(P = 0.031) and 1298A > C (P = 0.047) genotypes and the BNMN frequency (Fig. 2). None of the other analyzed polymorphisms was
related to the frequency of BNMN (data not shown). Regression
analysis pointed out an effect of the age (P = 0.046) on the BNMN
lymphocytes frequency in the total population (MDS + controls)
(data not shown).
None of the six studied polymorphisms in folate metabolizing
genes resulted to be an independent risk factor for a DS offspring
at a maternal age < 35 years (Table 2). Concerning MTHFR 677C > T,
MTHFR 1298A > C and MTRR 66A > G polymorphisms this is in
agreement with previous studies in Italian populations [5,7,20,23].
Conicting results have been obtained in Italy for the MTR 2756A > G
variant [5,20], and present results do not suggest a role in DS risk.
At best of our knowledge, the present is the rst genetic association
study which evaluates the contribution of TYMS gene polymorphisms to DS risk and no independent contribution was observed.
We had no opportunity to measure blood folates and Hcy levels
after pregnancy for the individuals included in the present study,
since their recruitment was performed several years after conception. We could only have data (collected at sampling) on blood
folates and Hcy values from 10 MDS and 17 control mothers, and
failed to nd any signicant difference between the two groups

18

F. Copped et al. / Neuroscience Letters 449 (2009) 1519

Table 3
Genegene interactions (only signicant results are shown).
MTHFR 677C > T MTR 2756A > G 90 MDS (%) 111 Controls (%) OR (95% CI)
CC
CC
CC
CT
CT
CT
TT
TT
TT

AA
AG
GG
AA
AG
GG
AA
AG
GG

18 (20)
5 (5.6)
1 (1.1)
33 (36.7)
15 (16.6)
1 (1.1)
15 (16.6)
2(2.3)
0()

29 (26.2)
8 (7.2)
1 (0.9)
41 (36.9)
12 (10.8)
2 (1.8)
8 (7.2)
10 (9.0)
0 ()

1.0 (Reference)
1.01 (0.283.56)
1.61 (0.0927.4)
1.30 (0.622.73)
2.01 (0.775.26)
0.81 (0.079.54)
3.02 (1.078.55)a , c
0.32 (0.061.64)

MTHFR 1298A > C TYMS 28 bp 80 MDS (%) 103 Controls (%) OR (95% CI)
AA
AA
AA
AC
AC
AC
CC
CC
CC

2R/2R
2R/3R
3R/3R
2R/2R
2R/3R
3R/3R
2R/2R
2R/3R
3R/3R

14 (17.5)
17 (21.25)
9 (11.25)
3 (3.75)
21 (26.25)
11 (13.75)
2 (2.5)
1 (1.25)
2 (2.5)

8 (7.8)
23 (22.3)
15 (14.6)
16 (15.5)
17 (16.5)
18 (17.5)
2 (1.93)
1 (0.97)
3 (2.9)

1.0 (Referent)
0.42 (0.141.23)
0.34 (0.101.14)
0.11 (0.020.48)b , c
0.75 (0.252.22)
0.35 (0.111.10)
0.57 (0.074.88)
0.57 (0.0310.4)
0.38 (0.052.78)

P = 0.034.
P = 0.003.
c
The difference is signicant using P-value adjusted for inated error (adjusted
P = 0.037). We used the Bonferroni correction provided by Keppel [13] that corrects
for the number of Chi-square tests and the degrees of freedom (df) involved in each
test according to the formula: alfa (new) = 1 (1 alfa)df /number of comparisons.
The adjusted P-value was considered as the new cut-off to determine statistical
signicance.
b

(data not shown); however these data should be taken with caution since they have been collected from a very small subgroup of
our population. Data from medical records indicate that subjects
included in the present study were not under particular dietary
restriction during pregnancy, and that they had adequate energy
intake as well as an adequate intake of fruit and green vegetables,
thus covering the daily requirements of folate and vitamins.
Here, we observed a statistically signicant increased frequency
of BNMN lymphocytes in MDS respect to control mothers (Table 1).
Micronuclei can originate either from chromosome breakage or
chromosome malsegregation. Our previous analysis in a subgroup
of subjects enrolled in the present study, showed that micronuclei
were mainly originated from chromosome malsegregation events
[17]. Present results, obtained in a larger casecontrol population,
conrm that MDS have a tendency to chromosome damage and
malsegregation which is detectable in somatic cells.
We observed a signicant correlation between the MTHFR 677
genotype and BNMN lymphocytes, with the BNMN frequency
increasing with the increasing number of 677T alleles (Fig. 2a).
Present results conrm previous ndings by us on a smaller
MDScontrol group [6] and are in agreement with other studies
[1,14] suggesting a role for the MTHFR 677T allele in micronuclei
formation.
We also observed a signicant increased frequency of BNMN
lymphocytes in individuals bearing the MTHFR 1298AA genotype,
respect to those bearing at least one copy of the MTHFR 1298C genotype (Fig. 2b). This could be explained by the existence of a strong
linkage disequilibrium (LD) between MTHFR 677C > T and 1298A > C
polymorphisms; particularly the 677T allele has been nearly always
observed in cis with the 1298A allele [15].
Interestingly, MDS were found to have a ve-fold increased
risk to develop Alzheimers disease (AD), respect to the general
population [21]. We previously observed a preferential occurrence
of chromosome 21 malsegregation in peripheral blood lymphocytes of AD patients [18]. Recently Thomas and Fenech observed
an increased frequency of chromosome 17 and 21 aneuploidy in

Fig. 2. (a) The mean frequency of binucleated micronucleated (BNMN) lymphocytes

in relation to the MTHFR 677C > T genotypes in our population (MDS + controls). A
signicant correlation between the frequency of BNMN and the MTHFR 677C > T
genotypes was observed. Particularly, the mean BNMN frequency increases signicantly with the increasing number of MTHFR 677T alleles carried by an individual.
MTHFR: methylenetetrahydrofolate reductase; CC: MTHFR 677CC homozygous
wild-type genotype; CT: MTHFR 677CT heterozygous genotype; TT: MTHFR 677TT
mutant genotype. (b) The mean frequency of binucleated micronucleated (BNMN)
lymphocytes in relation to the MTHFR 1298A > C genotypes. We observed that
MTHFR 1298AA individuals have a signicant higher BNMN frequency than MTHFR
1298AC + CC carriers (we grouped together 1298AC and CC carriers, since only 2
subjects were 1298CC). MTHFR: methylenetetrahydrofolate reductase; AA: MTHFR
1298AA homozygous wild-type genotype; AC + CC: MTHFR 1298AC heterozygous
genotype + MTHFR 1298CC mutant genotype.

buccal cells of AD patients compared to matched controls [24].

Present and previous results by us [17], suggest that also MDS have
a defect in chromosome segregation leading to aneuploidy. Moreover, impairments in folate and Hcy metabolism and folate gene
polymorphisms have been associated also with AD risk [4]. The
present is a retrospective study since mothers were recruited some
years after the birth of their child. Further prospective studies could
be helpful to understand whether or not an increased chromosome
instability observed in somatic cells of young women is associated
with increased risk to have DS children and to develop age-related
neurodegenerative diseases later in life.
Since the frequency of both MTHFR 677C > T and 1298A > C polymorphisms did not differ between the two groups, the MTHFR
genotype alone cannot account for the increased chromosome
damage that we observed in MDS respect to controls, which is likely
the result of a complex interaction between MTHFR variants and
both environmental and other genetic factors.
Several previous studies suggest that interactions between
folate gene polymorphisms can modify the risk of having a DS child
[5,7,10,20]. Here, when evaluating genegene interactions, the combined MTHFR 677TT/MTR 2756AA genotype was associated with
increased DS risk, and the combined MTHFR 1298AC/TYMS 2R/2R
genotype with protection (Table 3). An interaction between MTHFR

F. Copped et al. / Neuroscience Letters 449 (2009) 1519

and MTR polymorphisms in increasing DS risk is plausible since

MTHFR and MTR work in the same pathway of DNA methylation
that can be altered if one or more enzymes are impaired (Fig. 1).
Moreover, both TYMS and MTHFR require 5,10-MTHF, the rst for
the DNA synthesis pathway, the latter for DNA methylation (Fig. 1),
and the majority of chromosome 21 nondisjunction events occurs
at maternal meiosis I during embryogenesis, when cells have a
high demand of DNA precursors and a high rate of divisions [2]. In
this context, interactions between polymorphisms associated with
MTHFR and TYMS enzyme activities might shift pools of 5,10-MTHF
from DNA methylation toward purine and DNA synthesis, or vice
versa. It is therefore likely that combinations of MTHFR and TYMS
genotypes might affect chromosome segregation and DS risk. Even
if the present is the largest casecontrol study performed to date
in Italy on women aged less than 35 years at conception results
should however be taken with caution; they are indicative of possible genegene interactions that require conrmation in further
studies empowered with increased sample size.
We observed an effect of the age (P = 0.046) on the frequency of
BNMN lymphocytes; results are consistent with previous studies
reporting a positive correlation between the increased frequency
of micronuclei in peripheral lymphocytes and the age, in particular
in women where the preferential involvement of X chromosome in
nondisjunction events has been demonstrated [29].
Concluding, present results suggest that polymorphisms in
folate metabolizing genes contribute to chromosome damage and
malsegregation events, possibly affecting the risk of having a DS
child.

[9]
[10]

[11]

[12]

[13]
[14]

[15]

[16]

[17]

[18]

[19]

Conict of interest
All authors conrm that there are no conicts of interest associated with this manuscript.

[20]

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[21]

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