2006 Wiley-Liss, Inc.

American Journal of Medical Genetics Part A 140A:1083 1091 (2006)

Folate Gene Polymorphisms and the Risk of Down

Syndrome Pregnancies in Young Italian Women
Fabio Coppede`,1 Giulia Marini,1 Stefania Bargagna,2 Liborio Stuppia,3,4 Fabrizio Minichilli,5
Ilaria Fontana,1 Renato Colognato,1 Guia Astrea,2 Giandomenico Palka,3,6 and Lucia Migliore1*
1

Department of Human and Environmental Sciences, University of Pisa, Pisa, Italy

2
Scientific Institute Stella Maris, Calambrone, Pisa, Italy
3
Department of Biomedical Sciences, G. DAnnunzio University Foundation, Chieti-Pescara, Italy
4
I.T.O.I. CNR, c/o IOR, Bologna, Italy
5
Department of Epidemiology, Institute of clinical Physiology, National Council of Research (C.N.R), Pisa, Italy
6
Human Genetic Division, Pescara Hospital, Pescara, Italy
Received 4 November 2005; Accepted 17 February 2006

Maternal impairments in folate metabolism and elevated

homocysteinemia are known risk factors for having a child
with Down syndrome (DS) at a young age. The 80G>A
polymorphism of the reduced folate carrier gene (RFC-1) has
been recently demonstrated to affect plasma folate and
homocysteine levels, alone or in combination with the
677C>T polymorphism in the methylenetetrahydrofolate
reductase (MTHFR) gene. We performed the present study
on 80 Italian mothers of DS individuals, aged less than 35 at
conception, and 111 Italian control mothers, to study the role
of the RFC-1 80G>A, MTHFR 677C>T, and MTHFR 1298A>C
genotypes to the risk of a DS offspring at a young maternal
age. When polymorphisms were considered alone, both
allele and genotype frequencies did not significantly differ
between DS mothers and control mothers. However, the

combined MTHFR677TT/RFC-1 80GG genotype was borderline associated with an increased risk (OR 6 (CI 95%: 1.0
35.9), P 0.05), and to be MTHF1298AA/RFC-1 80(GA or
AA) was inversely associated with the risk (OR 0.36 (CI 95%:
0.140.96), P 0.04). Present results seem to indicate that
none of the RFC-1 80G>A, MTHFR 677C>T, and MTHFR
1298A>C polymorphisms is an independent risk factor for a
DS offspring at a young maternal age; however, a role for the
combined MTHFR/RFC-1 genotypes in the risk of DS
pregnancies among young Italian women cannot be
excluded. 2006 Wiley-Liss, Inc.

Key words: Down syndrome; MTHFR; RFC-1; folate metabolism; folate gene polymorphisms; risk of DS

How to cite this article: Coppede` F, Marini G, Bargagna S, Stuppia L, Minichilli F, Fontana I, Colognato R,
Astrea G, Palka G, Migliore L. 2006. Folate gene polymorphisms and the risk of Down syndrome
pregnancies in young Italian women. Am J Med Genet Part A 140A:10831091.

INTRODUCTION

Down syndrome (DS) is a genetic disease resulting

from the presence and the expression of three
copies of the genes located on chromosome 21.
Since the discovery of Lejeune et al. [1959], the
phenotype of DS has been associated with trisomy
for chromosome 21. For the majority of DS cases
(92%), the extra chromosome stems from the failure
of a normal chromosome segregation during meiosis (meiotic nondisjunction) [Epstein, 1995], and the
nondisjunction is maternal in 95% of cases, occurring primarily during meiosis I in the maturing
oocyte, before conception [Antonarakis et al., 1998].
Only 5% of DS cases are due to translocations and the
remaining 3% of cases to mosaicism [Antonarakis,
1998]. The prevalence of DS births has decreased
over the past two decades from 1 in 700 to about 1 in

1000, likely due to the increased application of

prenatal diagnosis [Olsen and Cross, 1997]. The
molecular mechanisms underlying meiotic nondisjunction leading to trisomy 21 are still poorly
understood and the major risk factor for trisomy 21
is advanced maternal age at conception [Antonarakis, 1998], while paternal age seems to be insignificant in the etiology of DS [Gaulden, 1992]. After age
35 the risk of bearing a child with DS increases
substantially with increasing maternal age; however,
several mothers of DS individuals (DS mothers) are

*Correspondence to: Prof. Lucia Migliore, Department of Human and

Environmental Sciences, University of Pisa, Via S. Giuseppe 22, 56126
Pisa, Italy. E-mail: l.migliore@geog.unipi.it
DOI 10.1002/ajmg.a.31217

American Journal of Medical Genetics Part A: DOI 10.1002/ajmg.a

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COPPEDE` ET AL.

35 years or younger, suggesting a genetic susceptibility to early nondisjunction for chromosome 21

in such women [Schupf et al., 1994]. Moreover,
we recently observed that young mothers of DS
children, aged less than 35 at conception, are more
prone than control mothers to chromosome malsegregation events for both chromosomes 13 and 21
in peripheral blood cells, suggesting a genetic
susceptibility to chromosome malsegregation in
both somatic cells and gametes [Migliore et al.,
2005]. Impairments in folate and methyl metabolism
can result in DNA hypomethylation and abnormal
chromosomal segregation [Fenech, 2001; Wang
et al., 2004], and folate deficiency has been
associated with chromosomes 17 and 21 aneuploidy
in human lymphocytes [Wang et al., 2004]. Methylenetetrahydrofolate reductase (MTHFR) plays a
crucial role in regulating cellular methylation,
through the reduction of 5,10-methylentetrahydrofolate (5,10-MTHF) to 5-methyltetrahydrofolate
(5-MeTHF) the main circulatory form of folate and
one carbon donor for the remethylation of homocysteine to methionine, then converted to the
methylating agent S-adenosylmethionine (SAM)
[Bailey and Gregory, 1999]. Two common polymorphisms have been described which reduce
MTHFR activity: the T variant at nucleotide 677
(MTHFR 677C>T) and the C variant at nucleotide
1298 (MTHFR 1298A>C), this latter to a lesser extent
[Frosst et al., 1995; Weisberg et al., 1998]. Conflicting
results have been obtained in subsequent studies of
association between the MTHFR 677T and 1298C
variants, and the risk of giving birth to a child with DS
[Chadefaux-Vekemans et al., 2002; OLeary et al.,
2002; Stuppia et al., 2002; Bosco et al., 2003;
Boduroglu et al., 2004; Acacio et al., 2005; Chango
et al., 2005; da Silva et al., 2005], probably due to
both the different size of the case-control groups
considered and to geographic variations in allele
frequencies among different populations. However,
a role of an abnormal folate metabolism in the risk of
DS offsprings cannot be excluded, and interactions
between folate gene polymorphisms predisposing
to elevated homocysteine levels have emerged as
a risk factor for having a DS child at a young
age [Bosco et al., 2003]. The reduced folate carrier
(RFC-1) is responsible for the internalization of 5MeTHF within cells. A single nucleotide polymorphism 80G>A has been discovered in the RFC-1 gene
[Chango et al., 2000], this polymorphism has an
impact on folate status separately or in combination
with the MTHFR 677C>T genotype, and a significant
increase in plasma total homocysteine levels was
detected in doubly homozygous 80GG/677TT individuals compared with 80GG/677CC subjects
[Chango et al., 2000]. A link between DS and neural
tube defects (NTD) in the same family has been
observed [Barkai et al., 2003], and NTD and DS are
influenced by the same genetic determinants of

folate metabolism [Gueant et al., 2003]. The RFC-1

80GG genotype has been associated with the risk of
NTD in the Italian population [De Marco et al., 2003],
however, to the best of our knowledge, it has never
been studied as a risk factor for DS pregnancies
among young Italian women. In the present study
we examined the role of the MTHFR 677C>T,
MTHFR 1298A>C, and RFC-1 80G>A polymorphisms, separately or in combinations, in the risk of
having a DS child in young Italian women aged less
than 35 years at conception.
MATERIALS AND METHODS
Study Population

Peripheral blood samples were collected from 80

DS mothers aging less than 35 years at conception
(mean age 28.4
4.5), and from 111 control mothers
of the same mean age at conception, who had at least
one healthy child and no experience of miscarriages
or abnormal pregnancies. All mothers were white
Caucasians (Italians), residents of central Italy at
interview, and filled-in a detailed questionnaire on
their personal, occupational, and medical history
and lifestyle. Informed consent for participation to
the study was obtained from each subject, and the
study was approved by the Scientific Institute Stella
Maris Ethics Committee, according to the Helsinki
declaration. Blood samples (5 ml) were obtained in
EDTA tubes from all participants.
Genotyping

Genomic DNA was isolated from whole blood by

means of the QIAamp1 Blood Mini Kit (Qiagen,
Milan, Italy) following the manufacturers instructions. All genotype analyses were performed using
PCR-RFLP technique. The genotyping protocol for
the MTHFR 677C>T polymorphism was adapted
from Frosst et al. [1995]: a 198-bp product was
amplified using 1.25 Units of Taq DNA polymerase
(Invitrogen, Milan, Italy), 20 pmol of each primer
(Forward: 50 -TGA AGG AGA AGG TGT CTG CGG
GA-30 and Reverse: 50 -AGG ACG GTG CGG TGA
GAG TG-30 ), 0.2 mM of each dNTP, 2.5 mM MgCl2,
and 30 ng of genomic DNA in a total volume of 25 ml.
PCR conditions were 40 cycles of 30 sec at 948C,
30 sec at 628C, and 30 sec at 728C, preceded by an
initial denaturation of 2 min at 948C and followed by
a final extension of 7 min at 728C. Two hours
digestion at 378C with Hinf I (Fermentas, Milan, Italy)
resulted in 175- and 23-bp products for the 677T
allele, and in a 198-bp undigested product for the
677C allele. Digestion products were visualized after
electrophoresis on a 3% agarose gel with ethidium
bromide.
For the analysis of the MTHFR 1298A>C polymorphism we used a modification of a method from

American Journal of Medical Genetics Part A: DOI 10.1002/ajmg.a

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FOLATE METABOLISM AND THE RISK OF DOWN SYNDROME

Weisberg et al. [1998]: a 163-bp product was

amplified using 1.25 Units of Taq DNA polymerase
(Invitrogen), 10 pmol of each primer (Forward: 50 CTT TGG GGA GCT GAA GGA CTA CTA C-30 and
Reverse: 50 -CAC TTT GTG ACC ATT CCG GTT TG30 ), 0.1 mM of each dNTP, 3 mM MgCl2, and 50 ng of
genomic DNA in a total volume of 25 ml. PCR
conditions were 38 cycles of 1 min at 928C, 1 min at
608C, and 30 sec at 728C, preceded by an initial
denaturation of 2 min at 928C, and followed by a final
extension of 7 min at 728C. Three hours digestion
with Mbo II (Fermentas) resulted in 56, 31, 30, 28, and
18-bp fragments for the 1298A allele, and 84, 31, 30,
and 18-bp fragments for the 1298C allele. Digestion
products were separated through electrophoresis on
a 3% agarose gel containing ethidium bromide, and
the major visible bands were those corresponding to
the 84-bp and the 56-bp fragments.
The genotyping protocol for the RFC-1 80G>A
polymorphism was adapted from Chango et al.
[2000]: a 230-bp product was amplified using
1.25 Units of Taq DNA polymerase (Invitrogen),
10 pmol of each primer (Forward: 50 -AGTGTCACCTTCGTCCC-30 and Reverse: 50 -TCCCGCGTG
AAGTTCTTG-30 ), 0.15 mM of each dNTP, 1.5 mM
MgCl2, and 10 ng of genomic DNA in a total volume
of 25 ml. PCR conditions were 44 cycles of 30 sec at
948C, 30 sec at 528C, and 45 sec at 728C, preceded by
an initial denaturation of 2 min at 948C, and followed
by a final extension of 7 min at 728C. Three hours
digestion with Cfo I (Sigma, Milan, Italy) resulted in
three fragments of 125, 68 and 37-bp, in the presence
of the 80G allele. The 80A allele produced two
fragments of 162- and 68-bp. Digestion products
were visualized after electrophoresis on a 3% agarose
gel containing ethidium bromide.
To avoid genotyping errors, two replicates of the
PCR/RFLP procedures followed by independent
readings and comparison were performed for 20%
of our samples randomly chosen. In addition, control
samples from homozygous and heterozygous individuals, whose genotypes have been previously
confirmed, were always included in the PCR/RFLP
procedures and analyzed on each gel.
Statistical Analyses

Allele frequencies were calculated for each genotype, and the difference in allele frequencies
between DS mothers and control mothers were
determined using chi-square test. Unconditional
logistic regression was performed to obtain odds
ratios (ORs). We used ORs to quantify the association
between each polymorphism and the risk of having a
DS child at a young age, and to assess possible
interactions between polymorphisms. The statistical
significance of each association was tested by z-test.
Analyses were performed using the software STATA
8.0 SE.

RESULTS
Allele and Genotype Frequencies

The allele frequencies of MTHFR 677C>T, MTHFR

1298A>C, and RFC-1 80G>A in DS mothers and
control mothers are listed in Table I and are
consistent with previous reports in Caucasians [Rady
et al., 2001; Shi et al., 2003]. The variant allele
frequencies among DS mothers were 47.5% for the
MTHFR 677T allele, 25.4% for the MTHFR 1298C
allele, and 40% for the RFC-1 80A allele (Table I). The
variant allele frequencies among control mothers
were 40.5% for the MTHFR 677T allele, 30% for the
MTHFR 1298C allele, and 44% for the RFC-1 80A
allele (Table I). No statistically significant differences
in allele frequencies have been observed between
DS mothers and control mothers (Table I).
Genotype frequencies are listed in Table II. All
genotype frequencies among controls were consistent with HardyWeinberg equilibrium expectations. The frequencies of MTHFR 677C>T
genotypes (CC, CT, and TT) among DS mothers
were 25.3, 54.4, and 20.3%, respectively (Table II).
The corresponding frequencies among controls
were 35.1, 48.7, and 16.2% (Table II). There were
no significant differences in genotype frequencies
between the two groups. Combination of heterozygous and homozygous MTHFR 677 variant genotypes (CC or TT) did not show significant differences
between the case and the control groups (Table II).
The frequencies of MTHFR 1298A>C genotypes
(AA, AC, and CC) among case mothers were 53.6,
42.0, and 4.4%, respectively (Table II). The corresponding frequencies among controls were 46.0,
48.0, and 6.0% (Table II). There were no significant
differences in genotype frequencies between the
two groups. Combination of heterozygous and
homozygous MTHFR 1298 variant genotypes (AC
or CC) did not show significant differences between
the case and the control groups (Table II).
The frequencies of RFC-1 80G>A genotypes (GG,
GA, and AA) among case mothers were 39.1, 42.0,
and 18.9%, respectively (Table II). The corresponding frequencies among controls were 33.3, 45.2, and
21.5% (Table II). There were no significant differences in genotype frequencies between the two
TABLE I. Allele Frequencies of MTHFR 677C > T, 1298A > C, and
RFC-1 80G > A in Mothers of Down Syndrome Children (DS
Mothers) and Control Mothers
Genotype
MTHFR 677
MTHFR 1298
RFC-1 80

Allele
C
T
A
C
G
A

DS mothers Control mothers

alleles (%)
alleles (%)
83 (52.5)
75 (47.5)
103 (74.6)
35 (25.4)
83 (60)
55 (40)

132 (59.5)
90 (40.5)
140 (70.0)
60 (30.0)
104 (56)
82 (44)

w2

1.80

0.179

0.87

0.351

0.58

0.446

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COPPEDE` ET AL.

1086

TABLE II. Genotype Frequencies of MTHFR 677C > T, 1298A > C, and RFC-1 80G > A in Mothers of
Down Syndrome Children (DS Mothers) and in Controls Mothers
Genotype
MTHFR 677
CC
CT
TT
CT or TT
MTHFR 1298
AA
AC
CC
AC or CC
RFC-1 80
GG
GA
AA
GA or AA

Number (%)
of DS mothers

Number (%) of
control mothers

79 (total)
20 (25.3)
43 (54.4)
16 (20.3)
59 (74.7)
69 (total)
37 (53.6)
29 (42.0)
3 (4.4)
32 (46.4)
69 (total)
27 (39.1)
29 (42.0)
13 (18.9)
42 (60.9)

111 (total)
39 (35.1)
54 (48.7)
18 (16.2)
72 (64.9)
100 (total)
46 (46.0)
48 (48.0)
6 (6.0)
54 (54.0)
93 (total)
31 (33.3)
42 (45.2)
20 (21.5)
72 (66.7)

groups. Combination of heterozygous and homozygous RFC-1 80 variant genotypes (GA or AA) did
not show significant differences between the case
and the control groups (Table II).
MTHFR 677C>T and MTHFR 1298A>C
Genotype Interaction

We compared the genotype frequencies between

MTHFR 677CC, CT, TT and MTHFR 1298AA, AC, CC
within the case mothers and with the control
mothers. Results are shown in Table III. None of
the combined genotypes was associated with the risk
of having a child with DS (Table III).
MTHFR 677C>T and RFC-1 80G>A
Genotype Interaction

We compared the genotype frequencies between

MTHFR 677CC, CT, TT and RFC-1 80GG, GA, AA
within the case mothers and with the control

OR (95%CI)

1 (referent)
1.55 (0.793.04)
1.73 (0.734.11)
1.59 (0.843.02)

0.199
0.211
0.151

1 (referent)
0.75 (0.401.41)
0.62 (0.142.65)
0.74 (0.401.36)

0.375
0.521
0.330

1 (referent)
0.79 (0.391.59)
0.74 (0.311.78)
0.78 (0.411.49)

0.516
0.509
0.447

mothers. Results are shown in Table IV. The

677TT/80GG genotype was associated with a borderline significant increased risk of a DS offspring
compared with the 677CC/80GG genotype (OR 6
[1.0035.9] P 0.05) (Table IV). For the combined
677(CT or TT)/80GG, compared with the 677CC/
80GG, an increased OR was observed (OR 3.47
[0.9612.6] P 0.058), even if not statistically significant (Table IV).
MTHFR 1298A>C and RFC-1 80G>A
Genotype Interaction

We compared the genotype frequencies between

MTHFR 1298AA, AC, CC and RFC-1 80GG, GA, AA
within the case mothers and with the control
mothers. Results are shown in Table V. A significant
inverse association with the risk of having a child
with DS was observed for the combined 1298AA/
80(GA or AA) genotype compared with the 1298AA/
80GG genotype (OR 0.36 [0.140.96] P 0.04)
(Table V).

TABLE III. Interaction Between MTHFR 677C > T and 1298A > C Genotypes in Mothers of Down
Syndrome Children (DS Mothers) and Control Mothers
MTHFR677/1298 genotype
677CC/1298AA
677CT/1298AA
677TT/1298AA
CT or TT/AAa
677CC/1298AC
677CC/1298CC
677CC/AC or CCb
677CT/1298AC
CT or TT/AC or CCc

Number of DS
mothers (%)

Number of control
mothers (%)

OR

95% CI

68 (total)
3 (4.4)
18 (26.5)
16 (23.5)
34 (50.0)
8 (11.8)
3 (4.4)
11 (16.2)
20 (29.4)
20 (29.4)

100 (total)
11 (11.0)
18 (18.0)
17 (17.0)
35 (35.0)
16 (16.0)
6 (6.0)
22 (22.0)
31 (31.0)
32 (32.0)

1.0
3.67
3.45
3.56
1.83
1.83
1.83
2.37
2.29

Referent
0.8715.38
0.8114.67
0.9113.89
0.408.49
0.2812.06
0.427.95
0.599.54
0.579.23

0.076
0.094
0.067
0.438
0.528
0.418
0.226
0.243

Missing combinations are due to the absence of DS mothers and/or control mothers with that particular genotype.
a
Combined 677(CT or TT)/1298AA.
b
Combined 677CC/1298(AC or CC).
c
Combined 677(CT or TT)/1298(AC or CC).

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FOLATE METABOLISM AND THE RISK OF DOWN SYNDROME

TABLE IV. Interaction Between MTHFR 677C > T and RFC-1 80G > A Genotypes in Mothers of Down
Syndrome Children (DS Mothers) and Control Mothers
MTHFR677/RFC-180
Genotype

Number of DS
mothers (%)

Number of control
mothers (%)

OR

95% CI

677CC/80GG
677CT/80GG
677TT/80GG
CT or TT/GGa
677CC/80GA
677CC/80AA
CC/GA or AAb
677CT/80GA
677CT/80AA
677TT/80GA
677TT/80AA
CT or TT/GA or AAc

68 (total)
4 (5.9)
16 (23.5)
6 (8.8)
22 (32.3)
11 (16.2)
3 (4.4)
14 (20.6)
13 (19.1)
6 (8.8)
5 (7.4)
4 (5.9)
28 (41.2)

93 (total)
12 (12.9)
16 (17.2)
3 (3.2)
19 (20.4)
17 (18.3)
5 (5.4)
22 (23.7)
18 (19.4)
9 (9.7)
7 (7.5)
6 (6.5)
40 (43)

1.0
3.0
6.0
3.47
1.94
1.8
1.9
2.17
2.0
2.14
2.0
2.1

Referent
0.7911.3
1.0035.9
0.9612.6
0.497.58
0.2911.1
0.517.11
0.578.25
0.439.26
0.4310.74
0.3710.91
0.617.19

0.105
0.050
0.058
0.340
0.528
0.335
0.257
0.375
0.354
0.423
0.237

Combined 677(CT or TT)/80GG.

Combined 677CC/80(GA or AA).
Combined 677(CT or TT)/80(GA or AA).

b
c

DISCUSSION

Although maternal age is the major risk factor for

trisomy 21 and after age 35 the risk of bearing a child
with DS increases substantially with increasing
maternal age, many DS children are born to mothers
aged <35 years [James et al., 1994], suggesting the
existence of genetic or environmental factors
responsible for the formation of disomic gametes in
younger women. Our recent finding of an increased
occurrence of chromosome 13 and 21 malsegregation events in peripheral lymphocytes of young
mothers of DS children, aged less than 35 at
conception, seems to indicate that chromosomal
nondisjunction occurs also in somatic cells in such
women, suggesting a generalized susceptibility to
chromosomal malsegregation events taking place
either in meiotic and mitotic processes [Migliore et al.,
2005]. Impairments in folate metabolism resulting in

pericentromeric DNA hypomethylation, which is

associated with impaired segregation and aneuploidy [Fenech, 2002], have been largely studied as
a risk factor for human nondisjunction and folate
gene polymorphisms have been associated with an
increased risk for trisomy 21 in several such studies
[James et al., 1999; Grillo et al., 2002; Bosco et al.,
2003]. Folate deficiency has been linked to chromosomal instability and chromosome 21 aneuploidy
[Wang et al., 2004; Beetstra et al., 2005], and the
genome damaging effect of folate deficiency in
cultured lymphocytes is modulated by the MTHFR
genotype [Kimura et al., 2004]; moreover, elevated
homocysteinemia has been demonstrated to be a
potent risk factor for having a child with DS in young
Italian women [Bosco et al., 2003]. In our recent
study, we also observed an increased frequency of
binucleated cells with micronuclei (MNBN) in
lymphocytes of mothers of DS children aged less

TABLE V. Interaction Between MTHFR 1298A > C and RFC-1 80G > A Genotypes in Mothers of Down
Syndrome Children (DS Mothers) and Control Mothers
MTHFR1298/RFC1-80
Genotype

Number of DS
mothers (%)

Number of control
mothers (%)

OR

95% CI

1298AA/80GG
1298AC/80GG
1298CC/80GG
AC or CC/GGa
1298AA/80GA
1298AA/80AA
AA/GA or AAb
1298AC/80GA
1298AC/80AA
1298CC/80GA
AC or CC/GA or AAc

64 (total)
16 (25)
10 (15.6)
1 (1.6)
11 (17.2)
11 (17.2)
6 (9.4)
17 (26.6)
13 (20.3)
5 (7.8)
2 (3.2)
20 (31.3)

87 (total)
11 (12.6)
12 (13.6)
3 (3.45)
15 (17.05)
20 (23)
12 (13.8)
32 (36.8)
20 (23)
6 (6.9)
1 (1.15)
29 (33.35)

1.0
0.57
0.23
0.50
0.38
0.34
0.36
0.45
0.57
1.37
0.47

Referent
0.181.79
0.022.50
0.171.50
0.131.09
0.091.19
0.140.96
0.161.26
0.142.35
0.1117.09
0.181.23

0.337
0.227
0.219
0.073
0.093
0.041
0.128
0.440
0.804
0.126

Missing combinations are due to the absence of DS mothers and/or control mothers with that particular genotype.
a
Combined 1298(AA or CC)/80GG.
b
Combined 1298AA/80(AG or AA).
c
Combined1298(AC or CC)/80(GA or AA).

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COPPEDE` ET AL.

than 35 at conception, compared to control

mothers [Migliore et al., 2005], and folate deficiency
has been recently demonstrated to increase sensitivity to radiation-induced micronuclei in human
lymphocytes [Beetstra et al., 2005]. Results of the
present study are in agreement with previous studies
indicating that conditions affecting the folate status
and/or homocysteine levels may underlie susceptibility to nondisjunction and therefore trisomy 21
[Bosco et al., 2003; Wang et al., 2004; Beetstra et al.,
2005]. In agreement with previous studies by some of
us and others performed in Italian and other
European populations the MTHFR 677T allele alone
was not associated with an increased risk for having a
DS child [Chadefaux-Vekemans et al., 2002; Stuppia
et al., 2002; Chango et al., 2005]; moreover, when the
combined MTHFR 677/1298 genotypes were considered, we failed to find any significant association
with the risk of a DS offspring, and again the present
results are consistent with previous findings in
European populations [Bosco et al., 2003; Chango
et al., 2005]. The two polymorphisms 677C>T and
1298A>C in the MTHFR gene are known to be in
linkage disequilibrium (LD), with a measured D0
value of 0.945 in Europe [Shi et al., 2003]; particularly
the 677T allele has been nearly always observed in cis
with the 1298A allele, and the 677C allele has been
more often observed in cis with the 1298T allele [Shi
et al., 2003]. However, LD is not complete, and the
presence of some individuals with the 677TT/
1298AC genotypes in large sample-size studies
[Parle-McDermott et al., 2003; Shi et al., 2003],
indicates that the frequency of the rare MTHFR
677T/1298C haplotype among Europeans is not zero,
and that the two alleles have been separated during
evolution. In the present study, LD is indicated by the
deviation of the observed frequencies of MTHFR
677/1298 combinations (Table III) from those
expected assuming independence between the two
polymorphisms, derived by multiplying genotype
frequencies (data not shown); moreover, we did not
encounter individuals with the combined MTHFR
677TT/1298AC or MTHFR 677TT/1298CC genotype,
and this is probably due to the small sample size of
the study population that has not allowed to observe
the rare 677T/1298C haplotype.
Several studies have been published that aim to
evaluate the role of the MTHFR gene polymorphisms
in the risk of DS, and results are often conflicting
[James et al., 1999; Chadefaux-Vekemans et al., 2002;
OLeary et al., 2002; Stuppia et al., 2002; Boduroglu
et al., 2004; Chango et al., 2005; da Silva et al., 2005].
One limitation of all those studies, and of the present,
is the small size of young DS mothers and control
mothers analyzed, that reduces the power of the
statistical analyses. This is mainly due to the low
frequency of young mothers of DS individuals, and,
sometimes, to socio-psychological or other difficulties in their recruitment for such a kind of study. In

fact, it is not a coincidence that all the studies

performed to date to test for association between
folate gene polymorphisms and the risk of a DS
offspring, have been conducted in a number of DS
mothers ranging from 31 to 157 [Hobbs et al., 2000;
Takamura et al., 2004], and that even in the largest
studies several DS mothers were aged more than
35 years at conception [Boduroglu et al., 2004;
Chango et al., 2005; da Silva et al., 2005]. Using data
and results of the present study, obtained in a total of
190 subjects, we have estimated that an adequately
powered case-control study to determine only the
role of the MTHFR 677C>T polymorphism in the risk
of a DS offspring should include more than 850
subjects, and the number of individuals should be
more than doubled to include other polymorphisms
and interactions; such a number could probably be
obtained by pooling together DNA samples from
different laboratories. To give one example, one of
us recently participated in two of the largest studies
performed to date in order to clarify the contribution
of polymorphisms in five folate-metabolizing genes
to the risk of nonHodgkin lymphoma (NHL): the first
study was conducted on 1,068 individuals from
Northern California [Skibola et al., 2004], the latter on
1,344 English subjects [Lightfoot et al., 2005]; however, despite the large number of subjects included,
when the two studies were compared, results were
still controversial for several of the studied polymorphisms [Lightfoot et al., 2005]; we concluded
that, to better clarify the potential role of folate
metabolism in NHL risk, there is the need for larger
and more comprehensive international studies
[Lightfoot et al., 2005]. We think that similar studies
would be helpful also to clarify the role of folate gene
polymorphisms as potential risk factors to have a
child with DS. However, results obtained in small
sample-size populations are useful, they can both
underline particular associations in a defined population or be indicative of a more general potential
association so that, when evaluating together results
produced to date, some general information can be
drawn. For example, the MTHFR gene polymorphisms, mainly the 677C > T, have been associated with
an increased risk of having a DS child in the majority
of the studies performed both in Northern [James
et al., 1999; Hobbs et al., 2000] and Southern America
[Grillo et al., 2002; Acacio et al., 2005; da Silva et al.,
2005], while all the studies performed in Mediterranean populations [Chadefaux-Vekemans et al., 2002;
Stuppia et al., 2002; Bosco et al., 2003; Boduroglu
et al., 2004; Chango et al., 2005] and the one in a
Japanese population [Takamura et al., 2004], failed to
find such an association. An association with the risk
was observed for the combined presence of the
MTHFR 677T polymorphism and the methionine
synthase reductase variant (MTRR 66G) in Northern
American and Irish populations [Hobbs et al., 2000;
OLeary et al., 2002]. The contrast of these results

American Journal of Medical Genetics Part A: DOI 10.1002/ajmg.a

FOLATE METABOLISM AND THE RISK OF DOWN SYNDROME

could be due to variations in allele frequencies

among different populations or to different sizes
of the case-control studies; however, it could
be attributable to the contribution of geographic
environmental factors that further requires clarification, for example, increased homocysteine levels
have been observed in DS mothers compared to
control mothers in several populations, including
Americans and Europeans [Bosco et al., 2003; da Silva
et al., 2005]. Several previous studies aimed to clarify
the role of the combined MTHFR 677/1298 genotypes on plasma homocysteine levels reported a
major role for the 677T allele in affecting homocysteine concentrations, whereas the 1298A>C
polymorphism seemed not to influence plasma
homocysteine levels [Dekou et al., 2001; Strandhagen et al., 2004]; however, a correlation between the
MTHFR 677TT genotype and homocysteine levels
has not been observed in the Italian study conducted
by Bosco et al. [2003], and similar results have been
obtained in a French population study [ChadefauxVekemans et al., 2002], suggesting that the effect of
the Mediterranean diet could counteract the individual effect of the MTHFR 677C>T polymorphism.
Similar results have been obtained in a Japanese
population study, and the Japanese is another folaterich population [Takamura et al., 2004]. Even if
the contribution of the MTHFR polymorphisms alone
seems to be not relevant to the risk of DS in European
populations [Chadefaux-Vekemans et al., 2002;
OLeary et al., 2002; Bosco et al., 2003; Boduroglu
et al., 2004; Chango et al., 2005], other folate gene
polymorphisms and interactions between polymorphisms in the folate metabolic pathway have
emerged as risk factors for a DS pregnancy in
different European populations [OLeary et al.,
2002; Bosco et al., 2003], so that the role of the folate
metabolic pathway cannot be excluded in the risk of
DS pregnancy. Further studies aimed to explore the
contribution of known polymorphisms in other
genes of the same metabolic pathway are required.
We included the RFC-1 80G>A polymorphism in
the present study because there is evidence that
some mothers of infants with DS have abnormal
folate and methyl metabolism similar to mothers of
infants with NTD [Botto and Yang, 2000], and
sometimes DS and NTD occur in the same family,
suggesting that, at least in a proportion of cases, DS
and NTD could have a common etiological pathway
[Barkai et al., 2003]. The RFC-1 GG genotype was
associated with the risk of NTD [De Marco et al.,
2003]; moreover, increased total homocysteine levels
have been observed in subjects with the MTHFR
677TT/RFC-1 80GG genotype when compared with
those with the 677CC/80GG genotype, and higher
plasma folate levels in individuals who were RFC-1
80AA compared with those who were GG [Chango
et al., 2005]. To the best of our knowledge, the RFC-1
80G>A polymorphism has never been studied alone

1089

or in combination with MTHFR polymorphisms, as a

risk factor for a DS pregnancy in young Italian
women. Present results seem to indicate that the RFC1 80G>A polymorphism is not an independent risk
factor for DS, and are consistent with those recently
obtained in a French population study of similar size,
the only one to date, evaluating the contribution of
the RFC-1 80G>A polymorphism to the risk of DS
[Chango et al., 2005]. In the present study we
observed a borderline significant fivefold increased
risk (P 0.05) for having a DS child in mothers
bearing the 677TT/80GG genotype compared with
mothers bearing the 677CC/80GG genotype.
Although not statistically significant, there appeared
to be a trend, in the present study, towards higher risk
for having a DS child in individuals who were TT
for the MTHFR 677 polymorphism as compared to
those who were CT or CC in individuals, who were
GG for the RFC-1 80 polymorphism (OR 6, 3, 1,
respectively). Interestingly, even if not statistically
significant, Chango et al. [2005] observed an
increased frequency of certain MTHFR677C>T/
RFC-1 80G>A combined genotypes in French
mothers of DS individuals compared to control
mothers. Both the present and the French study
seem to indicate that a role of the MTHFR677C>T/
RFC-1 80G>A combined genotypes in the risk of DS
cannot be excluded and additional studies are
required. Moreover, we observed a significant
inverse association with the risk of a DS pregnancy
for the combined MTHFR1298AA/RFC-180(GA or
AA) genotypes compared with the 1298AA/80GG
genotype (OR 0.36, P 0.04). The result is interesting, mainly if considered together with similar
previous findings of an effect of the combined
MTHFR1298/RFC-180 genotypes in the risk of NTD
in the Italian population [De Marco et al., 2003]. The
combined effect of these two polymorphisms on the
folate status requires further clarification. However,
because of the incomplete linkage between MTHFR
677 and 1298 polymorphisms, there is the need of
a larger and adequately powered study to evaluate
the contribution of combinations of the three
MTHFR677/MTHFR1298/RFC-180 polymorphisms
to the risk of a DS pregnancy. Data obtained both
at interview and from medical records indicate that
subjects included in the present study were not under
particular dietary restrictions during pregnancy.
Moreover, the Mediterranean diet in regions of
central Italy is usually rich in fruit and green
vegetables providing an adequate amount of folate
and vitamins, so that folic acid fortification is not
common. Despite these considerations, one limitation of the present study is that blood homocysteine
and folate values were not available from the
majority of DS mothers and control mothers at the
birth of their children, in order to study whether or
not any effect of the MTHFR/RFC-1 combined
genotypes on their levels, similar to those already

American Journal of Medical Genetics Part A: DOI 10.1002/ajmg.a

COPPEDE` ET AL.

1090

reported in literature [Chango et al., 2000], was

observable in our study population.
In conclusion, the present study is the largest
performed in an Italian population to evaluate the
role of folate gene polymorphisms to the risk of DS,
and one of the largest performed to date in Europe
taking into account only mothers aged less than 35 at
the time of DS pregnancy outcome. Present results,
in agreement with previous findings in Mediterranean populations, seem to indicate that none of the
studied MTHFR 677C>T, MTHFR 1298A>C, and
RFC-1 80G>A polymorphisms is an independent
risk factor for a DS offspring at a young maternal
age; however, they are also indicative for a possible
role of combined MTHFR/RFC-1 genotypes in the
risk of DS pregnancies in young Italian women. It is
important to stress that results here provided have
been obtained in a relatively small sample-size
population, so that they must not be considered
conclusive, but only indicative of possible associations that require confirmation in largest and
adequately powered designed studies.
ACKNOWLEDGMENTS

Authors thank Francesca Migheli and Cosima

Natola for their help in processing DNA samples. A
special acknowledgement is due to both DS mothers
and control mothers for donating their blood for the
present study.
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