The Journal of International Medical Research

2009; 37: 1718 1724

Distribution of Common
Methylenetetrahydrofolate Reductase Gene
Mutations in Patients with Obstructive
Sleep Apnoea
TT BEKCI1, N KOCAK2 AND R KESLI3
1
Department of Pulmonary Medicine, 2Department of Genetics, and 3Department of
Microbiology, Konya Education and Research Hospital, Konya, Turkey

Homocysteine levels have been shown to have a causal role in the

investigated in patients with obstructive
sleep apnoea syndrome (OSAS), a
syndrome associated with a high level of
comorbid cardiovascular disease (CVD).
While significant increases in homo-
cysteine levels have been observed in OSAS
patients with CVD, no increases have been
noted in OSAS patients without CVD. This
study was designed to investigate the
methylenetetrahydrofolate reductase
(MTHFR) gene, which is essential for
homocysteine metabolism and has been
development of CVD. Eighty subjects, 30
diagnosed with OSAS by poly-
somnography and 50 controls (healthy
volunteers with no symptoms of OSAS)
were enrolled. Two mutations in the
MTHFR gene were investigated using
polymerase chain reactions and restriction
fragment length polymorphisms. No
significant differences were found in mean
age, body mass index, homocysteine levels,
or MTHFR allele or genotype distributions
between patient and control groups.

KEY WORDS: HOMOCYSTEINE; METHYLENETETRAHYDROFOLATE REDUCTASE; AMINO ACID; MUTATION;

GENOTYPE; OBSTRUCTIVE SLEEP APNOEA SYNDROME

Introduction Among these, increased homocysteine levels

It has previously been reported that nitric
oxide formation is reduced, insulin resistance
is increased and the risks for cardiovascular
disease (CVD), stroke and dementia are
increased in patients with obstructive sleep
apnoea syndrome (OSAS).1 9 Attention has
focused on the biochemical and genetic
processes, including apolipoprotein E,
angiotensin converting enzyme, nitric oxide
and homocysteine, which are considered to
play an active role in these pathologies.
are known to influence endothelial
dysfunction, arterial intimalmedial wall
thickening and prothrombotic processes.10 13
Elevated homocysteine levels have also been
associated with several pathologies, such as
coronary heart disease, hypertension,
arteriosclerosis and dementia, which are
regarded as complications of OSAS.10,14,15
Lavie et al.,16 found that homocysteine levels
were higher in OSAS patients with
cardiovascular pathology compared with

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 T Bekci, N Kocak, R Kesli
MTHFR gene mutations in obstructive sleep apnoea
controls; however, no significant difference
between OSAS patients without
cardiovascular problems and controls was
reported. Increased homocysteine levels
associated with CVD have been observed in
patients with later stage OSAS,17 suggesting
that some compensatory mechanisms play
an active role early in OSAS.
There are two possible biochemical
pathways for homocysteine; it is either
transformed into methionine via methionine
synthase or into cystathionine via
cystathionine synthase.17 This suggests that
compensatory mechanisms may exist and,
therefore, abnormalities in gene expression
or enzyme levels in this pathway may lead to
an increase in the substrate level not being
observed until advanced age.
One of the main enzymes involved in the
folate pathway, in which homocysteine is
formed, is methylenetetrahydrofolate
reductase (MTHFR).18 This enzyme catalyses
the transformation of 5,10-methylene-
tetrahydrofolate into 5-methyltetra-
hydrofolate which, in turn, eliminates
homocysteine through transformation into
methionine. The methionine is transformed
into S-adenosylmethionine and serves an
epigenetic role, especially through DNA
methylation.19 21
Investigation of the genes that encode
enzymes involved in the homocysteine
folate pathway might provide clues to the
relationship between OSAS and
homocysteine. A role of epigenetic
mechanisms has been reported for the
development of craniofacial morphology as
well as for a role in obesity.22 24 Thus, the
present study was designed to investigate the
common mutations of the MTHFR gene
(C677T and A1298C)18 which are thought to
play an active role in increases in
homocysteine levels and in DNA
methylation, and to search for clues
1719
regarding the role of these common
mutations in the development of OSAS.
Patients and methods
PATIENTS
This observational study included randomly
selected patients who had been newly
diagnosed with OSAS and had been
admitted to the Konya Education and
Research Hospital Sleep Laboratory, Konya,
Turkey, between January 2008 and January
2009. Patients with pathologies which may
co-exist with increased homocysteine levels,
including CVD, recurrent abortion, neural
tube defects, smoking, and folic acid and
vitamin B12 use, were excluded. The control
group was selected from healthy volunteers
who had no symptoms of OSAS and who
scored 0 on the Epworth sleepiness scale.25
The body mass index (BMI) of all subjects
was calculated based on height and body
weight measurements. Informed consent was
obtained from all subjects verbally and in
writing before inclusion in the study and
ethical approval was provided from the
Ethics Committee of Meram Medical School
of Selcuk University, Konya, Turkey.
POLYSOMNOGRAPHY
At least one full-night polysomnography was
performed in all subjects. Electro-
encephalography, submental electromyo-
gram (EMG), leg EMG, electro-oculogram,
and electrocardiography recordings were
obtained. Air flow was measured by nasal
cannula, oxygen saturation was measured
using a pulse oximeter, and chest and
abdominal respiratory movements were
monitored. Discontinuation of nasal air flow
for at least 10 s was regarded as apnoea. A
reduction in oxygen saturation to 4%, or
the occurrence of symptoms of physiological
awakening, following 50% reduction in air
flow for a minimum of 10 s, was considered
 T Bekci, N Kocak, R Kesli
MTHFR gene mutations in obstructive sleep apnoea
to be hypopnoea. Patients apnoea
hypopnoea index (AHI) was manually
scored according to standard criteria,26 and
individuals with an AHI > 5 were diagnosed
as having OSAS and were included in the
study.
DETERMINATION OF MTHFR GENE
MUTATIONS
Genomic DNA of patients and controls was
isolated from peripheral blood samples using
the QIAamp DNA Blood Mini Kit (Qiagen,
Valencia, CA, USA). The extracted genomic
DNA samples then underwent polymerase
chain reaction (PCR) to screen for and
amplify two MTHFR gene mutations; one
involving base pair 677 and the other
involving base pair 1298. The PCR reaction
mix contained 5 l genomic DNA, 5 l 10
PCR buffer (P-2192; Sigma-Aldrich, St Louis,
MO, USA), 5 l of deoxynucleotide
triphosphate mixture (Promega, Madison,
WI, USA) containing 0.2 mM of each
nucleotide, 5 l 1 mM TrisHCl, 5 l 5 mM
KCl, 0.2 l of Taq polymerase enzyme (D-
6677; Sigma-Aldrich) and 4 pmol each of
forward and reverse primers for each region.
The volume was made up to 50 l with
double distilled water. The primers for each
region were: base pair 677 forward 5-AGG
ACG GTG CGG TGA GAG TG-3, reverse 5-
TGA AGG AGA AGG TGT CTG CGG GA-3;
base pair 1298 forward 5-CTT TGG GGA
GCT GAA GGA CTA CTA C-3, reverse 5-CAC
TTT GTG ACC ATT CCG GTT TG-3. The PCR
thermal cycling conditions for each region
were: base pair 677, an initial denaturation
step at 95 C for 3 min, followed by 35 cycles
of 94 C for 1 min, 61 C for 1 min, 72 C for
1 min, and one last extension at 72 C for 7
min; base pair 1298, an initial denaturation
step at 92 C for 2 min, followed by 35 cycles
of 92 C for 2 min, 60 C for 1 min, 72 C for
30 s, and one last extension at 72 C for 7
1720
min.
The PCR products for the base pair 677
and 1298 mutations underwent restriction
enzyme digestion: for base pair 677, 15 l of
the 198 base pair PCR product was added
into a reaction mixture containing 7 l of
double distilled water, 5 U of Hinfl enzyme
(Fermentas UAB, Vilnius, Lithuania) and 2.5
l of Red (R+) Buffer (Fermentas UAB); for
base pair 1298, 20 l of the 163 base pair
PCR product was added into a reaction
mixture containing 1 U of Mboll enzyme
(Fermentas UAB) and 0.5 l of Blue (B+)
buffer (Fermentas UAB). Both reactions were
incubated at 37 C for 16 h. To visualize the
fragments formed after cleavage, 25 l of
digested PCR products for each base pair
(677 and 1298) were loaded onto 2% or 3%
ultra-pure agarose gel, respectively, and
electrophoresed for 90 min to identify the
alleles for each base pair mutation. For the
base pair 677 mutation, the samples
containing only the 198 base pair fragments
were identified as 677 CC, the samples
containing 198, 175, and 23 base pair
fragments were identified as 677 CT, and the
samples containing 175 and 23 base pair
fragments were identified as 677 TT. For the
base pair 1298 mutation, the samples
containing 56, 31, 20, 28 and 18 base pair
fragments were identified as 1298 AA, the
samples containing 84, 56, 31, 30, 28 and 18
base pair fragments were identified as 1298
AC, and the samples containing 84, 31, 30
and 18 base pair fragments were identified
as 1298 CC.
MEASUREMENT OF HOMOCYSTEINE
LEVELS
Patient blood samples were obtained in the
morning between 09:00 and 10:00 h
following the sleep study. Homocysteine
levels were measured by competitive
immunoassay using an Immulite 2000
 T Bekci, N Kocak, R Kesli
MTHFR gene mutations in obstructive sleep apnoea

analyser (Diagnostic Products Corp., Los no statistically significant difference between

Angeles, CA, USA).
STATISTICAL ANALYSIS
The data were analysed using the SPSS
statistical package, version 12.0 (SPSS Inc.,
Chicago, IL, USA) for Windows. The
KolmogorovSmirnov test was used to test for
normal distribution of the results and a one-
way analysis of variance was used to
determine the homogeneity of the data.
Independent sample t-tests were used for
parametric comparisons and an
independent 2 test was used for non-
parametric comparisons. A P-value < 0.05
was considered statistically significant.
Results
A total of 80 subjects (30 patients with OSAS
and 50 healthy controls), were included in
the study and their baseline demographic
and clinical characteristics, and homo-
cysteine levels are shown in Table 1. There
were no significant differences between the
patient and control groups with regard to
age, BMI or homocysteine levels.
There were no statistically significant
differences in genotype distributions of the
C677T or A1298C alleles between the patient
and control groups (Table 2). There were also
the groups in terms of C and T allele
frequencies (C677T) or A and C allele
frequencies (A1298C), nor were there any
differences in genotype distributions with
either allele (Table 2).
Discussion
The lack of difference in homocysteine levels
and MTHFR genotype or allele distributions
between the OSAS and control groups in the
present study suggests that there is no
association between the presence of OSAS
and these two mutations. The prevalence of
these mutations in the general population
and their role in biochemical processes
should, however, be taken into account
when considering their potential
associations with specific diseases. The
MTHFR C667T and A1298C polymorphisms
are common in the general population,27
and were found at similar frequencies in the
OSAS and control groups in the present
study. This is important when considering
the potential relationship between diseases
and these mutations, particularly for MTHFR
which plays an essential role in DNA
methylation and homocysteine metabolism,
both of which are which involved in key
cellular pathways. When the functional

TABLE 1:
Demographic and clinical characteristics, and homocysteine levels of obstructive sleep
apnoea syndrome (OSAS) patients and controls
Controls OSAS
(mean SD) (mean SD) Statistical
(n = 50) (n = 30) significancea
Age (years) 44.4 8.1 48.7 11.3 NS
BMI (kg/m2) 26.7 3.1 29.7 4.8 NS
AHI NA 46.9 24.5
Homocysteine (mol/l) 14.09 0.5 13.9 0.7 NS
aIndependentsample t-test.
BMI, body mass index; AHI, apnoeahypopnoea index; NA, not applicable; NS, not statistically significant
(P > 0.05).

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 T Bekci, N Kocak, R Kesli
MTHFR gene mutations in obstructive sleep apnoea

TABLE 2:
Comparison of genotype and allele frequencies of the two polymorphisms in the
methylenetetrahydrofolate reductase gene between patients with obstructive sleep apnoea
syndrome (OSAS) and non-affected controls
Controls, n (%) OSAS, n (%) Statistical
(n = 50) (n = 30) significancea
Allele (C677T) NS
CC 21 (42.0) 13 (43.3)
CT 27 (54.0) 14 (46.7)
TT 2 (4.0) 3 (10.0)
C 69 (69.0) 40 (67.7)
T 31 (31.0) 20 (33.3)
Allele (A1298C) NS
AA 23 (46.0) 15 (50.0)
AC 23 (46.0) 11 (36.7)
CC 4 (8.0) 4 (13.3)
A 69 (69.0) 41 (68.3)
C 31 (31.0) 19 (31.7)
Genotype NS
TT/AA 1 (2.0) 3 (10.0)
TT/AC 1 (2.0) 0 (0)
CC/CC 4 (8.0) 4 (13.3)
CC/AA 8 (16.0) 3 (10.0)
CC/AC 9 (18.0) 6 (20.0)
CT/AA 14 (28.0) 9 (30.0)
CT/AC 13 (26.0) 5 (16.7)
a
Independent 2 test.
NS, not statistically significant (P > 0.05).

effects of these mutations on DNA underlying pathology in many genetic

methylation are taken into account, the
extent of their impact can be better
understood, since nearly all genomic
processes involve DNA methylation. It is
possible that the high prevalence of MTHFR
gene mutations in the general population
may lead to an increased sensitivity for
extrinsic and intrinsic factors. Conditions
that result in changes in genetic activity also
bring epigenetic re-organizations, such as
hyper- or hypo-methylation, into
consideration. Stable inheritance of the
normal DNA methylation pattern is
essential for the maintenance of highly
differentiated cell functions.28 Disturbance or
loss of the DNA methylation pattern is the
diseases and leads to initiation of several
chronic diseases seen in the elderly, such as
cancer.29 Stable inheritance of DNA
methylation among cell generations is
maintained by DNA methyltransferases.
Thus, it is possible that functional changes in
the genome may have an impact on the
MTHFR pathway. Mutations that do not
cause any problems in normal conditions
may trigger pathological processes under
certain circumstances. Several conditions,
including obesity, sleep disorders or
sedentary lifestyle, may affect the general
metabolism and may put a strain on these
hot spots of the genome.28
In addition, diseases that are thought to

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 T Bekci, N Kocak, R Kesli
MTHFR gene mutations in obstructive sleep apnoea

affect general metabolism, such as OSAS, possibly provide novel perspectives in

can also induce an increased metabolic load
that can have a further detrimental effect. It
has been observed in several studies that,
although infrequent in the early stages of
OSAS, there is a significant increase in
predisposition to CVD in the later stages of
OSAS.30 33 The prevalence of CVD increases
in parallel with OSAS disease duration.
The lack of a significant difference in
MTHFR gene mutations between the OSAS
and control groups in the present study does
not necessarily indicate that these mutations
do not lead to a predisposition for these
diseases. Comparison of variations between
the two groups might aid in evaluation of
the critical balance between the
environment and the genome. Comparative
studies using similar variables that are
known to have general effects on
metabolism, such as BMI and age, could
determining these relationships.
Genotype results obtained from allele
distributions do not indicate the exact
locations in the genotype. Determining
compound and wild-type alleles, especially in
cases of heterozygosity for both the 677 and
1298 mutations, may aid in evaluating the
functionality of this gene at the protein level.
It should be kept in mind that these
numerically similar heterozygotic conditions
might have different structural implications.
These differences might lead to significant
differences and functional consequences at the
enzyme level. Thus, a precise determination of
genotype structure using methods, such as
sequence analysis, is necessary.
Conflicts of interest
The authors had no conflicts of interest to
declare in relation to this article.

Received for publication 9 July 2009 Accepted subject to revision 14 July 2009
Revised accepted 29 October 2009
Copyright 2009 Field House Publishing LLP

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Authors address for correspondence

Dr Taha Bekci
Department of Pulmonary Medicine, Konya Education and Research Hospital, Meram
Yeniyol, 42100 Meram-Konya, Turkey.
E-mail: tahabekci@yahoo.com

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