2007 Wiley-Liss, Inc.

American Journal of Medical Genetics Part A 143A:2006 2015 (2007)

Polymorphisms in Folate and Homocysteine

Metabolizing Genes and Chromosome Damage in
Mothers of Down Syndrome Children
Fabio Coppede`,1 Renato Colognato,2 Alessia Bonelli,2 Guja Astrea,3 Stefania Bargagna,3
Gabriele Siciliano,1 and Lucia Migliore2*
2

1
Department of Neurosciences, University of Pisa, Pisa, Italy
Department of Human and Environmental Sciences, University of Pisa, Pisa, Italy
3
Scientific Institute Stella Maris, Calambrone (Pisa), Italy

Received 1 September 2006; Accepted 8 May 2007

We recently observed an association between combinations

of polymorphisms in the methylenetetrahydrofolate reductase (MTHFR 677C > T or 1298A > C) and reduced folate
carrier (RFC-1 80G > A) genes and the risk of a Down
syndrome (DS) pregnancy in young Italian women. Others
have observed an association between a methionine synthase (MTR 2756A > G) gene polymorphism and the risk of a
DS offspring in Italy. Moreover, in a separate study, we
observed an increased frequency of both binucleated
micronucleated cells (BNMN) and chromosome malsegregation events in peripheral lymphocytes of mothers of DS
individuals aged less than 35 years at conception (MDS) in
respect to controls. The aim of the present study was to
evaluate chromosome damage, measured by means of the
micronucleus assay, in peripheral lymphocytes of a group of
women (n 34) who had a DS child in young age
(<35 years) and in a control group (n 35), and to correlate
them with MTHFR 677C > T and 1298A > C, RFC-1 80G > A

and MTR 2756A > G polymorphisms. We observed an

increased frequency of BNMN in the MDS group compared
to the control group (17.13
8.31% vs. 10.28
4.53%;
P < 0.001), and, in the general population, a correlation
between years of age and BNMN frequency (P 0.05). A
significant correlation between the frequency of BNMN and
the MTHFR 677C > T polymorphism (P 0.038) was also
found. Present results indicate that MDS are more prone to
chromosome damage than control mothers; moreover the
contribution of folate and homocysteine metabolizing gene
polymorphisms seems to have an effect on the baseline
frequency of BNMN lymphocytes. 2007 Wiley-Liss, Inc.

Key words: micronuclei; MTHFR; RFC-1; MTR; folate

and homocysteine metabolism; gene polymorphisms; Down
syndrome

How to cite this article: Coppede` F, Colognato R, Bonelli A, Astrea G, Bargagna S, Siciliano G, Migliore L. 2007.
Polymorphisms in folate and homocysteine metabolizing genes and chromosome damage in mothers of Down
syndrome children. Am J Med Genet Part A 143A:20062015.

INTRODUCTION

The risk of a Down syndrome (DS) pregnancy is a

function of maternal age, and after age 35 years it
increases substantially with increasing maternal age
[Antonarakis, 1998]. However, several women aged
less than 35 years at conception have a child with DS,
suggesting a predisposition to early chromosome
malsegregation events in such women [Schupf et al.,
1994]. To better address this question we recently
performed a study aimed at evaluating cytogenetic
characteristics, measured by means of the micronucleus assay coupled with FISH technique, on
peripheral lymphocytes of mothers of DS individuals
(MDS), aged less than 35 years at conception; we
observed a significant increased frequency of both

binucleated micronucleated (BNMN) cells and chromosome 13 and 21 malsegregation events in the MDS
group compared to the control group, indicating that
MDS are more prone to chromosome damage than
controls, and that this tendency to chromosome
damage is observable even in somatic cells, such as
peripheral lymphocytes [Migliore et al., 2006].

*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.31886

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

FOLATE METABOLISM AND CHROMOSOME DAMAGE

Folate is an important nutrient which is required for

both DNA synthesis and methylation. The conversion of dietary folate into active intracellular forms
requires receptors for intestinal uptake, reduction
and methylation into the liver to form 5-methyltetrahydrofolate (5-methylTHF), release into the
blood, and cellular uptake; then it can be used for
the synthesis of DNA precursors or for the conversion
of homocysteine to methionine and its subsequent
conversion into the main DNA methylating agent Sadenosylmethionine (SAM) [Fowler, 1998]. Several
studies performed on human cell cultures, in vivo
studies in humans and studies involving animal
models have demonstrated that folate depletion from
the media, or inadequate folate dietary intake, result
in DNA hypomethylation, chromosome breakage,
increased frequency of micronuclei (MN) and
aneuploidy [Fenech, 2001]. For this reason it has
been postulated that impairments in folate
and homocysteine metabolism due to genetic polymorphisms of metabolic enzymes could predispose
an individual to chromosome damage events and act
as risk factors for a DS pregnancy [James et al., 1999].
Since 1999, several epidemiological studies have
been performed on MDS, aimed at evaluating the
role of folate and homocysteine gene polymorphisms in the risk of a DS offspring. Despite the
conflicting results often due to the use of different
populations and their limited sample size, several
associations have been observed [James et al.,
1999; Hobbs et al., 2000; Grillo et al., 2002; OLeary
et al., 2002; Bosco et al., 2003; Acacio et al., 2005;
Coppede` et al., 2006; Scala et al., 2006].
The reduced folate carrier (RFC-1) facilitates the
internalization of 5-methylTHF from the blood into
peripheral cells. Methylenetetrahydrofolate reductase (MTHFR) plays a pivotal role in regulating
cellular methylation, through the reduction of 5,10methylentetrahydrofolate (5,10-MTHF) to 5-methylTHF, the main circulatory form of folate, and
one carbon donor for the remethylation of homocysteine to methionine mediated by the activity of
methionine synthase (MTR) [Bailey and Gregory,
1999]. A single nucleotide polymorphism RFC-1
80G > A replaces an arginine with a histidine in the
protein, and the 80AA variant has been associated
with higher plasma folate levels; moreover an
increase in plasma total homocysteine levels was
detected in doubly homozygous RFC-1 80GG/
MTHFR 677TT individuals [Chango et al., 2000].
Two common polymorphisms have been described
which reduce MTHFR activity: a T variant at
nucleotide 677 (MTHFR 677C > T) and a C variant
at nucleotide 1298 (MTHFR 1298A > C), this latter to a
lesser extent. The MTHFR 677C > T polymorphism,
associated with reduced MTHFR enzyme activity,
shifts the pools of 5,10-MTHF from DNA methylation
toward DNA synthesis, thus leading to DNA hypomethylation [Frosst et al., 1995; Weisberg et al., 1998].

2007

We have recently observed that combinations of

MTHFR 677C > T and RFC-1 80G > A, and RFC-1
80G > A and MTHFR 1298A > C gene polymorphisms are associated with the risk of a DS pregnancy
in young Italian women from central Italy [Coppede`
et al., 2006]. Subsequently others observed several
associations and interactions between MTHFR
677C > T, MTHFR 1298A > C and RFC-1 80G > A
polymorphisms and the risk of DS offspring in
Southern Italy [Scala et al., 2006]. Moreover, among
homocysteine metabolizing enzymes, the MTR
2756A > G gene polymorphism, which has been
predicted to alter enzyme activity that may affect
DNA methylation processes, has been associated,
alone or in combination with other genetic and
environmental factors, with the risk of a DS offspring
in Southern Italy [Bosco et al., 2003].
There is increasing evidence of associations
between polymorphisms in folate and homocysteine
metabolizing genes and the levels of chromosome
damage in blood cells from different human populations [Andreassi et al., 2003; Botto et al., 2003;
Kimura et al., 2004; Ishikawa et al., 2006]; thus, the
aim of the present study was to evaluate chromosome damage, measured by means of the micronucleus assay, in peripheral lymphocytes of a group
of women (n 34) who had a DS child in young age
(<35 years) and in a control group (n 35), and to
correlate them with four of the major polymorphisms
in folate and homocysteine metabolic enzymes
which have been associated to the risk of a DS
pregnancy in Italian populations, i.e. MTHFR
677C > T and 1298A > C, RFC-1 80G > A and MTR
2756A > G.
MATERIALS AND METHODS
Study Population

The study was based on 34 recruited mothers

who had children affected by DS below 35 years of
age. MDS have been enrolled by the medical
personnel of the Scientific Institute Stella Maris
(Pisa, Italy), and the chromosome findings of the
children (primary trisomy) was confirmed by cytogenetic analysis carried out by the Hospitals where
diagnoses were originally performed. Discrimination
between a nondisjunction event occurred at maternal meiosis I or II was not performed. The control
group was composed of 35 mothers who had no
miscarriages or children affected by genetic disorders in their life, and at least one healthy child before
age 35 years; they have been recruited by us among
women employed in the Hospital and in the
University of Pisa.
The individuals included in the study have been
selected after the administration of a detailed
questionnaire, designed to document their previous
conditions in order to apply the adopted exclusion

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

COPPEDE` ET AL.

2008

criteria. Recent exposure to radiographs or nuclear

magnetic resonance, viral infections and inflammatory disorders experienced during the last 3 months,
or the current use of pharmacological products
known to interfere with the micronucleus frequency,
such as cytostatic drugs, anti-cancer drugs, antiinflammatory drugs or anti-epileptic drugs, were
used as exclusive criteria.
The majority of the women included in the study
was healthy and not taking drugs; however we
enrolled a few subjects (two MDS and seven
controls) using anti-hypertensive drugs, because
several investigators have observed that they do
not exert significant genotoxicity in human lymphocytes, or when metabolic enzymes are added to
in vitro genotoxicity tests [Chlopkiewicz, 2001; Telez
et al., 2001]. One MDS and a control mother were
using eye drops (collyrium) for the treatment of
glaucoma; they have been included in the study
because anti-glaucoma drugs are likely to exert their
toxic effects mainly on the cells of the ocular surface
[De Saint Jean et al., 2000]. One MDS and a control
mother were taking contraceptive pills; we decided
to include them in the study because of evidence
suggesting that long-term treatment with common
contraceptive pills does not induce micronuclei in
peripheral blood human lymphocytes [Loncar et al.,
2004].
Smoking habits have been recorded; only three
MDS were smokers and we included age-matched
controls with similar smoking habits. There was no
record of occupational exposure to chemicals
known to induce MN among subjects included in
the study. For instance, women handling anti-neoplastic drugs, such as those working in the hospital
pharmacy department and nurses exposed to anticancer compounds, have not been enrolled as
controls.
Five milliliters of blood for the subsequent cytogenetic analysis was collected by venipuncture
in heparinized tubes, and the experimental work
was carried out within 45 hr after the blood draw.
Blood samples (5 ml) were obtained in EDTA tubes
from all participants in order to extract the DNA for
genotyping. All mothers (MDS and controls) were
white Caucasians and residents of central Italy at
interview. 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.
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. Genotyping for the MTHFR
677C > T, MRHFR 1298A > C and RFC-1 80G > A

polymorphisms was performed according to Coppede` et al. [2006]. Briefly the MTHFR 677C > T
polymorphism was genotyped by amplifying a 198bp product using the Forward: 50 -TGA AGGAGAAGGTGTCTGCGGGA-30 and the Reverse: 50 AGGACGGTGCGGTGAGAGTG-30 primers; digestion with HinfI (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.
For the analysis of the MTHFR 1298A > C polymorphism a 163-bp product was amplified using
the Forward: 50 -CTTTGGGGAGCTGAAGGACTACTAC-30 and the Reverse: 50 -CACTTTGTG ACCATTCCGGTTTG-30 primers, and digested with MboII
(Fermentas) resulting in 56-, 31-, 30-, 28-, and 18bp fragments for the 1298A allele, and 84-, 31-, 30-,
and 18-bp fragments for the 1298C allele.
Genotyping for the RFC-1 80G > A polymorphisms
was obtained by amplifying a 230-bp product using
the Forward: 50 -AGTGTCACCTTCGTCCC-30 and the
Reverse: 50 -TCCCGCGTGAAGTTCTTG-30 primers;
digestion with CfoI (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.
The genotyping protocol for the MTR 2756A > G
polymorphism was adapted from Skibola et al.
[2002]: a 211-bp product was amplified using
1.25 Units of Taq DNA polymerase (Invitrogen,
Milan, Italy), 10 pmol of each primer (Forward: 50 TGTTCCCAGCTGTTAGATGAAAATC-30 and Reverse:
50 -GATCCAAAGCCTTTTACACTCCTC-30 ), 0.15 mM of
each dNTP, 1.5 mM MgCl2, and 20 ng of genomic DNA
in a final volume of 25 ml. PCR conditions were
40 cycles of 30 sec at 948C, 30 sec at 608C, 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 with HaeIII (Sigma)
resulted in an undigested fragment of 211 bp, in
the presence of the 2756A allele. The 2756G
allele produced two fragments of 131- and 80-bp,
respectively.
All the digestion products were visualized after
electrophoresis on a 3% agarose gel containing ethidium bromide. To avoid genotyping errors 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. Moreover, two
replicates of the PCR/RFLP procedures followed by
independent readings and comparison were performed for 20% of our samples randomly chosen.
Cytokinesis-Block Micronucleus Assay (CBMN)

The CBMN assay was performed according to the

procedure previously reported [Migliore et al., 1999].
Briefly, two paired, independent lymphocyte cultures were set up for each subject. Standard medium

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

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FOLATE METABOLISM AND CHROMOSOME DAMAGE

was composed by RPMI 1640 (Gibco BRL, Milan,

Italy) supplemented with 20% fetal bovine serum
(Gibco BRL), 1.5% phytohemagglutinin (Gibco BRL)
and 1% penicillinstreptomycin (Gibco BRL). Cytochalasin B (Sigma) at the final concentration of 6 mg/
ml, was added to each tube 44 hr after the starting of
the cultures in order to block the cytokinesis of
dividing cells [Fenech, 2000]. Lymphocyte cultures
were harvested after 72 hr. Cells were then treated
with an hypotonic solution (0.075 M KCl) to lyse
erythrocytes, prefixed in 3:5 methanol:acetic acid,
washed once with methanol and subsequently fixed
twice with 7:1 methanol:acetic acid fixative solution.
The cell solution was finally dropped onto a cold
glass slide. Staining procedure was performed by
immersion of the air-dried slides in a 4% Giemsa
solution. Two thousand binucleated cells for each
individual were examined, 1,000 from each independent culture replicate, following the scoring
criteria adopted by the Human MicroNucleus Project
[Bonassi et al., 2001]. We evaluated the binucleated
micronucleated lymphocytes (BML) frequency
(BNMN%) as number of binucleated lymphocytes
containing one or more micronuclei per 1,000
binucleated cells. The ratio of the percentage of
binucleated cells to the total cells scored (BN%) was
also evaluated [Fenech, 2000].
Statistical Analysis

Data were analyzed using the STATGRAPHICS

Plus software package for Windows (SWGIN,
version 5.1). Data obtained from the micronucleus
test, expressed as mean and standard deviation (SD)
for each individual were not normally distributed;
they have been normalized, and the natural logarithmic-transformed data have been elaborated
through the multifactorial variance analysis (MANOVA) by including as covariance, the age and as a
categorical variable, the condition of being an MDS.
This method led us to evaluate the statistical
significance of the difference in the BML frequency
between MDS and controls, taking into consideration
the bias which is known to influence the analysis of
CBMN test. MANOVA was also used to test associations between the BML frequency and the particular
genotype for each of the four polymorphisms under
study. The relationship between the age and the BML

frequency was tested by regression analysis. The

level of significance was P < 0.05.
RESULTS
Binucleated Micronucleated Lymphocytes
Frequency

Demographic characteristics of the study population are listed in Table I. The mean (
SD) age of MDS
was 49.09
10.15 years, comparable with that of
control mothers 48.09
7.02.
Table I also shows the results obtained from the
CBMN assay. The binucleated micronucleated lymphocytes frequency lays within a range of 6.0
40.5%, with a mean of 17.13
8.31%, in MDS group;
instead, the control group shows a frequency range
between 4.0 and 21.0% with a mean of 10.28

4.53%. The mean frequency of BML in MDS is
clearly higher if compared to the control group; this
evidence is statistically significant by the MANOVA
analysis (P < 0.001) (Table I).
MTHFR 677C > T, MTHFR 1298A > C, RFC-1
80G > A, and MTR 2756A > G Gene Frequencies

Table II summarizes the distributions of folate

and homocysteine metabolic enzyme gene polymorphisms and their allele frequencies. The
genotype and allele frequencies for the four polymorphisms analyzed were found to be in Hardy
Weinberg equilibrium in the MDS group and in
controls. The variant MTHFR 677T and 1298C allele
frequencies were 0.53 and 0.28 in MDS, and 0.385
and 0.33 in controls, respectively. The variant RFC-1
80A and MTR 2756G allele frequencies were 0.29 and
0.13 in MDS, and 0.53 and 0.15, respectively. Allele
frequencies were in agreement with previously
reported frequencies for these polymorphisms in
Italian populations [Stuppia et al., 2002; Coppede`
et al., 2006; Scala et al., 2006].
Effects of Folate and Homocysteine Metabolic
Enzyme Genotypes on the BML Frequency

MANOVA analysis for the relationship between the

BNMN frequency and the MTHFR 677C > T, MTHFR
1298A > C, RFC1 80G > A and MTR 2756A > G

TABLE I. Study Population and CBMN Assay Results

Age (years
SD)

Age < 45 years (number of mothers)
Age 4555 years (number of mothers)
Age > 55 years (number of mothers)
BML%a (mean
SD)

MDS (n 34)

Controls (n 35)

49.09
10.15
11
11
12
17.13
8.31*

48.09
7.02
14
11
10
10.28
4.53

a
Frequency of micronucleated binucleated lymphocytes calculated analyzing 2,000 cells.
*MANOVA: P < 0.001, MDS versus controls.

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TABLE II. Distributions of Four Folate and Homocysteine Metabolic Enzyme Gene Polymorphisms and
Their Allele Frequencies in the Present Study
Polymorphism

MDS no.

Allele frequency

Controls no.

MTHFR 677C > T

CC: 5
CT: 22
TT: 7
AA: 16
AC: 17
CC: 1
GG: 15
GA: 17
AA: 1
AA: 23
AG: 8
GG: 0

C: 0.47
T: 0.53

CC: 12
CT: 19
TT: 4
AA: 12
AC: 19
CC: 1
GG: 7
GA: 14
AA: 9
AA: 24
AG: 8
GG: 1

MTHFR 1298A > C

RFC-1 80G > A
MTR 2756A > G

genotypes revealed that the MTHFR 677C > T genotype was the only significant contributor to the
BNMN frequency. Figure 1 shows the mean BNMN
frequencies in relation to the MTHFR 677C > T
genotypes in MDS and controls, separately. In both
groups a nonsignificant increment in the mean
BNMN frequency with the increasing number of the
MTHFR 677T allele is observable (Fig. 1); however
when data where collected together (MDS concontrols), a statistically significant correlation
between the frequency of BNMN and the MTHFR
677C > T genotypes was observed (P 0.038)
(Fig. 2). Figure 2 shows that in the total population
(MDS controls, 69 individuals) subjects with the CC
genotype for MTHFR 677 have a significantly lower

A: 0.72
C: 0.28
G: 0.71
A: 0.29
A: 0.87
G: 0.13

Allele frequency
C: 0.615
T: 0.385
A: 0.67
C: 0.33
G: 0.47
A: 0.53
A: 0.85
G: 0.15

mean BNMN frequency than CT or TT individuals,

and that the mean BNMN frequency increases
significantly with the increasing number of MTHFR
677T alleles carried by an individual (P 0.038).
None of the other analyzed polymorphisms was
related to the frequency of BNMN (data not shown).

Regression Analysis Assessing the Relationship

of BML Frequency With Age

Regression analysis pointed out that an effect of the

age (P 0.05) on the increase of the BNMN
lymphocytes frequency is observable in the total
population (MDS controls); the effect of the age

FIG. 1. The mean frequency of binucleated micronucleated (BNMN) lymphocytes in relation to the MTHFR 677C > T genotypes in mothers of Down syndrome
individuals (MDS) and controls (Ctrl), separately. In both groups a nonsignificant increment in the mean BNMN frequency with the increasing number of the MTHFR
677T allele is observable, meaning that for each group MTHFR 677CC individuals have a lower mean damage than CT ones, and MTHFR 677CT individuals have a lower
mean damage than TT ones. However, for each of the MTHFR 677C > T genotypes MDS have an increased mean BNMN lymphocytes frequency than controls.

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

FOLATE METABOLISM AND CHROMOSOME DAMAGE

2011

FIG. 2. The mean frequency of binucleated micronucleated (BNMN) lymphocytes in relation to the MTHFR 677C > T genotypes in the total population under study
(mothers of Down syndrome individuals controls). A statistically significant increment in the mean BNMN frequency with the increasing number of the MTHFR 677T
allele is observable, meaning that MTHFR 677CC individuals have a lower mean damage than CT ones, and MTHFR 677CT individuals have a lower mean damage than
TT ones.

was higher in controls than in MDS, but the difference

was not statistically significant (data not shown).
DISCUSSION

In the present study we investigated the role of

MTHFR 677C > T, MTHFR 1298A > C, RFC-1 80G > A
and MTR 2756A > G gene polymorphisms in modulating the baseline frequency of BML in a group of 34
MDS and 35 controls. In agreement with previous
results by us [Migliore et al., 2006] we observed a
significant increased frequency of BML in the MDS
group compared to controls (P < 0.001), suggesting
the existence of a tendency to chromosome damage
and malsegregation events in MDS. Among the
studied polymorphisms we observed a significant
correlation (P 0.038) between the MTHFR 677C > T
polymorphism and the frequency of BNMN lymphocytes in the total population (MDS controls), and a
trend when the MDS and the control groups were
considered separately which was not statistically
significant likely due to the low number of individuals for each group.
Present results indicate that the MTHFR 677C > T
polymorphism affects the baseline frequency of
BNMN, and are in agreement with previous studies
performed in coronary artery disease patients
[Andreassi et al., 2003; Botto et al., 2003], and in
cultured human lymphocytes [Kimura et al., 2004].
All these studies clearly showed significant higher

levels of lymphocytes with MN in MTHFR 677TT

individuals than in CT or CC ones [Andreassi et al.,
2003; Botto et al., 2003; Kimura et al., 2004];
moreover here we observed that the mean frequency
of BNMN cells increases with the increasing of copy
numbers of the MTHFR 677T allele carried by an
individual.
All together results from the present and the above
quoted studies point out a role for the MTHFR
677C > T genotypes on the baseline frequency of
MN; however others failed to find any correlation
between the frequency of MN and the MTHFR
677C > T polymorphism [Crott et al., 2001; Zijno
et al., 2003], thus the question is still open for debate.
As indicated in Figure 1, despite a trend between
the BNMN frequency and the MTHFR 677C > T
genotypes observable either in MDS or controls for
each of the MTHFR 677C > T genotypes MDS have an
increased mean BNMN frequency compared to
controls, suggesting that other factors together with
the MTHFR 677T variant are responsible for the
increased baseline frequency of BNMN lymphocytes
observed in MDS compared to controls.
Interestingly Martinez-Frias et al. [2006] have
recently observed that the same genotype combinations of polymorphisms in folate metabolizing genes
could have different effects on maternal homocysteine levels of mothers of DS individuals and control
mothers, suggesting that the interaction between
different polymorphism may greatly modify their

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

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COPPEDE` ET AL.

individual effect, and that some of those effects are

different in mothers of live born DS children, and in
control mothers.
We suggested a role for combinations of MTHFR
677C > T and MTHFR 1298A > C with RFC1 80G > A
polymorphisms in the risk of having a child with DS
in Italy [Coppede` et al., 2006]; others recently
obtained complementary results on a larger sample
size Italian population [Scala et al., 2006], thus
strengthening the evidence of a role for these three
polymorphisms in the risk of DS in Italy. In the
present study we failed to find any trend or
correlation between the MTHFR 1298A > C or the
RFC-1 80G > A polymorphisms and the frequency of
BNMN lymphocytes. Concerning the MTHFR
1298A > C polymorphism, present results are in
agreement with previous studies indicating no
correlation between the MTHFR 1298A > C genotypes and the baseline frequency of MN [Botto et al.,
2003; Ishikawa et al., 2006]; whereas, to the best of
our knowledge there are no studies evaluating the
correlation between the BNMN frequency and the
RFC-1 80G > A polymorphism. The number of
individuals included in the present study is comparable or even greater than those of subjects included
in several previous casecontrol studies aimed at
evaluating the contribution of polymorphisms of
metabolic enzymes (GSTT1, GSTM1, GSTP1, EPXH1,
CYP2E1) or DNA repair enzymes (XRCC1, XRCC3) to
the baseline frequency of micronuclei [Godderis
et al., 2004; Teixeira et al., 2004], so that present
results can be considered indicative; however, the
present study was not designed to evaluate the
contribution of combinations of polymorphisms to
the measured endpoint. Thus we strongly suggest
that further studies on larger sample-sized populations are required to better evaluate the contribution
of MTHFR 1298A > C and RFC-1 80G > A polymorphisms, and that of combinations of them with
other gene polymorphisms including the MTHFR
677C > T one, to the baseline levels of chromosome
damage.
The two polymorphisms 677C > T and 1298A > C
in the MTHFR gene are known to be in strong linkage
disequilibrium (LD), particularly the 677T allele has
been nearly always observed in cis with the 1298A
allele. However, LD is not complete, and individuals
with the 677TT/1298AC genotype have been
observed when several hundreds of samples have
been genotyped, indicating that the frequency of the
rare MTHFR 677T/1298C haplotype among Europeans is not zero [Shi et al., 2003]. Due to the very low
frequency of the MTHFR 677T/1298C haplotype, it is
presumable that the majority (if not even all) of the
women included in the present study and bearing the
MTHFR 677T mutant allele, were also bearing in cis
the MTHFR 1298A wild-type allele.
The contribution of MTHFR haplotypes to the
BNMN frequency is a matter of great interest, but

requires larger casecontrol groups to be evaluated;

this was not the purpose of the present study.
The MTR 2756A > G polymorphism was associated
with the risk of having a child with DS in Southern
Italy [Bosco et al., 2003]; however this association has
not been recently confirmed [Scala et al., 2006]. In the
present study we failed to find correlations between
the MTR 2756A > G polymorphism and the baseline
level of chromosome damage measured by means of
the MN assay in peripheral lymphocytes. Present
results are in agreement with others [Ishikawa et al.,
2006], suggesting no effect of this polymorphism on
MN frequency. However, because of the MTR
2756AG genotype was found to interact with the
methionine synthase reductase (MTRR) 66A > G
polymorphism in the risk of DS [Bosco et al., 2003],
additional studies designed to evaluate the contribution of combinations of polymorphisms are
required prior to exclude a role for this polymorphism to the baseline levels of chromosome damage.
Micronuclei are small accessory nuclei of chromatine which can be observed in the cytoplasm of a cell;
they can originate either from chromosome breakage or chromosome malsegregation, when the
broken fragment or the entire chromosome is not
incorporated in the main nucleus of the cell. Several
studies indicate that impairments in folate
and homocysteine values reduce SAM and dTMP
synthesis, causing DNA hypomethylation and excessive uracil incorporation in DNA, which may generate point mutations and can lead to chromosome
breakage and micronucleus formation [Blount et al.,
1997; Duthie and Hawdon, 1998]. Moreover hypomethylation of centromeric DNA regions can result in
chromosome loss, and it has been observed that folic
acid or 5-methylTHF deficiency can increase the rate
of aneuploidy of chromosomes 17 and 21 in cultured
human lymphocytes [Wang et al., 2004]. Indeed our
recent analysis of MN in BNMN lymphocytes in MDS
and controls using specific probes for the centromeric
region of chromosomes 13 and 21, showed a statistically significant increased rate of aneuploidy of
chromosomes 13 and 21 in BML of MDS, respect to
controls [Migliore et al., 2006]. In the present study
we have observed a correlation between the MTHFR
677T allele and the baseline frequency of BNMN
lymphocytes, suggesting a direct link between the
amount of chromosome damage observed in blood
cells and a variant of a gene whose product is involved
in DNA methylation and homocysteine metabolism.
The question on what is the mechanism by which
alterations in folate metabolism would be related to
an increased risk for DS has been recently evaluated
by Martinez-Frias et al. [2006] in light of the recent
advances in this field. These authors concluded that
there are at least two other possible answers together
with that of alterations in the DNA methylation: the
first related to the fetal viability, and the second
related to the altered chromosomal segregation itself.

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

FOLATE METABOLISM AND CHROMOSOME DAMAGE

Concerning fetal viability the majority of trisomy 21

conceptions (about 90%) end in spontaneous failure,
and only some fetuses with trisomy 21 reach birth.
Because several of the genes related to homocysteine
and folate metabolism are located in chromosome
21, and are therefore overexpressed in DS fetuses,
it has been suggested that the maternal and fetal
genotypes, together with the maternal level of
homocysteine, play a key role in determining why
only some fetuses with trisomy 21 can survive up to
birth [Martinez-Frias et al., 2006]. Moreover, all the
nine possible combinations of the MTHFR 677 C > T
and MTHFR 1298 A > C polymorphisms have been
observed in fetuses, but those involving the formation of the MTHFR 677T1298C haplotype were
absent in neonates [Isotalo et al., 2000], suggesting
that the recombination between these two polymorphisms of the MTHFR gene is often critical for
embryo viability or fetal survival, and explaining the
strong LD existing between the MTHFR 677 and 1298
polymorphisms in live births.
Concerning the altered chromosomal segregation
itself, altered genetic recombination has been
identified to correlate with chromosome nondisjunction, and has been found for all meiosis I derived
trisomies studied to date, including trisomy 21. In
addition the trisomy 21 meiosis II derived cases were
highly associated with pericentromeric exchanges,
leading to the hypothesis that many of them were
actually the result of errors initiated in meiosis I
[Lamb et al., 1996]. It has been recently observed that
altered patterns of chromosome 21 recombination
appear to exert their greatest effects on nondisjunction at a younger age, leading to the hypothesis that
for a young woman the greatest risk factor for
nondisjunction is the lack of recombination or the
altered placement of recombination; whereas as a
woman ages, her meiotic machinery accumulates
errors becoming less efficient and more error prone
[Lamb et al., 2005]. Based on this recent evidence,
it has been suggested that a correct pattern of DNA
methylation could be critical for chromosome
stability and for a correct pattern of recombination;
whereas the hypothesis that an altered pattern of
DNA methylation could lead to a different pattern of
recombination and to the subsequent nondisjunction of chromosomes in oocytes of young women,
particularly of chromosome 21, cannot be excluded
[Martinez-Frias et al., 2006].
Therefore, the recent findings by Lamb et al. [2005]
suggests that a different mechanism is responsible for
chromosome 21 nondisjunction in young women
respect to older women, but both mechanisms can
be affected by an altered pattern of DNA methylation.
This last observation could also partially explain why
some of the polymorphisms in genes of the folate
metabolic pathway, which have been associated
with the risk of a DS offspring, seem to play a
different role in women aged less or more than

2013

35 years at conception [Coppede` et al., 2006; Scala

et al., 2006].
Present findings, together with other recent observations by us in women aged less than 35 years at the
moment of the conception of a DS child [Migliore
et al., 2006], seem to indicate that MDS have
increased frequencies of chromosome damage and
malsegregation events in peripheral lymphocytes.
These increased frequencies could be partially
explained by genetic factors affecting, as the MTHFR
677T variant does, the processes of chromosome
methylation and stability, and those of chromosome
recombination and segregation. These genetic factors, acting in combination with other factors either
in somatic cells or in oocytes, could be responsible
for the formation of trisomic somatic cells and/or
disomic gametes.
One of the limits of the present study is the lack of
data concerning the blood values of folate and
homocysteine for the majority of the subject under
study; thus we were unable to evaluate any correlation
between them and the frequency of BML, taking into
account the contribution of genetic variants.
We observed an effect of the age (P 0.05) on the
increase of the BML frequency in the total population; many previous studies have reported a positive
correlation between the increased frequency of MN
in peripheral lymphocytes and the age [Migliore
et al., 1991; Bolognesi et al., 1997], in particular in
women, where the preferential involvement of X
chromosome in nondisjunction events has been
demonstrated [Richard et al., 1994; Zijno et al.,
1996; Catalan et al., 1998].
In conclusion, results here indicate that MDS are
more prone than controls to chromosome damage
and malsegregation events, and suggest that, among
four of the major folate and homocysteine metabolizing gene polymorphisms previously associated
with the risk of a DS pregnancy in Italy [Bosco et al.,
2003; Coppede` et al., 2006; Scala et al., 2006], the
MTHFR 677T variant seems to account for an
increased baseline level of BNMN lymphocytes.
However, it is clear that the increased frequency of
BNMN cells observed in MDS respect to controls
cannot be explained only by this polymorphism,
and likely several other genetic and environmental
factors interact with each other to determine this
observed increased damage. We strongly suggest the
need for further studies of larger sample size aimed at
better elucidating the contribution of the interacting
factors determining the increased frequency of
chromosome damage in MDS.
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