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ROLE OF CELL FREE FETAL DNA IN MATERNAL BLOOD - A PROSPECTIVE ROLE OF CΒS AND MTHFR GENE AS ANTENATAL GENETIC MARKER IN PREECLAMPSIA PATIENTS

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International Journal of Genetics
International Journal of Genetics
ISSN: 0975–2862 & E-ISSN: 0975–9158, Vol. 3, Issue 2, 2011, PP-62-65
Online available at: http://www.bioinfo.in/contents.php?id=25
ROLE OF CELL FREE FETAL DNA IN MATERNAL BLOOD - A PROSPECTIVE
ROLE OF CΒS AND MTHFR GENE AS ANTENATAL GENETIC MARKER IN PRE-
ECLAMPSIA PATIENTS
GARIMA GUPTA, JYOTI GUPTA, SULEKHA PANDEY, PANDEY L.K., SAXENA A.K.*
Human Cytogenetic and Molecular Genetic laboratory, Center of Experimental Medicine and Surgery, Department of
Obstetrics and Gynecology, Institute of Medical sciences, Banaras Hindu University, Varanasi-221005, India
*Corresponding Author: Email- draksaxena1@rediffmail.com
Received: October 04, 2011; Accepted: November 30, 2011
Abstract- The pathophysiology of cell - free fetal DNA in maternal blood of pre eclampsia patients remain inexplicable
because of two main reasons- first presence of very minute quantity of cell free fetal DNA in plasma required
sophisticated equipments & techniques for its identification and second is the variation in gestational age of the patients.
Because of lacking molecular biology and associated “risk factors” involvement of CβS and MTHFR gene mutation is remain
unidentified in such patients. Hence, the curiosity has been developing to understand the role of these genes in such cases
to explore etiology of increasing risk in patients. In the present study, n=151 case were selected and 80% cases showed
Sry positive (Y-chromosome specific sequence) used as marker for confirmation of cell free fetal DNA in maternal blood.
Genetic study reveals, >20% case showing mutation of CβS gene and MTHFR C677T gene polymorphism showing lack of
transition between C/T alleles. Statistical analysis showing significant difference due to hence, these findings could be useful
to assess as prenatal diagnosis marker in genetics lead to altered single gene folic acid metabolism (homocysteine) and
may increased as an independent “risk factor” in pre eclampsia patients.
Key words- Cell free fetal DNA, Cystathionine β Synthase, Methylene Tetrahydrofolate reductase and Pre eclampsia.
Short Title- Cell free fetal DNA in maternal blood as genetic marker in pre-eclampsia patients
Introduction
Globally, a hypertensive disorders such as pre-
eclampsia of pregnancy responsible for 50,000 maternal
and 9, 00,000 prenatal deaths annually [1]. In human
pregnancy, it seems to be a unique disease and require
extensive investigations whose underlying etiology at
molecular level remains obscure. Approximately 3% of
pregnant women in the western world suffer from pre-
eclampsia condition, a potentially life-threatening multi-
system disorder [2]. Pre-eclampsia & eclampsia remains
one of the leading causes of prenatal mortality and
morbidity selected as model for such clinical study.
Clinical feature includes hypertension, loss of protein in
the urine, headache and visual disturbances. The burden
of pre-eclampsia on reproductive health care resources
and substantial progress in the prevention and treatment
will require advance knowledge to our understanding of
the pathophysiology of the disease at the molecular
level. Because pre-eclampsia resolves postpartum,
premature delivery of the baby may be essential to
safeguard the mother’s life. The relationship between the
entry of fetal nucleated cells into maternal circulation and
cell- free fetal DNA remains to be elucidated. Several
studies have been shown that both intact fetal cells and
cell-free fetal DNA cross the placenta circulate in blood
stream of mother [3]. Placental membrane is separating
the fetal and maternal circulations during pregnancy.
Numerous evidence has accumulated that placenta
forms an impermeable barrier between mother and child.
The bidirectional cellular trafficking of genomic materials
between the fetus and the mother during pregnancy
becomes an essential prenatal genetic marker for clinical
research as non-invasive assessment methods [4,5].The
tremendous knowledge of human genetics has been
accumulated dramatically for the last two decades on
pre eclampsia has been accumulated regarding analysis
of circulating fetal DNA in maternal blood plasma or
serum as a metabolic markers was not realized until
1996, when DNA with tumor-specific characteristics was
demonstrated in cancer patients[6].
Methylenetetrahydrofolate reductase (MTHFR), an
important regulatory enzyme of folate metabolism
associated plasma homocysteine concentration
increased risk factor for atherosclerosis and other clinical
lesions. MTHFR regulate folic acid metabolism and to
maintain the genomic instability during DNA methylation
[7]. Epidemiological studies of hyperhomocysteinemia
have been reported as a risk factor during pregnancy
complication in pre eclampsia patients [8-11].
Environmental factors including intake of folate as dietary
supplements are essentials molecules for growing fetus
and polymorphic variation in MTHFR alleles may
Garima Gupta, Jyoti Gupta, Sulekha Pandey, Pandey IK, Saxena AK
63
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increase “risk factor” for severe disorders such as
neonatal congenital malformations of neural tube
defects, trisomy-21, Alzheimer disease, cardiovascular
disorders and cancer [12, 13]. However, apart from its
biological complications and risk associated gene
mutation or polymorphic variation of allele’s frequency
may raise the possibility of circulating DNA in maternal
blood to prove non - invasive tool as a genetic marker for
prenatal diagnosis.
Materials and Methods
In the present study we selected n=151 case attending
antenatal clinic, Department of Obstetrics &
Gynecology, S.S Hospital, I.M.S., B.H.U.,Varanasi in age
group of 20-30 yrs with live intrauterine pregnancy and
clinically diagnosed pre-eclampsia & eclampsia
(according to Williams Obstetrics 22nd edition) with their
respective controls (n=50), Maternal plasma was
collected from pregnant patients for cell free fetal DNA.
Maternal blood (10 ml) was collected into EDTA sterile
vials and centrifuged within 4 hrs at 1500 rpm for 15min.
Isolated plasma was transferred into another vial of
polypropylene and immediately stored at –80°C till
further study. The study was dually approved by ethical
committee of the Institute of Medical Sciences and
samples were collected after written consent either from
the patients or their attendant.
Analyses of Cell free fetal DNA (cffDNA) from
maternal blood
The identification and characterization of cell free DNA
from maternal blood is a very difficult task. The genomic
DNA was extracted from maternal blood (plasma) using
a standard protocol Miller et al. 1994 of chloroform -
phenol method [14]. Starting volume and experimental
conditions fluctuate during standardization of our
protocol was as follows - initially 400 µl of plasma
sample were collected from maternal blood (10 ml)
samples and out of which 0.5µg-1µg (20 µl) DNA was
finally extracted for genomic study, Sry gene (a single
copy Y- chromosome specific sequence) primers (F=5’-
GGTAAGTGGCCTAGCTGGTG-3’ and R=5’-
CACAGAGAGAAATACCCGAA-3’) was used as a
specific marker to determined the fetal DNA in maternal
blood. For CβS gene forward=5’-
GTTGTTAACGGCGGTATTGC-3 and reveres 5’-
GTTGTCTGCTCCGTCTGGTT-3’ primers were selected.
For MTHFR C677T allele analysis selected primers are
F=5’-TGA AGG AGA AGG TGT CTG CGG GA-3’ and
R=5’-TGA GAG TGG GGT GCA GGG AGC TT-3’. We
have developed PCR specific strategies using forward
and reverse primers in total volume of 25 µl contain 50-
100 ng of plasma DNA , 20 pmole of each primer,
200µM of each dNTPs mix with Taq buffer (10mM Tris
HCl pH 8.3, 50mM KCl), 3.0mM MgCl2 and 3 unit of Taq
polymerase (New England Biolab). PCR conditions were
4 min at 94ºC for initial denaturation, 58 ºC/1min for
annealing followed by 35 cycles and 72 ºC/7min for final
extension. RFLP analysis was carried out for the
polymorphism analysis of MTHFR C677T allele. PCR
product (5 µl) were digested at 37ºC for 3hr. in reaction
volume of 25 µl containing 1U of Hinf-I restriction
enzyme (New England, Biolabs) and NEB buffer (2.5 µl).
The digested product of RFLP was separated on 3%
agarose gel stained with Et.Br and visualized on Gel Doc
system (SR Biosystem).
Statistical Analysis was carried out between cases and
controls using chi square fisher exact probability test to
find out the level of significance differences (p<0.05).
Results
We have selected (n=151) pre-eclampsia cases for
isolation of cell free fetal DNA and 62.75 % cases were
evaluate successfully for the identification and
characterization of cell free fetal DNA using Sry specific
primer of Y- chromosome sequences corresponding to
male DNA serve as internal control . Those patient’s
(35.29%) having lack of clinical history of pre-eclampsia
or eclampsia symptom were excluded from the present
study. Figure 1A & B shows 80% cases were Sry
positive while 20 % were Sry negative confirming
females serve as negative controls. In the present study,
we have also evaluate the mutation of 171 bp DNA
fragment of CβS gene as one of the important
candidate gene for pre-eclampsia with variability in
expression in different cases of pre eclampsia, up
regulation (increased intensity of band) as observed in
lane -2, while down regulation i.e. decreased intensity of
band was observed in lane 3-6 as shown in fig-2.The
most interesting finding were 3.6 % patients showing
complete disappearance of 171bp amplified product of
CβS gene as shown in lane-1(arrow head). These
findings were repeated thrice to confirm the
disappearances of 171bp DNA fragment of CβS gene.
Simultaneously, the polymorphic variation of MTHFR
C677T gene was also evaluated to identify the genotype
frequency in pre eclampsia cases. The highest frequency
of CT allele was 37.5% in pre eclampsia cases and
24.0% frequency was observed in controls as shown in
fig-3. The individual frequency of T allele (0.187%) was
calculated using Hardy Weinberg Equilibrium (HWE).
Besides this we have also noticed first time that
complete disappearance of 198 bp fragment of MTHFR
gene in one case (0.18 %) confirming the role of folate
metabolism associated risk factor in pre eclampsia
patients. Statistical analysis were carried out between
cases and controls using fisher exact probability test
shows significant (p<0.05) differences.
Discussion
The anatomy of cffDNA in reproductive medicine is quite
relevant. The presence of fetal DNA in maternal plasma
was first discovered in 1997 through the detection of Y-
chromosome specific sequences in the plasma of
women who were carrying male fetuses [15]. Maternally
inherited fetal DNA and native maternal DNA is being
used as a diagnostic obstruction. A comparable
equivalent to Y-specific DNA that could serve as a facile
internal control to verify presence of fetal DNA in male
while lacking consider as negative control (female fetus)
as present study significantly (80 %) documented Sry
Role of cell free fetal DNA in maternal blood - a prospective role of cβs and mthfr gene
64
International Journal of Genetics
ISSN: 0975–2862 & E-ISSN: 0975–9158, Vol. 3, Issue 2, 2011
positive as shown fig-1A. The presence of fetal DNA in
maternal plasma was through the detection of Y-
chromosome specific sequences (primers) in the plasma
of women who were carrying male fetuses [15]. The
numbers of copies of Sry (a single-copy of Y
chromosome-specific sequence) and the ratio of fetal to
maternal DNA was increased (775–970 fold) in the
plasma than amount of DNA derived from intact fetal
cells. Several reports have confirmed that gestational
age correlates positively with amount of fetal DNA in
plasma and concentrations to be low in the first trimester
rising in the second and third trimester [16].
Inability of PCR to distinguish readily between maternally
inherited fetal DNA and native maternal DNA is clearly a
diagnostic obstacle. Identification of circulating cell free
fetal DNA can lead to selection or creation of more
effective methods of enrichment during DNA isolation
from plasma for improved clinical application. Therefore,
understanding the molecular structure and dynamics of
fetal DNA in plasma of normal individuals will be
necessary to achieve further advancement in the field of
reproductive medicine. Fetal cells appear early in the
maternal circulation during the first trimester and
continue to be present throughout gestation till birth [3]
The origin of the cffDNA has remained uncertain and
evidence suggest that the placenta is also the most liable
source. Three sources of circulating DNA can be
plausibly hypothesized- (i) dying cells (necrotic or
apoptotic); (ii) active secretion of DNA; (iii) terminal
differentiation. Present study dealt with the variation in
CBS gene expression might be due to different
gestational age of the pre eclampsia cases correlates
positively with amount of fetal DNA exist in plasma [17].
In recent studies, represent that 3-6 % of total DNA in
maternal plasma release during 2nd -3rd trimester.
However, the isolation of cell free fetal DNA from the
maternal blood circulation is stills a difficult task which
requires extensive exercise / labor [3].
Another gizmo has been added to further confirmation of
cffDNA to evaluate the polymorphism of MTHFR alleles
in pre eclampsia patients because, a common C to T
transition at nucleotide 677 (C677T) associated as a risk
factor for various clinical lesions including pre-eclampsia
[18]. Interestingly, the transition between 677C -> T of
MTHFR gene C677T polymorphisms was unable to
observed in the present study due to either small
sample size or unknown biological factors such variation
of gestational age/or ethnicity. Embryonic development
of homozygous pregnant female lacking the enzyme
appears to be normal but there is an increased mortality
during postnatal development in third trimester.
Cystathionine ß-synthase gene mutation not only
induces complications during pregnancy but extend
their effects to increase “risk factor” for congenital neural
tube defects [19]. However, the present study confirm
the disappearance of 171bp fragment of CBS gene in
two patients might have responsible for increasing as
an independent “risk factor” in pre eclampsia patients.
Epidemiological studies have shown that consumption of
vegetables / fruit as dietary supplements (rich in folate)
might have reduced risk of pre- eclampsia due to
protective effect [20]. However, the high degree of
genetic heterogeneity is quite striking in the present
study, each serve as complexity of mutation has been
present in only one or two family of pre eclampsia. These
findings in conjunction with observed clinical variability
suggested genetic heterogeneity in such cases.
Earlier, it was observed that variation the frequency of
alleles of MTHFR gene between homozygous (TT),
mutant subjects had significantly higher concentration of
plasma total homocysteine, whereas in CC alleles and in
heterozygous (CT) subjects were indistinguishable [19].
Such diversity in the frequency of alleles between
homozygous (CC) / heterozygous (CT) of MTHFR may
be either due to ethnic variation in populations or
unknown biological reason/ factors in neural tube
defects, early pregnancy loss and venous thrombosis
[21] .
Inability of PCR to distinguish readily between maternally
inherited fetal DNA and native maternal DNA is clearly a
diagnostic impediment. However, studies of epigenetic
changes resulting in distinguishable patterns of DNA
methylation between fetal and maternal DNA provide
promise in the development of unique gene or global
fetal specific sequence detection assays [22]. Although,
our observations could be useful to assess future studies
on genetic determinants of folate and homocysteine
levels with an independent “risk factor” in patients like
preeclampsia. Thus, an important goal in this study
should be to identify the predominant form of cell free
fetal DNA in maternal plasma among normal
pregnancies and its correlation to the other gene
mutation as non- invasive tool for genetic marker in
prenatal diagnosis .
Conclusion
We conclude from the present study that - (1) Cell free
fetal DNA from maternal blood should be used as
“genetic marker” for programmed cell - death, (2) Single
gene mutation (CβS and MTHFR genes) may also help
to assess as an independent risk factor” for several
disorders such as pre-eclampsia & eclampsia condition,
and (3) cffDNA may act as non- invasive device for
antenatal diagnosis.
Acknowledgement
Authors are thankfully acknowledged to the patients who
participated in the present study to make it successful.
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A
B
Fig.1- Identification of cell frees fetal DNA in maternal plasma using Sry marker. Lane (M) ladder 100 bp, lane 1 to 7
showing all patients are SRY positive (Fig. A) while lane-1& 3 showing absence of Sry gene act as negative control
while lane- 2, 4,5,6 & 7are Sry positive (fig.B).
Fig.2- Representative photograph of CβS gene in pre-eclamsia patients. M - 100bp ladder, Lane-1 showing mutation of CβS
gene (indicated by arrow), Lane-2-4 cases showing differential expression of CβS gene, Lane-5 & 6 act as controls.
Fig.3- MTHFR-C677T gene analysis in pre-eclampsia patients. M-ladder 100bp, lane 1& 2 showing CC genotype. Lane 3 &
4 CT genotype.
... In human, MTHFR gene is thermolabile in nature and present on chromosome 1p3 6.3. The polymorphic variation of C677T allele (rs1801133) , the missense (point) mutation is responsible for the reduction enzymatic activity (30% -70%) followed by increase "risk factor" in the variety of disease in heterozygous condition other than CHDs, such as neural tube defects (NTD), mental retardation, miscarriage and cancers [12][13][14][15][16][17]. Only few studies have been demonstrated regarding variations of MTHFR allele in CHD cases in Indian population [18,19]. ...
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  • S Daly
  • A Cotter
  • A E Molloy
Daly S., Cotter A., Molloy A.E. et al. (2005) Vasc. Med., 5, 190-200.
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