Experimental Gerontology 81 (2016) 83–91

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Experimental Gerontology

journal homepage: www.elsevier.com/locate/expgero

Homocysteine metabolism and the associations of global DNA

methylation with selected gene polymorphisms and nutritional factors in
patients with dementia
Małgorzata Bednarska-Makaruk a,⁎, Ałła Graban b, Agata Sobczyńska-Malefora c, Dominic J. Harrington c,
Michael Mitchell c, Kieran Voong c, Letian Dai c, Wanda Łojkowska b, Anna Bochyńska b, Danuta Ryglewicz b,
Anna Wiśniewska a, Hanna Wehr a
a
Department of Genetics, Institute of Psychiatry and Neurology, Warsaw, Poland
b
First Department of Neurology, Institute of Psychiatry and Neurology, Warsaw, Poland
c
The Nutristasis Unit and the Molecular Haemostasis and Thrombosis Laboratory, Viapath, St. Thomas' Hospital, London, UK

a r t i c l e i n f o a b s t r a c t

Article history: Epigenetics (particularly DNA methylation) together with environmental and genetic factors, are key to under-
Received 27 December 2015 standing the pathogenesis of many diseases including dementia. Disturbances in DNA methylation have already
Received in revised form 27 April 2016 been implicated in dementia. Homocysteine metabolism, with folate and vitamin B12 as essential cofactors, is in-
Accepted 6 May 2016
tegral to methylation processes.
Available online 7 May 2016
We evaluated in a case-control study the association of global DNA methylation, homocysteine, folate and vita-
Keywords:
min B12 status with dementia. Selected polymorphisms of genes previously associated with dementia develop-
Dementia ment and the influence of various factors on DNA methylation were also investigated.
Global DNA methylation 102 patients with dementia (53 with Alzheimer's disease, 17 with vascular dementia and 32 with mixed demen-
Homocysteine tia) were recruited. The non-demented controls consisted of 45 age-matched subjects without dementia and 47
Folate individuals with mild cognitive impairment. Global DNA methylation was determined by Imprint Methylated
Plasma 5-methyltetrahydrofolate DNA Quantification Kit MDQ1 (Sigma-Aldrich, Gillingham, Dorset, UK). Plasma homocysteine, serum folate
Vitamin B12 and vitamin B12 were determined by chemiluminescence. Plasma and erythrocyte 5-methyltetrahydrofolate
Plasma methylmalonic acid
and plasma methylmalonic acid (markers of folate and vitamin B12 status) were measured by HPLC. APOE,
PON1 p.Q192R, MTHFR 677CNT (c.665CNT) and IL1B-511CNT polymorphisms were identified using PCR-RFLP
methods.
Patients with dementia had significantly higher concentrations of homocysteine (p = 0.012) and methylmalonic
acid (p = 0.016) and lower folate (p = 0.002) and plasma 5-methyltetrahydrofolate (p = 0.005) than non-
demented subjects. There was no difference in DNA methylation between patients and controls. A non-
significant tendency to higher DNA methylation in patients with vascular dementia (p = 0.061) was observed.
Multivariate regression analysis of all recruited individuals demonstrated a significant positive association be-
tween DNA methylation and folate (p = 0.013), creatinine (p = 0.003) concentrations and IL1B-511 T (p =
0.002) and PON1 192R (p = 0.049) alleles and negative association with fasting glucose (p = 0.004).
The biochemical results showed significantly lower folate and vitamin B12 status in demented patients than con-
trols. Global DNA methylation was associated with markers of folate status, creatinine, glucose and PON1 and IL1B
polymorphisms.
© 2016 Elsevier Inc. All rights reserved.

1. Introduction
Dementia is caused by defects in cerebral structure and function,
often manifesting as a progressive deterioration of memory and other
cognitive functions. In the elderly Alzheimer's disease (AD) is the
⁎ Corresponding author at: Institute of Psychiatry and Neurology, Department of
Genetics, Sobieskiego 9, 02-957 Warsaw, Poland.
E-mail address: makaruk@ipin.edu.pl (M. Bednarska-Makaruk).
most prevalent cause of dementia and results in the irreversible loss of
neurons, particularly in the cortex and hippocampus (Sosa-Oritz et al.,
2012). Pathological hallmarks of AD include extracellular β-amyloid
plaques and intraneuronal neurofibrillary tangles of
hyperphosphorylated forms of microtubule-associated protein tau. AD
is classified into early onset AD (EOAD, b65 years of age) accounting
for 1–5% of all cases, and late-onset AD (LOAD, N65 years of age) ac-
counting for N95% of all those affected. Causal mutations in three
genes: amyloid precursor protein (APP), presenilin 1 (PSEN1) and

http://dx.doi.org/10.1016/j.exger.2016.05.002
0531-5565/© 2016 Elsevier Inc. All rights reserved.
84 M. Bednarska-Makaruk et al. / Experimental Gerontology 81 (2016) 83–91
presenilin 2 (PSEN2) have been identified in the early-onset forms, es-
tablishing the central role of amyloid in Alzheimer's disease. Most
late-onset cases are sporadic, with genetic and environmental factors
likely to contribute to development. The presence of the ε4 allele of
the Apolipoprotein E (APOE) gene is known to increase susceptibility
to AD. Additionally, recent genome-wide association studies (GWASs)
have identified several genes that might be potential risk factors for
LOAD (Tosto and Reitz, 2013). These studies have pointed to probable
new pathways in the pathogenesis of AD including APP metabolism, im-
mune response, inflammation, lipid metabolism and cell signalling
(Reitz and Mayeux, 2014; Zou et al., 2014). Many recognized vascular
risk factors for ischemic heart disease or stroke (diabetes, hypertension,
smoking and obesity) have all been found to increase the risk of demen-
tia (Reitz et al., 2011; Beydoun et al., 2014). An increasing number of
studies have suggested a possible role for epigenetic changes in AD eti-
ology (Chouliaras et al., 2010).
Epigenetics relates to stable and not necessarily heritable patterns of
gene expression and genomic function that are not caused by changes in
DNA sequence. DNA methylation is a crucial epigenetic modification of
the genome that is involved in regulating many cellular processes. DNA
methylation is the post-replication addition of methyl groups to the 5′
position of cytosine residues that predominantly form cytosine-
guanine sequences, named CpG dinucleotides (Pufulete et al., 2003). A
non-CpG DNA methylation, which mainly occurs in CpA dinucleotides
and is particularly frequent in the brain, has recently been reported
(Sanchez-Mut and Gräff, 2015).
Methyl groups are provided by active methionine (S-Adenosyl me-
thionine – SAM) which is converted into S-Adenosyl-homocysteine –
SAH which is then hydrolysed to homocysteine. Methylation is
catalysed by DNA methyltransferases and occurs throughout the ge-
nome but is most relevant in cytosine – guanidine dinucleotides rich re-
gions, called CpG islands, usually located in the promoter region of
nearly all housekeeping genes and some tissue-specific genes. Cytosines
in CpG islands are usually not methylated, whereas CpGs outside CpG
islands are methylated. Methylation of CpG islands in the promoter re-
gion is associated with the silencing of gene expression (Pogribny and
Beland, 2009; Lu et al., 2013; Tammen et al., 2013).
DNA methylation status is tightly connected to homocysteine and
folate metabolism. Efficient homocysteine metabolism is dependent
on the availability, and efficient utilization, of several B vitamins (mainly
folates and vitamin B12) and is thought to play an important role in the
prevention of Alzheimer's disease (Ulrey et al., 2005). 5-Methyltetrahy-
drofolate (5MTHF), the predominant form of folate in plasma and red
cells, is a substrate for the methionine synthase and vitamin B12 medi-
ated conversion of homocysteine to methionine. 5-
Methylenetetrahydrofolate reductase (MTHFR) – an enzyme assisting
in the conversion of 5,10 methylene tetrahydrofolate to 5-MTHF, has
several polymorphic forms. The T form of the 677CNT mutation is ther-
molabile. In TT homozygotes the synthesis of 5-MTHF may be reduced,
homocysteine remethylation is less efficient and may become elevated
(Frosst et al., 1995).
Elevated total plasma homocysteine (tHcy), low vitamin B12 and
folate have been associated with AD (Clarke et al., 1998; Seshadri
et al., 2002; Wehr et al., 2009) and are suggestive of dysregulation
in the homocysteine-methionine pathway leading to disturbances
in the availability of SAM for DNA methylation. In addition, ele-
vated homocysteine leads to the elevation of SAH, a potent inhibi-
tor of SAM in a reversible reaction, resulting in DNA
hypomethylation (Seshadri et al., 2002; Mulder et al., 2005;
Scarpa et al., 2006). However, it is still unclear if elevated homocys-
teine have a causal connection to dementia or it is a biomarker
reflecting an underlying pathogenic biochemical process. The re-
sults of vitamin intervention trials have not been conclusive: sev-
eral, but not all vitamin intervention trials have documented
improvement in cognitive functions (reviewed in Clarke et al.,
2014 and Gillette-Guyonnet et al., 2013).
With advancing age, DNA methylation patterns become destabilized
with resulting hypomethylation of some regions and hypermethylation
of the others (Dong et al., 2002). Lower genomic DNA methylation has
been observed in common age-related diseases e.g. cancer, atheroscle-
rosis and macular degeneration (Pogribny and Beland, 2009).
The epigenetic contribution has been suggested to be involved in the
etiology of a number of human neurodegenerative diseases including
Alzheimer's, Parkinson's and Huntington disease and amyotrophic lat-
eral sclerosis (Lu et al., 2013). Global and gene-specific DNA methyla-
tion changes have been reported by several research groups, but no
clear conclusion about global DNA methylation changes in AD can be
drawn from the previous studies. The results were quite variable with
some suggesting hypermethylation, other hypomethylation, and some
without obvious differences. (Lu et al., 2013; Cacabelos and Torrellas,
2015; Bennett et al., 2015; Sanchez-Mut and Gräff, 2015). In a recent
epigenome-wide association studies, many genes in brain tissue
showed methylation differences between AD patients and controls,
but only two genes have been reported to be hypermethylated in repli-
cation studies (Sanchez-Mut and Gräff, 2015).
So far, studies investigating DNA methylation changes in AD have
been mainly conducted in human postmortem brain tissues. The search
for peripheral biomarkers of AD is of particular interest for early diagno-
sis of the disease. A few studies investigating changes in DNA methyla-
tion in peripheral blood leukocytes in AD, have also obtained
heterogeneous findings - the increased global DNA methylation in AD
patients (Bollati et al., 2011; di Francesco et al., 2015) or no differences
in methylation levels between AD patients and controls (Hernandez
et al., 2014).
Currently, it is not known whether the global DNA methylation
changes contribute to AD development. The aim of the present work
was to study changes in global methylation of DNA derived from
blood leukocytes and measured using an ELISA method in patients
with various types of dementia and to evaluate the possible association
of global DNA methylation with tHcy, folate and vitamin B12 status. We
also explored the association of global DNA methylation with selected
genetic and non-genetic factors known to be involved in dementia
development.
2. Material and methods
2.1. Participants
This case-control study was carried out on a total of 194 unrelated
elderly individuals (69 males and 125 females) with a mean age of
71.1 ± 7.56 years old. The group with dementia included 102 consecu-
tive dementia patients admitted to the First Department of Neurology,
Institute of Psychiatry and Neurology in Warsaw between November
2008 and June 2009. There were 53 subjects with probable AD - 11
males and 42 females, with a mean age of 70.9 ± 8.26 years; 17 patients
with dementia of vascular origin (VaD) - 6 males and 11 females, with a
mean age 69.6 ± 6.85 years; 32 patients with mixed form of dementia
(MD) - 9 males and 23 females, with a mean age 74.5 ± 6.14 years.
The non-demented controls included 45 individuals without symptoms
of dementia and in good general health, matched to dementia patients
for age, 20 males and 25 females, with a mean age 71.8 ± 6.46 years
and 47 individuals with a mild cognitive impairment (MCI) - 23 males
and 24 females, with a mean age 69.0 ± 8.18 years. The non-
demented subjects were mainly recruited from healthy volunteers,
healthy spouses of demented patients as well as from patients referred
to hospital with memory problems, in whom dementia was excluded
basing on MMSE and clock drawing tests (diagnosed as normal cogni-
tion or mild cognitive impairment, MMSE score ≥ 26 points). Subjects
on folate and vitamin B12 supplementation were excluded from the
study. Other exclusion criteria were as follows: malignant disease, hy-
pothyroidism, alcohol abuse, severe depression and other mood
disorders.
 M. Bednarska-Makaruk et al. / Experimental Gerontology 81 (2016) 83–91
Dementia was recognized according to ICD-10 and DSM-IV
criteria. Mini Mental State Examination and a Clock Drawing Test
were used as screening tests for existing dementia in all participants.
All patients were subjected to a general medical and neurological
evaluation, neuroimaging examinations: computer tomography
(CT) and magnetic resonance (MR) and comprehensive neuropsy-
chological tests. The type of dementia was diagnosed based on the
NINCDS-ADRDA scale for AD and NINDS-AIREN for VD. Differential
diagnosis of AD, VaD and MD was also based on the Hachinski scale
(Hachinski et al., 1975). Significant radiological evidence of cerebro-
vascular disease in CT or MRI suggested coexisting cerebrovascular
disease in AD hence these patients were included into the mixed de-
mentia group. For MCI diagnosis, the Petersen criteria were used
(Petersen and Negash, 2008).
The study was approved by the Ethics Committee of the Institute of
Psychiatry and Neurology. Subjects gave their informed consent either
directly or received it from their guardians.
2.2. Methods
The blood samples for biochemical and molecular analyses were ob-
tained after an overnight fast. Genomic DNA was extracted from periph-
eral blood leukocytes by phenol/chloroform extraction and ethanol
precipitation (Maniatis et al., 1982).
Global DNA methylation was determined by an Imprint Methylated
DNA Quantification Kit MDQ1 (Sigma-Aldrich, Gillingham, Dorset U.K.).
Plasma tHcy, serum folate and vitamin B12 were determined by chemi-
luminescent methods using Siemens kits and the Immulite analyzer.
Plasma and erythrocyte 5-MTHF concentrations were measured by
HPLC with fluorescence detection by an in-house method based on
Pfeiffer et al. (Pfeiffer et al., 2004; Sobczynska-Malefora et al., 2014a).
Plasma methylmalonic acid (MMA), a functional marker of vitamin
B12 deficiency, was measured by HPLC with fluorescence detection
(Babidge and Babidge, 1994). Hyperhomocysteinemia was defined as
tHcy N14 μmol/L (Selhub et al., 1993). Folate deficiency was defined as
serum folate concentration b 10 nmol/L and vitamin B12 deficiency as
serum vitamin B12 b150 pmol/L according to WHO recommendations
(de Benoist, 2008). Plasma 5-MTHF b6.6 nmol/L and red cells 5-MTHF
b223 nmol/L were considered low (Sobczynska-Malefora et al.,
2014a). An MMA concentration N360 nmol/L was considered to be ele-
vated (Sobczynska-Malefora et al., 2014b).
Serum glucose levels were determined using an enzymatic method.
Insulin concentration was assayed using the ELISA kit (DRG Instruments
GmbH, Germany). The HOMA-IR - homeostatic model assessment index
was calculated as follows: fasting glucose (mmol/L) × fasting insulin
(mU/L)/22.5 (Muniyappa et al., 2008).
Serum apolipoprotein E level was determined by the
electroimmunodiffusion method using Hydragel LP E kits (Sebia,
France).
Paraoxonase 1 activity was determined spectrophotometrically
based on the Kitchen method (Kitchen et al., 1973) using phenylacetate
as a substrate. One unit of activity equaled to 1 μmol of phenol liberated
per minute by 1 mL of serum.
Serum creatinine was determined using a colorimetric kinetic test
based on the Jaffe method.
Selected polymorphisms of four genes APOE, MTHFR, PON1 and
IL1B, which are known to be associated with dementia or other
age-dependent diseases, were identified using the PCR - Restriction
Fragment Length Polymorphism (PCR-RFLP) methods. APOE ε2/ε3/
ε4 polymorphism was investigated using the Hixon and Vernier
(1990) procedure. MTHFR 677C NT polymorphism was identified by
the Frosst et al. (1995) method, PON1 p.Q192R by Humbert et al.
(1993) with minor modifications described by Hasselwander et al.
(1999) and for IL1B-511C N T polymorphism Takamatsu et al.
(2000) method was used.
85
2.3. Statistical analysis
All statistical analyses were performed using Statistica version 10
(Statsoft, Poland). The normality of data distribution was checked by
the Kolmogorov-Smirnov and Shapiro-Wilk tests. Variables that were
skewed were ln-transformed or nonparametric tests were used. Quan-
titative data was expressed as medians with interquartile ranges
(IQR). Differences between the groups with and without dementia
were tested using the nonparametric Kruskal-Wallis analysis of vari-
ance (ANOVA) followed by a post hoc test for multiple comparisons. Dif-
ferences between the two groups with and without dementia were
tested using the Mann-Whitney test. The statistical significance of the
differences in frequencies of the qualitative variables was evaluated
using a Pearson's χ2 test. Correlations were evaluated using Spearman's
test. Multivariate forward stepwise regression analysis was performed
to assess the independent effects of the different factors on global
DNA methylation. The following variables (chosen on the basis of previ-
ous univariate analyses) were included in the model: age, sex, MMSE,
plasma homocysteine, serum folate, 5-MTHF in red cells, serum vitamin
B12, serum creatinine, fasting glucose, fasting insulin, serum apolipopro-
tein E level, and PON1p.Q192R polymorphism, IL1B-511CNT polymor-
phism and MTHFR 677C NT polymorphism. p-Values b 0.05 were
considered statistically significant.
3. Results
The global methylation status of the studied groups is shown in Fig.
1. There was no difference in global DNA methylation between de-
mented and control individuals (p = 0.599). There was only a tendency
for higher global DNA methylation in patients with vascular dementia
(p = 0.061) in comparison to other groups of investigated subjects.
The biochemical markers of folate and vitamin B12 status are shown
in Table 1. The median tHcy (p = 0.012) and MMA (p = 0.016) concen-
trations were significantly higher and folate (p = 0.002) and plasma 5-
MTHF (p = 0.005) concentrations were significantly lower in patients
with dementia than in control subjects. There was a tendency for
lower 5-MTHF in erythrocytes and serum vitamin B12 concentration in
demented subjects (p = 0.056 and p = 0.086, respectively). The preva-
lence of hyperhomocysteinemia and elevated MMA was significantly
higher in patients with dementia than in control subjects (Table 1).
This combination of biochemical results is strongly indicative of lower
folate and vitamin B12 status in dementia compared to control
individuals.
The association of global DNA methylation with various factors in-
volved in homocysteine metabolism and dementia development in
the whole investigated group is shown in Table 2. A significant positive
correlation of global DNA methylation with creatinine and a negative
correlation with vitamin B12, fasting glucose and the APOE level were
found. Borderline negative correlation was observed with the tHcy
and HOMA-IR index.
Fig. 2 shows the association of global DNA methylation with selected
alleles of four genes APOE, MTHFR, PON1 and IL1B, which polymor-
phisms are known to be associated with dementia development. In
the whole investigated group, global DNA methylation was significantly
higher in the PON1 192R allele (p = 0.021) and IL1B-511T allele (p =
0.004) carriers in comparison to non-carriers of those alleles. We also
found a tendency for higher global DNA methylation in non-carriers in
comparison to carriers of the MTHFR 677 T allele (p = 0.090). There
was no association of global DNA methylation with APOE
polymorphism.
Multivariate regression analysis [Table 3] performed in the whole
group of investigated individuals demonstrated significant positive as-
sociations between global DNA methylation and creatinine (p =
0.003), folate (p = 0.013), and IL1B-511T (p = 0.002) and PON1 192R
(p = 0.049) rare alleles and negative associations with fasting glucose
86 M. Bednarska-Makaruk et al. / Experimental Gerontology 81 (2016) 83–91

Fig. 1. Global DNA methylation in various groups of patients and controls. AD - Alzheimer's disease; MCI - mild cognitive impairment; MD - mixed dementia; VaD - vascular dementia.

(p = 0.004). It was shown that the variables included in the model ex-
plained 18.2% of the variance of global DNA methylation.
4. Discussion
In this study we found significantly higher functional markers of fo-
late and vitamin B12 status (tHcy and MMA) and lower folate and 5-
MTHF concentrations in patients with dementia in comparison to con-
trol subjects. Also the prevalence of hyperhomocysteinemia and MMA
N360 nmol/L were significantly higher in dementia than in control sub-
jects. This combination of biochemical findings strongly suggests that
folate and vitamin B12 status in dementia patients may have been
compromised.
Plasma MMA concentration is considered as a functional indicator of
vitamin B12 status and is more sensitive for identifying early changes in
vitamin B12 status i.e. subclinical vitamin B12 deficiency (Green, 2008).
As expected, in the present study the difference in vitamin B12 status be-
tween demented and control subjects were more pronounced by
plasma MMA than serum vitamin B12 concentrations. The differences
in folate status between the investigated groups were more evident
when expressed by serum folate and plasma 5-MTHF (the main circula-
tory form of folate) concentrations than red blood cell 5-MTHF concen-
tration. Our results confirm the importance of folate and vitamin B12
markers associated with homocysteine metabolism in dementia ob-
served by other authors. Moderately elevated levels of tHcy are com-
mon in the population and increase with aging (Selhub et al., 1993)
and have been reported in patients with clinical diagnoses of AD or
VaD (Joosten et al., 1997; Seshadri et al., 2002; Graban, 2005; Wehr
et al., 2009). Low blood concentrations of folate and vitamin B12, and
elevated tHcy levels were also seen in AD (Clarke et al., 1998) and
VaD patients (Malaguarnera et al., 2004).
The mechanisms by which Hcy may damage the brain are not fully
understood. Recent studies have focused on the role of homocysteine
in causing hypomethylation, endothelial dysfunction, oxidative stress
and the role of Hcy thiolactone protein homocysteinylation in brain
damage (Herrmann and Obeid, 2011; Petras et al., 2014).
However it is still unclear, if elevated homocysteine has a causal con-
nection to dementia or it is a biomarker reflecting an underlying patho-
genic biochemical process. Because the elevated homocysteine is
usually associated with folate and vitamin B12 status, it is unclear
whether hyperhomocysteinemia or vitamin deficiency, either alone or
in combination, is responsible for cognitive decline. The risk of develop-
ing cognitive decline could be modified by the dietary folate and vitamin
B-complex intake. Several, but not all vitamin intervention trials have
documented improvement in cognitive functions. The study by Blasko
et al. demonstrated that supplementation with folate with vitamin
B12 in MCI subjects had a protective effect on the brain atrophy associ-
ated with hyperhomocysteinemia and predicted a lower conversion
rate to dementia (Blasko et al., 2012). Similarly, in the randomized con-
trolled VITACOG study it was shown that lowering of homocysteine
with B vitamins slowed global and regional brain atrophy and
prevented cognitive decline in elderly subjects with mild cognitive im-
pairment, in particular in those with elevated homocysteine at baseline
(Smith et al., 2010). However, a meta-analysis of 11 large trials in 22,000
participants revealed that Hcy lowering by using B vitamins had no sig-
nificant effect on individual cognitive domains or global cognitive func-
tion or on cognitive aging (Clarke et al., 2014). The critical review of this
meta-analysis was carried out recently by McCaddon and Miller (2015),

Table 1
Median concentrations with interquartile ranges for homocysteine (tHcy), folate and 5-methyltetrahydrofolate (5-MTHF), vitamin B12 and methylmalonic acid (MMA) in subjects with
and without dementia and the frequencies of these markers outside the cut-offs.

Dementia Non-demented group p-Value Cut-off suggesting Dementia Non-demented group p-Value
sub-optimal status
(n = 102) (n = 92) (n = 102) (%) (n = 92) (%)

n n
tHcy [μmol/L] 102 14.1 [11.0–17.7] 91 12.4 [9.3–14.5] 0.012 N14 μmol/L 51.0 33.0 0.012
Folate [nmol/L] 102 15.4 [10.6–21.3] 90 19.0 [13.1–29.4] 0.002 b10 nmol/L 21.6 11.1 0.052
5-MTHF plasma [nmol/L] 102 13.45 [9.84–19.76] 92 16.89 [11.66–26.59] 0.005 b6.6 nmol/L 7.8 1.1 0.058
5-MTHF erythrocytes [nmol/L] 99 464.4 [394.1–680.4] 92 571.6 [411.5–800.1] 0.056 b223 nmol/L 9.8 7.6 0.589
Vitamin B12 [pmol/L] 102 222.9 [174.7–269.0] 90 242.5 [177.6–340.5] 0.086 b150 pmol/L 20.6 11.1 0.075
MMA plasma [nmol/L] 102 226.3 [141.1–354.1] 92 186.8 [99.8–308.3] 0.016 N360 nmol/L 23.5 12.0 0.036

Values on left side of the table are presented as median [interquartile range - IQR]; non-demented group - controls and subjects with mild cognitive impairment; MMA - methylmalonic
acid; 5-MTHF - 5-methyltetrahydrofolate; tHcy - total homocysteine.
 M. Bednarska-Makaruk et al. / Experimental Gerontology 81 (2016) 83–91 87

Table 2 Table 3
Spearman's correlation coefficients (R) of global DNA methylation levels with various in- Factors associated with global DNA methylation based on multivariate stepwise regression
dicators of homocysteine and glucose metabolism traits in the whole group of investigated analysis in the whole group of investigated elderly individuals.
elderly individuals.
Variables β ΔR2 p
Variable n R p
Creatinine 0.242 0.048 0.003
Age 186 0.074 0.318 IL1B -511T allele 0.198 0.050 0.002
tHcy 185 −0.132 0.073 Fasting glucose# −0.179 0.040 0.004
Folate 185 0.102 0.165 Folate# 0.238 0.029 0.013
5-MTHF plasma 186 0.054 0.465 PON1 192R allele 0.147 0.018 0.049
5-MTHF erythrocytes 183 −0.028 0.704 5-MTHF erythrocytes# −0.170 0.014 0.079
Vitamin B12 185 −0.154 0.036 Vitamin B12# −0.119 0.010 0.147
MMA plasma 186 −0.108 0.142 MTHFR 677T allele −0.080 0.008 0.181
Fasting glucose 186 −0.165 0.024 tHcy# −0.085 0.005 0.297
Fasting insulin 183 −0.113 0.127 R2 adj = 0.182
HOMA index 183 −0.139 0.061 p b 0.000001
Creatinine 185 0.227 0.002
# - logarithmically transformed in statistical analysis; β - the standard regression coeffi-
apoE level 185 −0.156 0.033
cient; R2 (adj) - the multiple coefficient of determination (adjusted); IL1B -511CNT poly-
PON1 activity 186 −0.001 0.984
morphism (T allele carrier i.e. CT or TT genotype = 1; CC genotype = 0;); IL1B - gene
MMSE 186 0.028 0.706
coding interleukin 1 beta; PON1 p.Q192R polymorphism (R allele carrier i.e. QR or RR
tHcy - total homocysteine; 5-MTHF - 5-methyltetrahydrofolate; MMA - methylmalonic genotype = 1; QQ genotype = 0); PON1 - gene coding paraoxonase1, enzyme of antiox-
acid; HOMA index - homeostatic model assessment index; apoE - apolipoprotein E; idant activity; MTHFR c.677CNT polymorphism (T allele carrier i.e. CT or TT genotype = 1;
PON1 - paraoxonase 1; MMSE - Minimental State Examination. CC genotype = 0); MTHFR - gene coding methyltetrahydrofolate reductase; 5-MTHF - 5-
methyltetrahydrofolate; tHcy - total homocysteine.

who concluded that careful examination of the trials in the meta-

analysis of Clarke et al. indicates that no conclusion can be made regard-
ing the effects of homocysteine-lowering on cognitive decline, since the
trials typically did not include individuals who were experiencing such
decline and there is a need for further trials in older adults experiencing
cognitive decline (McCaddon and Miller, 2015).
Although in the present study the investigated biochemical compo-
nents of the homocysteine remethylation pathway indicated sub-
optimal status in the patients with dementia, we were unable to dem-
onstrate the differences in the global methylation of DNA derived
from blood leukocytes measured using an ELISA method between pa-
tients with and without dementia. This could be explained by the
small number of patients in various dementia groups, in particular in
vascular dementia group. The other reason could relate to interactions
between mixed pathologies combining both neurodegenerative and
vascular components (Korczyn, 2005). The neurodegenerative
symptoms are dominating in AD whilst the contribution of degenerative
and vascular factors is similar in MD.
Several studies have shown altered DNA methylation in AD. Most of
them have analyzed methylation status of the promoter regions associ-
ated with a priori candidate genes i.e. genes related to AD pathology. In
early reports a hypomethylation in the promoter region of the beta am-
yloid precursor (APP) gene in the temporal cortex of AD patients (West
et al., 1995) as well as of presenilin 1 (PSEN1) gene (Fuso et al., 2005)
were detected in a patient with AD. Another study showed that al-
though DNA methylation was decreased in the promoter region of the
microtubule-associated protein tau (MAPT) gene in the parietal cortex,
its transcription was down-regulated (Tohgi et al., 1999).
However, subsequent studies find no significant difference in meth-
ylation level of APP gene in frontal cortex, parietal cortex, and hippo-
campus of AD patients as compared with controls (Wang et al., 2008;

Fig. 2. Effect of APOE, MTHFR, PON1 and IL1B genes polymorphisms on global DNA methylation in the whole group of investigated elderly individuals. APOE - apolipoprotein E; IL1B -
interleukin 1 beta; MTHFR - methylenetetrahydrofolate reductase; PON1 - paraoxonase 1. APOE ε4 allele: carriers n = 63; non-carriers n = 119 (four individuals with ε4/2 genotype
were excluded because of the opposite effect of ε4 and ε2 allele on dementia risk); MTHFR T allele carriers n = 94; non-carriers n = 92; PON1 192 R allele carriers n = 93; non-
carriers n = 93; IL1B-511 T allele carriers n = 101; non-carriers n = 85.
88 M. Bednarska-Makaruk et al. / Experimental Gerontology 81 (2016) 83–91
Barrachina and Ferrer, 2009). Similarly, PSEN1 gene hypomethylation
and significant differences in DNA methylation in the promoter of
MAPT gene have not been observed in frontal cortex and hippocampus
of post-mortem AD samples (Wang et al., 2008; Barrachina and Ferrer,
2009). In the recent study Tannorella et al. (2015) showed no difference
in the mean methylation levels of six candidate AD genes involved in
amyloid-beta peptide production (PSEN1 and BACE1), in DNA methyla-
tion (DNMT1, DNMT3A and DNMT3B), and in one-carbon metabolism
(MTHFR) between 120 LOAD patients and 115 neurologically healthy
controls (Tannorella et al., 2015). However, Rao et al. demonstrated
that gene-specific CpG methylation in AD brains is not always represen-
tative of changes occurring at global levels (Rao et al., 2012).
Although it is clear that on the whole-genome scale DNA methyla-
tion is altered in AD cases, no firm conclusion about the global DNA
methylation changes in AD brain tissues can be drawn from the previ-
ous studies as both hypo- and hypermethylation and no significant dif-
ferences have been reported (Lu et al., 2013; Sanchez-Mut and Gräff,
2015).
Using antibodies that recognize methylated DNA, a loss of DNA
methylation has been observed in the entorhinal cortex (Mastroeni
et al., 2010) and the hippocampus of post-mortem samples of AD
(Chouliaras et al., 2013). In contrast, in the study of Lashley et al. no sig-
nificant differences were found between AD and control cases in global
DNA methylation levels in the entorhinal cortex using immunohisto-
chemistry and enzyme-linked immunosorbent assays (Lashley et al.,
2015). Conversely, other studies reported even increased level of DNA
methylation in the frontal and the temporal cortex (Coppieters et al.,
2014) and the hippocampus of AD samples (Bradley-Whitman and Lov-
ell, 2013). Interestingly DNA hypomethylation tendency in AD brain tis-
sues is mainly supported by immunoassays, but the opposite tendency
has been observed through bisulfite treatment.
Recent advances in microarray and genomic sequencing technolo-
gies allow performing the studies of the epigenome, particularly for
DNA methylation, across much larger sample and loci collections at
the same time. However, so far the published epigenome-wide associa-
tion studies (EWAS) showed many genes in brain tissue with methyla-
tion differences between AD and controls, with almost every single
study reporting a different subset of altered genes (Sanchez-Mut and
Gräff, 2015). Only two different genes have been reported to be
hypermethylated by two independent groups, namely SORBS3
(Siegmund et al., 2007; Sanchez-Mut et al., 2013) and ANK1 (De Jager
et al., 2014; Lunnon et al., 2014).
The recently published study of complete human DNA methylomes
of the prefrontal cortex of Alzheimer's disease, dementia with Lewy
bodies, Parkinson's disease and Down's syndrome patients at base reso-
lution using the whole-genome bisulfite sequencing (WGBS) showed
that those neurodegenerative diseases share similar aberrant CpG
methylation shifts targeting a defined gene set (Sanchez-Mut et al.,
2016).
So far, studies investigating DNA methylation changes in AD have
been mainly conducted in human postmortem brain tissues. Moreover,
DNA methylation pattern in human brain changes during the lifespan
(Siegmund et al., 2007). Therefore exploring methylation markers of
AD in ante-mortem peripheral blood cells is of great interest. A limited
number of studies have attempted to assess the methylation profiles
in dementia using peripheral blood derived DNA samples and the re-
sults are conflicting. A study of Bollati et al. (2011) shows a very small
increase in DNA methylation in repetitive elements of Alu, LINE-1 and
SAT-α sequences measured by pyrosequencing method in patients
with AD as compared to controls. Unexpectedly, the group with the
best cognitive performances in MMSE showed higher levels of methyl-
ation in comparison to the group with worst performances (Bollati
et al., 2011). The increase in genome-wide DNA methylation, measured
by luminometric methylation assay, in 37 LOAD subjects as compared to
44 healthy controls was also found by Di Francesco et al. (2015). On the
contrary, Hernandez et al. did not find any significant difference in
global LINE-1 DNA methylation status between patients with late
onset of AD and controls assayed using a methylation-sensitive high-
resolution melting quantitative assay (Hernandez et al., 2014). Wang
et al. assayed DNA methylation across the 12 potential Alzheimer's sus-
ceptibility loci including APP, PSEN1, BACE1, MTHFR and APOE genes in
brain tissue and peripheral leukocytes. Although no significant differ-
ences in individual gene-specific DNA methylation between AD and
controls were detected the combined analysis revealed the epigenetic
distance from the norm (the median methylation of the healthy control
individuals) was higher in brains of people with AD than in healthy con-
trols, and that this ‘epigenetic distance’ increased with age. It was an in-
teresting finding that lymphocytes in people with AD also showed an
age-specific epigenetic drift, although the differences were smaller
than in brain tissue. The authors speculated that the age-related differ-
ences in methylation state and epigenetic drift between lymphocytes
and brain tissue resulted from the more frequent renewal of lympho-
cytes, which may be associated with a more efficient removal of
epimutations (Wang et al., 2008).
The absence of differences in the global DNA methylation in whole
blood cells in our population of subjects with and without dementia
does not imply the lack of alterations in DNA methylation in specific
loci. A limitation of this study is that we didn't analyze the methylation
status of the promoters of genes involved in the pathophysiology of
dementia.
In our study, out of the investigated polymorphisms of four genes
APOE, MTHFR, PON1 and IL1B, which are known to be associated with
dementia or homocysteine metabolism, only the rare alleles T and R
of IL1B-511C N T and PON1 p.Q192R polymorphisms were associated
with global DNA methylation, both in univariate and multivariate
analyses.
Interleukin-1beta is a potent proinflammatory cytokine and is over-
expressed in Alzheimer's disease (Mrak and Griffin, 2001). It was shown
that the T allele of IL1B-511CN T promoter polymorphism determined
enhanced expression of the gene and increased levels of the cytokine
in the plasma. The T allele was considered to be a risk factor in
Alzheimer's disease (McGeer and McGeer, 2001). We found a positive
association of IL1B -511 T allele with global DNA methylation in the
whole group of investigated subjects.
Our previous observations showed that paraoxonase 1 (PON1) activ-
ity, an antioxidant enzyme associated with HDL, was decreased in the
forms of dementia with prevailing neurodegenerative symptoms. It
was shown that 192Q allele of the p.Q192R polymorphism in the coding
region of the gene is associated with more effective protection against
LDL oxidation than 192R allele (Aviram et al., 1998). Although we
found no correlation of global DNA methylation with PON1 activity we
found a positive association PON1 192R allele with global DNA methyl-
ation in whole group of investigated subjects.
The APOE ε4 allele plays a significant disadvantageous role in AD and
other types of dementia development (Saunders et al., 2000; Wehr
et al., 2000). It has been suggested that AD risk correlates with APOE
gene transcription activity (Lahiri, 2004) and serum apoE concentration
could indicate the extent of the APOE gene expression. Although we
found no association of global DNA methylation with APOE polymor-
phism, we found a negative correlation of global DNA methylation
with serum apoE level in univariate analysis. In hypomethylation state
the increased level of disadvantageous apolipoprotein E4 isoform in
the brain of Alzheimer's disease subjects could increase the develop-
ment of AD pathology.
In recent work Pearce et al. (Pearce et al., 2012) quantified DNA
methylation in Long Interspersed Nucleotide Element 1, which is an
index of global methylation, in a population-based sample of 228 indi-
viduals. They evaluated its association with various biomarkers of met-
abolic health such as fasting glucose and lipid levels which were
anticipating the presentation of some diseases. Unexpectedly those
markers which are known to be the risk factors of vascular disease
were positively associated with DNA methylation though it has been
 M. Bednarska-Makaruk et al. / Experimental Gerontology 81 (2016) 83–91
repeatedly shown that a characteristic feature of vascular disease is DNA
hypomethylation. The authors proposed a coexistence of multiple con-
founding influences. In the present study fasting serum glucose level,
which is a well-known risk factor in vascular disease, type 2 diabetes
and also dementia, was negatively associated with DNA methylation,
both in univariate and multivariate analyses. Other indices of insulin re-
sistance i.e. serum insulin and HOMA-IR were not significantly associ-
ated with global DNA methylation. The similar association of DNA
hypomethylation, measured as global leukocyte DNA methylation,
with hyperglycemia was stated by Luttmer et al. (2013) in the
population-based group of 738 subjects.
Our study suggests that the association of various biochemical pa-
rameters with global DNA methylation may give a proxy estimate
DNA methylation intensity. We found the serum creatinine and folate
levels to be independently positively associated with global methyla-
tion in our group of elderly subjects.
Serum creatinine level shows a particularly high association with
DNA methylation because its precursor, creatine synthesis is one of
the main methylation pathways. Creatine is a constituent of a normal
diet and is also synthesized endogenously. Creatine synthesis is esti-
mated to consume between 40% to 70% of available methyl groups pro-
vided by SAM. The primary physiological function of creatine is to buffer
energy concentrations in tissues with significant and fluctuating energy
demands, especially in muscles and the brain. It is becoming increas-
ingly evident that endogenous creatine plays a critical role in a range
of cognitive functions (Allen, 2012). However the creatinine level can-
not be used as a global DNA methylation index because serum creati-
nine level depends significantly on renal excretion and kidney
function. Creatine level could possibly be a better index of efficiency of
methylation pathways in the body.
The evidence from the recent studies suggests that low folate status
is associated with decreases in global DNA methylation (Crider et al.,
2012). The folate is the most important source of the one carbon
group used to methylate DNA. Only several studies have examined the
effects of controlled folate status on global DNA methylation in leuko-
cytes in humans. For example Rampersaud et al. (2000) found that
moderate folate depletion increases plasma homocysteine and de-
creases global DNA methylation in postmenopausal women, while
Friso et al. (2002) showed that the association between lower global
DNA methylation and lower plasma folate concentration was MTHFR
genotype-dependent. The results of our study confirmed the indepen-
dent positive association between serum folate and global DNA methyl-
ation in the group of elderly subjects with and without cognitive
impairment. It seems that serum folic acid level could possibly serve
as an indicator of DNA methylation intensity.
The present study has some limitations, some were already men-
tioned earlier. As we had no previous data about the level of the global
DNA methylation determined using the ELISA-based method in patients
with various types of dementia, we were not able to determine the
number of participants in each group to detect statistically significant
differences in mean methylation levels. The calculated power of the
ANOVA used to compare the global DNA methylation level between
the investigated groups is approximately 50%. Assuming that the
power of the test should be at least 80%, the size of the groups should ex-
ceed 28 individuals in each group. We did not have sufficient number of
patients in the VaD group (n = 17) to reach such power of the test. Be-
cause this study had a small sample size, it's results should be regarded
as preliminary. We recommend the design of larger case-control studies
to further address this issue.
The limitation of the global DNA methylation approach used in the
present study should also be underlined. Leukocyte DNA has been
used for measurements, but the results may not be the same for DNA
from other tissue sources, including brain. Our finding of a lack of differ-
ence in leukocyte global DNA methylation in patients with various types
of dementia does not rule out altered methylation of specific promoter
regions involved in the etiology of the disease. Further studies of
89
genome-wide DNA methylation in specific cell types to analyze its pos-
sible role in the expression of particular genes would be of importance.
5. Conclusions
Global DNA methylation was associated with markers of folate sta-
tus, creatinine, glucose and PON1 and IL1B polymorphisms.
The biochemical results showed significantly lower folate and vita-
min B12 status in demented patients than controls.
Although biochemical markers associated with methylation pro-
cesses indicated sub-optimal status, we were unable to demonstrate
the diagnostic utility of the global DNA methylation ELISA assay in pa-
tients with dementia.
Conflict of interest
There is no conflict of interest regarding the manuscript.
Acknowledgments
This work was supported by statutory subsidies from the state bud-
get of Ministry of Science and Higher Education, Poland.
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