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This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/aogs.12640 This article is protected by copyright. All rights reserved.
Received Date : 15-Jul-2014 Accepted Date : 29-Mar-2015 Article type : Main Research Article The obesity related FTO gene variant associates with the risk of recurrent
miscarriage
Running title: FTO gene variant in recurrent miscarriage
Prabha H Andraweera1,2, Gustaaf A Dekker1,3, Rohan W Jayasekara2, Vajira HW
Dissanayake2 & Claire T Roberts1
1School of Paediatrics and Reproductive Health, Robinson Research Institute, University of
Adelaide, Adelaide, Australia 2Human Genetics Unit, Faculty of Medicine, University of Colombo, Colombo, Sri Lanka 3Women’s and Children’s Division, Lyell McEwin Hospital, Elizabeth Vale, South Australia,
Australia
Correspondence
Prabha Andraweera
School of Paediatrics and Reproductive Health, Robinson Research Institute, University of
Adelaide, Adelaide, Australia
Email: prabha.andraweera@adelaide.edu.au
Conflicts of interest
All authors declare no conflicts of interest
Abstract
Objective: To investigate the association of the fat mass and obesity associated gene (FTO)
rs9939609 single nucleotide polymorphism (SNP) with recurrent miscarriage (RM). Design:
Candidate gene association study. Setting: Human Genetics Unit, Colombo, Sri Lanka.
Population: 202 Sinhalese women with two or more first trimester miscarriages and no living
children (cases) and 202 age and ethnicity matched women with no history of miscarriage
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and having two or more living children (controls). Methods: Peripheral blood was collected
from the participants and DNA was extracted. Genotyping was performed at the Australian
genome Research Facility using the Sequenom MassARRAY system. Genotype and allele
frequencies of cases were compared with controls using chi-squared testing. Main outcome
measures: The prevalence of the SNP in cases and controls. Results: The mean age of the
women in the RM group was 31.9 ± 0.4 years and that of the control group was 32.3 ± 0.3
years. Of the women in the RM group, 140 (69.3%) had experienced ≥3 first trimester
miscarriages. The prevalence of the AA genotype [p = 0.0002, OR (95% CI) = 3.8 (1.8-8.0)]
and A allele [p = 0.002, OR (95% CI) = 1.6 (1.2-2.2)] of the FTO rs9939609 SNP were
increased in women in the RM group compared to the control group. Conclusion: The obesity
related FTO rs9939609 SNP associates with RM. This finding warrants further investigation
with controlling for important factors such as body mass index, diabetes and cardiovascular
disease status. The SNP may be useful in predicting the risk of recurrent miscarriage.
Key words
recurrent miscarriage, FTO rs9939609, polymorphism
Abbreviations
RM, recurrent miscarriage;
FTO, Fat Mass and Obesity;
SNP, single nucleotide polymorphism
OR, odds ratio
BMI, body mass index
Key message
The FTO rs9939609 SNP which is well known for the associations with obesity and vascular
and metabolic phenotypes, is associated with increased risk of recurrent miscarriage. The
SNP may be useful in predicting the risk of recurrent miscarriage.
Introduction
Spontaneous miscarriage is involuntary pregnancy loss before the 20th week of gestation and
affects approximately 15% of normal couples. Of these, 1-5% experience recurrent
miscarriage (RM). At present the definition of RM is debated with the American definition
being the loss of two or more consecutive pregnancies, while the European definition is the
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loss of three or more consecutive pregnancies (1, 2). A genetic contribution to RM is
suggested by studies that have shown that the prevalence of RM among first degree relatives
is two- to seven-fold higher compared to the general population and that the prevalence of
miscarriage among siblings of those experiencing RM is approximately double that of the
general population (3, 4).
Women who experience RM are at increased risk of later life vascular and metabolic
disorders including coronary artery disease and diabetes mellitus and parents of women who
experience RM are also at a higher risk of developing coronary artery disease (5-7). These
findings suggest that these families may have shared genetic, epigenetic and environmental
risk factors that predispose them to RM and later life vascular disease. Among the many
mechanistic pathways that have been proposed to contribute to the above findings, one
plausible mechanism is the presence of phenotypic variants that predispose to metabolic
syndrome, including obesity, higher fasting insulin, glucose and triglycerides and lower high
density lipoproteins (8).
A few years ago, genome wide association studies identified a strong association between a
common single nucleotide polymorphism (SNP rs9939609) in the fat mass and obesity
associated gene (FTO) on chromosome 16q12.2 and the risk of obesity. Since then the FTO
rs9939609 SNP has been investigated in relation to many metabolic diseases and molecular
pathways and is one of the most extensively investigated SNPs. There is strong and
consistent evidence that has been replicated by many groups on the association of this SNP
with adverse metabolic traits, diabetes mellitus and coronary artery disease (9). However, this
polymorphism has not been studied in RM. The aim of our study was to investigate the
association of the FTO rs9939609 SNP with RM in Sinhalese women in Sri Lanka.
Material and methods
This study was conducted on 404 DNA samples that comprise a biobank of samples extracted
from peripheral blood of 404 women recruited to the “Recurrent Miscarriage Study” which
was conducted with the aim of identifying genetic variants that are associated with RM. We
have investigated a total of 100 SNPs in different molecular pathways that are implicated in
the pathogenesis of RM and some of the findings have previously been published (10, 11).
The FTO rs9939609 SNP was the only obesity-related polymorphism that was investigated in
this cohort and the findings are discussed in this manuscript.
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The participants were consecutively recruited at two tertiary care maternity hospitals and at
the Human Genetics Unit of the Faculty of Medicine, University of Colombo, Sri Lanka
between January 2006 and December 2008. The study population consisted of 202 women in
the RM group and 202 women in the control group. The eligibility criteria to be recruited into
the RM group included women who had experienced at least two consecutive first trimester
pregnancy losses and having no living children. All eligible women and their partners
underwent karyotyping. If a chromosome abnormality was detected in the woman or her
partner, the woman was not eligible to participate in the study. Only Sinhalese women were
eligible to participate. Women of mixed ethnicity and those who had pregnancies fathered by
non Sinhalese men were not recruited to ensure ethnic homogeneity. All women in the RM
group had been assessed by an obstetrician and had undergone routine testing to identify a
cause for RM including endocrine disorders, structural abnormalities of the reproductive
tract, thrombophilias and antiphospholipid antibody syndrome. Those with an identifiable
cause for RM were not eligible to participate in the study. The control group consisted of 202
ethnicity and age matched women who had two or more living children and who did not have
a past history of miscarriage. The women in the control group were recruited at postnatal
wards of the same maternity hospitals as the women in the RM group.
The study was approved by the Ethics Review Committee of the Faculty of Medicine,
University of Colombo, Sri Lanka (EC/05/003, 10/02/2005) and the Human Ethics
Committee of the University of Adelaide, Australia (H147-2008, 13/11/2008). All women
provided written informed consent.
Peripheral blood samples were collected from the women and DNA was extracted using
QiaAmp blood midi DNA extraction kits (Qiagen, Limberg, Netherlands). Genotyping was
performed at the Australian Genome Research Facility (Brisbane, Australia) using the
Sequenom MassARRAY system (Sequenom Inc, San Diego, California). The primer
sequences were ACGTTGGATGTGTCTGAATTATTATTCTAG (1st PCR primer),
ACGTTGGATGTAGAGTAACAGAGACTATCC (2nd PCR primer) and
GGTACTTGCGACTGCTGTGAATTT (extend primer). As a quality control measure 300
independent samples which were genotyped in house for the same SNPs using real time-PCR
and high resolution melting (HRM) analysis were genotyped using the Sequenom
MassARRAY system at the Australian Genome Research Facility. The concordance rate of
the RT-PCR results and MassARRAY results was 100%.
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The chi-squared test was used to test the genotypes for Hardy-Weinberg Equilibrium and to
compare categorical variables. All data analyses were performed using PASW version 17.02
(SPSS, Chicago, IL, USA). The genotype and allele frequencies of cases were compared with
controls using chi-squared analyses. Results were reported as number and percent [n (%)] or
mean ± standard deviation where appropriate. To account for multiple testing, the Bonferroni
correction was applied. Significant associations were defined at a two-tailed p value
<0.05/100 = 0.0005. The prevalence of the SNP in the general population was determined by
genotyping 85 randomly selected Sinhalese men and women. On the basis of prevalence of a
polymorphism in 20% in the general population, a ratio of one control subject to one case,
200 women with RM and 200 control women have 80% power to detect an odds ratio (OR)
of 2.0 (β = 80%, α = 0.05).
Results
The mean age of the women in the RM group was 31.9 ± 0.4 years and the mean age of the
control group was 32.3 ± 0.3 years. Of the 202 women in the recurrent pregnancy loss group,
140 (69.3%) had experienced 3 or more first trimester pregnancy losses and 114 (56.4%) had
experienced only first trimester pregnancy losses. The details of pregnancy loss are shown in
Table 1. All cases and controls were non-smokers. The FTO rs9939609 SNP was in Hardy-
Weinberg Equilibrium in both cases and controls. Genotype data of 7 (3.5%) women in the
RM group and 9 (4.5%) women in the control group were not available due to genotyping
failure. The prevalence of the AA genotype [p = 0.0002, OR (95% CI) = 3.8 (1.8 - 8.0)] and
A allele [p = 0.002, OR (95% CI) = 1.6 (1.2-2.2)] of the FTO rs9939609 SNP were increased
in women in the RM group compared to the control group (Table 2). The association of the
AA genotype of the FTO rs9939609 SNP with RM was significant after Bonferroni
correction. As a post-hoc analysis, we sub-categorized the women in the RM group into those
who have had three or more first trimester miscarriages and those who have had two first
trimester miscarriages and compared the prevalence of the genotypes with the control group.
We observed similar results in the two groups (Table 3).
Discussion
This is the first study to investigate the prevalence of the FTO rs9939609 SNP in women who
experience RM. Our findings demonstrate a significant increase in the prevalence of the
variant allele of the SNP in women who experience RM.
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There is increasing evidence linking RM with later life coronary artery disease and diabetes
mellitus (5, 6, 12). A recent systematic review and meta-analysis demonstrated that women
with a history of miscarriage are at a 45% higher risk and those with a history of RM at a
two-fold risk of developing coronary artery disease (8). Women with a history of RM are also
shown to be at two times higher risk of developing diabetes mellitus in later life (6). These
associations can be explained by two plausible theories. One theory is that RM and later life
vascular and metabolic disorders share the same etiology and the second theory is that
pathological processes initiated due to the pregnancy loss may predispose a woman to future
vascular and metabolic disorders.
Interestingly, Smith and colleagues recently reported that the prevalence of coronary artery
disease is also higher among parents of women with RM. The risk was shown to be 25% for
parents of women experiencing two miscarriages and 56% for parents of women
experiencing three or more miscarriages (7). These findings suggest that these families may
have shared genetic, epigenetic and environmental risk factors that predispose them to RM
and later life coronary artery disease, supporting the theory of shared genetic etiology among
these disorders.
The FTO rs9939609 SNP is an ideal candidate polymorphism in this setting. This SNP
gained much research interest when in 2007, three well-powered genome wide association
studies identified it as a strong contributor to both childhood and adult obesity (13-16). Since
then, it has been one of the most extensively studied polymorphisms with consistent evidence
of its association with many metabolic and vascular phenotypes.
The molecular mechanism of FTO action in metabolism is yet to be determined but the FTO
protein is highly expressed in the central nervous system and is considered to be implicated in
energy balance and regulation of appetite (17). The variant allele (A allele) of the SNP has
been shown to have a robust and consistent association with elevated body mass index
(BMI), central fat distribution and obesity (18). It has also been shown to be associated with
higher fasting insulin, glucose, triglycerides and lower high density lipoprotein cholesterol
(19). A strong and consistent association between the variant allele and the risk of type 2
diabetes mellitus and coronary artery disease has also been reported (9, 20, 21). Until
recently, the association between the SNP and these clinical phenotypes was thought to be
mediated via its association with elevated BMI and obesity. However, recent evidence
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demonstrates an independent association of the SNP with disease risk. Two recent meta-
analyses on the association of the FTO rs9939609 SNP with type 2 diabetes among 24 987
and 95 551 patients demonstrated a significant association independent of BMI (22, 23).
Another recent systematic review and meta-analysis of 19 153 patients with coronary artery
disease also reported that the association between the SNP and coronary artery disease risk is
independent of BMI and other conventional coronary artery disease risk factors (23).
In our study, we found that homozygosity for the variant allele of this SNP was associated
with a 3.8-fold risk of RM in Sinhalese women providing further evidence for potential
shared genetic factors between RM, coronary artery disease and diabetes mellitus. When the
miscarriage cohort was sub-categorized into those with three or more and two first trimester
miscarriages, we found a marginal increase in the odds ratio in the group with three or more
miscarriages. However, the subgroups do not have sufficient power to test for significant
differences between the two groups.
We could not investigate the interaction between the polymorphism and the woman's BMI,
cardiovascular status and the presence of diabetes mellitus since we did not have information
on these variables. We consider this a limitation of our study. However, the presence of
strong evidence from meta-analyses demonstrating the potential role of this polymorphism
independent of BMI, warrants further investigation of this polymorphism in other groups of
women with RM for whom more clinical and environmental data are available.
In addition, most consistent associations between this polymorphism and clinical variables
were initially shown in studies of Europeans with less consistent evidence shown among
Asians. Recently, a few groups have found strong associations between this polymorphism
and disease risk in Asian populations demonstrating a potential risk independent of ethnicity
(22, 23). Our findings add further evidence to the association of the FTO rs9939609 SNP
with disease risk among South Asians.
Conclusion
Our study suggests that the FTO rs9939609 SNP can be a risk factor for recurrent
miscarriage. This finding warrants further investigation with controlling for important factors
such as BMI, diabetes and cardiovascular disease status.
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Funding
This study was funded by the National Health and Medical Research Council Australia
(NHMRC) Project Grant ID519225 awarded to CTR and GAD. CTR is supported by a
NHMRC Senior Research Fellowship APP1020749. PHA is supported by a NHMRC Peter
Doherty Postdoctoral Fellowship (GNT1090778). The recruitment, characterisation and
establishment of the RM sample collection in Sri Lanka was funded by a grant from the
National Science Foundation (NSF), Sri Lanka awarded to VHWD and RWJ (Grant No:
SIDA/BT/2005/04). The study sponsors had no role in study design, data analyses, and
interpretation or writing this report.
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Table 1. Characteristics of the study population.
Characteristic Controls RM
(n=202) (n=202)
Age (years) 32.3 ± 0.33 31.9 ± 0.41
Pregnancy loss n (%)
T1 only 0 114 (56.4)
T1 and T2 0 75 (37.1)
T1 and T3 0 09 (4.5)
T1, T2 and T3 0 4 (2.0)
≥ 3 T1 losses 0 140 (69.3)
RM, recurrent miscarriage; T1, first trimester; T2, second trimester; T3, third trimester.
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Table 2. Genotype and allele frequency distribution of FTO rs9939609 SNP in Sinhalese
women with recurrent miscarriage and controls.
FTO rs9939609 Controls RM p value OR (95% CI)
n (%) n (%)
Genotype
TT 94 (48.7) 78 (40.2) ref 1.0 (ref)
TA 88 (45.6) 81 (41.8) 0.6 1.1 (0.7-1.7)
AA 11 (5.7) 35 (18.0) 0.0002 3.8 (1.8-8.0)
Allele
T 276 (71.5) 237 (61.1) ref 1.0 (ref)
A 110 (28.5) 151 (38.9) 0.002 1.6 (1.2-2.2)
RM, recurrent miscarriage; p values and OR (95% CI) in bold are significant after Bonferroni
correction.
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Table 3. Genotype and allele frequency distribution of FTO rs9939609 SNP in RM sub
groups and controls.
FTO rs9939609 Controls RM P value OR (95% CI)
n (%) n (%)
3 or more T1 miscarriages
Genotype
TT 94 (48.7) 54(40.6) ref 1.0 (ref)
TA 88 (45.6) 54(40.6) 0.8 1.1 (0.7 - 1.7)
AA 11 (5.7) 25(18.8) 0.0003 3.9 (1.8 - 8.7)
Allele
T 276 (71.5) 163 (60.8) ref 1.0 (ref)
A 110 (28.5) 105 (39.2) 0.005 1.6 (1.2 - 2.2)
2 T1 miscarriages
Genotype
TT 94 (48.7) 24 (39.3) ref 1.0 (ref)
TA 88 (45.6) 27 (44.3) 0.6 1.2 (0.6 - 2.2)
AA 11 (5.7) 10 (16.4) 0.007 3.6 (1.4 - 9.4)
Allele
T 276 (71.5) 75 (61.5) ref 1.0 (ref)
A 110 (28.5) 47 (38.5) 0.04 1.1 (1.0 - 2.4)
RM, recurrent miscarriage; T1, first trimester; p values and OR (95% CI) in bold are
significant after Bonferroni correction.