Asian Journal of Psychiatry 12 (2014) 5862

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Asian Journal of Psychiatry

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

Reduced telomere length in subjects with dementia and diabetes

mellitus type 2 is independent of apolipoprotein E4 genotype
Lakshmi Narayanan Kota, Srikala Bharath *, Meera Purushottam, Pradip Paul,
Palanimuthu Thangaraju Sivakumar, Mathew Varghese, Sanjeev Jain
Department of Psychiatry, National Institute of Mental Health and Neurosciences, Bangalore, India

A R T I C L E I N F O A B S T R A C T

Article history: Apolipoprotein E4 gene is associated with increased risk of dementia with comorbid diabetes mellitus.
Received 28 November 2013 Both dementia and diabetes mellitus type 2 are independently associated with telomere shortening. We
Received in revised form 10 June 2014 assessed relative telomere length and apolipoprotein E genotype in subjects with dementia (n = 70) and
Accepted 14 June 2014
cognitively normal control groups (n = 55) with and without comorbid diabetes mellitus type 2. Relative
telomere length was highest in the control group (Q2 = 0.91) followed by dementia (Q2 = 0.48) and
Keywords: dementia with comorbid diabetes mellitus type 2 (Q2 = 0.39). Apolipoprotein E4 allele frequency was
Apolipoprotein E4
highest in dementia with comorbid diabetes mellitus type 2 (0.26). Apolipoprotein E4 allele was not
Dementia with diabetes
signicantly associated with telomere attrition in both dementia and cognitively normal group
Telomere
irrespective of comorbid diabetes mellitus type 2 (P > 0.05). The ndings suggest that relative telomere
length is unrelated to apolipoprotein E4 genotype in dementia and cognitive normal subjects with or
without comorbid diabetes mellitus type 2.
2014 Elsevier B.V. All rights reserved.

called telomerase (Greider and Blackburn, 1985, 1987). The

1. Introduction
Telomeres are repetitive DNA sequences at the ends of
eukaryotic chromosomes (Moyzis et al., 1988). Without telo-
meres, the ends of chromosomes would be recognized as
double stranded DNA breaks by DNA repair machinery. During
S phase of every cell replication, the telomeres replicate
incompletely and become shortened by a few base pairs
(Olovnikov, 1971, 1973; Watson, 1972). Under normal circum-
stances, this shortening is corrected by an RNA enzyme complex
Abbreviations: AD, Alzheimers disease; Ct, cycle threshold; DM, diabetes mellitus
type 2; ICD-10, international classication of diseases-10; PBL, peripheral blood
leukocyte; PGC1a and PGC1b, PPARg co-activators; PPARg, peroxisome prolif-
erator activated receptors; Relative T/S, relative telomere/single copy gene; RTPCR,
real time polymerase chain reaction.
* Corresponding author at: National Institute of Mental Health and Neuros-
ciences (Deemed University), Bangalore 560 029, India. Tel.: +91 80 2699 5271;
fax: +91 80 2656 2121/4830.
E-mail addresses: lakshmineuroscience@gmail.com (L.N. Kota),
srikala.bharath@gmail.com (S. Bharath),
meera.purushottam@gmail.com (M. Purushottam),
paul.pradipp@gmail.com (P. Paul),
sivakumar.nimhans@gmail.com (P.T. Sivakumar),
mat.varg@yahoo.com (M. Varghese), sjain.nimhans@gmail.com (S. Jain).
telomerase and telomere associated shelterin proteins are
suggested to have an important role in telomere homeostasis
inuenced by specic haplotypes representing these proteins
(Codd et al., 2010). Short telomeres are associated with aging and
degenerative phenotypes like dementia, diabetes and atheroscle-
rosis (Von Zglinicki et al., 2000; Thomas et al., 2008; Lof-Ohlin
et al., 2008; Kume et al., 2012; Honig et al., 2012; Balasubrama-
nyam et al., 2007).
Apolipoprotein E4 allele is an established risk factor for
developing dementia (Bharath et al., 2010). Apolipoprotein E4
isoform is involved in the build-up of insoluble Ab amyloid protein
in neurons (Jiang et al., 2008a) and also increased plasma
cholesterol levels (Lane and Farlow, 2005). These events end in
disruption of cellular homeostasis as the effects of increased
amyloid or plasma cholesterol contribute to degenerative pheno-
type leading to neuronal loss or atherosclerosis.
As dementia is associated with apolipoprotein E4 genotype
and low telomere length, it is important to explore inuence of
apolipoprotein E4 genotype on telomere length. Earlier studies
had suggested signicant association to no association of
telomere attrition with apolipoprotein E4 status in dementia
and cognitively normal group (Takata et al., 2012; Honig et al.,
2006; Starr et al., 2008; Jacobs et al., 2013). One study had

http://dx.doi.org/10.1016/j.ajp.2014.06.012
1876-2018/ 2014 Elsevier B.V. All rights reserved.
 L.N. Kota et al. / Asian Journal of Psychiatry 12 (2014) 5862
suggested longer relative telomere length with apolipoprotein E4
genotype in cognitively normal (Wikgren et al., 2012). These
ndings suggest that the relationship between telomere length
and apolipoprotein E genotypes may not be linear and warrants
further scrutiny. Further studies could unravel intermediate
cellular pathways and other phenotypes linking apolipoprotein
E gene with telomere length explain whether longer or shorter
telomere length observed is a consequence of low replicative
potential (as replication events tend to shorten telomere) or
merely reect higher replicative capacity (longer telomere mean
higher replicative potential).
Diabetes mellitus type 2 (DM) is associated with dementia
(Peila et al., 2002) and telomere attrition (Balasubramanyam et al.,
2007; Adaikalakoteswari et al., 2005). The Indian population has
higher rates of DM (Diabetes, 2006) but lower frequencies of
apolipoprotein E4 compared to western populations (Bharath
et al., 2010). Our earlier study had shown higher rates of DM in
dementia subjects with apolipoprotein E4 allele (Kota et al., 2012).
With increasing rates of dementia (Shaji et al., 2010) and DM
(Diabetes, 2006) in India, it is necessary to understand the
molecular basis of these diseases.
Peripheral blood leukocyte telomere length (PBL) is sug-
gested to be a surrogate for global cellular replicative capacity
(Sahin and DePinho, 2012) and altered PBL cellular viability
was established in subjects with dementia (Yoon et al., 2010).
We studied apolipoprotein E4 allele frequencies and PBL relative
telomere length in cognitively normal subjects & people with
dementia with and without comorbid DM from a predominantly
southern Indian population.
2. Subjects and methods
2.1. Subjects
Subjects (N = 125) were recruited from the geriatric clinical
services of the Department of Psychiatry, National Institute of
Mental Health and Neurosciences, Bangalore. Dementia cases
(n = 70) were diagnosed clinically by psychiatrists using interna-
tional classication of diseases (ICD-10) criteria. Hindi Mental
Status Examination (Ganguli et al., 1995) was used to assess
cognitive ability of the subjects. Cognitively normal subjects
(n = 55) with no lifetime history of neuropsychiatric illness or
family history of neuropsychiatric illness were recruited after age
group matching with dementia subjects. Comorbid DM, hyperten-
sion and ischemic heart disease were ascertained by clinical
history.
2.2. Methods
The study was approved by institutional ethics committee.
After informed written consent, ten milliliters of venous blood
was collected, and DNA was isolated using salting out method
(Miller et al., 1988). The real time quantitative polymerase
chain reaction (RTPCR) was setup for relative telomere length
estimation using Applied Biosystems 7500 real time PCR
system (Cawthon, 2002). The telomere sequence and a single
copy gene in a known quantity of DNA were amplied
simultaneously and the results were recorded as number of
cycles of RTPCR required to reach threshold uorescence (Ct or
cycle threshold). The Ct value is inversely related to the
initial copy number of telomere or single copy gene. The T/S
ratio (or DCt = Ct of telomere amplication  Ct of single copy
gene amplication) reects number of copies of telomere in
each cell in an assay. The relative T/S ratio which was calculated
as difference between the DCt of unknown sample and
normalized for DCt of reference DNA enables comparison of
59
relative T/S ratio across assays. The following formula was used
to calculate the relative T/S ratios. DCt (unknown sample)  DCt
(reference sample) = DDCt; rT=S ratio 2DDC t .
The reference sample was an arbitrary sample that was
included in all experimental assays. Each sample was assayed in
duplicate and Ct values which showed s.d. > 1 between the
technical replicates were not considered for analysis. Experi-
ments were repeated after recoding the DNA samples to
eliminate sample bias.
To detect the apolipoprotein E genotype, polymerase chain
reaction (PCR) was done using sequence-specic primer PCR
methodology (Pantelidis et al., 2003). The presence of 173-bp
band was indicative of the specic apoliopoprotein E haplotype.
The results were corroborated with RTPCR results for 10% of the
samples in every experiment (Kota et al., 2012).
2.3. Reproducibility of the measured relative T/S ratios
Replicate assays of sample and reference were setup at different
times to calculate the inter assay variation. The coefcient of
variation (square root of variance/mean) as calculated by
measuring relative T/S ratios of a sample repeated over four
different assays was 6.7%. Samples differing in average telomere
length by as little as 13% (1.96  s.d.) should be distinguishable by
this method at the 95% condence interval (Cawthon, 2002).
2.4. Statistical analysis
Statistical tests were done using R software (Fox, 2005) and
QTI-plot (Vasilief, 2011). Chi square test (x2) was used for
comparing categorical variables. Mann Whitney U test (U, two
tailed p) and Kruskal Wallis H test were used for comparison
between groups of continuous variables as the relative T/S ratios
and log transformed relative T/S ratios calculated were not
normally distributed and non-parametric tests are most useful
for small samples (Fagerland, 2012; Hart, 2001). Spearmans
rank correlation (rs, two tailed p) and partial correlation (r, two
tailed p) coefcients were used for correlation analysis.
3. Results
3.1. Comparison of relative T/S ratios: age, gender and phenotype
The compared groups (Table 1) were age group and gender
matched. There was a trend of age related decline of relative T/S
ratios in the cognitively normal group (rs = 0.218; p = 0.11).
There was a trend of lower relative T/S ratios in those who
develop dementia earlier in their lives (rs = 0.104, p = 0.39).
Dementia group showed signicantly lower relative T/S ratio
compared to the cognitively normal group after adjusting for
age, gender, hypertension, diabetes and ischemic heart disease
(p = 0.001; Fig. 1A). The relative T/S ratios compared between
subtypes of dementia or severity of dementia showed no
signicant association within the sub-groups (p > 0.05). Relative
T/S ratios in DM group did not differ signicantly from non-
diabetic group (Fig. 1A). Relative T/S ratios were signicantly
lower in dementia with co-morbid DM (Fig. 1B).
3.2. Relative T/S ratios comparison: apolipoprotein E4 and phenotypes
The apolipoprotein E4 allele frequency was signicantly higher
in dementia group (0.21) compared to the cognitively normal
group (0.055). Dementia subjects with comorbid DM had higher
apolipoprotein E4 allele frequency (0.26) compared to cognitively
normal with DM (0.07) though dementia group had signicantly
low comorbid DM compared to cognitively normal group. There
60 L.N. Kota et al. / Asian Journal of Psychiatry 12 (2014) 5862

Table 1
Clinical characteristics and apolipoprotein E genotype of subjects recruited for the study. The dementia and cognitively normal group are age
group and gender matched. The dementia group had signicantly lower rates of DM and higher apolipoprotein E4 allele frequency compared to
the cognitively normal group.

N = 125 Dementia* (n = 70) Cognitively Statistical tests

normal (n = 55)

Age at blood draw 65.9  6.4 64.6  10.1 U = 1755, P = 0.39

Gender M:F 35:35 26:29 x2 = 0.09, df = 1, P = 0.76
HMSE 11.16  7.7 30.78  0.66 U = 0, P < 0.0001
DM 19 28 x2 = 7.4, df = 1, P = 0.006
Hypertension 31 40 x2 = 10.2, df = 1, P = 0.001
IHD 4 12 PF = 0.002
Apo E status (n)
E 23 3 5 PF = 0.008
E 33 41 44
E 24 2 0
E 34 20 6
E 44 4 0
ApoE4 allele frequency 0.21 0.055 P < 0.0001

DM, diabetes mellitus type 2; IHD, ischemic heart disease; F, Fishers exact test; U, MannWhitney test; x2, Pearsons Chi Square test.
*
Predominantly of Alzheimers disease (AD, n = 57).

Fig. 1. Box plot comparing relative T/S ratios (A) Between individual phenotypic groups. *P adjusted for age, gender, hypertension, diabetes and ischemic heart disease by
partial correlation analysis. **P adjusted for age, gender, dementia, hypertension and ischemic heart disease by partial correlation analysis. (B) With or without comorbid DM.
(C) Within dementia and cognitively normal groups with respect to apolipoprotein E4 homozygote, heterozygote and non-ApoE4 status. The lower, middle and upper
horizontal lines of the box represent 25th, 50th and 75th percentiles respectively (Q1, Q2, and Q3). DM, diabetes mellitus type 2; Apo E4, apolipoprotein E4.
 L.N. Kota et al. / Asian Journal of Psychiatry 12 (2014) 5862 61

Table 2
Relative T/S ratios in subjects with and without apolipoprotein E4 allele. Presence of apolipoprtein E4 allele is not signicantly associated with low relative T/S ratio in any of
phenotypic groups. Q1, Q2 and Q3 represent 25th, 50th and 75th percentile. DM, diabetes mellitus type 2.

1 Apo E4 allele No Apo E4 allele Statistical test

n Relative T/S ratio n Relative T/S ratio

Q1 Q2 Q3 Q1 Q2 Q3

Total, n = 125 32 0.36 0.62 0.97 93 0.37 0.71 1.25 U = 1310.5, P = 0.315
Dementia, n = 70 26 0.31 0.56 1.01 44 0.35 0.46 0.9 U = 539, P = 0.69
Cognitively normal, n = 55 6 0.51 0.78 1 49 0.51 0.98 1.38 U = 118, P = 0.45
DM, n = 47 9 0.33 0.52 1.06 38 0.35 0.58 1.04 U = 162, P = 0.821
No DM, n = 78 23 0.37 0.65 0.9 55 0.37 0.87 1.34 U = 523, P = 0.23
Dementia with DM, n = 19 5 0.25 0.35 0.87 14 0.25 0.41 0.72 U = 34, P = 0.964
Cognitively normal with DM, n = 28 4 0.56 0.86 1.19 24 0.48 0.77 1.33 U = 47, P = 0.975

was no signicant correlation between the presence of apolipo-
protein E4 and relative telomere length in dementia, diabetes and
cognitively normal as individual groups or combination groups
(Table 2). Homozygosity for apolipoprotein E4 allele did
not correlate with telomere attrition in dementia group (Fig. 1C).
4. Discussion
Our data conrms the association of apolipoprotein E4 with
dementia, compared to cognitively normal group (Bharath et al.,
2010; Kapur et al., 2006; Luthra et al., 2004; Ganguli et al., 2000;
Chandak et al., 2002). We also observed telomere attrition in PBL
of subjects with dementia compared to cognitively normal
group, like earlier studies (Von Zglinicki et al., 2000; Thomas
et al., 2008; Lof-Ohlin et al., 2008; Kume et al., 2012). Dementia
subjects with comorbid DM had signicant telomere attrition
(P = 0.046). However, the telomere attrition seemed to be
independent of apolipoprotein E4 allele in both dementia and
cognitively normal groups, irrespective of comorbid DM.
Takata et al. (2012) did not nd telomere attrition in AD
group (n = 74) compared to cognitively normal elderly (n = 35),
but a strong association of apolipoprotein E4 homozygosity
with telomere attrition in AD group was observed. We
observed telomere attrition in subjects with dementia (pre-
dominantly AD, n = 57) and there was no signicant correlation
with apolipoprotein E4 homozygosity (Fig. 1C). This may be
expected as apolipoprotein E4 allele frequencies and the
strength of its association with dementia depend upon
the levels of penetrance (Genin et al., 2011). Honig et al.
(2006) associated dementia with telomere attrition (P = 0.034),
but no correlation was found between telomere attrition and
apolipoprotein E4 status per se. However, low telomere
length in subjects (aged 83  8 years) with apolipoprotein E4
was correlated with signicantly increased odds of mortality on
18-month follow-up. Our dementia subjects were younger, aged
66  6 years and our study was cross sectional.
Other studies that link apolipoproein E4 allele with telomere
length were done on cognitively normal groups. Starr et al.
(2008) had reported shortened telomere with apolipoprotein E4
carriage in cognitively normal subjects aged 79 years. Jacobs
et al. (2013) had observed telomere attrition with apolipopro-
tein E4 carriage in postmenopausal women that improved by
hormonal therapy during follow-up. Wikgren et al. (2012) had
associated longer relative telomere length with apolipoprotein
E4 status in cognitively normal, however the telomere attrition
rates per year were highest in the presence of apolipoprotein E4
allele in that study. Our study did not nd correlation between
relative telomere length and apolipoprotein E4 status in
cognitively normal and had lower apolipoprotein E4 carrier
rates (11% vs. 29%) and higher DM rates (51% vs. 4%) compared
to Wikgren et al. (2012).
Adaikalakoteswari et al. (2005) had shown correlations of
telomere attrition with Asian Indian type 2 DM. We did not observe
this in our cognitively normal subjects with DM. However, subjects
with both dementia and comorbid DM had shown signicant
telomere attrition, thus perhaps linking DM with dementia at sub-
cellular level. This observation adds to other cellular mechanisms
that suggests Alzheimers disease as type 3 diabetes mellitus (De la
Monte and Wands, 2008).
The reduced telomere length observed in dementia and DM is
not inuenced by apolipoprotein E4 genotype in our study
suggesting a complex relationship between apolipoprotein E4
genotype, DM, dementia and relative telomere length. However,
these observations need to be validated with larger sample size of
subjects with dementia of Alzheimers type and co-morbid DM.
One limitation of this study is that diagnosis of DM is by clinical
history alone. A larger cohort of cognitively normal groups with
and without DM is required for comparison.
The apolipoprotein E4 haplotype is related to accelerated
apoptosis (Cash et al., 2012), and hence a short telomere is
expected. However the effects of apolipoprotein E4 may be
partly routed through modier genes like peroxisome prolif-
erator activated receptors (PPARg) (Combarros et al., 2011).
Apolipoprotein E4 genotype is associated with increased plasma
cholesterol and triglycerides consequent to impaired clearance
of chylomicron VLDL and LDL remnants. PPARg can be activated
by these elevated lipid levels (Jiang et al., 2008b).
Sahin et al. (2011) suggested repression of PPARg co-activators
(PGC1a and PGC1b) by P53 activation brought about by telomere
dysfunction. Recently, telomere associated shelterin proteins like
repressor activator protein 1 (RAP1) is identied to modulate lipid
metabolism by binding PPARa and PGC-1a (Martnez et al., 2013).
PPARg modulation inuence metabolic diseases such as hyperlipi-
daemia, insulin resistance, and diabetes (Terauchi and Kadowaki,
2005; Rangwala and Lazar, 2004). These observations may support
telomere dysfunction in subjects with dementia and DM in our study.
The complex interdependence of these molecular pathways
necessitates further studies on factors related to lipid metabolism
for understanding relation between telomere homeostasis in
dementia and DM. The relative telomere length measurements
may be useful clinically in complex diseases like dementia and DM
only if these underlying mechanisms inuencing the telomere
homeostasis are thoroughly explored.
Role of funding source
The study was funded by Indian Council of Medical Research
(ICMR project number: 53/6/2009-BMS). Fellowship for LNK was
supported under MD-PhD programme of ICMR.
62 L.N. Kota et al. / Asian Journal of Psychiatry 12 (2014) 5862
Conict of interest statement
All the authors declare no actual or potential conict of interest
in study undertaken. They further declare that there are no
nancial and personal relationships that might bias their work.
Acknowledgements
We would like to thank Muralidharan J for technical support.
The authors would also like to thank all the patients, their family
members and volunteers for their participation and cooperation.
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