Mutation Research/Reviews in Mutation Research: Fabio Coppede'

Mutation Research 682 (2009) 5470
Contents lists available at ScienceDirect
Mutation Research/Reviews in Mutation Research

journal homepage: www.elsevier.com/locate/reviewsmr
Community address: www.elsevier.com/locate/mutres
Review
The complex relationship between folate/homocysteine metabolism and risk

of Down syndrome
Fabio Coppede` *
Department of Neuroscience, Neurological Clinic, University of Pisa, Via Roma 67, 56126 Pisa, Italy
A R T I C L E I N F O
A B S T R A C T
Article history:
Received 16 April 2009
Received in revised form 2 June 2009
Accepted 3 June 2009
Available online 11 June 2009
Folates are essential nutrients that are required for one-carbon biosynthetic and epigenetic processes. A
deciency in cellular folates results in aberrant DNA methylation, point mutations, chromosome
breakage, defective chromosome recombination and aneuploidy. In 1999 it was rst reported that
impairments in folate/homocysteine metabolism, due to genetic polymorphisms of metabolic enzymes,
could increase the risk for having an infant with Down syndrome (DS). That paper stimulated
considerable investigation into the possible role of folate/homocysteine metabolism in the risk of having
a DS child and several studies have been performed so far in different countries to better address this
issue. However, despite 10 years of active research, the question is still unsolved. Overall, both in vitro
and in vivo studies indicate that an impaired folate/homocysteine metabolism can result in chromosome
21 nondisjunction; however, the birth of a DS child seems to be the result of the interplay of several
factors of genetic, epigenetic, environmental, and stochastic origin, making it difcult to discriminate the
single contribution of each of them. My opinion is that it is now time for the design of a collaborative
study large enough to have the power to separate trisomy 21 into all its component parts and to test for
the contribution of folate/homocysteine gene polymorphisms to each of them. This study should be
paralleled by in vitro and in vivo studies aimed at clarifying the contribution, if any, of folate/
homocysteine metabolism to the methylation pattern of regions involved in recombination and
segregation of chromosome 21. Further studies are also required to address the possible contribution of
both the paternal diet and the maternal grandmother dietary habits to chromosome 21 nondisjunction
events.
2009 Elsevier B.V. All rights reserved.
Keywords:
Down syndrome (DS)
Folate metabolism
Homocysteine (Hcy)
Gene polymorphisms
Epigenetics
MTHFR
MTRR
MTR
RFC1
CBS
Contents
1.
2.
3.
4.
5.
6.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Folate/homocysteine/methionine metabolism and impact on DNA methylation or synthesis. .
Methylenetetrahydrofolate reductase (MTHFR) and DS risk . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.
The MTHFR 677C > T polymorphism and the risk of having a DS child . . . . . . . . . . . . .
3.2.
The MTHFR 1298A > C polymorphism and the risk of having a DS child . . . . . . . . . . . .
3.3.
MTHFR gene polymorphisms, chromosome damage and aneuploidy . . . . . . . . . . . . . . .
The methionine synthase-methionine synthase reductase (MTR-MTRR) complex and DS risk .
4.1.
The MTR 2756A > G polymorphism and the risk of having a DS child . . . . . . . . . . . . . .
4.2.
The MTRR 66A > G polymorphism and the risk of having a DS child . . . . . . . . . . . . . . .
4.3.
MTR and MTRR gene polymorphisms, chromosome damage and aneuploidy. . . . . . . . .
The reduced folate carrier (RFC1 or SLC19A1) and DS risk . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.1.
The RFC1 80G > A polymorphism and the risk of having a DS child . . . . . . . . . . . . . . . .
5.2.
Trisomy for RFC1 in DS individuals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Cystathionine b-synthase (CBS) and DS risk . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.1.
The CBS 844ins68 polymorphism and the risk of having a DS child . . . . . . . . . . . . . . . .
6.2.
Trisomy for CBS in DS individuals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
* Tel.: +39 050993347; fax: +39 050992748.

E-mail address: f.coppede@geog.unipi.it.
1383-5742/$ see front matter 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.mrrev.2009.06.001
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F. Coppede` / Mutation Research 682 (2009) 5470
7.
8.
9.
Other genes participating in folate/homocysteine metabolism and DS risk . . . . . . . . . . . . . . . . . . . .

7.1.
Thymidylate synthase (TYMS) and DS risk . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.2.
Methylenetetrahydrofolate dehydrogenase (MTHFD1) and DS risk . . . . . . . . . . . . . . . . . . . . .
7.3.
Transcobalamin (TC) and DS risk . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.4.
Other genes located on chromosome 21 involved in folate/homocysteine metabolism . . . . .
Environmental factors and DS risk: dietary factors and age at conception. . . . . . . . . . . . . . . . . . . . .
8.1.
Homocysteine, folate, vitamin B12 and related micronutrients in MDS and control mothers
8.2.
Maternal age at conception and DS risk: is there a link with folate metabolism? . . . . . . . . .
8.3.
The paternal contribution: diet, age at conception, and DS risk. . . . . . . . . . . . . . . . . . . . . . . .
8.4.
Alcohol consumption and its relevance to folate metabolism and DS risk . . . . . . . . . . . . . . .
Discussion and conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction
Members of the family of B9 vitamins are commonly known as
folates. They are essential nutrients that are required for one-carbon
biosynthetic and epigenetic processes. Folates are derived entirely
from dietary sources, mainly from the consumption of green
vegetables (such as spinaches, broccoli, asparagus and lettuce), fruits
(mainly oranges, lemons, strawberries and kiwi), cereals, beans and
liver. Folic acid is the synthetic form added to foods and found in
dietary supplements. After intestinal absorption, folate metabolism
requires reduction and methylation into the liver to form 5-
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methyltetrahydrofolate (5-methylTHF), release into the blood and

cellular uptake; then it can be used for the synthesis of DNA and RNA
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). Folic acid is converted to
a natural biological form of the vitamin as it passes through the
intestinal wall, with enzymatic reduction and methylation resulting
in the circulating form of the vitamin, 5-methylTHF [1,2].
A deciency in cellular folates results in aberrant DNA
methylation, point mutations, chromosome breakage and increased
frequency of micronuclei (MN), defective chromosome recombina-
Fig. 1. Simplied overview of the human folate metabolic pathway. Enzymes: CBS: cystathionine b-synthase; GART: phosphoribosylglycineamide transformylase; MTHFD1:
methylenetetrahydrofolate dehydrogenase; MAT: methionine adenosyltransferase; MTHFR: methylenetetrahydrofolate reductase; MTR: methionine synthase; MTRR:
methionine synthase reductase; RFC1: reduced folate carrier; TC: transcobalamin; TYMS: thymidilate synthase. Metabolites: DHF: dihydrofolate; GSH: glutathione; THF:
tetrahydrofolate; dTMP: deoxythymidine monophosphate; dUMP: deoxyuridine monophosphate; SAH: S-adenosyl homocysteine; SAM: S-adenosylmethionine. Cofactors:
B12: vitamin B12 or cobalamin.
56
tion and aneuploidy [3]. Impaired folate metabolism, resulting from

the presence of common functional polymorphisms of genes
encoding for metabolic enzymes, has been associated with several
human diseases including various kinds of cancer [47], cardiovascular diseases [8,9], neurodegenerative diseases [10,11] and neural
tube defects [12,13].
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
[14]. In 1999 James et al. [15] suggested that impairments in folate
metabolism due to genetic polymorphisms of metabolic enzymes
could increase the risk for having an infant with DS. They reported
that the variant 677T allele of the methylenetetrahydrofolate
reductase gene (MTHFR) might be a maternal risk factor for having
a child with DS in a North American population, and observed
increased plasma Hcy levels in mothers of DS individuals (MDS),
respect to mothers of healthy children [15]. That paper stimulated
considerable investigation into the possible role of folate
metabolism in the risk of having a DS child and several studies
[1540] have been performed so far in different countries to better
address this issue, which is still unsolved (Table 1). Conicting
results have often emerged when evaluating each single polymorphism as an independent DS risk factor (Table 1) and the
current opinion is that the presence of a single polymorphism of a
gene participating in the folate/Hcy metabolic pathway might be
insufcient to increase the risk of having a child with DS, whereas
the combined presence of two or more of them in the genome
could increase DS risk [16,17,19,21,25,2730,34,35,39] (Table 2).
Some investigators also suggest a complex interaction between
genetic and environmental factors, particularly dietary factors, in
the formation of eggs carrying two copies of chromosome 21
[15,17,25,31,35]. Table 3 lists all the genetic association studies
available in pubmed on April 15th 2009 and reporting measured or
estimated plasma values of Hcy, folates, vitamin B12 (vitB12) and/
or related micronutrients in both MDS and control mothers,
including, when available, indication of possible interactions with
polymorphisms of metabolic genes.
Another complexity to our understanding of the role of folate
metabolism in DS risk is the fact that several metabolic genes,
including among others those encoding for the reduced folate
carrier (SLC19A1 or RFC1) and for cystathionine beta synthase
(CBS), are located on chromosome 21. Trisomy for these genes
might increase folate demand or availability in developing DS
foetuses. Therefore, the complex relationship between folate/Hcy
metabolism and human trisomy 21 has been nowadays revised
and the current opinion is that maternal and embryonic
combinations for variants in folate metabolizing genes, coupled
with the maternal nutritional and life style status during
pregnancy, may strongly inuence the probability that some
embryos with trisomy 21 survive to the birth [31]. Moreover, since
most of chromosome 21 nondisjunctions occurs during maternal
embryogenesis in the grandmother body, also the maternal
grandmother genotype and nutritional status during pregnancy
might strongly affect the probability that some maternal eggs will
carry two copies of chromosome 21 [40,41].
Advanced maternal age at conception represents the major risk
factor for trisomy 21 and after age 35 years the risk of a DS
pregnancy increases proportionally to increasing maternal age
[14]. However, most of DS cases are born from women aging less
than 35 years and different mechanisms seem to be responsible for
chromosome 21 nondisjunction in young women respect to older
ones, each of them potentially affected by an impaired folate/Hcy
metabolism [42]. Therefore, the contribution of maternal age at
conception adds complexity to the investigation of the role of
folate metabolism in DS risk and recent ndings also suggest that
the maternal age effect is different for errors occurring at maternal

meiosis II than it is for errors occurring at maternal meiosis I [43].
Moreover, it has been hypothesized that also maternal trisomy 21
ovarian mosaicism might provide another DS risk factor [44].
Overall, results emphasize the fact that human nondisjunction is a
multifactorial trait that must be dissected into its component parts
to identify specic associated risk factors [4244]. Alcohol
consumption was associated with low serum folate levels, and
with impaired methionine synthase (MTR) activity (one of the
enzymes required for the production of SAM, see the next section).
Therefore, alcohol could be another dietary factor to be taken into
account in DS risk association studies [45].
Only a few of DS cases (less than 5%) are due to errors occurring
during paternal meiosis and little is known about the effect of
paternal nutrition and aneuploidy in sperms. Recently, Young and
colleagues observed that men with high folate intake had lower
frequencies of sperm with disomy 21 compared with men with
lower intake, providing additional evidence for the importance of
folates in human nondisjunction events [46].
Aim of this article is to review the literature which has been
produced in the last decade (19992009) on the possible role of
folate and Hcy metabolism in the risk of Down syndrome,
discussing the validity and the limits of the studies performed
so far, and providing some suggestions on what investigators can
now do to help unravel this issue.
2. Folate/homocysteine/methionine metabolism and impact on
DNA methylation or synthesis
As summarized in Fig. 1 cellular folates can be used either for
DNA methylation processes than for the synthesis of nucleic acid
precursors (Fig. 1). Folates are highly hydrophilic molecules that do
not cross biological membranes by diffusion alone, so it is not
surprising that sophisticated membrane transport systems have
evolved for facilitating their uptake by mammalian cells and
tissues. Folates use several genetically distinct and functionally
diverse transport systems to enter the cells: the reduced folate
carrier (RFC1), folate receptors (FR) and the proton-coupled folate
transporter (PCFT). By far the best-characterized folate transporter
is the ubiquitously expressed reduced folate carrier [47,48].
Whereas RFC1 exhibits a relatively wide pattern of tissue
expression, the expression of the additional folate uptake systems
is rather restricted to a limited number of tissues. Indeed, given its
widespread tissue expression, RFC1 must be considered the major
transport system in mammalian cells and tissues, where it
participates in the uptake of folate cofactors from the blood [49].
Methylenetetrahydrofolate reductase (MTHFR) plays a pivotal
role in regulating DNA methylation, through the reduction of 5,10methylentetrahydrofolate (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). MTR is a key enzyme
in the methionine cycle since it catalyzes the transmethylation of
Hcy to methionine in a cobalamin-dependent reaction that utilizes
methyl-THF as a methyl group donor. Indeed, MTR transfers a
methyl group from 5-methylTHF to Hcy forming methionine and
tetrahydrofolate (THF). Cobalamin (or vitamin B12) is a cofactor in
this reaction. Mehionine synthase reductase (MTRR) is required for
the maintenance of MTR in its active state (Fig. 1). Methionine is an
essential amino required for the formation of SAM in a reaction
catalyzed by methionine adenosyltransferase (MAT). Then, most of
the SAM generated is used in transmethylation reactions, whereby
SAM is converted to S-adenosyl homocysteine (SAH) by transferring the methyl group to diverse biological acceptors, including the
DNA, in a reaction catalyzed by methyltransferase enzymes. SAH is
then converted to Hcy and adenosine in a reversible reaction
catalyzed by SAH hydrolase [1]. Therefore, Hcy can be converted to
Table 1
Genetic association studies between folate/homocysteine metabolizing gene polymorphisms and maternal risk of having a DS baby.
Reference
Country
MDS
(n)
Controls
(n)
MTHFR 677C > T
MTHFR 1298A > C
MTRR
66A > G
MTR
2756A > G
RFC-1
80G > A
CBS 844
ins68
Others
1999
2000
James et al. [15]

Hobbs et al. [16]
USA
USA Canada
57
157
50
144
Pb OR = 2.6 (1.25.8)c
Pa OR = 1.9 (1.23.0)c
2002
OLeary et al. [17]
Ireland
48
192
Na,b OR = N.A.
2002
France*
85
107
N OR = N.A.
2002
2002
2003
Chadefaux-Vekemans
et al. [18]
Grillo et al. [19]
Stuppia et al. [20]
Bosco et al. [21]
Pa OR = 2.6
(1.35.0)c
Pa OR = 10.5
(1.478.6)c
2003
2004
2004
2005
Sheth and Sheth [22]

Takamura et al. [23]
Boduroglu et al. [24]
da Silva et al. [25]
Na OR = N.A.
2005
2005
Chango et al. [26]

Acacio et al. [27]
2006
Coppede` et al. [28]
Brazil
Italy (central)*
Italy (Sicily)*
36
64
63
200
112
72
P OR = N.A.
N OR = N.A.
N OR = N.A.
P OR = N.A.
N OR = N.A.
Na OR = N.A.
India
Japan
Turkey*
Brazil
(Sao Paulo)
France*
Brazil
(Sao Paulo)
Italy
(central)*
India
Italy*
(southern)
Spain*
N.A.
31
152
154
N.A.
60
91
158
N OR = N.A.
N OR = N.A.
N OR = 1.3 (0.82.3)c
Pa,b OR = 1.4 (1.02.1)c
N OR = 0.7 (0.41.3)c
Na OR = N.A.
Na OR = N.A.
Pa OR = 3.5
(1.210.9)c
Na OR = N.A.
119
70
119
88
N OR = N.A.
Na OR = 0.8 (0.23.0)c
N OR = N.A.
Na OR = 0.7 (0.13.5)c
N OR = N.A.
N OR = N.A.
N OR = N.A.
N OR = N.A.
80
111
Na OR = 1.6 (0.83.0)c
Na OR = 0.7 (0.41.4)c
N OR = 0.9
(0.61.3)c
Pb OR = N.A.
N OR = 0.8
(0.51.3)c
Na OR = 0.8
(0.41.5)c
Pa OR = 1.5
(1.02.1)c
N OR = 0.9
(0.41.8)c
MTHFD1 1958G > A Na

OR = 0.9 (0.71.4)c
Na OR = 1.6
(0.55.8)c
Na OR = 1.4
(0.63.0)c
149
94
165
264
P OR = 7.7 (1.735.1)
Na OR = 0.9 (0.71.4)c
91
90
N OR = N.A.
Pb OR = N.A.
China
Egypt*
China
100
42
64
100
48
70
Pb OR = 3.4 (1.48.4)c
P OR = 4.1 (1.05.7)c
Pa OR = 3.8 (1.88.5)c
P OR = 31.5 (3.5282.)c
Biselli et al. [35]
Brazil
72
194
Na OR = 1.7 (0.64.6)c
Na,b OR = 1.5 (0.54.9)c
Pa OR = 5.2
(1.914.2)c
2008
Kohli et al. [36]
104
109
N OR = N.A.
2008
103
108
N OR = 0.6 (0.51.4)c
N OR = 0.9 (0.51.5)c
Brazil
Italy
(central)*
67
94
113
113
Na OR = 1.2 (0.81.8)c
Na OR = 0.8 (0.51.3)c
N OR = 0.7
(0.41.2)c
N OR = 1.2
(0.81.8)c
2008
2009
Santos-Reboucas
et al. [38]
Biselli et al. [37]
India
(northern)
Brazil
Na OR = 0.9
(0.51.5)c
N OR = N.A.
2009
Pozzi et al. [40]
Italy
(northern)*
74
184
N OR = 0.9 (0.42.2)c
P OR = 2.2
(1.14.4)c
TC 776C > G N OR = N.A.

TYMS 28 bp rep. Na
OR = 0.9 (0.61.3)c 1494 del6 N
OR = 1.1 (0.71.6)c
2006
2006
Rai et al. [29]

Scala et al. [30]
2006
2007
2008
2008
Martnez-Fras
et al. [31]
Wang et al. [32]
Meguid et al. [33]
Wang et al. [34]
2008
P OR = 4.4 (1.413.3)
Pa OR = 1.5 (1.02.1)c
Year
P = association between the polymorphism and increased DS risk; N = no association between the polymorphism and increased DS risk; N.A. = not available; () not studied; MDS = mothers of Down syndrome (DS) individuals.
a
Genegene interaction (described in Table 2).
b
Geneenvironment interaction (described in Table 3).
c
OR = odds ratio (given with 95% condence interval).
*
Mediterranean countries.
57
58
Table 2
Interactions between polymorphisms in folate/homocysteine metabolizing genes and maternal risk of having a DS baby.
Year
Reference
Country
MTHFR + others
Other interactions
b
2000
2002
2002
2003
Hobbs et al. [16]

OLeary et al. [17]
Grillo et al. [19]
Bosco et al. [21]
USA, Canada
Ireland
Brazil
Italy (Sicily)
MTHFR 677T + MTRR 66G " OR = 4.1 (1.98.6)

MTHFR 677T + MTRR 66G " OR = 2.9 (1.27.5)b
MTHFR 677CT + MTHFR 1298AC " OR = N.A.
2005
Brazil
2005
2006
Acacio et al. [27]

Brazil
Italy (central)
2006
2006
Rai et al. [29]

Scala et al. [30]
India
Italy (southern)
MTHFR 677T + MTHFR 1298C + MTR 2756G + MTRR 66G + CBS

844 ins68 " (complex a) OR = 1.2 (1.01.6)b
MTHFR 677CT + MTHFR 1298AC " OR = 5.7 (1.718.8)b
MTHFR 677TT + RFC1 80GG " OR = 6.0 (1.035.9)b
MTHFR 1298AA + RFC1 80GA/AA # OR = 0.4 (0.10.9)b
MTHFR 677CC + MTHFR 1298CC " OR = 4.0 (1.213.6)b
MTHFR 677TT + MTHFR 1298CC/CA " OR = 7.2 (1.447.2)b
2008
2008
Wang et al. [34]

Biselli et al. [35]
China
Brazil
2009
Italy (central)
MTR 2756AG + MTRR 66AG "

OR = 5.0 (1.124.1)b
RFC1 80 GG + MTHFD 1958 AA "

OR = 4.4 (1.217.9)b
MTHFR 1298CC/CA + RFC1 80 GG/GA " OR = 2.6 (1.16.3)b

MTHFR 677T-1298C haplotype " OR = N.A.
MTHFR 677TT/CT + MTRR 66GG " OR = 6.0 (2.017.5)b
MTHFR 677T + MTHFR 1298C + RFC1 80G + MTR 2756G "
(complex a) OR = 1.7 (1.03.0)b
MTHFR 677TT + MTR 2756AA " OR = 3.0 (1.08.5)b
MTHFR 1298AC + TYMS 2R/2R # OR = 0.11 (0.10.5)b
"=associated with maternal increased DS risk; #=associated with maternal reduced DS risk.
a
Mothers of Down syndrome individuals (MDS) showed a signicant increased number of mutant alleles than control mothers.
b
OR = Odds ratio (given with 95% condence interval).
Table 3
Homocysteine (Hcy), folates, vitamin B12 and related micronutrients in mothers of DS individuals (MDS) and control mothers, and their interaction with folate/homocysteine
metabolizing gene polymorphisms.
Year
Reference
Country
Plasma homocysteine (Hcy) concentration
1999
James et al. [15]
USA
2002
OLeary et al. [17]
Ireland
2002
Chadefaux-Vekemans
et al. [18]
France
2003
Bosco et al. [21]
Italy (Sicily)
2003
2004
Sheth and Sheth [22]

Takamura et al. [23]
India
Japan
Increased Hcy levels in MDS

Interaction between Hcy levels and the
MTHFR 677C > T polymorphism
No difference in Hcy levels between MDS
and control mothers
MTHFR 677C>T polymorphism
and control mothers
No interaction between Hcy levels and the
No interaction between Hcy levels and
MTHFR 677C > T, MTR 2756 A > G and
MTRR 66A > G polymorphisms
2005
Brazil
2006
Scala et al. [30]
2006
Martnez-Fras et al. [31]
Italy
(southern)
Spain
2997
Wang et al. [32]
China
2008
2008
Meguid et al. [33]

Biselli et al. [35]
Egypt
Brazil
2008
Kohli et al. [36]
2008
Santos-Reboucas et al. [37]
India
(northern)
Brazil
2009
Italy
(central)

and control mothers
Complex interactions between
Hcy levels, MTHFR 1298A > C
polymorphism and MTRR 66A > G
polymorphism
Increased Hcy levels in MDS Interactions
between Hcy levels and the
MTHFR 1298A > C polymorphism
Decreased Hcy levels in MDS

and control mothersa
Data obtained from a limited number of MDS and control mothers.
Other nutrients
Plasma folate and vitamin B12 levels resulted signicant

predictors of Hcy levels
No difference in folate levels between MDS and control mothers

No difference in vitamin B12 levels between MDS and
control mothers
Decreased serum folic acid levels in MDS

No difference in vitamin B6 and B12 levels between MDS
and control mothers
Folate intake from foods was signicantly lower than the

Recommended Daily Allowance in MDS
Folate intake from foods was lower than the Recommended

Daily Allowance in both MDS and controls
MDS had an estimative signicantly lower intake of zinc and
methionine respect to control mothers
No difference in folate levels between MDS and control mothersa
No difference in vitamin B12 levels between MDS and
control mothersa
methionine by MTR. Alternatively, Hcy can be condensed with

serine to form cystathionine in a reaction catalyzed by cystathionine b-synthase (CBS), which requires vitamin B-6 as a cofactor.
Cystathionine can be then utilized to form the antioxidant
compound glutathione (GSH). Indeed, SAM allosterically regulates
the activity of CBS. When MTR is compromised by exposure to
ethanol, a methyl group is transferred from betaine to homocysteine to form methionine in a reaction catalyzed by betainehomocysteine methyltransferase [45].
Depending on cellular demands 5,10-MTHF, which is the
substrate of MTHFR in the DNA methylation pathway, can be used
in DNA synthesis processes. Thymidylate synthase (TYMS)
converts deoxyuridine monophosphate (dUMP) and 5,10-MTHF
to deoxythymine monophosphate (dTMP) and dihydrofolate (DHF)
in the de novo synthesis of pyrimidines (Fig. 1). Methylenetetrahydrofolate dehydrogenase (MTHFD1) is a trifunctional enzyme
that interconverts tetrahydrofolate derivatives for purine, methionine and thymidylate synthesis. The enzyme consists of three
activities: 5,10-MTHF dehydrogenase, 5,10-methenyltetrahydrofolate cyclohydrolase and 10-formyltetrahydrofolate synthetase,
respectively, and catalyzes three sequential reactions in the
interconversion of THF derivatives. Once generated by the 10formyltetrahydrofolate synthetase activity, 10-formyltetrahydrofolate can be utilized for the production of purines by the
phosphoribosylglycineamide transformylase (GART) enzyme [50].
Overall, folate metabolism requires several enzymes and cofactors
to fulll the cellular metabolic demands (Fig. 1).
3. Methylenetetrahydrofolate reductase (MTHFR) and DS risk
Two common polymorphisms are known to reduce MTHFR
activity: MTHFR 677C > T (Ala222Val) and MTHFR 1298A > C
(Glu429Ala) [51]. Numerous studies have shown that the MTHFR
677T allele is associated with increased total plasma Hcy (tHcy)
and decreased serum folate, mainly in 677TT homozygous subjects
[5254]. The impact of the MTHFR 1298A > C polymorphism on
enzyme activity is less than that of the 677C > T [55], and most
studies have not detected signicant differences in tHcy or serum
folate levels between different MTHFR 1298A > C genotypes [56].
MTHFR 677C > T and 1298A > C polymorphisms are in strong
linkage disequilibrium (LD), particularly the 677T allele has been
nearly always observed in cis with the 1298A allele. A study
suggested that the 677T variant arose later than the 1298C variant
on a chromosome harbouring 1298A [57]. LD is not complete;
however frequencies below 0.005 have generally been reported for
the rare 677T1298C haplotype [58].
The effects of these two polymorphisms on the properties of the
MTHFR enzyme in humans have been described in recent years
[59,60]. Yamada et al. [59] proposed that the MTHFR enzyme is
constituted by dimers in humans, each of which has the catalytic
domain harbouring position 222 and the regulatory domain
harbouring position 429. According to this model the monomers
associate head to tail to form the catalytically active enzyme
dimer. The catalytic domain binds the avin-adenosine-dinucleotide (FAD) cofactor and folate, while the regulatory domain binds
SAM, suggesting that an allosteric interaction between the
regulatory domain of one subunit and the catalytic domain of
the other modulates the afnity for FAD. The same authors
observed that the dimer dissociates, with loss of enzyme activity,
upon heat treatment and enzyme dilution, but is stabilised by
physiological levels of folates, FAD and SAM [59].
Previous studies in Escherichia coli had shown that physiological
levels of folates protect the wild-type MTHFR enzyme against
avin loss, but the MTHFR 677T mutation decreases the afnity of
the enzyme for its FAD cofactor and the mutant enzyme requires
higher folate levels than the wild-type for the stabilization of the
59
binding of FAD [61]. In this context, McNulty et al. observed that

high dietary intake of riboavin (FAD) for a 12-week period
reduced Hcy levels in MTHFR 677TT healthy humans, partially
correcting the negative effect of the MTHFR 677T mutation [62].
A biological explanation for the LD existing between the two
different MTHFR polymorphisms has been recently suggested
[60,63]. Ulvik and colleagues [60] proposed that in humans the
stability of the dimer depends on what aminoacid is present at
position 222 and what at position 429, resulting from the MTHFR
677/1298 genotype. They genotyped 10,034 subjects observing
that no individual in this large sample carried the double
homozygous 677TT/1298CC genotype, and only a few subjects
(0.05%) had genotypes with three mutations (677TT/1298AC or
677CT/1298CC), suggesting that the presence of more than two
mutations in these genotypic combinations results in severe
enzyme instability likely because of the head to tail association
of the monomers is compromised [60]. This hypothesis might
explain the very low frequency of the 677TT/1298CC genotype in
living individuals, while it has been observed with a higher
frequency in foetuses [64]. Ulvik et al. also observed that tHcy
levels increased as a function of the decrease in folate, but this was
also depending on the different MTHFR genotypes and was
stronger for the 677TT/1298AA genotype [60].
3.1. The MTHFR 677C > T polymorphism and the risk of
having a DS child
In 1999, on the basis of evidence that abnormal folate and
methyl metabolism can lead to DNA hypomethylation and
abnormal chromosomal segregation, James et al. hypothesized
that the MTHFR 677C > T polymorphism might be a risk factor for
maternal meiotic nondisjunction and DS in young mothers. They
performed their study on 57 MDS and 50 age-matched control
mothers from North America observing a signicant increase in
plasma Hcy concentrations and lymphocyte methotrexate cytotoxicity in MDS, consistent with abnormal folate and methyl
metabolism. Moreover, the MTHFR 677T allele was associated with
a 2.6-fold higher risk of having a DS child. The results of that initial
study indicated that folate metabolism is abnormal in MDS and
that this might be explained, in part, by a mutation in the MTHFR
gene [15]. Several association studies [1540] have been
performed in the last decade (19992009) aimed at clarifying
the role of folate and methyl metabolism in DS risk, and the MTHFR
677C > T polymorphism has been investigated almost in all of
them (Table 1).
The MTHFR 677C > T polymorphism resulted to be an
independent risk factor for a DS offspring in the USA-Canadian,
Egyptian and Chinese populations [15,16,3234]. Conicting
results have been obtained in Brazil [19,25,27,35,37] and India
[22,29,36]. None of the studies performed in Europe [17,18,20,
21,26,28,30,31,39,40], nor the ones performed in Turkey and Japan
[23,24] found it to be an independent DS risk factor (Table 1). The
contrast of these results could be due to variations in allele
frequencies among different populations or to the different sizes of
the casecontrol studies; however, it could also largely result from
the contribution of environmental factors, such as dietary factors
[18,26,28].
An example is given by the observation that all the studies
performed in Mediterranean countries [18,20,21,24,26,28,30,31,
39,40] (Table 1), with the exception of the one performed in Egypt
[33], failed to support an independent contribution for the MTHFR
677T allele to DS risk, suggesting that the effect of the Mediterranean
diet, usually rich in folates, could counteract the individual effect of
the MTHFR 677T allele [17,26,28]. Moreover, the Egyptian study of
Meguid et al. was performed in a group of MDS with low folate intake
from food (below the Recommended Daily Allowance), providing
60
additional evidence of interactions between low folate intake and

the MTHFR genotype [33].
However, when gene-gene interactions have been evaluated,
several studies suggest an increased risk for having a DS child for
those women carrying combinations of the MTHFR 677T allele (or TT
genotype) with both the MTHFR 1298C variant [19,25,27,29,30,35]
and/or other genetic polymorphisms of different genes participating
in folate and Hcy metabolism, including the reduced folate carrier
(RFC1) [28], methionine synthase (MTR) [39] and methionine
synthase reductase (MTRR) [16,17,25,34] (Table 2). Moreover,
interactions between three or more polymorphisms, including the
MTHFR 677C > T allele, have been suggested to increase DS risk
[25,35,65], and there is also indication that Hcy levels in MDS result
from complex interactions between polymorphisms of different
genes participating in folate metabolism [31]. Overall, these data
suggest that the presence of the MTHFR 677T allele alone is not
sufcient to increase the risk of having a DS child; however it could
play a role in increasing DS risk when combined with low folate
intake and/or other polymorphisms of the same metabolic pathway.
3.2. The MTHFR 1298A > C polymorphism and the risk of
having a DS child
The MTHFR 1298A > C polymorphism has been studied less
extensively than the 677C > T in DS risk (Table 1). The rst evidence
of an association between the 1298C allele and DS risk was obtained
in 2002 in a Brazilian population [19]. The same authors observed
interaction between MTHFR 1298A > C and 677C > T polymorphisms in increasing DS risk [19,27]. Similar results have been obtained
in India [29] and in Southern Italy [30]; there is also indication that
both variants could play a role in DS risk in Egypt [33].
Four [19,25,27,35] of the ve [19,25,27,35,37] studies performed to date in Brazil suggest that combinations of the MTHFR
1298C variant with other polymorphisms in genes of the folate
metabolic pathway, including the MTHFR 677C > T allele, result in
increased DS risk (Table 2).
We rst suggested that combinations of the MTHFR 1298AA
genotype with the RFC1 genotype (80AA or 80AG) might result in a
decreased risk for having a child with DS in Italian women aging
less than 35 years at conception [28], and this result was later
conrmed in a pooled analysis of Italian studies [66]. Others
observed that the MTHFR 1298C allele, as well as the RFC1 80G
allele, are associated with increased risk of having a DS child in
Italian women aging more than 34 years at conception [30]. The
same authors observed interactions between MTHFR 677C > T
and 1298 A > C polymorphisms, with the 677T-1298C haplotype
resulting in a risk factor for Down syndrome [30]. Mart`nez-Fràs
et al. observed complex interactions between the MTHFR
1298A > C polymorphism and Hcy levels in Spanish MDS [31].
We recently observed that homozygozity of the thymidylate
synthase (TYMS) 28 bp repeat polymorphism (2R/2R) combined
with the MTHFR 1298AC genotype results in reduced DS risk in a
population of Central Italy [32]. Overall, results are similar to
those obtained for the MTHFR 677C > T polymorphism, indicating
that complex interactions between different polymorphisms of
the folate metabolic pathway, rather than the presence of a single
one, might modulate DS risk. There are however four studies that
failed to nd association between DS risk and the MTHFR 1298
A > C polymorphism (either alone or combined with other
variants) [21,24,26,37].
3.3. MTHFR gene polymorphisms, chromosome damage and
aneuploidy
We recently measured chromosome damage and malsegregation events by means of the micronucleus assay coupled by
uorescence in situ hybridization (FISH) technique in peripheral

lymphocytes of MDS aging 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 [67]. There is also increasing evidence of association
between polymorphisms in folate metabolizing genes (mainly
MTHFR 677C > T) and the amount of chromosome damage and
malsegregation measured in human blood cells [6870]. Particularly, we recently observed that the presence of the MTHFR 677T
allele accounts for an increased baseline level of BNMN lymphocytes in MDS and in control mothers, and that BNMN lymphocytes
are increased in MTHFR 1298AA carriers compared with MTHFR
1298AC+CC genotypes [39,71]. The MTHFR 1298A > C polymorphism, but not the MTHFR 677C > T allele, has been recently related
to chromosomal aneuploidy leading to Turner Syndrome [72].
Hassold et al. searched for possible associations of the MTHFR
677C > T polymorphism with other trisomies other than trisomy
21, observing a signicant increase in the MTHFR 677T variant
allele only in mothers of trisomy 18 individuals [73]. Moreover,
meiotic studies on the pachytene stage of spermatogenesis have
demonstrated that infertile men have impaired chromosome
synapsis and decreased frequency of recombination [74]. There is
indication of association between low folate intake and high
frequency of sperm with aneuploidy [46] and the MTHFR 677TT
genotype was associated with male infertility [75]. Overall, these
studies point to a possible contribution for MTHFR variants to
human aneuploidy, thus strengthening the hypothesis that the
MTHFR genotype might contribute to the risk of having a DS child
[15].
4. The methionine synthase-methionine synthase reductase
(MTR-MTRR) complex and DS risk
Methionine synthase is the enzyme that catalyzes the
transmethylation of Hcy to methionine and, on the basis of
sequence similarity with E. coli cobalamin-dependent methionine
synthase (MetH), human MTR comprises four discrete functional
modules that bind from the N- to C-terminus, respectively, Hcy, 5methylTHF, cobalamin, and SAM. The C-terminal activation
domain also interacts with MTRR [76]. Human MTRR, a NADPHdependent diavin enzyme, is required for the reductive activation
of the cobalamin-dependent MTR [77].
A common polymorphism MTR 2756A > G (Asp919Gly) is
known, and there is indication from large scale population studies
that it can have an effect on Hcy levels [78]. However, results are
still conicting and the contribution of the MTR 2756A > G
polymorphism to Hcy concentrations has not been fully claried.
Some studies reported increased Hcy levels in the presence of the
wild type (MTR 2756A) allele [79,80], whereas others observed
increased Hcy levels in the presence of the mutant (MTR 2756G)
allele [81,82]. There is also indication that the heterozygous
genotype MTR 2756AG is associated with increased Hcy concentrations in DS individuals [83]. These apparent discrepancies might
be explained by recent evidence suggesting that the interaction
between different polymorphisms may totally modify their
individual effect [31,84], and that the same genotype combinations
could have different effects on maternal Hcy levels in different
individuals, depending on interactions with nutritional and
lyfestile factors [84].
A common polymorphism in the MTRR gene, MTRR 66A > G
(Ile22Met), has been largely investigated for its possible contribution on tHcy levels. Even though recent large scale population
studies do not support an independent role of this polymorphism
on tHcy levels [78,85], some of them suggest interactions between

the MTRR 66A > G polymorphism and the MTHFR genotype in
determining tHcy levels [31,85]. There is also indication that
combinations of low levels of cobalamin and the MTRR genotype
might be associated with increased Hcy levels in pregnant women
[86].
4.1. The MTR 2756A > G polymorphism and the risk of having a DS
child
In 2003 Bosco et al. [21] provided the rst evidence of a
possible contribution for the MTR 2756A > G polymorphism to DS
risk in 63 MDS from Sicily. After adjustment for age, tHcy and the
MTR 2756 AG/GG genotype were signicant risk factors for having
a DS child. Moreover, also the double heterozygosity MTR
2756AG/MTRR 66AG genotype resulted a signicant DS risk
factor. Authors concluded that both tHcy and the MTR genetic
polymorphism were two potent risk factors for mothers to have a
DS child in Sicily [21]. Subsequent studies performed in different
countries, including Brazil [25,35], France [26], Central [28] and
Southern Italy [30], have failed to nd an independent associations between the MTR 2756A > G polymorphism and DS risk
(Table 1). However, a study performed in Brazil suggests that
when MTHFR 677T, MTHFR 1298C, MTR 2756G, MTRR 66G and CBS
844ins68 alleles are evaluated together, MDS tend to have a higher
number of uncommon alleles than the mothers of healthy child
[25]. Similar results were obtained in Brazil by Biselli et al. when
evaluating together MTHFR 677T, MTHFR 1298C, MTR 2756G and
RFC1 80G variant alleles [35]. We recently observed association
between the combined MTHFR 677TT/MTR 2756AA genotype and
increased DS risk in Italy; the combined MTHFR 677TT/MTR
2756GG genotype was not observed in our casecontrol population [39]
4.2. The MTRR 66A > G polymorphism and the risk of having a DS
child
The MTRR 66A > G polymorphism has been the second
polymorphism, after the MTHFR 677C > T allele, to be associated
with an increased risk for having a DS child [16]. In 2000 Hobbs
et al. performed a study in a North American population observing
that the MTHFR 677T allele was more prevalent among MDS than
among control mothers. In addition, the homozygous MTRR 66GG
genotype was independently associated with an increase in DS
risk, and the combined presence of both polymorphisms was
associated with a greater risk of having a DS child than was the
presence of either alone [16]. Similar results have been obtained by
Wang et al. in China [34] and by OLeary et al. who observed that
the MTRR 66G variant was more common in Irish MDS than in
control mothers and that combinations of both MTRR 66G and
MTHFR 677T variants increased the risk of producing offspring with
DS [17]. Bosco et al. observed that the MTR 2756AG/MTRR 66AG
combined genotype resulted in increased DS risk in Sicily, but
failed to nd interactions between MTRR and MTHFR variants in DS
risk [21]. Da Silva et al. proposed that DS risk might result from a
complex interaction between MTHFR 677T, MTHFR 1298C, MTR
2756G, MTRR 66G and CBS 844ins68 variant alleles [25]. MartinezFràs et al. observed a role for the MTRR 66A > G polymorphism in
determining tHcy levels in MDS, particularly in interaction with
the MTHFR 1298 A > C variant [31]. More recently Pozzi et al.
observed association between the MTRR 66AG and GG genotypes
and DS risk in Italy, however no interaction with the MTHFR
677C > T polymorphism was found [40]. There are however four
published studies that failed to nd association between DS risk
and the MTRR 66 A > G polymorphism (either alone or combined
with other variants) [26,30,37,39].
61
4.3. MTR and MTRR gene polymorphisms, chromosome damage and

aneuploidy
Little is known concerning evidence of associations between
MTR or MTRR gene polymorphisms and chromosome damage and
aneuploidy in humans. Hassold et al. failed to nd association
between the MTRR 66A > G polymorphism with human trisomies
other than trisomy 21, including sex-chromosome trisomy and
autosomal trisomies [67]. We failed to nd association between
the MTRR 66A > G polymorphism and the frequency of BNMN
lymphocytes in MDS [39]. However, there are some reports of a
possible association between the MTRR 66A > G polymorphism
and chromosomal damage in peripheral lymphocytes of healthy
subjects, but results are still controversial since Zijno et al. [87]
observed an increased frequency of micronuclei (MN) in MTRR
66GG individuals, whereas others observed that the MTRR 66AA
genotype acts to increase MN frequency in smokers [88]. The MTR
2756A > G polymorphism was not associated with increased MN
frequency either in MDS [71] or in healthy subjects [71,88].
Overall, the possible contribution of MTR 2756A > G and MTRR
66A > G gene polymorphisms to human nondisjunction and DS
risk remains controversial. However, since several authors suggest
interactions of both polymorphisms with the MTHFR 677T variant
in increasing DS risk [16,17,34,39], further studies are required to
clarify this issue.
5. The reduced folate carrier (RFC1 or SLC19A1) and DS risk
The reduced folate carrier is the major transport system in
mammalian cells and tissues, where it participates in the uptake of
folate cofactors from the blood [49]. There is also indication of a
role for RFC1 in specialized tissue functions such as absorption
across the luminal epithelium in intestine, transplacental transport
of folates, folate uptake across the blood-brain-barrier and
transport across the basolateral membrane of renal tubules [89].
The analysis of the human RFC1 amino acid sequence predicts a
12 transmembrane domain (TMD) integral membrane protein
with internally oriented amino- and carboxyl termini and a large
connecting loop between TMDs 16 and TMDs 712. Although the
detailed mechanism of RFC1 transport is not rmly established,
RFC1 shows a striking structural homology to bacterial transporters. By analogy with these bacterial transporters, transport of
folates into mammalian cells would be expected to involve a
physical translocation of the carrier within the plasma membrane
driven by the extrusion of anions down a large (intra- to
extracellular) concentration gradient [89].
The rst report of a RFC1 gene polymorphism was in 2000 by
Chango et al. [90] who described a high frequency 80G > A single
nucleotide polymorphism resulting in replacement of Arg27 by His
(Arg27His) in TMD1. Authors found a moderate, but signicant,
increase in tHcy levels in doubly homozygous RFC1 80GG/MTHFR
677TT subjects as compared to RFC1 80GG/MTHFR 677CC or CT
subjects. In addition, individuals who were RFC1 80AA/MTHFR
677CT had higher plasma folate levels than those who were RFC1
80GG/MTHFR 677CT [90]. Further studies provided conicting
results, therefore the effect of the RFC1 80G > A polymorphism on
plasma folate and Hcy levels is still debated [78,9194]; however
others observed increased Hcy levels in doubly homozygous RFC1
80GG/MTHFR 677TT individuals [92].
5.1. The RFC1 80G > A polymorphism and the risk of having a DS child
Based on the initial report of a possible effect of the RFC1
80G > A polymorphism (in combination with the MTHFR 677C > T)
on plasma folate and Hcy levels [90], several studies have been
performed to explore the clinical relevance of the RFC1 80G > A
62
polymorphism to a range of human conditions, including the risk of

having a child with DS [26,28,30,35,38]. The majority of the studies
performed so far (Table 1) agree that the RFC1 80G > A
polymorphism is not an independent risk factor for having a DS
child [26,28,35,38]. Only one study performed in Southern Italy
suggests that the RFC1 80G allele might increase DS risk in mothers
aging more than 34 years at conception [30].
We rst suggested a possible interaction between RFC1 and
MTHFR loci to DS risk in Italy [28]. Particularly, we observed a
borderline signicant increased DS risk for the combined RFC1
80GG/MTHFR 677TT genotype and a signicant protective effect for
the RFC1 80(GA or AA)/MTHFR 1298AA genotype [28]. Further
studies on Italian MDS and a pooled analysis of the data obtained in
Italy conrmed our observation and provided complimentary data,
suggesting that combinations of the RFC1 80A allele with the
MTHFR 1298A allele decrease DS risk, while combinations of the
RFC1 80G allele and the MTHFR 1298C allele increase DS risk
[30,66]. We then hypothesized that DS risk could be affected by the
combined presence of MTHFR 677T, MTHFR 1298C and RFC1 80G
mutant alleles in the genome, but had no statistical power enough
to test for this hypothesis [65]. Recently, Biselli et al. provided
evidence supporting our hypothesis, observing that the presence of
three or more mutant alleles for MTHFR 677C > T, MTHFR
1298A > C, MTR 2756A > G, and RFC1 80G > A are maternal risk
factors for DS in Brazil [35]. Only the study performed by Chango
et al. [26] in France failed to observe association between the RFC1
80G > A polymorphism, alone or combined, and DS risk.
populations, with the variant allele being prevalent in African,

European and North American populations [101103].
The 844ins68 polymorphism consists of the insertion of 68 bp
within exon 8 of the CBS gene and results in a duplication of the 30
splice site at the intron 7/exon 8 junction, generating a proximal
and a distal 30 splice site in relation to the 50 splice site in exon 7.
However, the 844ins68 allele generates mostly a normal transcript
because of the distal 30 splice site is preferentially selected, such
that the 68-bp insertion is skipped in the mature mRNA derived
from the 844ins68 allele [104].
A transition mutation 833T > C is known to cosegregate in cis
with the 844ins68 polymorphism in all individuals bearing the
insertion. The 833C > T mutation results in an Ile278Thr change in
the mutant protein and is associated with mild hyperhomocysteinemia [105,106]. There is indication that the 833T > C/844ins68
double mutation might be a neutral polymorphism in the CBS gene
since the splicing of the intron 7 eliminates the mutant allele
carrying the 833T > C, suggesting that the insertion was an
evolutionary event that allowed the rescue of the wild-type
sequence, so preventing protein function [107].
Several studies report that the CBS 844ins68 polymorphism
alone has not a relevant effect on tHcy concentrations [108,109].
However, there is indication that interactions between diverse
polymorphisms, including MTHFR 677C > T, MTHFR 1298A > C,
MTR 2756A > G and the CBS 844ins68, probably inuence Hcy
levels [110112].
6.1. The CBS 844ins68 polymorphism and the risk of having a DS child
5.2. Trisomy for RFC1 in DS individuals

The RFC1 gene is located on chromosome 21 and likely
overexpressed in DS individuals. In 2003 Lubec et al. observed
an increased RFC1 expression in the foetal DS brain [95]. Trisomy
for RFC1 may be hypothesized to increase folate demand in DS
individuals and it has been therefore suggested that a dosage effect
of RFC1 and a functional effect of the 80G > A polymorphism could
contribute to an imbalance in one-carbon metabolism [95,96]. It
has been speculated on trisomy for RFC1 in developing DS foetuses,
since trisomy for RFC1 is likely to increase the availability of folate
units [31]. Mice transgenic for the human reduced folate carrier
(hRFC) have been recently produced and utilized to study the
impact of hRFC overexpression on cognitive functions, behaviour
and locomotor activity [97]. There is indication that mice
containing a hRFC construct, in addition to two mouse copies of
the reduced folate carrier gene, show cognitive and behavioural
changes similar to human DS [97].
6. Cystathionine b-synthase (CBS) and DS risk
Human cystathionine b-synthase is a hemoprotein which
catalyzes the condensation of Hcy and serine to form cystathionine
(Fig. 1). Human CBS is a multidomainal protein with three
distinguishable modules: the heme domain, the catalytic core
domain and a regulatory domain at the C terminus. The full-length
enzyme exists as a tetramer. Insufciency in CBS activity may lead
to hyperhomocysteinemia and a gross deciency in CBS activity is
associated with homocystinuria, an inborn recessive metabolic
disorder [98,99]. The CBS gene is known to have a large number of
mutations, including missense and nonsense ones, as well as some
insertion, deletion and splice site variants, some of which are
polymorphic in nature [98].
The identication of an 844ins68 insertion in the CBS gene was
rst reported in a patient affected by homocysteinuria due to CBS
deciency [100]. Subsequent studies have revealed that this
insertion is not a disease causing mutation but rather a common
polymorphism whose frequency is largely different among human
None of the three published studies aimed at investigating the

role of the CBS 844ins68 polymorphism as a possible risk factor for
having a child with DS found it to be an independent DS risk factor
[25,26,30]. Only da Silva et al. suggested that when MTHFR 677T,
MTHFR 1298C, MTR 2756G, MTRR 66G and CBS 844ins68 mutant
alleles were evaluated together MDS tended to have a higher
number of uncommon alleles than control mothers, suggesting
polygenic interactions in DS risk [25].
6.2. Trisomy for CBS in DS individuals
The gene for CBS maps on human chromosome 21, and it has
been observed that the levels of CBS are approximately three times
greater in DS brains than in control brains [113]. It has been also
suggested that CBS over-expression in the DS brain may be one of
the cause of developmental abnormality in cognition in DS
children and of dementia later in life [113]. There is also indication
that CBS over-expression in lymphocytes of DS children signicantly alter Hcy metabolism and may contribute to the metabolic
alterations of this complex genetic disorder [114]. Mice expressing
the human CBS gene have been produced to analyze the possible
role of CBS in DS [115]. Several authors have speculated on trisomy
for CBS in the possible role of enzyme polymorphisms and dietary
response to folates in both developing DS foetuses and DS
individuals [26,31]. If trisomy for RFC is likely to increase the
availability of folate units in developing DS foetuses, that for CBS
could create a functional folate deciency [41]. Therefore, folate
metabolism in DS foetuses likely results from complex interactions
between trisomy for the genes on chromosome 21 and the
maternal nutrition and genotype.
7. Other genes participating in folate/homocysteine
metabolism and DS risk
A few other polymorphic genes participating in folate/Hcy
metabolism have been recently evaluated for their possible
contribution to the risk of having a DS child. They have been
studied less extensively than MTHFR, MTR, MTRR, RFC1 and CBS and,
in most of the cases, only by a single research group. None of them
resulted to be an independent DS risk factor, but for some variants
there is indication of possible interactions with MTHFR or RFC1
polymorphisms in affecting DS risk [30,39].
7.1. Thymidylate synthase (TYMS) and DS risk
Folates are required for the de novo synthesis of nucleotides:
5,10-MTHF is reduced by MTHFR in the DNA methylation pathway,
however it is also used by thymidylate synthase for the conversion
of deoxyuridine monophosphate (dUMP) to deoxythymine monophospate (dTMP) in the de novo synthesis of pyrimidines (Fig. 1).
Therefore, polymorphisms associated with reduced MTHFR
enzyme activity shift pools of 5,10-MTHF from DNA methylation
toward DNA synthesis, whereas polymorphisms affecting TYMS
activity might shift the pools of 5,10-MTHF from DNA synthesis
toward DNA methylation [7]. 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 (6 bp/6 bp+) at position 1494 (1494del6) in
the 30 -untranslated region (30 -UTR); the rst inuences TYMS
mRNA expression, the latter is thought to inuence mRNA
expression and/or stability [116,117].
We have recently investigated both TYMS 28 bp repeat and
1494del6 polymorphisms as possible risk factors for having a DS
child and none of them resulted to be independently associated
with DS risk. However, the combined MTHFR 1298AC/TYMS 2R/2R
genotype resulted in a signicant decreased DS risk respect to the
reference MTHFR 1298AA/TYMS 2R/2R genotype [39]. The majority
of chromosome 21 nondisjunction events occurs at maternal
meiosis I during maternal embryogenesis in the grandmother body
when cells have a high demand of DNA precursors and a high rate
of divisions [118]. In this context, it is likely that interactions
between polymorphisms associated with MTHFR and TYMS
enzyme activities might impair DNA methylation with consequences on chromosome segregation and DS risk [39].
7.2. Methylenetetrahydrofolate dehydrogenase (MTHFD1) and DS
risk
Methylenetetrahydrofolate dehydrogenase is a trifunctional
enzyme that interconverts tetrahydrofolate derivatives for purine,
methionine and thymidylate synthesis. The enzyme possesses
three enzymatic properties: 5,10-MTHF dehydrogenase, 5,10methenyltetrahydrofolate cyclohydrolase and 10-formyltetrahydrofolate synthetase, respectively, and catalyzes three sequential
reactions in the interconversion of THF derivatives (Fig. 1) [50]. A
common MTHFD1 1958G > A polymorphism (Arg653Gln) reduces
the enzyme activity and stability and has been associated with
increased risk of several human diseases, including neural tube
defects, congenital heart defects and unexplained second semester
pregnancy loss [119121].
Scala et al. investigated the possible contribution of the MTHFD1
1958G > A polymorphism as a maternal risk factor for having a DS
child and observed positive interactions for the combined MTHFD1
1958AA/RFC1 80GG genotype [30].
7.3. Transcobalamin (TC) and DS risk
Vitamin B12, in the form of methylcobalamin, serves as a
coenzyme for methionine synthase during the remethylation of
Hcy to methionine (Fig. 1). In circulation, vitamin B12 is bound to
two plasma proteins: transcobalamin or haptocorrin. Transcobalamin is the transport protein required for cellular uptake of
vitamin B12. Specic membrane receptors recognize the tranco-
63
balamin-vitamin B12 complex, whereas free vitamin B12 or

haptocorrin-bound vitamin B12 is not taken up by the cell
[122,123]. A common TC 776 C > G polymorphism results in the
replacement of proline with arginine and negatively affects
vitamin B12 metabolism, thus increasing plasma Hcy levels [124].
Recently, Biselli et al. investigated the distribution of the TC 776
C > G polymorphism among 67 Brazilian MDS and 113 control
mothers, observing no association between the polymorphism and
the maternal risk of DS [38].
7.4. Other genes located on chromosome 21 involved in folate/
homocysteine metabolism
Together with RFC1 and CBS several other genes involved in
folate/Hcy metabolism are located on chromosome 21. Their
overexpression in DS foetuses could result in increased demand or
availability of one carbon metabolites, thus affecting the complex
relationship between the maternal nutrition during pregnancy and
the foetal genotype. Among them the gene encoding phosphoribosylglycineamide transformylase (GART) which is a protein
required for purine synthesis, the formiminotetrahydrofolate
cyclodeaminase (FTCD) gene, and those encoding for methyltransferases or methyltransferase-like proteins: protein arginine
N-methyltransferase 1 like-1 (HRMT1L1, PRMT2), DNA methyltranferase 3-like (DNMT3L) and the putative N6-DNA methyltransferase (NAMT1, PRED28). Further studies are required to
clarify their possible contribution to DS pathogenesis [41].
8. Environmental factors and DS risk: dietary factors and
age at conception
Table 3 lists all the genetic association studies between
maternal folate gene polymorphisms and DS risk reporting
measured or estimated Hcy, folate, vitamin B12 and related
micronutrients in MDS and control mothers. Data are often
conicting and inconclusive (Table 3). In the present section we
will discuss them in light of the complex interactions between the
maternal and the maternal grandmother dietary factors, the
maternal metabolism and the foetal requirements, in both
formation and survival of DS foetuses [31,39,41]. Then, we will
discuss the possible role of an impaired folate metabolism on the
origin of maternal chromosome 21 nondisjunction events, in light
of recent evidence and hypothesis of literature, taking into account
the two major DS risk factors: maternal age at conception and
impaired chromosome 21 recombination [4244]. Moreover, the
possible contribution of the paternal diet in the formation of
diploid 21 sperms [45] is also discussed.
8.1. Homocysteine, folate, vitamin B12 and related
micronutrients in MDS and control mothers
Several investigators reported increased Hcy levels in MDS
respect to control mothers [15,2123,25,31,32,35], while others
observed no difference between the two groups [17,18,30,39] or
even decreased Hcy levels in MDS [36] (Table 3). There is also
evidence that polymorphisms in folate metabolizing genes might
increase plasma Hcy levels [15,17,25,31,32,35], but again results
are conicting [18,21]. A few investigators observed decreased
serum folate values in MDS [23] or estimated a low dietary intake
of folate and related micronutrients, such as zinc and methionine,
in MDS [33,37], but results are still inconclusive including either
positive and negative ndings [21,39]. No difference in vitamin
B12 levels has been observed between MDS and control mothers
[21,23,30] (Table 3).
Recently, it has been suggested that there could be at least three
possible explanations for the conicting results obtained when
64
considering the maternal diet as a possible risk factor for having a

child with DS [31,39,41].
A rst possible explanation for the conicting results observed
so far (Table 3) derives from the fact that the studies have been
performed in different populations, some of them with adequate
dietary folate intake and others with low dietary folate intake
[21,33,37,39]. Moreover, several investigators have measured
plasma Hcy, folate and vitamin B12 values during or soon after
pregnancy, while others some years after conception and/or in a
restricted group of subjects [15,16,39]. Others have failed to report
plasma Hcy values since they had no available data soon after
conception [26]. Moreover, in some cases, the intake of folates and
micronutrients has not been measured, but only estimated from
food questionnaires [37].
The second explanation comes from the observation that most
of the nondisjunction events leading to DS occurs at maternal
meiosis I during the foetal development of the mother in the
maternal grandmother body [14]. Therefore, rather than the
maternal diet, it would be the maternal grandmother whose diet
might be signicant for the formation of eggs carrying two copies
of chromosome 21 [39,41]. This could also partially explain why
several authors have failed to observe a decline in the occurrence of
trisomy 21 following folic acid fortication of the mother during
peri-conception [125129]. If it is the grandmother whose diet is
important, it is likely that an effect of the folic acid fortication in
reducing DS incidence, if any, would appear only in the second
generation. Unfortunately, there is still no available data concerning maternal grandmother dietary habits and DS risk [41].
A third explanation comes from the observation that several
genes participating in folate/Hcy metabolism are located on
chromosome 21 and therefore over-expressed in developing
foetuses with DS [31,41]. This could lead to complex interactions
between the maternal diet (and genotype) and the foetal folate
demand resulting from its trisomic genotype. These interactions
could be relevant in selecting those embryos that will survive up to
the birth [31]. Indeed, Martinez Fràs et al. recently observed that
the same maternal genotype combinations could have different
effects on maternal Hcy levels of MDS and control mothers [31].
Moreover, it is estimated that 1 in 150 conceptions has trisomy 21
and that 80% of these are lost during early pregnancy [130].
Therefore, it is likely that at least for those errors occurring at
maternal meiosis I the maternal grandmother diet might be
relevant for trisomy 21 formation, whereas the maternal diet for
the selection of those trisomic foetuses that will reach up to the
birth [31,39,41]. Fig. 2 summarizes the discussed transgenerational effects.
Concluding, since Hcy levels vary as a function of several
factors, including pregnancy, folate intake and combinations of
genetic polymorphisms of metabolic enzymes, the conicting
results obtained so far in different populations are not surprising
(Table 3), but rather reect this multifactorial nature.
8.2. Maternal age at conception and DS risk: is there a link with folate
metabolism?
Advanced maternal age at conception represents the major risk
factor for trisomy 21, and after age 35 years the risk for a DS
pregnancy increases proportionally to increasing maternal age
[118]. However, the cellular and molecular mechanisms that
underlie meiotic nondisjunction in DS are still largely unknown.
Studies of trisomy 21 have shown that altered levels of
recombination are associated with maternal nondisjunction
occurring at both meiosis I and meiosis II [131,132]; therefore,
advancing maternal age and the location of genetic recombination
represent the two most important risk factors for chromosome 21
nondisjunction that have been identied so far.
To determine whether an association does exist between

maternal age and altered chromosome 21 recombination patterns,
Lamb et al. examined 400 trisomy 21 cases of maternal meiosis I
origin, grouped by maternal age [42]. That study suggested that for
an old woman chromosome 21 nondisjuntion could be the result of
the age-related accumulation of errors making her meiotic
machinery less efcient and more error prone, leading to
malsegregation of oocytes with stable chromosome exchange
patterns; on the contrary, chromosome 21 nondisjunction in a
young woman would be caused by the lack or the altered
placement of recombination (close to either the centromere or the
telomere) during meiosis [42]. A recent study based on the
examination of meiosis II errors, suggests that the presence of a
single exchange within the pericentromeric region of 21q interacts
with maternal age-related risk factors. In contrast, analysis of
maternal meiosis I errors indicates that a single telomeric
exchange imposes the same risk for nondisjunction, irrespective
of the age of the oocyte [43].
It was suggested that stable centromeric DNA chromatin may
depend on the epigenetic inheritance of specic centromeric
methylation patterns and on the binding of specic methylsensitive proteins to maintain the higher order DNA architecture
necessary for kinetochore assembly [133]. Therefore, a defect in
DNA methylation has been hypothesized as a potential causative
mechanism of meiotic nondisjunction [15]. According to this
hypothesis pericentromeric hypomethylation, resulting from
impaired folate/Hcy metabolism, would lead to anomalies in the
formation of the kinetochore resulting in impaired chromosomal
segregation [15]. Several studies have linked folate deciency with
human aneuploidy; particularly, it has been observed that folate
deciency can increase the rate of aneuploidy of both chromosomes 17 and 21 in cultured human lymphocytes [134], and that
lowered dietary folate intake increases the amount of aneuploidy
in sperms, including chromosome 21 disomy [46].
Interestingly, Waterland and Jirtle [135] observed that maternal methyl donor supplementation during gestation can alter the
offspring phenotype by methylating the epigenome. It is therefore
likely that a grandmother reduced intake of folates or impairments
in the folate metabolic pathway during maternal gestation might
alter the pattern of methylation of several chromosome regions,
including pericentromeric regions and regions involved in
chromosome recombination, thus resulting in an increased
formation of aneuploid gametes.
DNA methylation at CpG islands in the primordial germ cells
establishes monoallelic expression of imprinted genes which
exhibit monoallelic expression throughout the lifetime of an
organism, maintains retrotransposons in an inactive state and
inactivates one of the two X chromosomes. However, recent
evidence also suggests that in addition to direct transcriptional
silencing, DNA methylation is important for suppression of
recombination [136]. Crossovers (COs) are essential for meiosis
and improve the efciency of selection. COs are not randomly
distributed but are found at specic regions, or COs hotspots,
which are highly dynamic, as shown by differences in activity
between individuals, populations and closely related species. It has
been proposed a role for DNA methylation in preventing the
formation of COs, a regulation that might explain, in part, the
correlation between recombination rates and GC content in
mammals [137]. Therefore, it is not unlikely that altered DNA
methylation, resulting from impairments in folate/Hcy metabolism, might contribute to altered chromosome 21 recombination
during meiosis, resulting in increased DS risk. Moreover, if
different mechanisms are responsible for chromosome 21 nondisjunction in young women, respect to older ones [42], it is also
presumable that the effect of polymorphisms in folate metabolizing genes might be more or less pronounced in the rst group,
65
Fig. 2. Diagram showing the possible contribution of folate/homocysteine metabolism to the formation of chromosome 21 disomic gametes, leading to Down syndrome (DS). (A)
Most of the maternal meiotic errors leading to chromosome 21 nondisjunction occurs at maternal meiosis I, during maternal embryogenesis in the maternal grandmother body. In
this context the maternal grandmother diet and genotype during pregnancy might affect chromosome 21 methylation and segregation during meiotic and mitotic processes in the
developing female foetus (that, in adulthood, could originate a DS child). (B) The maternal nutrition status and genotype during peri-conception might affect some of the
chromosome 21 malsegregation events at maternal meiosis II. C) The paternal diet and genotype could result in aberrant chromosome 21 segregation and aneuploidy during
spermatogenesis, potentially resulting in DS cases of paternal origin. D) After conception, the selection of those trisomic foetuses (DS foetuses) that will survive up to birth is
believed to be the result of complex interactions between the maternal intake and metabolism of folates and the requirements of the developing DS foetus.
respect to the latter, or vice versa. Recent ndings support this

hypothesis [28,30,66]. Therefore, maternal age at conception is
likely to be another factor that could explain conicting results
obtained in DS risk association studies.
A recent paper by Hulte`n et al. suggests that maternal trisomy
21 ovarian mosaicism might provide another causative factor for a
DS pregnancy. The authors used uorescence in situ hybridization
with two chromosome 21-specic probes to determine the copy
number of chromosome 21 in ovarian cells from eight female
foetuses at gestational age 1422 weeks. All eight phenotypically
normal female foetuses were found to be mosaics, containing
ovarian cells with an extra chromosome 21. Trisomy 21 occurred
with about the same frequency in cells that had entered meiosis as
in pre-meiotic and ovarian mesenchymal stroma cells. Therefore,
the authors suggested that eggs containing two copies of
chromosome 21 could originate from trisomic 21 ovarian premeiotic cells, since trisomy for chromosome 21 could lead to
altered chromosome recombination and nondisjunction during
meiosis [45]. Hulte`n et al. also suggested that the maternal age
effect is caused by a differential selection of these cells during
foetal and postnatal development until ovulation, and that the
exceptional occurrence of high-grade ovarian mosaicism might
explain why some women have a child with DS already at young
age as well as an associated increased incidence at subsequent
conceptions [44]. A recent retrospective analysis of 151 families
with a DS baby found eight families with a carrier of gonadal
mosaicism. In all cases, the mother was younger than 35 years old
and the prevalence of parental mosaicism in young couples was
estimated to be 6.5% [138].
The ndings by Hulte`n et al. [44] are not in contrast with our
recent observations in peripheral lymphocytes of MDS aging less
than 35 years at conception, since we observed a signicant
increased frequency of both chromosome 13 and 21 malsegregation events as compared to a control group [67]. These ndings
point that nondisjunction events also occur in the somatic cells of
these women, suggesting a generalized susceptibility to abnormal
66
chromosomal segregation both in meiotic and mitotic processes

[67]. More recently, we observed association between both MTHFR
677C > T and 1298A > C polymorphisms and chromosome
damage measured in blood cells of MDS, suggesting that folate
metabolism might partially account for the increased susceptibility to abnormal chromosomal segregation observed in women
who had a DS child in young age [39,71]. Overall, there is emerging
evidence suggesting that human nondisjunction is a multifactorial
trait that must be dissected into its component parts to identify
specic associated risk factors.
8.3. The paternal contribution: diet, age at conception, and DS risk
Since most of DS cases are due to errors occurring during
maternal meiosis [118], little is known about the paternal
contribution to DS risk, and, at best of our knowledge, there are
no published genetic association studies between paternal folate
gene polymorphisms and DS risk. Although the association between
maternal age and the risks of birth defects has been well established,
it is known that spontaneous mutations in germ cells increase with
male age, but an association between paternal age and congenital
malformations, including DS, is not yet well established [139,140].
The analysis of the frequency and distribution of numerical and
structural chromosomal abnormalities in spermatozoa from normal
men points that chromosome 21 and sex chromosomes have a
greater tendency to suffer segregation errors than the rest of the
autosomes. Moreover, an association between advancing donor age
and increased frequency of numerical and structural chromosome
abnormalities has been reported in spermatozoa of normal men
[141]. Recent ndings support a weak association between paternal
age at conception and DS, but it appears to play only a small role in
the aetiology of the disease [139,140].
Little is still known about the effect of paternal nutrition and
aneuploidy in sperms. Recently, Young and colleagues observed
that men with high folate intake had lower frequencies of sperm
with disomies X, 21 and sex nullisomy compared with men with
lower intake, providing evidence for the importance of folates in
human nondisjunction in sperms [46]. Moreover, there is
indication that polymorphisms in folate-related enzymes (MTHFR,
MTR, MTRR) are associated with non obstructive male infertility
[142], and abnormal meiotic recombination in infertile men is
associated with sperm aneuploidy [143]. Overall, there is
indication suggesting that folate metabolism might be relevant
for male aneuploidy in sperms (Fig. 2).
8.4. Alcohol consumption and its relevance to folate metabolism
and DS risk
Alcohol consumption represents another important risk factor
during pregnancy for malformations in the offspring. Indeed,
frequent alcohol drinking during pregnancy may result in facial
dysmorphism, growth retardation and central nervous system
decits in infants ranging from Fetal Alcohol Effects (FAE) to Fetal
Alcohol Syndrome (FAS) [144]. However, despite public health
campaigns and clinical interventions that encourage women to
abstain from alcohol during pregnancy, some women continue to
drink while pregnant [145]. Several studies have suggested that
chronic alcohol exposure impairs folate absorption by inhibiting
expression of the reduced folate carrier and decreasing the hepatic
uptake and renal conservation of circulating folate [146]. Moreover, it reduces MTR activity and the production of SAM [147,45].
Therefore, frequent alcohol consumption during pregnancy might
alter DS risk through an impaired folate metabolism. To evaluate
the possible effects of maternal alcohol consumption on the
occurrence of a recognized DS pregnancy, data from a casecontrol
study of 997 liveborn infants or fetuses with DS ascertained in
California from 1991 to 1993 and 1,007 liveborn controls without a

birth defect, were analyzed. Interestingly, this study revealed that
high alcohol consumption (4 drinks/week) in the rst month of
pregnancy was associated with reduced risk for a recognized DS
conceptus (odds ratio (OR) = 0.54; 95% condence interval (CI):
0.34, 0.85) [148]. Another study performed on 687 infants with DS
suggested that maternal use of alcohol during pregnancy was not
associated with any specic defect in the DS baby [149]. Others
failed to observe association between maternal alcohol consumption at peri-conception with congenital heart defects in DS infants
[150], and a more recent paper failed to nd a correlation between
non-disjunction of chromosome 21 and parental alcohol consumption [151]. However, it was observed that maternal
hyperhomocysteinemia, and not global genomic hypomethylation,
is a risk factor for having a child with congenital heart diseases. On
the contrary, maternal global hypomethylation seems to be
associated with offspring having both congenital heart diseases
and DS [152]. Interestingly, paternal alcohol consumption during
pre-conception was associated with increased risk of childhood
leukaemia in children with DS [153]. Overall, there is conicting
evidence suggesting that parental alcohol consumption during
conception alters the risk for chromosome 21 nondisjunction.
However, it could result in transmissible mutations or epigenetic
modications affecting some of the DS associated defects in the
offspring [153]. Moreover, alcohol consumption during pregnancy
might alter folate metabolism in the mother, impairing the balance
between maternal folate availability and the folate demand of a
foetus bearing three copyes of chromosome 21, and affecting the
selection of which foetus will survive up to the birth. In this context
alcohol contribution might be similar and/or additive to that of
polymorphisms in folate-related genes, and should be considered
as a possible interacting factor in genetic association studies.
Further studies are required to clarify this point.
9. Discussion and conclusions
After 10 years (19992009) of active research in the eld the
question of whether or not polymorphisms in folate/Hcy
metabolizing genes are associated with increased DS risk is still
largely debated in literature, and none of the studied polymorphisms can be rmly considered as an independent DS risk factor
[1540]. Even if MTHFR 677C > T, MTHFR 1298A > C and MTRR
66A > G gene polymorphisms gave positive results in several
independent studies, results are still conicting and inconclusive
for each of them (Table 1). A recent cumulative meta-analysis of
casecontrol studies relating MTHFR 677C > T, MTHFR 1298A > C
and MTRR 66A > G gene polymorphisms to DS risk showed a trend
toward an association between the MTHFR 677C > T allele and
disease risk as the amount of data increased; however, the
recursive cumulative meta-analysis indicated that there was
insufcient evidence for claiming or denying an association for
all gene polymorphisms [154]. Several studies suggest that
interactions between different polymorphisms in folate/Hcy
metabolizing genes might modify DS risk, particularly interactions
between MTHFR 677C > T and MTRR 66A > G, MTHFR 677C > T and
MTHFR 1298A > C, MTHFR 1298A > C and RFC1 80G > A have been
repeated by independent research groups and deserve further
investigation, but again results are conicting (Table 2). There are
also some reports suggesting more complex interactions involving
three or more polymorphisms in the folate/Hcy metabolic pathway
as putative DS risk factors [25,35]. However, one of the major limits
of all the studies performed so far is the limited sample size which
largely reduces the statistical power to test for gene-gene
interactions. Unfortunately, for several reasons, it is not so easy
to obtain DNA from an adequate number of MDS, and this is
reected by all the genetic association studies performed so far,
which have often been performed in case groups of 100 MDS or less
[1540]. There are at least two possible options to overcome the
limits of the reduced sample size: the rst requires pooled analysis
and meta-analysis of published data which are often tedious
processes since only few tabular data are usually provided by the
authors in the published manuscripts, making it difcult to
perform meta-analysis. The second option would be the creation of
an international collaborative research group that could easily
recruit an adequate number of MDS to test for gene-gene
interactions with enough statistical power. Such an international
collaborative research is highly desirable since it seems almost
impossible for each single research unit to collect a number of
samples large enough to perform such a study. Moreover, the
researchers could exchange all their data on MDS and this would
be really helpful to reduce the bias due to other risk factors.
As largely discussed in section 8, several recent studies suggest
that chromosome 21 nondisjunction is a multifactorial trait that
must be dissected into its component parts in order to understand its
causes. Only an international collaborative research group could
obtain such an adequate number of MDS to separate DS cases due to
ovarian mosaicism from those caused by a meiotic failure, errors
occurred at meiosis I from those occurred at meiosis II, nondisjunction events caused by impaired recombination from those due to the
aging of the meiotic machinery. Such a separation could help to
better clarify the contribution of impaired folate metabolism to each
of the processes leading to disomy 21 in gametes.
Based on the genetic association studies performed so far [15
40], on the minor allele frequencies of the studied polymorphisms,
and on the odds ratios generated in those studies (Table 1), I have
estimated that a casecontrol study of at least 350 individuals each
would be required to detect with enough statistical power (> 80%)
an independent association between each single polymorphism
and the risk of having a DS child, resulting in an odds ratio of 1.5 or
higher. However, this number should be increased to test for genegene interactions. For example, I have estimated that to test with
enough statistical power an interaction between two different
polymorphisms, the rst having a low frequency of 1516%
(obtained in one of my papers for the MTR 2756G allele [39]), and
the latter a high frequency of 4647% (obtained in the same paper
for the MTRR 66G allele [39]), a casecontrol group of at least 650
subjects each would be required to detect odds ratios of 1.5 or
higher. Therefore, we could assume that 650 MDS and 650 control
mothers would be enough to test with power for the individual
contribution of each single polymorphism to DS risk and for the
majority of the gene-gene interactions. However, this number
should be further increased to take into consideration additional
factors. For example, it should be doubled to discriminate between
errors occurred at maternal meiosis I and meiosis II and/or to
discriminate between conceptions at early (< 35 years) or late (>
35 years) maternal age. Moreover, the required number of subjects
increases also as a function of the number of dietary factors to be
included (for example plasma folate and Hcy levels). Therefore, I
would assume that only a casecontrol study of at least 1500
individuals each could have enough power to unravel the effect of
genotype and nutrition on DS risk, and that such a number can only
be achieved trough collaborative research. Such a study could also
evaluate the tendency to generate chromosome 21 aneuploid cells
in peripheral lymphocytes in relation to folate/Hcy values and
metabolic polymorphisms.
Studies performed in cell cultures have clearly demonstrated
that folate deciency from the media is able to induce chromosome
21 aneuploidy [132]. Moreover, we have recently observed that
MTHFR gene polymorphisms correlate with chromosome damage
and aneuploidy in blood cells obtained from MDS [39]. However,
several chromosome 21 nondisjunction events are likely to be
caused by the lack or the altered placement of recombination [42].
67
There is also evidence from cancer studies that impaired folate

metabolism and polymorphisms of folate metabolic genes might
alter the methylation pattern at several CpG islands [155,156].
Within this context, it is my opinion that the design of studies
aimed at elucidating a possible contribution of folate gene
polymorphisms on the methylation status of CpG islands involved
in chromosome 21 recombination events, would be really helpful
to validate the hypothesis of a possible contribution to DS risk.
Prospective studies covering at least three different generations
are required to clarify the contribution of both the maternal diet
and the maternal grandmother diet to DS prevalence [41]. Even in
this case a separation of errors occurred at maternal meiosis I from
those occurred at maternal meiosis II would be desirable to
discriminate between the two dietary contributions.
Recent evidence supports a role for folate deciency on paternal
chromosome 21 nondisjunction [45]. This is an interesting eld of
research that is worth to be better addressed in further studies,
given that all the studies performed so far only explored the
maternal contribution of folate gene polymorphisms to DS risk.
However, such a study would require the selection of an adequate
number of DS cases of paternal origin.
Concluding, several studies performed in the last decade
suggest a possible contribution of an impaired folate metabolism
to DS risk, and the current opinion is that the combined presence of
two or more mutant alleles in the genome might affect disease risk
(Table 2). However, the studies performed so far have often
provided conicting results [1540] and the question is still
unsolved. Moreover, trisomy 21 seems to be the result of the
interplay of several factors of genetic, epigenetic, environmental
and stochastic origin. In this context all the studies performed so
far [1540] have provided some putative DS risk factors, but it is
now time for the design of studies large enough to separate trisomy
21 into all its component parts and to test for the contribution of
folate/Hcy gene polymorphisms to each of them.
Conicts of interest
There are no conict of interest associated with this manuscript.
Acknowledgements
Dr. Coppede` acknowledges all MDS and control mothers that in
the last few years gave their blood samples and their consent for
the performance of a few studies [28,39,65,67,71], making it
possible to put a little stone in the complex understanding of the
origin of Down syndrome.
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Contents lists available at ScienceDirect

Mutation Research/Reviews in Mutation Research

The complex relationship between folate/homocysteine metabolism and risk

* Tel.: +39 050993347; fax: +39 050992748.

F. Coppede` / Mutation Research 682 (2009) 5470

Other genes participating in folate/homocysteine metabolism and DS risk . . . . . . . . . . . . . . . . . . . .

methyltetrahydrofolate (5-methylTHF), release into the blood and

F. Coppede` / Mutation Research 682 (2009) 5470

tion and aneuploidy [3]. Impaired folate metabolism, resulting from

the maternal age effect is different for errors occurring at maternal

MTHFR 677C > T

MTHFR 1298A > C

James et al. [15]

OLeary et al. [17]

Sheth and Sheth [22]

Chango et al. [26]

Coppede` et al. [28]

MTHFD1 1958G > A Na

Biselli et al. [35]

Na,b OR = 1.5 (0.54.9)c

Kohli et al. [36]

Pozzi et al. [40]

TC 776C > G N OR = N.A.

Rai et al. [29]

F. Coppede` / Mutation Research 682 (2009) 5470

F. Coppede` / Mutation Research 682 (2009) 5470

Hobbs et al. [16]

MTHFR 677T + MTRR 66G " OR = 4.1 (1.98.6)

da Silva et al. [25]

Acacio et al. [27]

Rai et al. [29]

MTHFR 677T + MTHFR 1298C + MTR 2756G + MTRR 66G + CBS

Wang et al. [34]

Coppede` et al. [39]

MTR 2756AG + MTRR 66AG "

RFC1 80 GG + MTHFD 1958 AA "

MTHFR 1298CC/CA + RFC1 80 GG/GA " OR = 2.6 (1.16.3)b

Plasma homocysteine (Hcy) concentration

James et al. [15]

OLeary et al. [17]

Bosco et al. [21]

Sheth and Sheth [22]

Increased Hcy levels in MDS

da Silva et al. [25]

Scala et al. [30]

Martnez-Fras et al. [31]

Wang et al. [32]

Meguid et al. [33]

Kohli et al. [36]

Santos-Reboucas et al. [37]

Coppede` et al. [39]

Increased Hcy levels in MDS

No difference in Hcy levels between MDS

Data obtained from a limited number of MDS and control mothers.

Plasma folate and vitamin B12 levels resulted signicant

No difference in folate levels between MDS and control mothers

Decreased serum folic acid levels in MDS

Folate intake from foods was signicantly lower than the

Folate intake from foods was lower than the Recommended

F. Coppede` / Mutation Research 682 (2009) 5470

methionine by MTR. Alternatively, Hcy can be condensed with