analysis of methylenetetrahydrofolate reductase (mthfr ... article- … · 1. 70 % of the mthfr...

Nessa Publishers| www.nessapublishers.com

Journal of Gynecology

Volume 1| Issue 4

Review Article Open Access

Analysis of Methylenetetrahydrofolate Reductase (MTHFR) Polymorphisms (C677t & A1298c) in

Recurrent Pregnancy Loss (RPL)

Balasubramaniam, A. 1 *, Kotalawala, S. 2 and Amarasekara, R.3

1, 2, 3 BMS School of Science, Galle Road, Colombo 6, Sri Lanka

*Corresponding author: Balasubramaniam, A., BMS School of Science, Galle Road, Colombo 6, Sri Lanka;

Email: [email protected]

Citation: Balasubramaniam, A. (2017) Analysis of Methylenetetrahydrofolate Reductase (MTHFR) Polymorphisms

(C677t & A1298c) in Recurrent Pregnancy Loss (RPL): Nessa J Gynecology

Copyright: © 2017 Balasubramaniam, A., Kotalawala, S. and Amarasekara, R. et al. This is an open-access article

distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution,

and reproduction in any medium, provided the original author and source are credited.

Abstract

Introduction and Objectives

Recurrent Pregnancy Loss (RPL) is a multifactorial disorder responsible for 15 % of pregnancy failures. The

underlying etiology of RPL remains unknown and literature suggests that MTHFR polymorphisms to be the primary

genetic risk factors for RPL. Accordingly aim of this study was to find out the frequencies of MTHFR C677T and

A1298C MTHFR polymorphisms associated with RPL.

Method: From the eligible studies 17,780 cases and 22,346 controls were considered as study subjects.

Results: A positive MTHFR polymorphisms associated with RPL were seen in 75 % (26/35) of the study (p = 0.002).

Among the positive studies, 35 % had both C677T and A1298C polymorphisms. C677T polymorphism was the only

polymorphism associated with RPL in 50 % of cases, while 15 % found A1298C polymorphism associated with RPL.

Analysis also showed the prevalence of MTHFR-C677T polymorphism in the partners of women suffered from RPL.

Conclusion: The study confirms that the MTHFR C677T polymorphism is significantly associated with RPL (p =

0.043). Accordingly the C677T polymorphism could be used as a genetic marker for early diagnosis of RPL. Study

further suggests paternal screening is equally important as maternal screening in the early diagnosis of RPL.

Keywords: RPL, Recurrent Pregnancy Loss, Spontaneous Abortion, Methylenetetrahydrofolate reductase, MTHFR,

MTHFR Polymorphisms.

mailto:[email protected]


Journal of Gynecology Volume 1| Issue 4

1. Introduction

1.1 Recurrent Pregnancy Loss (RPL), etiology, prevalence

Recurrent Pregnancy Loss (RPL) or spontaneous pregnancy loss is a multifactorial distressing disease that leads to 15 %

of pregnancy failures (Chen et al., 2015). “WHO defined miscarriage as loss of the fetus or embryo weighting less than

500 g which would normally be at 20-22 complete weeks of gestation” (Vanilla et al., 2015). Many scientific studies

reveal that 1 % to 2% of the female population suffer from RPL (Tiwari et al., 2015). The etiology underlying RPL is

still unknown and pathogenesis is complicated. Several reliable published data state that the risk of miscarriages could

be due to subsequent pregnancies and accounts up to 30 % following 2 losses and 33% following 3 losses without a

history of live birth (Zonouziet et al., 2012). RPL is thought to be a multifactorial complex disease with the involvement

of genetic, immunological, environmental, endocrine, and physio-pathological factors (Yousefian et al., 2014; Cao et al.,

2013).

Maintaining a normal pregnancy requires a series of differential gene expressions. Number of gene mutations are

suggested to be reasons for RPL, few of which are: the MTHFR gene, the CYP Family genes, and the Factor V Leiden

(FVL) mutation (Park and Chang, 2014; Nair et al., 2013). Finding abnormal expression of these genes may outline

general health during pregnancy (Chaithra et al., 2011).

Numerous investigations have revealed the possible association of MTHFR polymorphisms with recurrent spontaneous

miscarriages and reproductive failures (Nan and Li, 2015; Vanilla et al., 2015; Yalcintepe et al., 2014; Ocak and Ozyurt,

2013; Isotalo, et al., 2000).

In addition, in the year 2000, Isotalo and his colleagues stated the existence of human MTHFR gene mutation has its

potential role in compromised fetal viability. Almost 30 % of women with unexplained pregnancy loss showed MTHFR

polymorphisms (Cao et al., 2014).

1.2 MTHFR Gene polymorphisms in RPL

Folate pathway and progesterone pathway are the two most critical pathways contributing to a successful pregnancy

(Tiwari et al., 2015). Folate plays a key role in biosynthetic processes. Folate dependent mechanisms have shown to be

vital for the fetal growth and development, and also for the maternal well-being (D’Elia et al., 2014; Puri et al., 2013).

Therefore, any folate mechanism deficiency can influence various pregnancy complications.

Folate mechanism is regulated by the enzyme called 5-Methylenetetrahydrofolate reductase (MTHFR). The MTHFR

gene is located at position 36.3 on the short (p) arm of chromosome 1 (Figure 1) with 11 exons and the cDNA is 2220 in

length (Nan and Li, 2015). The best characterized single nucleotide polymorphisms (SNPs) are at the mRNA positions

677 and 1298 (Ren and Wang, 2006).



Figure 1: Molecular location of MTHFR Gene (Nan and Li, 2015)

MTHFR catalyzes the conversion of 5, 10-Methyleneterahydrofolate to 5-methylenetetrahydrofolate, the circulatory form

of folate. Mutations in the MTHFR gene lead to hyperhomocysteinemia and decreased activity of the enzyme (Aarabi et

al., 2015; Yousefian et al., 2014). Since several mutations were described in MTHFR gene, C677T and A1298C

polymorphisms are the two most common mutations involved in RPL (Cao et al., 2014). Table 1 describes the

characteristic features of these polymorphisms.

Table 1: Comparison of C677T and A1298C polymorphisms in MTHFR gene

C677T Polymorphism A1298C polymorphism

1. 70 % of the MTHFR mutation - 35 % of MTHFR mutation

2. Missense mutation - Point mutation

3. Occurs in the exon 4 of the gene - Occurs in the exon 7 of the gene

4. Converts an alanine to a valine codon - Glutamate to alanine substitution

5. Mutation at codon 222 in the N-terminal

catalytic domain of the protein

- At codon 429 within the C-terminal

regulatory domain of the protein

6. Mutation leads to thermolabile protein - No thermolability changes

7. Decreases the enzymatic activity – 50 to

70 % loss of activity in homozygotes

(677TT) and 30 % of loss in

heterozygotes (677CT).

- Decreases the enzymatic activity

C677T and A1298C polymorphisms decrease the conversion of homocysteine into methionine, hence homocysteine

accumulates in blood and results in hyperhomocysteinemia (HHcy) (Wiwanitkit, 2005). Hyperhomocysteinemia may

further influence common pregnancy outcomes, preterm delivery or intrauterine growth restriction, vasculopathy in the

placental vasculature which can lead to RPL (Hubacek et al., 2015; Li et al., 2015). Hyperhomocysteinemia may also

damage the coagulation system and influencing uteroplacental circulation selectively, which is closely related to the

http://ghr.nlm.nih.gov/dynamicImages/chromomap/MTHFR.jpeg



occurrence of premature delivery (Parveen et al., 2013). MTHFR polymorphisms are also associated with fetal loss at

early stage of pregnancy (Varga et al., 2005).

However the role of MTHFR polymorphisms in RPL is still controversial based on the varying results that have been

obtained over a span of many years. Most of the studies are associated MTHFR with RPL while others have revealed no

association. Thus, this literature review is conducted to study the frequency of MTHFR polymorphisms in

hyperhomocysteinemia leading to RPL, and further examine respect to it.

2. Materials and Methodology

2.1 Eligible Studies

37 published reports were identified among which 2 that discussed about MTHFR mutation and non-pregnancy

complications were excluded. Hence, 35 reports were selected for this review. 29 case control studies, 4 meta-analysis

and one nested case control study and one case control study plus meta-analysis were taken into account for this study.

Further, 30 articles were on URPL, 3 on Hcy level and treatment, and one each on pre-matured delivery and pre-

eclampsia complications. All the URPL studies included women with cases of a minimum of two or more early fetal

losses. Studies were carried out with focus on worldwide, as elaborated in the table (Table 2) below. Table 2: Summarize

data of eligible studies

Country Number of Studies Year

1. China 07 2015, 2015, 2015, 2014, 2012,

2013, 2007

2. India 06 2015, 2015, 2013, 2013, 2013,

2012

3. Iran 04 2014, 2012, 2012, 2010

4. Turkey 04 2015, 2014, 2013, 2012,

5. Egypt 02 2012, 2011

6. Brazil 02 2015, 2014

7. Korea 02 2014, 2011

8. Italy 02 2008

9. Avicenna 01 2006

10. Slovenia 01 2015

11. Tunisia 01 2006

12. Mexico 01 2009

13. Canada 01 2000

14. USA 01 2007

15. Unknown 01 2005



Although the search was restricted to the last 10 years, one article from 2000 was used for the analysis as it was the first

ever evidence about ‘MTHFR polymorphisms and RPL’.

2.3 Data Extraction

From each study, year, authors, journal name, place of study, sample size and source, methods and detections, mutation

found, and mutation type and analysis information were extracted. Therefore from the eligible studies, 17,780 samples as

cases and 22,346 controls were taken into the analysis.

Year Aut

hor

Journ

al

Plac

e of

stud

y

Sample

Size

Type of

Sample

Sour

ce

Method

s &

Detectio

n

Mutatio

n

Found

Mutation Type and Analysis

2013

Cao

et

al.,

Gene

514

(2013)

105-11

China

46

reports

3559

URPL

cases,

5097

healthy

controls

Maternal

Meta-

analysis

MTHFR

C677T

# 68 % TT verus Total genotypes

# 34 % T verus total alleles

# East Asians people are higher

risk of URPL with 677TT

genotype.

# At a higher level TT and CT

genotypes influence the overall

association.

# 1298CC mutation was 9% in

both samples

2015

Tiwa

ri et

al.,

Meta

Gene

3

(2015)

31-42

Nort

h

East

Indi

a

209 PTD

cases

403

controls

Pregnant

women

18 - 45

years

whol

e

Bloo

d

PCR-

RFLP

analysis

of

MTHFR

polymor

phism

MTHFR

# MTHFR found in 198 bp band

# Preterm delivery was

significantly at a higher

distribution.

# significantly associated with

extremely

as well as moderately preterm

delivery

# For more than 3 folds, MTHFR

significantly correlates with

negative pregnancy outcome.


2015

Vani

lla et

al.,

Bioche

mistry

section

9(2),

BC15-

BC18

Kola

r

Distr

ict -

Sout

h

Wes

t

Indi

a

15

couples

from each

control

and

sample

Blood

sample

from

male and

female

Whol

e

Bloo

d

PCR-

RFLP

Based

Screenin

g

MTHFR

C677T

# In RSA couple,

CC ( 80% M & 86.6 % F)

CT ( 13.3 M & 13.3 F)

TT ( 6.67 M & 0 Female)

# In healthy couples there is no

significant difference observed

with homozygous wild type CC,

CT, TT.

# In RSA cases of male partners

are at a higher frequency of

getting the mutant allele.

2012

Zono

uzi

et

al.,

Obstet

rics

and

Gynec

ology

Nort

hwe

st of

Iran

139

women

( 89 RM,

50 C)

Maternal

Bood

leuko

cyte

PCR-

RFLP

Based

Screenin

g

ARMS-

PCR

Based

Screenin

g

MTHFR

C677T

MTHFR

A1298C

# 677 C/T - 33.7 % (P)

# 677 C/T - 44 % ©

# 677 T/T - 6.74 % (P)

# 677 T/T - 2% ©

# In both groups there is no

statistical significant difference in

the frequency of A1298CC

2013

Ocak

and

Ozy

urt

Africa

n

Health

Scienc

es

13 (2)

447-

452

Turk

ey

495

couples

Maternal

and

paternal

Bloo

d

(leuk

ocyte

)

Karyoty

ping

PCR-

RFLP

Screenin

g

MTHFR

C677T

# MTHFR polymorphism

(67.9%) is due to frequent

abnormality for heterozygote

mutation.

2015

Nan,

and

Li,

Int J

Clin

Exp

Pathol

8(6),

7397-

7402)

Chin

a

108

premature

delivery

pregnant

women

and

108

control

Maternal

Veno

us

blood

PCR-

RFLP

MTHFR

C677T

MTHFR

A1298C

#Than in controls TT allele

C677T is higher in cases.

# There is a lower distribution CC

allele A1298C in cases than

controls.

#Premature delivery risk could be

due to TT of MTHFR C677T.

# Premature delivery protection is

influenced by CC of A1298.


2006

Coul

am

et

al.,

Am J

Repro

ud

immun

ol

55(5),

360-8

150 RPL

women

20 fertile

controls

Maternal

Bucc

al

swab

s

PCR-

RFLP

10 gene

mutatio

ns

were

found

# In the frequency of the specific

gene there is no difference.

#homozygous mutation with RM

was significantly higher than

normal

# 59 % found in RM and 10 % in

control

2015

Chen

et

al.,

Arch

Gynec

ol

Obstet

Chin

a

16 articles

1420 RPL

cases

1408

controls

Maternal

Meta-

analy

sis

Meta-

analysis

MTHFR

C677T

MTHFR

A1298C

# C677T was significantly

associated with RPL risk under

dominant

( TT + CC)

# Sensitivity analysis excluding

studied that deviated from HWE

did not change the direction of

effect

#A1298C - no significant

association was found

2005

Wiw

anitk

it, V

Clin

Appl

Throm

b

Hemos

t

11(3),

pp.343

-5

8 case

control

studies

752

patients

625

controls

Maternal

Meta-

analy

sis

Meta-

analysis

MTHFR

C677T

# 53% of subjects with T allele

have RPL while 55 % of subjects

without T allele have RPL

# Ethnicity of the study do not

correlate with pattern of MTHFR

C677T.

2015

Hub

acek,

J.A

et

al.,

Clin

Chim

Acta

440

p.104-

7.

Slav

onia

464

samples

486

controls

aborted

samples

Tissu

e

PCR-

RFLP

MTHFR

C677T

MTHFR

A1298C

# Carriers with dominant allele of

these

polymorphisms were associated

with

higher risk of abortion.

# Distinct combination of

MTHFR could be associated with

higher risk of abortions in

Caucasians.

2015 Li et

al.,

Gynec

ol

Obstet

Invest

79(2),

pp.107

-112

Chin

a

7,812

women Maternal

2,928

pregnant

women

were

tested

with

Taqman-

MGB

for

genotyp

e

4,884

women

MTHFR

C677T

MTHFR

A1298C

#MTHFR C677T were 49.1 %

and 50.9 %

#MTHFR A1298C were 80.2 %

and 19.8 %

#After folic acid supplement, the

complication rate in different age

groups were significantly reduced


without

folic

acid

supplem

entation

(control)

2013

Parv

een

et

al.,

Arch

Gynec

ol

Obstet

,

288(5)

, 1171-

7

Nort

h

Indi

a

200

patients

300

controls

Maternal

Perip

heral

blood

sampl

es

PCR-

RFLP

MTHFR

MTHFD

PAI-1

# MTHFR C677T, A1298C,

MTHFD G1958A variant alleles

were found to be significantly

more prevalent than control

# MTHFR C677T is only twofold

and further decreased to only one

fold, and MTHFD-1958 lost its

significance upon meta-analysis

2012

Idali

et

al.,

Am J

Repro

ud

immun

ol

68(5),

400-7

Iran

177 RPL

women

100

control

Maternal

Perip

heral

blood

sampl

es

PCR-

RFLP

MTHFR

A1298C

PAI-1

4G/5G

#MTHFR A1298C and PAI-1

4G/5G

mutations in Iranian Women

suffering

from RPL with and without

PCOS

2012

Nair

et

al.,

Repro

d Sci,

19(2),

210-5

Nort

h

Indi

a

Case

control

Study

106 RPL,

140

control

Maternal Bloo

d

PCR-

RFLP

and

sequenci

ng

MTHFR

C677T

# statically significant association

was

found in RPL and the mutant T

allele

# homo and heterozygosity for

MTHFR

C677T polymorphism confer

increased risk of idiopathic REPL

2015

Boas

et

al.,

Revist

a

Brasile

ira de

Ginec

ologia

e

Obstet

rícia,

37(2),

Braz

il

Case

control

Study

89 RPL

women

150

healthy

women

Maternal Bloo

d

Serum

Folate

and B12

- ELISA

PCR-

RFLP

for DNA

analysis

MTHFR

C677T

MTHFR

A1298C

# No statistical difference

between in the frequency of the

genotypes between the studied

groups

#In study population

66.3% of presented with

C677T polymorphism

64 % presented with A1298C

polymorphism

# In control population

65% of presented with C677T

polymorphism

53.4 % f presented with

C677T polymorphism


2007

Ren

and

Wan

g

Fertilit

y and

Sterilit

y

86(6)

Chin

a

Meta-

analysis

pregnant

women

18 - 45

years

2120

URPL

cases

2949

Healthy

controls

Meta-

analysis

MTHFR

C677T

#Certain scientific studies

revealed out a strong correlation

between C677T and URPL only

in Chinese studies.

#Except in Chinese population,

MTHFR mutation is not genetic

risk factor.

2011

Park

et

al.,

Clinic

al

Experi

mental

Repro

ductiv

e

Medici

ne,

38(3)

Kore

a

39

patients

with

RM

50 control

women

Maternal

Veno

us

blood

#

Biochem

istry

measure

ment

#

MTHFR

genotypi

ng with

PCR

machine

# Flow

cytometr

ic

analysis

of pbNK

cells

MTHFR

C677T

# Homocysteine levels was

elevated in individual with TT

variant than those with CC and

CT variants in RM group.

# Between two groups' mean

plasma homocysteine level there

was no difference observed.

# Therefore it is very hard to use

plasma homocysteine as the

predictive marker of RM


2000

Isota

lo et

al.,

The

Ameri

can

Journa

l of

Huma

n

Geneti

cs,

67(1),

Can

ada

119

samples

161

controls

119

neonatal

cord

blood

161 fetal

tissue

Neon

atal

PCR-

RFLP

#

MTHFR

677CC

677CT

677TT

#

MTHFR

1298AA

1298AC

1298CC

# First article described about

MTHFR mutation

# There is an evidence, fetal

viability compromization is due

to combination of MTHFR

C677T and A1298C mutation.

# In neonatal group, the combined

677CT / 1298CCand 677TT /

1298CC genotype with three or

four mutant alleles were not

observed

2008

D'Uv

a et

al.,

Biolog

ics:

Target

s &

Therap

y

Italy

115

UPRL

women

75

healthy

age-

matched

controls

Maternal

1st

and

2nd

blood

sampl

e

PCR

amplific

ation

with

specific

primer

and the

light

cycler

apparatu

s

MTHFR

C677T

FVL

PTHRA

20210G

among the study group 30% were

presented with MTHFR C677T

homozygosity (highest)

2014

D'Eli

a et

al.,

Repro

ductiv

e Bio

Medici

ne

Online

28(6):

733-8

Braz

il

82

women

1432 IVF

cycles

were

performe

d

maternal

and

paternal

Sper

m,

Bloo

d

Sperm

Count

Ovarian

Stimulat

ion

Genotyp

e

determin

ation by

Taqman

SNP

genotypi

MTHFR

C677T

MTHFR

A1298C

#MTHFRpolymorphism was

observed with a significant

decrease in oocytes maturity.

# Elevated homocysteine in

plasma which occurs individually

seems is not due to the presence

of MTHFR A1298C both in CC

and AC.

# When compared to homozygous

TT and CC genotypes, patients

with heterozygous CT genotypes

has high percentage of better

quality embryos and increase


ng chance of pregnancy.

2014

Yous

efian

et

al.,

Iranian

Red

Cresce

nt

Medic

al

Journa

l,

16(7),

Iran

204 RPL

women

116

controls

Maternal Bloo

d

Reverse

hybridiz

ation

method

MTHFR

C677T

MTHFR

A1298C

# The prevalence of 677TT

patient - 8.8%

control- 8.6%

# The prevalence of 1298CC

Patient - 12.3%

control- 8%

#Between RPL patients and

healthy control subjects, MTHFR

C677T and A1298C did not

indicate a significant differences.

2014

Park

and

Chan

g

Vascul

ar

Specia

list

Interna

tional,

30(4),

Kore

a

146

samples

Arterial

and

venous

thrompisi

s

Real-

time

PCR for

screenin

g of

MTHFR

C677T

Fluoresc

ence

polarizat

ion

assay for

plasma

Hcy

level

measure

ment

MTHFR

C677T

# With high level of Hcy,

MTHFR C677 TT genotype gets

interacted.

2006

Mitr

aoui

et

al.,

Repro

ductio

n, 131,

Tuni

sia

200 RPL

patients

200 age

matched

controls

Maternal Bloo

d

PCR-

RFLP

Fasting

Hcy

level

measure

d by

ELISA

MTHFR

C677T

MTHFR

A1298C

# It was similar among patients

and controls higher Hcy levels

# In patients, there is a significant

higher frequency of MTHFR

677T/T and 1298C/C genotypes.

# In late and late-early RPL, there

is a higher prevalence of MTHFR

677T/T

# Higher prevalence of 1298C/C

was seen only in early-late RPL

# Between controls and patients

there is no statistical significant in

heterozygosity.

#Irrespectively for elevated total

Hcy level and risk factors of

RPLs is due to homozygosity of

MTHFR C677T/T and A1298C/C


2007

Alto

mare

et

al.,

Throm

bosis

Journa

l,

5(17),

USA

3 of

whom

were

actively

pregnant

(were

referred

with

history of

RPL)

Bloo

d

MTHFR

C677T

#Relationship between recurrent

fetal loss and C677Tmutation an

evident support must be provided.

# To achieve a full term

pregnancy and reduce risk of

recurrent abortion in future, the

hopes of therapeutic intervention

were suggested by lower dose of

lower molecular weight (LMW)

of heparin.

2013

Puri

et

al.,

Journa

l of

Prenat

al

Medici

ne,

1(41)

Nort

h-

Indi

a

107

URPL

women

343

controls

Maternal Bloo

d

PCR-

RFLP

MTHFR

C677T

# Within case and control, there is

no significant difference in

MTHFR genotypic distribution.

# In case group there is a C677T

found in increased Hcy

# T allele is detrimental with B12

deficiency

2013

Nair

et

al.,

Fertilit

y and

Sterilit

y,

99(5)

Indi

a

129 RPL

Patients

220

controls

+

For meta-

analysis

1080

cases and

709

controls

Maternal,

aborted

embryos

Bloo

d

PCR-

RFLP

MTHFR

A1298C

# Genetic risk factor for RPL is

MTHFR A1298C polymorphism.

# Increased risk of RPL could be

due to presence of rare allele "C"

and heterozygous and rare

homozygous genotypes.

# increased risk of carriers of AC

and CC genotypes showed due to

meta-analysis of both RPL and in

spontaneously aborted embryos.

2012

Wu

et

al.,

Geneti

c

Testin

g and

Molec

ular

Bioma

rkers,

16(7)

Chin

a

2427

cases and

3118

controls

Meta-

analysis

Meta-

analysis

MTHFR

C677T

# Except Caucasian, the increased

risk of RPL is associated with

idea of MTHFR C677T.

#Increase risk of RPL had

homozygous TT allele


2012

Ozde

mir

et

al.,

Geneti

c

Testin

g and

Molec

ular

Bioma

rkers,

16(4)

Turk

ey

543 RPL

women

and 327

male

partners

(870

individual

s with

RPL)

and 106

fertile

couples as

control

group

Maternal

and

paternal

Bloo

d

Real-

Time

PCR

Reverse

Hybridiz

ation

method

#FVL

#Factor

V

H1299R

#Factor

II

prothro

mbin

G20210

A

#PAI-1

#GPIIIa

L33P

#MTHF

R

C677T

#MTHF

R

A1298C

#ACE

I/D

#Apo B

R3500Q

#Apo E

Genes

# RPL is associated with

heterozygous or homozygous

point mutation in FVL, FVR2,

ApoE2, PAI-1, MTHFR C677T,

MTHFR A1298C and ACE

genes.

# In women, homozygosity of

MTHFR C677T and 4G in PAI-1

gene play a critical role in RPL

also considered as a risk factor.

2012

Ibrah

im et

al.,

Middl

e East

Fertilit

y

Societ

y

Journa

l, 17

Egy

pt

44 pre-

eclamptic

women

44 normal

pregnanci

es women

Maternal Bloo

d

SSP-

PCR

MTHFR

C677T

#TT allele frequency is

significantly high in study group.

# In Egyptian pregnant women,

the development of pre-eclampsia

is due to TT genotypes risk

factor.

2010

Jeddi

-

Tehr

ani

et

al.,

Ameri

can

Journa

l of

Repro

ductio

n

Immu

nology

,

2011,

Iran

100 RPL

women

and

100

healthy

women

Maternal Bloo

d

PCR-

RFLP

PAI-I

BF

ITGB3

MTHFR

C677T

MTHFR

A1298C

# BF, MTHFR C677T, MTHFR

A1298C were found positive

# RPL risk increases due to

presence of both mutations of

MTHFR gene.


2011

Setti

n et

al.,

Geneti

c

Testin

g and

Molec

ular

Bioma

rkers’,

15(12)

Egy

pt

70 RPL

cases

136

controls

Maternal Bloo

d

PCR-

RFLP

MTHFR

C677

TT

MTHFR

1298

CC

677T/12

98C

# Higher frequency of

homozygous mutant MTHFR

677TT, 1298CC genotypes are

showed in URPL.

# Haplotype 677T/1298C does

not reach statistical significance.

# For URPL mutation related to

MTHFR gene is not only the risk

factor but there are genetic

varient interactions might aid to

be as a risk factor.

2009

Rodr

igue

z-

Guill

en et

al.,

Salud

Public

a Mex,

51(1),

Mex

ico

Nested

case

control-

study

23 SA

women

and

74

controls

women

Maternal Bloo

d

# PCR-

RFLP

# Serum

homocys

teine by

HPLC

MTHFR

C677TT

MTHFR

A1298C

# Increased risk of SA is shown

by carriers of MTHFR C677TT

and A1298C genotypes

respectively.

# Risk of SA could be due to

smoking and paternal occupation

# Non-significant protection of

SA is shown by maternal of

young age and higher education.

# Than heterozygous,

homozygous alleles carried a high

risk factor of SA.

2014

Cao

et

al.,

Genes

Nutriti

on, 9,

Chin

a

82 RPL

women

166

Healthy

Women

Maternal

perip

heral

blood

#

Sequeno

m

MassArr

ay

# PCR

MTHFR

A1298C

MTHFR

677C

MTRR

SLC19

A1

# A significant association

between A1298C and URPL is

shown by the result

# RPL is associated with MTHFR

677C - MTHFR 1298C allele

combination.

# SLC19A1, 80GSLC had lower

association with RPL.

2014

Ince

biyik

et

al.,

Obstet

rics

and

Gynec

ology

Scienc

e,

57(6),

Turk

ey

1507 RPL

women Maternal

Bloo

d

# FV-

PTH-

MTHFR

Strip A

kit

#

Hybridiz

ation of

PCR

amplifie

d DNA

products

FVL

Prothro

mbine

MTHFR

C677T

# FVL

Homozygous - 0.2%

Heterozygous - 5.51%

# Prothrombine Analysis

Homozygous - None


# MTHFR

Homozygous - 8.29%


# Screening seems to be

controversial


Although the search was restricted to the last 10 years, one article from 2000 was used for the analysis as it was the first

ever evidence about ‘MTHFR polymorphisms and RPL’.

2.3 Data Extraction

From each study, year, authors, journal name, place of study, sample size and source, methods and detections, mutation

found, and mutation type and analysis information were extracted. Therefore from the eligible studies, 17,780 samples as

cases and 22,346 controls were taken into the analysis.

3 Results Analysis

From the analysis, 75 % (p = 0.002) of the studies (n=26) accepted that MTHFR polymorphisms effect recurrent

pregnancy loss. Whereas, 24 % of studies did not confirm this polymorphisms in RPL (Figure 2).

Figure 2: Ratio of the case studies

2015

Yalc

intep

e et

al.,

Interna

tional

Journa

l of

Molec

ular

and

Cellul

ar

Medici

ne,

4(2)

Turk

ey

23

abortion

materials

22

women

with

URPL

22 control

subjects

Maternal

and fetal

Spont

aneou

sly

abort

ed

fetal

mater

ials,

their

moth

ers

and

fertile

wom

en

Real

time-

PCR

FVL

Prothro

mbin-

G20210

A

MTHFR

C677T

PAI-1

4G/5G

ACE

I/D

eNOS

E298D

Apo E

E2/E3/E

4

# Compare to the mothers and

control group, PAI-1 4G/5G,

eNOS E2980, ApoE E2 / E3 / E4

genotypes and T' alleles were

spontaneous in aborted fetal

material

# Due to small sample size,

MTHFR C677T, ACE I / D were

not statistically significant.

75%

25%

Agree Disagree



Among the positive studies, 9 studies (35 %) discussed about both polymorphisms (C677T and A1298C) in RPL,

whereas 50 % of the cases specifically confirming the relationship between the C677T polymorphism in RPL and, 15 %

of studies discussed about the A1298C polymorphism’s specific role in RPL (Figure 3).

Figure 3: Percentage of frequency of both polymorphisms in RPL

4 Discussions

4.1 Hyperhomocysteinemia and risk for RPL

A successful pregnancy outcome is determined by several factors; maternal immunity, fetal vasculature and hemostatic

balance. Homocysteine metabolism cofactors; vitamin B12, B6, and folate are some of the cofactors that are essential for a

healthy pregnancy. Hyperhomocysteinemia (HHcy) is one of the leading factor, which has been stated as an independent

risk factor for several pregnancy-related complications, some are placental abruption, neural tube defects, pre-eclampsia,

and recurrent miscarriages. Hyperhomocysteinemia can occur as a result of folate deficiency when the dietary folate is

low (Nair et al., 2013). Now it was identified that in patients with chronic renal failure elevated level of creatinine can

cause an increase of plasma homocysteine levels (Park and Chang, 2014).

Low serum folate level in Rhesus monkeys has been related with granulosa cell impairment and with decreased estradiol

and progesterone level. Delayed ovulation and reduction of follicle growth are the indicators of embryonic growth and

malformation (Jangbloet et al., 2008). Thus folate metabolism is regulated by MTHFR gene, this makes the

polymorphisms in encoding MTHFR enzyme gene an important biomarker to assess the risk of RPL (Incebiyik et al.,

2014). Numerous latest studies are consistent with this fact and explains the significance of these polymorphisms with

RPL risk.

C677T MTHFR polymorphism

in RPL

50%

A1298C MTHFR polymorphism in RPL

15%



4.2 MTHFR in RPL

In 2000, Isotalo and his colleagues provided the very first evidence to suggest “combined MTHFR 677T and 1298C

mutations may be responsible for compromised fetal viability”. Furthermore, they explained, the role of human MTHFR

677T/1298C and 677TT/ 1298CC genotypes in fetal viability.

Unfortunately, they couldn’t figure out how these polymorphisms present at 1298 and 677 positions interact with

pregnancy complications. Because, most of the recurrent miscarriages occur before the delivery, the real influence of

MTHFR polymorphisms to fetal viability is difficult to determine (Isotalo et al., 2000). However, they further suggested

that, combined MTHFR polymorphisms is believed to cause decreased fetal viability, especially when couple with folate

deficiency.

The hypothesis which was left by Isotalo and his colleagues was proved by Incebiyik and his mates in 2014. The cis

mutation identification is more significant, because it allows more than two mutant alleles to be present in the genome of

fetus. If c is MTHFR configuration happened, they would result in selection disadvantages because of the expression of

severe phenotypes include spontaneous abortion (Incebiyik et al., 2014). The probability for embryos with TT or CT

genotypes to terminate an early stage has been advanced as an explanation for the ‘unique distribution’ of 677CT and

677TT genotypes in spontaneously aborted embryos, irrespective of chromosomal integrity (Jangbloet et al., 2008).

Pregnancy complications were categorized into three stages: early (5-12 weeks), late (13-30 weeks), and combined early-

late (Gupta et al., 2014). Homozygous variant of both SNPs (677T/T and 1298C/C) plays a significant role in pregnancy

complications.

Homozygosity C677T is associated in combined early-late and late RPL, while A1298C homozygosity is related to

combine early-late idiopathic RPL (Mitraoui et al., 2006).

With the presence of various genes in pregnancy regulations, MTHFR is believed to have a high tendency with the RPL.

Earlier it was suggested that FVL, prothrombin (G20210A) might cause RPL, however later it was described to be cause

by mutation in the MTHFR gene particularly C677T single nucleotide polymorphism (SNP) (Mitraoui et al., 2006).

Further, Jeddi-Tehrani and his colleagues' research in 2010 showed homozygous and heterozygous frequency of MTHFR

A1298 and C677T were significantly higher in cased compared to the control group and suggested an association of

these polymorphisms with RPL. This study also confirmed that combined heterozygosity for MTHFR mutations 677C/T

and 1298A/C may present as a genetic marker for placental abruption.

In 2012, Ozdemir et al., confirmed that, among the 12 Thromphilic mutations studied, MTHFR polymorphisms were the

second common mutation to be involved in RPL. In 2014, Incebiyik and his team conducted a research in Turkey to

identify the prevalence of thromogenic gene mutations in miscarried women. MTHFR polymorphisms were detected in

49 % cases, whereas other related mutations failed to produce a significant amount. The same result was in line with the

study conducted by Ocak et al., in 2013 in Turkey.



Subsequently, several researches have stated the existence of the MTHFR polymorphisms to be associated with the

increased risk of RPL (Hubacek et al., 2015; Li et al., 2015; Tiwari et al., 2015; D’Elia et al., 2014; Ozdemir et al.,

2012; Zonouzi et al., 2012; Settin et al., 2011; Rodriguez-Gillen et al., 2009; Mitraoui et al., 2006; Wiwanitkit et al.,

2005; Isotalo et al., 2000).

On contrary, studies from different authors showed no significance difference in MTHFR polymorphisms in RPL (Boas

et al., 2015; Incebiyik et al., 2014; Yalcintepe et al., 2014; Yousefian et al., 2014; Puri et al., 2013; Coulam et al., 2006).

Variations of the frequency of MTHFR polymorphisms in different ethnic population were observed and this may be due

to the fact that some studies included women with 3 or more pregnancy losses and some studies included women with 2

or more pregnancy losses. Meanwhile MTHFR polymorphisms only result in the second and third trimester fetal loss

(Yousefian et al., 2014). Further, the gestational weeks used in defining pregnancy loss was different among the studies.

4.3 C677T in RPL

C677T polymorphism has been denoted as a risk factor for RPL because of expand tHcy. Homozygous TT genotype was

significantly higher in the RPL women in china (p = 0.003), and in East Asia (Chen et al., 2015; Nan and Li, 2015;

Vanilla et al., 2015; Park and Chang, 2014; Cao et al., 2013; Praveen et al., 2013; Ibrahim et al., 2012; Nair et al.,

2012; Wu et al., 2012; Park et al., 2011; Altomare et al., 2007; Ren and Wang, 2007).

C677T polymorphism is associated with hyperhomocysteinemia, and increased risk of arterial stiffness. Women with

hyperhomocysteinemia express an important association with defective chronic villous vascularization.

In embryogenesis, the survival and the growth of the embryo is stimulated by its own blood supply through angiogenesis.

A noble interchange between mother and fetus is necessary to ensure normal fetal growth; impaired chronic villous

vascularization can result in embryonic death and leading to miscarriages (Yalcintepe et al., 2014).

Yila et al., (2012) state, that MTHFR 677T allele has an association with low folate status. D’Elia et al., (2014) declared

that there is a statistically significant decrease in oocyte maturity when the MTHFR C677T was detected. Rodriguez-

Guillen et al., (2009) detected an increased risk of recurrent miscarriages among female carriers of MTHFR 677T.

Prevalence of the MTHFR genotype was higher among Mexicans (38-48 %), and a much lower frequency of 1298C

allele has been observed in Mexicans (0 - 2.4 %) than in Europeans (Rodriguez-Guillen et al., 2009). In 2010, Jeddi-

Tehrani and his team revealed MTHFR 677C/T was significantly more frequent in Iranian women with early pregnancy

loss. It was the highest MTHFR mutation rate found in patients with RPL.

Wu et al., (2012) state, MTHFR-C677 gene mutation was more prevalent in paternal side than maternal. MTHFR

paternal T-allele was connected with 2.37 fold risk, whereas maternal T-allele did not show any interference in risk of

RPL. Subsequently, high frequency of MTHFR T-allele was absorbed in recurrent spontaneous abortions (RSA)

couples’ paternal side in a study carried out in India (Vanilla et al., 2015). Accordingly, both studies suggest, paternal



screening is equally important with maternal screening. When both parents are carrying mutant allele the chance of

inheriting the mutation in the offspring is much higher, which thereby influence the risk of miscarriages.

4.4 A1298C in RPL

Some of the individual studies have detected an increased risk of pregnancy loss in Indian, Turkish, and the Tunisian

population with 1298AC and 1298 CC polymorphisms (Li et al., 2015; Cao et al., 2014; Cao et al., 2013; Nair et al.,

2013; Idali et al., 2012; Zonouzi et al., 2012; Jeddi-Tehrani et al., 2010 ). Mean plasma homocysteine was elevated

among 1298AA homozygotes as compared with 1298C allele and was associated with RPL (Yila et al., 2012). MTHFR

1298 AC genotype allele has increased risk of spontaneous abortion along with MTHFR-C677T (Rodriguez-Guillen et

al., 2009).

MTHFR A1298C mutation was seen in Iranian women suffering from RPL (p< 0.001) (Idali et al., 2012). Meanwhile,

the frequency of both allele of A1298 were significantly higher in Iranian population and suggested an association with

RPL (p< 0.001) (Jeddi-Tehrani et al., 2010). D’Elia et al., (2014) found women with MTHFR 1298CC with In vitro

fertilization (IVF) were less probable to become pregnant than those with 1298AA genotype in Brazil.

4.5 MTHFR polymorphisms in other pregnancy complications

Maternal MTHFR 677T homozygosity was associated with lower birth weight among female infants in Japan (Yila et

al., 2012). Ibrahim and his colleagues study in 2012 showed, C677TT and the T allele polymorphisms were higher in

Egypt. TT allele of MTHFR gene was found to be associated with pre-eclampsia and other pregnancy complications.

MTHFR C677TT is considered as a good diagnostic test as it is highly sensitive and specific (Ibrahim et al., 2012).

Study of Nan and Li (2015) showed, C677TT is a candidate factor for premature delivery, and might increase the risk by

1.853 times than A1298C allele. Therefore A1298 polymorphisms act as a protective factor in premature delivery. A

study carried out in India also confirmed that, MTHFR polymorphism increased the preterm delivery by more than 2

folds. Further, MTHFR polymorphism was also focused to be significantly associated with fetal death and increased the

negative outcome by more than 3 folds (Tiwari et al., 2015).

4.6 Limitations

MTHFR mutation is a rare genetic mutation in leading to RPL, to obtain literature survey is difficult in its recent

publishing. Due to its pathetic conditions, therefore analysis was restricted for the past ten years from 2005 to 2015.

Since, MTHFR is related with other disease conditions the number of articles of MTHFR polymorphisms related to RPL

is limited.



4.7 Suggestions, Treatments, Future aspects

Literatures suggest, carrying out a routine test for the defection of C677T MTHFR polymorphism gene using simple

technique such as Single Specific Primer-PCR (SSP-PCR) as a marker for pregnancy complication susceptibility

(Ibrahim et al., 2012). Zonouzi et al., (2012) study confirm that both Amplification Refractory Mutation System –PCR

(ARMS-PCR) and Restriction Fragment Length Polymorphism-PCR (RFLP-PCR) methods would detect the MTHFR

polymorphisms with the same specificity. Therefore, to detect large number of samples they proposed ARMS-PCR for

the detection of MTHFR polymorphisms as a diagnostic test since it is easy and cost effective.

Screening of pregnant women for MTHFR-C677T polymorphism during the first trimester can be used as a prognostic

marker in women who have a risk for miscarriages (Tiwari et al., 2015). Patients with 677CT genotype have a higher

proportion of better quality embryos and increased chances of a healthy pregnancy, when compared to the homozygous

allele. Therefore, it is recommended to detect for the MTHFR polymorphism prefer to perform an IVF (D’Elia et al.,

2014).

Women with folate deficiency have been identified to be at higher risk of early pregnancy loss compared to the women

having a higher or normal level of folate (D’Uva et al., 2008). RPL patients with MTHFR polymorphisms should be

advised to take a balanced diet with adequate intake of Vitamin B12, and folic acid to prevent RPL and poor pregnancy

outcomes (Nair et al., 2013; Ibrahim et al., 2012).

Several folate acid supplementation studies were concluded with positive outcomes in pregnant women with a risk for

RPL. A study carried out on 7,812 Chinese women confirmed that, periconceptional folic acid supplementation and

health care following gene polymorphism testing could be a powerful measure to decrease pregnancy complications (Li

et al., 2015).

Study carried out in Italy showed, in moderate hyperhomocysteinemia, Hcy concentration was reduced after folate-

enriched diet, and 5-MTHF or folic acid supplementation (p<0.001) (Zappacosta et al., 2013). Diverse supplementation

by enriched-diet (200 µg/ day), folic acid (200 µg/ day, 340 DFE) or 5-MTHF (200 µg/ day, 340 DFE) produced

significant decrease in Homocysteine concentration (Anderson et al., 2014).

Recent genetics studies predict that allosteric regulation of MTHFR gene structure plays a significant role in fetal

growth. In folate deficiency, cells get arrested in the S-phase of the cell cycle, which would increase the uracil

disincorporation and cause DNA damage. This would lead to complications in pregnancy and reduced fetal growth.

According to the 5, 10-MTHFR gene structure, the A1298C is located on the C-terminal regulatory domain, containing

protein retention signals that stop delivery of proteins to the secretory pathway. It has now been shown that allosteric

inhibitory interplay in the s-adenosyl methionine (SAM) and s-adenosyl-homocysteine (SAH) cycle is implicated in the

functional behavior of this SNP relative to folate level and mediation of fetal growth (Yila et al., 2012).



5 Conclusions

This review analyzed the relationship of MTHFR polymorphisms with RPL. Based on the review the following

conclusions were drawn: Since 40 % of RPL cases are idiopathic, genetic factors tend to be highly associated with

miscarriages. Compared to other genetic mutations, MTHFR polymorphisms are the primary genetic risk factor for RPL

and MTHFR C677T and A1298C are the two most frequently occurring polymorphisms. Among both the

polymorphisms, C677T polymorphism is significantly associated with RPL (p = 0.043) compared to A1298C. MTHFR

polymorphisms could be easily and cost effectively detected using ARMS-PCR instead of RFLP-PCR. Screening of

pregnant women for MTHFR C677T polymorphism during the first trimester can be used as a prognostic marker in

women who have a risk for miscarriages. Hence, the C677T polymorphism can act as a genetic marker for early

diagnosis of RPL. Literature also strongly emphasizes that, MTHFR-C677 polymorphism is also prevalent in paternal

testing. Therefore, this review firmly concludes, paternal screening is also equally important along with maternal

screening.



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analysis of methylenetetrahydrofolate reductase (mthfr ... article- … · 1. 70 % of the mthfr...

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