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Current Alzheimer Research, 2016, 13, 1-10 1

RESEARCH ARTICLE

Lack of Association between Genetic Polymorphism of Circadian Genes

(PER2, PER3, CLOCK and OX2R) with Late Onset Depression and Alz-
heimer's Disease in a Sample of a Brazilian Population (Circadian Genes,
Late-Onset Depression and Alzheimer's Disease)

Patricia Araújo Pereiraa,*, António Alvim-Soaresa, Maria Aparecida Camargos Bicalhoa,b,

Edgar Nunes de Moraesa,b, Leandro Malloy-Dinizb, Jonas Jardim de Paulaa,
Marco Aurélio Romano-Silvaa and Debora Marques Miranda

a
INCT de Medicina Molecular, Faculdade de Medicina, Universidade Federal de Minas Gerais, Av Alfredo Balena,
190, Belo Horizonte-MG, CEP 30130-100, Brazil; bCentro Jenny Faria de Assistência à Saúde do Idoso e da Mulher,
Hospital das Clínicas, Universidade Federal de Minas Gerais (UFMG), Brazil

Abstract: Objectives: This study aims to evaluate the association between poly-
morphisms in circadian genes and Alzheimer’s disease (AD) and/or late-onset de-
pression (LOD). AD pathology leads to circadian disturbances, with clear negative
influence on quality of life. In addition, there is an increasing evidence that regula- Please provide
corresponding author(s)
tors of circadian system have effects on AD and LOD pathology. Design and Sub- photograph
ARTICLE HISTORY
jects: An exploratory case-control study designed to evaluate SNPs in the PER2, size should be 4" x 4" inches

Received: January 18, 2016 PER3, CLOCK and OX2R genes in a sample composed by 249 AD, 222 LOD and
Revised: April 10, 2016
Accepted: April 27, 2016 112 healthy individuals. Measures: The participants were evaluated using DSM-IV
DOI: 10.2174/15672050136661606030
criteria for LOD and NINCDS-ADRDA for AD. Results: In allelic analysis, the
05630 OX2R SNP, rs2134294, showed an association of allele C with LOD (p =0.02,
OR= 1.6) and AD (p=0.04, OR =1.5). The rs2134294 also showed a genotypic association C/C (p
=0.01) for higher risk to develop LOD compared to the control group, with an odd’s ratio of 2.7. The
rs9370399 (OX2R) has also shown an association between A allele (p=0.03, OR= 1.4) and AD. These
results do not persist after a 1,000 permutations test. For other markers of the OX2R gene and for all
other markers of this study no association was found. Conclusion: In conclusion, the present study
found that the investigated Circadian Genes (PER2, PER3, CLOCK and OX2R) polymorphisms were
not associated with LOD or AD.
Keywords: Single-nucleotide polymorphism (SNP), Late-onset depression (LOD), Alzheimer’s Disease (AD), gene, Period
Homolog (PER), CLOCK, Orexin Receptor 2 (OX2R).

1. INTRODUCTION depression and risk for AD being controversial [6], several

Alzheimer’s disease (AD) is a neurodegenerative disease
that accounts for most cases of dementia. AD incidence is
approximately 1.5% around 65 years, reaching 30% on aver-
age around 80 years [1, 2, 3]. AD is characterized by an in-
crease in amyloid β protein (Aβ) aggregated into plaques
deposited in the brain [4]. A meta-analysis report showed
that history of depression is a risk factor for dementia [5].
However, no data clearly explains this association. The two
most likely hypotheses were that (a) depression may be a
prodrome of dementia and (b) depression is a risk factor for
development of dementia. Despite the relationship between
*Address correspondence to this author at the INCT de Medicina Molecular,
Faculdade de Medicina, Universidade Federal de Minas Gerais, Av Alfredo
Balena, 190, Belo Horizonte-MG, CEP 30130-100, Brazil;
Tel.: +55 3134099753; E-mail: patriciabiomol@yahoo.com.br.
studies present depression as a dementia symptom [7]. The
signs and symptoms of depression often precede cognitive
decline and dementia [8; 9; 10; 11], but the relation between
depression and dementia is far from clear.
Circadian dysfunction has been described in many psy-
chiatric disorders. In AD, circadian deregulation may exac-
erbate pathology [12]. In depression, symptoms such as sleep
disruption, diurnal mood swings, and elevated nocturnal
body temperature are usually found [12]. Genetic variants in
circadian genes could result in phenotypes predisposing to
lose compensatory mechanisms of the circadian cycle. The
impact of the genetic variation is corroborated by recent re-
search showing that variation in CLOCK and PER3 genes
are associated with differences in sleep-wake pattern [12,
13]. The CLOCK/BMAL1 complex activates transcription of
other core genes: CRY, PER. In turn, CRY and PER proteins

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2 Current Alzheimer Research, 2016, Vol. 13, No. 10
form a complex, which translocates to the nucleus, and sup-
presses transactivation by the CLOCK/BMAL1 complex,
resulting in oscillatory transcription of clock-controlled
genes [13, 14].
Two hypothalamic neuroexcitatory peptides, orexin A
and orexin B (also known as hypocretin 1 and 2, respec-
tively), are strongly associated to the maintenance of wake-
fulness, modulation of the sleep-wake cycle and other
physiological functions with diurnal variations [15], and
may be associated with AD. Both orexins exert their actions
interacting with two G-protein coupled receptors. The
orexin-1 receptor (OX1R) is selective for orexin A, while the
orexin-2 receptor (OX2R) is not. The OX1R and OX2R are
coupled to Gq and Gi proteins [15].
The accumulation of Aβ peptide in the brain is an early
process in AD [16]. A study in mice expressing the human
Aβ precursor protein [4] identified a diurnal variation of ex-
pression in the levels of Aβ at cerebrospinal fluid. These
cyclical changes might be modulated by orexin, as intracere-
broventricular (ICV) infusion of Orexin-A increased the lev-
els of Aβ and 8-week systemic treatment with almorexante,
an orexin-receptor antagonist, led to a significant reduction
of Aβ plaques in various brain regions [4]. In another study,
the ICV administration of Orexin-A, just after a learning
trial, improved the consolidation of learning and retrieval
processes in rats, in a dose-dependent manner [17].
Clearly, AD and depression are associated with circadian
dysfunction, but how these changes modulate the disease
pathogenesis remains unclear. We hypothesized that the neu-
rodegenerative process in AD might be affected by the
orexinergic system, which would contribute to sleep distur-
bances in AD patients. In face of that, this study evaluated
association of circadian genes polymorphisms in the PER2,
PER3, CLOCK and OX2R genes with AD and/or LOD.
2. METHODS
2.1. Clinical Data
A total of 249 AD patients, 222 LOD patients and 112
controls older than 65 years were recruited from the Outpa-
tients Clinic of the Instituto Jenny de Andrade Faria for the
Elderly, Universidade Federal de Minas Gerais (UFMG)
Brazil, between 2006 and 2011. All subjects underwent an
extensive diagnosis and behavioral assessment by trained
professionals.
Based on clinical report, patients were analyzed consider-
ing the following clinical variables: arterial hypertension
(AH), diabetes mellitus (DM), dyslipidemia, coronary artery
disease (CAD), atrial fibrillation, chronic obstructive pulmo-
nary disease (COPD), smoking, alcoholism, cancer, cardiac
failure and osteoarthritis.
All participants underwent an extensive diagnosis and
behavioral assessment by trained professionals. They were
submitted to the same cognitive and functional assessments.
All the instruments were translated and validated to Brazilian
Portuguese. The University’s Ethics committee approved the
protocols. All subjects (patients and/or their caregivers) gave
written informed consent to participate in the study, which
was conducted in accordance with the provisions of the Hel-
sinki Declaration.
Pereira et al.
2.1.1. Alzheimer’s Disease
249 AD patients were included in the study. AD diagno-
sis were performed according to the National Institute of
Neurological and Communicative Disorders and Stroke-
Alzheimer’s Disease and Related Disorders Association
(NINCDS-ADRDA) classification criteria for probable AD
[18].
The evaluation of patients was performed by medical and
family histories, neurological examination, routine blood
tests (as hematology, biochemistry, thyroid-stimulating hor-
mone, vitamin B12 levels, syphilis and HIV serology de-
pending on history), and brain imaging studies (CT or MRI).
The cognitive screening tests and functional assessment in-
cluded the Mini-Mental State Examination [19], CERAD
Protocol [20] and Clock Drawing Test [21] and assessment
of basic and instrumental activities of daily living [22]. All
patients were classified according to the Clinical Dementia
Rating Scale [23]. To avoid inclusion of Vascular Dementia
cases we excluded patients that scored over two points in the
Hachinski Ischemic Scale [24]. Psychiatric or behavioral
disorders were assessed by direct evaluation of the subjects
and by anamnesis with the main caregiver in every day con-
tact with the patient. The Neuropsychiatric Inventory Scale
(25), a structured informer interview was also carried out.
The adopted exclusion criteria were: (a) a history of psy-
chotic disorders, drug use disorder or intellectual disability
in accordance to DSM-IV; (b) cerebrovascular diseases, hy-
drocephalus, and intra-cranial mass, evidenced by neuroi-
maging; (c) abnormal thyroid hormone, serum folate and
Vitamin B12 levels or sexually transmitted diseases serol-
ogy; (d) history of traumatic brain injury or any other neuro-
logical disease; (h) lack of caregiver that could inform prop-
erly patient’s functional capacity.
2.1.2. Late-Onset Depression
222 individuals whose first depressive episode begun af-
ter 65 years were selected to compose the LOD group All of
them underwent a comprehensive diagnostic and behavioral
evaluation by experienced professionals. LOD diagnosis was
performed when the first depressive episode occurred after
65 years, and according to the Geriatric Depression Scale
(GDS; Portuguese validation done by Sheikh & Yesavage,
with a cutoff of 6/15) [26], Diagnostic and Statistical Manual
IV (DSM-IV, Text Revision criteria) and MINI-plus Brazil-
ian Portuguese version (Amorim, 2000) [19]. Patients were
also submitted to the same cognitive and functional assess-
ments as AD patients.
Individuals diagnosed with other psychiatric disorders,
reactive depressive symptoms following a significant life
event, medical conditions or medication, and those with neu-
rological diseases were excluded. All LOD patients were
monitored for at least two years. The cases were discussed
and reviewed regularly by a group of psychiatrists, geriatri-
cians and neuropsychologists.
2.1.3. Comparison Group
112 subjects with no personal or family (first-degree rela-
tives) history of neuropsychiatric disorders were carefully
selected in the same clinic and included as the comparison
Lack of Association between Genetic Polymorphism
group. The healthy subjects were also submitted to all diag-
nostic instruments applied to the other groups..
2.2. Genotyping
Peripheral blood was collected in vacuum tubes from all
individuals and genomic DNA isolated using the high salt
method [27] The selected probes corresponded to the follow-
ing SNPs: OX2R: rs9370399, rs3134705, rs9349768,
rs7741664, rs2134294 e rs2653349 (exonic, Ile/Val); PER2:
rs934945 (exonic, Gly/Glu); PER3: rs10462020 (exonic,
Val/Gly), rs228697 (exonic, Pro/Ala); CLOCK: rs6855837
(exonic, Leu/Ile). All SNPs were chosen using the Hapmap
database (www.hapmap.org) with Minor Allele Frequency
(MAF) of 0.2%. Genotyping was done by PCR-Real Time
using allelic discrimination - Strategene Mx3005 equipment,
(MxPro QPCR System, 2007 Software, La Jolla, CA). PCR
protocols were accomplished according to the TaqMan®
Genotyping Master Mix manufacturer’s instructions (Ap-
plied Biosystems, Foster City, CA).
2.3. Statistical Analysis
Allele, haplotype and genotype frequencies have been
compared among the groups with the χ2 test by using UN-
PHASED software (v.3.0.13) [28]. HAPLOVIEW 4.1 soft-
ware was employed to assess pairwise linkage disequilib-
rium (LD) matrices between each SNP to investigate the LD
block diagram and Hardy-Weinberg equilibrium (cutoff
0.05) [29]. Odds ratio and permutations were obtained by
UNPHASED. 1,000 Permutations test (post test) were done
for each test to estimate the global significance for all analy-
ses and to validate the expectation-maximization values. All
tests were two tailed and significance was set at p<0.05.
Categorical variables were analyzed by the chi-square test,
and continuous variables, by analysis of variance (ANOVA).
After Bonferroni correction, the significance level was estab-
lished at 0.0025 for the genotype x group comparisons.
3. RESULTS
In order to identify genetic association with AD, LOD
and the Circadian Cycle, polymorphisms in the OX2R,
PER2, PER3 and CLOCK genes were genotyped. A total of
ten SNPs from HapMap were genotyped in a sample of 249
AD patients, 222 LOD and 112 comparison groups. All se-
lected SNPs showed a minor allele frequency higher than
10% indicating a good penetration of the alleles in the popu-
lation.
Comparison group showed a mean age of 78 (±7.3)
years, 67,8% were female and MMSE of 24,3 (±4.0). AD
patients have mean age of 79 (±7.5) years, 68,2% were fe-
male and MMSE of 13.1 (±4.7). Comparison group and AD
subjects showed no statistically significant differences con-
cerning educational level (M = 3,09 SD = 2.6 years of formal
education; M = 2.89 SD = 3.1 years of formal education,
respectively). Among 222 individuals with LOD, the mean
absolute number of educational years was 3,18 (± 2.9). The
GDS mean was 9.5 ± 3.7 for LOD.
The participants showed significant differences in sex,
age, cognitive performance and depressive symptoms when
the three groups were compared. For the sociodemographic
Current Alzheimer Research, 2016, Vol. 13, No. 10 3
measures, effect sizes for age (Cohen’s d) were small (com-
parison x AD = 0.01), or small-to-moderate (comparison x
LOD = 0.33, LOD x AD = 0.42) and for sex (Crámer’s V)
were small (0.181); which are unlikely to influence the fol-
lowing analysis. Effect sizes for cognitive performance and
depressive symptoms were large. (Table 1).
In allelic analysis, the OX2R rs2134294, showed an asso-
ciation with LOD (p=0.02, OR= 1.6) and AD (p=0.04, OR
=1.5) for C allele in relation to comparison group. The
rs2134294 also showed a genotypic association C/C (p
=0.01) for the risk of developing LOD compared with the
comparison group, with an odds ratio of 2.7. The allele A of
rs9370399 (OX2R) also showed an association with AD
(p=0.03, OR= 1.4). However, these results did not persist
after 1,000 permutations test. For other markers of the OX2R
gene and for markers of other genes, none association was
observed (Table 2).
We estimate the statistical power for the analysis using
the g*power software [30]. In a post hoc design, computing
the achieved power based in the sample size and degree of
freedoms for chi-square tests we have 99% power for the
identification of a large or moderate effect size as 45% do
detect a small effect size.
4. DISCUSSION
In this association study, we evaluated gene polymor-
phism in late onset depression and Alzheimer’s disease aim-
ing to identify common susceptibility genes for changes on
sleep-wake cycle. A total of 10 SNPs were investigated in
AD, LOD and comparison groups.
It is estimated that more than 24 million people have de-
mentia worldwide, and about 1 million in Brazil [31]. Cur-
rently, increasing the knowledge of the genetic and molecu-
lar biology of AD is necessary [32]. Several risk factors for
AD have been identified, among these factors, depression
deserves special mention [33]. Late onset depression is regu-
larly linked to cognitive impairment and could be an initial
manifestation of dementia or even a prodrome of dementia
[34,35,33].
Orexin receptors are known as potential therapeutic tar-
get for treatment of AD [4], eating disorders [36] and as
cognitive enhancement [37]. It is important to mention
that stress reactions, through stimulation of the hypothala-
mus–pituitary–adrenal axis, in the presence of orexin defi-
ciency, may trigger mood disturbances and psychological
alterations through diverse pathways [38]. Studies in humans
have reported orexin involvement in depression. 24-hour
cerebrospinal fluid’s orexin A concentrations were signifi-
cantly different in depressed patients compared to compari-
son groups [39], while orexin A concentrations in cerebro-
spinal fluid are significantly decreased in suicidal patients
with major depressive disorder [40]. The orexinergic system
projects to all monoaminergic nuclei, including raphe nuclei
(5-HT), the ventral tegmental area (dopamine) and locus
coeruleus (norepinephrine), [41,42], supporting the presence
of a functional positive feedback loop between these differ-
ent systems [43]. Furthermore, intra-cerebroventricular ad-
ministration of Orexin-A induces an antidepressant-like ef-
fect and hippocampal cell proliferation [44], acting as anti-
depressant in a condition of calories restriction [45].
4 Current Alzheimer Research, 2016, Vol. 13, No. 10 Pereira et al.

Table 1. Clinical and social demographic data between the groups.

Comparison
Variable Patients LOD Patients AD P value*
Group

Total 112 222 249

Females 76 (67.9%) 173 (77.9%) 170 (68.3%) 0.06

Age (mean ± SD) 78.3 (± 7.3) 75.7 (± 8.1) 79 (± 7.5) <0.01a

Education in years (mean ± SD) 3.09 (± 2.6) 3.18 (± 2.9) 2.89 (± 3.1) 0.16

MMSE (mean ± SD) 24.3 (± 4.0) 21.36 (± 2.6) 13.16 (± 4.7) <0.001

GDS (mean ± SD) 2.3 (± 1.4) 9.5 (± 3.7) _ <0.05

Arterial hypertension (%) 75 77.8 67.7 0.18

Diabetes mellitus (%) 11.8 22.6 16.3 0.1

Dyslipidemia (%) 36.6 26.3 25.4 0.06

Coronary artery disease (%) 7.2 10.7 8.3 0.61

Atrial fibrillation (%) 9.2 4.8 5.2 0.76

COPD (%) 9.2 9.7 5.5 0.72

Smoking (%) 30.3 33.3 27 0.3

Alcoholism (%) 7.9 5.5 7.9 0.14

Cancer (%) 2.6 8.1 6.8 0.52

Cardiac Failure (%) 3.9 10.2 8.4 0.48

MMSE: Mini-Mental State Examination COPD: Chronic Obstructive Pulmonary Disorder; *among all comparisons, except when indicated; a between LOD
and AD; b comparing LOD to AD and LOD

Table 2. Genotypic and Allelic SNPs PER2, PER3, CLOCK and OX2R in AD and LOD.

Comparison
Case 249 Group p-value
AD (%) 112 (%) Odds-R 95% Lo 95% Hi X2 Df (1.000 permut.)

PER3

rs10462020

GTC⇒ GGC Val⇒Gly

T/T 179(71) 62(55) 1 (ref) 2.3 2 0.31

T/G 52(20) 14(12) 1.2 0.65-2.38

G/G 3(1) 0(0) 5.30E+07 3E+07 - 9.3E+07

T* 410(82) 138(61) 1 (ref) 1.2 1 0.27

G 58(11) 14(6) 1.3 0.7-2.5

rs228697

CCT ⇒ GCT Pro⇒Ala

C/C 200(80) 67(60) 1 (ref) 1.8 2 0.40

C/G 29(11) 13(11) 0.74 0.36-1.52

+08
G/G 2(0.8) 0(0) 1.16E 5.80E+07 2.33E+08
Lack of Association between Genetic Polymorphism Current Alzheimer Research, 2016, Vol. 13, No. 10 5

(Table 2) contd…..

Comparison
Case 249 Group p-value
AD (%) 112 (%) Odds-R 95% Lo 95% Hi X2 Df (1.000 permut.)

C* 429(86) 147(66) 1 (ref) 0.2 1 0.68

G 33(6) 13(5) 0.86 0.44-1.69

PER2

rs934945

GGA⇒ GAA Gly⇒Glu

G/G 168(67) 55(50) 1 (ref) 0.1 2 0.97

A/G 67(27) 23(21) 0.95 0.54-1.67

A/A 7(3) 2(2) 1.14 0.23-5.67

G* 403(80) 133(60) 1 (ref) 0.0 1 0.96

A 81(16) 27(12) 0.99 0.62-1.59

CLOCK

rs6855837

CTT⇒ ATT Leu⇒Ile

C/C 3(1) 1(1) 1 (ref) 1.5 2 0.46

C/A 37(15) 9(8) 1.8 0.14-22.5

A/A 193(78) 83(74) 1.1 0.10-12.7

C* 43(9) 12(6) 1 (ref) 1.1 1 0.30

A 423(85) 177(80) 0.6 0.34-1.40

OX2R

rs9370399

A/A 80(32) 25(23) 1 (ref) 4.1 2 0.09

A/C 93(35) 34(31) 0.8 0.47-1.7

C/C 35(14) 23(21) 0.4 0.21-0.95

A* 253(51) 84(38) 1 (ref) 4.4 1 0.03 (0.2)

C 163(33) 80(35) 0.65 0.43-0.97

rs3134705

A/A 120(50) 50(45) 1 (ref) 2.5 2 0.28

A/G 40(16) 9(8) 1.8 0.82-4.06

G/G 47(20) 19(17) 1.0 0.54-1.93

A* 282(57) 109(50) 1 (ref) 0.2 1 0.63

G 134(27) 47(21) 0.9 0.60-1.35

rs9349768

A/A 109(44) 28(25) 1 (ref) 0.2 2 0.91

A/T 61(25) 16(14) 0.9 0.49-1.95

T/T 6(3) 1(1) 1.5 0.17-13.3

A* 279(56) 72(32) 1 (ref) 0.0 1 0.87

6 Current Alzheimer Research, 2016, Vol. 13, No. 10 Pereira et al.

(Table 2) contd…..

Comparison
Case 249 Group p-value
AD (%) 112 (%) Odds-R 95% Lo 95% Hi X2 Df (1.000 permut.)

T 73(15) 18(8) 1.04 0.58-1.8

rs7741664

T/T 124(50) 42(38) 1 (ref) 1.6 2 0.44

C/T 79(31) 37(33) 0.7 0.44-1.20

C/C 15(6) 7(6) 0.8 0.27-1.90

T* 327(66) 121(54) 1 (ref) 1.3 1 0.24

C 109(22) 51(23) 0.8 0.58-1.22

rs2134294

T/T 53(21) 18(16) 1 (ref) 4.3 2 0.11

C/T 105(60) 28(25) 1.2 0.6-2.4

C/C 59(24) 8(7) 2.3 1.0- 6.3

T* 211(42) 64(30) 1 (ref) 3.9 1 0.04 (0.2)

C 223(44) 44(21) 1.5 1.0- 2.3

rs2653349

ATT⇒ GTT Ile⇒Val

G/G 182(73) 65(60) 1 (ref) 0.0 1 0.97

A/G 61(25) 22(20) 0.9 0.57-1.70

A/A 0(0) 0(0) 0 0

G* 425(85) 152(68) 1 (ref) 0.0 1 0.97

A 61(12) 22(10) 0.9 0.59-1.63

Comparison
Case 222 group
LOD (%) 112 (%) Odds-R 95%Lo 95%Hi x2 Df p-value

PER3

rs10462020

GTC⇒ GGCVal⇒Gly

T/T 142(64) 62(55) 1 (ref) 5.1 2 0.07

T/G 50(23) 14(13) 1.5 0.80-3.02

G/G 5(2) 0(0) 3.28E+08 2.10E+08 -5.12E+08

T* 334(75) 138(62) 1 (ref) 3.4 1 0.06

G 60(2) 14(6) 1.7 0.94-3.22

rs228697

CCT⇒GCT Pro⇒Ala

C/C 168(76) 67(60) 1 (ref) 3.2 2 0.19

Lack of Association between Genetic Polymorphism Current Alzheimer Research, 2016, Vol. 13, No. 10 7

(Table 2) contd…..

Comparison
Case 249 Group p-value
AD (%) 112 (%) Odds-R 95% Lo 95% Hi X2 Df (1.000 permut.)

C/G 19(9) 13(12) 0.58 0.27-1.24

1.621E+07
G/G 2(1) 0(0) 3.25E+07 6.534E+07

C* 355(75) 147(65) 1 (ref) 0.7 1 0.39

G 23(5) 13(6) 0.73 0.36 - 1.48

PER2

rs934945
GGA⇒GAAGly⇒Glu

G/G 137(62) 55(50) 1 (ref) 0.1 2 0.94

A/G 52(23) 23(21) 0.9 0.50-1.62

A/A 5(2) 2(2) 1.0 0.18-5.32

G* 326(75) 133(60) 1 (ref) 0.1 1 0.79

A 62(14) 27(12) 0.93 0.57-1.53

CLOCK

rs6855837

CTT⇒ ATT Leu⇒Ile

C/C 2(1) 1(1) 1 (ref) 0.3 2 0.88

C/A 25(12) 9(8) 1.7 0.13 - 21.25

A/A 183(83) 83(75) 1.4 0.13 - 16.41

C* 29(7) 12(6) 1 (ref) 0.1 1 0.84

A 391(88) 177(80) 0.9 0.46 - 1.87

OX2R

rs9370399

A/A 58(26) 25(23) 1 (ref) 2,0 2 0.37

A/C 98(45) 34(31) 1.24 0.67-2.28

C/C 42(19) 23(21) 0.78 0.39-1.57

A* 214(50) 84(37) 1 (ref) 0.4 1 0.54

C 182(41) 80(36) 0.89 0.62-1.28

rs3134705

A/A 106(50) 50(45) 1 (ref) 4.5 2 0.10

A/G 43(20) 9(8) 2.25 1.01-4.98

G/G 47(21) 19(17) 1.16 0.62-2.19

A* 255(58) 109(50) 1 (ref) 1.2 1 0.27

G 137(31) 47(21) 1.24 0.83-1.85

rs9349768

A/A 81(37) 28(25) 1 (ref) 1.2 2 0.53

 
8 Current Alzheimer Research, 2016, Vol. 13, No. 10 Pereira et al.

(Table 2) contd…..

Comparison
Case 249 Group p-value
AD (%) 112 (%) Odds-R 95% Lo 95% Hi X2 Df (1.000 permut.)

A/T 38(17) 16(14) 0.82 0.39-1.69

T/T 7(3) 1(1) 2.42 0.28-20.54

A* 200(45) 72(32) 1 (ref) 0.0 1 0.89

T 52(11) 18(8) 1.04 0.57-1.89

rs7741664

T/T 121(54) 42(37) 1 (ref) 3.3 2 0.18

C/T 67(30) 37(33) 0.6 0.40-1.0

C/C 12(6) 7(6) 0.6 0.25-1.6

T* 309(70) 121(54) 3,0 1 0.10

C 91(20) 51(23) 0.6 0.49-1.0

rs2134294

T/T 52(24) 18(16) 1 (ref) 7.8 2 0.01 (0.1)

C/T 73(33) 28(25) 0.9 0.44-1.8

C/C 63(28) 8(7) 2.7 1.02-6.7

T* 177(40) 64(29) 1 (ref) 5,0 1 0.02 (0.2)

C 199(45) 44(20) 1.6 1.1-2.5

rs2653349 ATT⇒GTT
Ile⇒Val

G/G 168(76) 65(58) 1 (ref) 0.7 2 0.39

A/G 44(20) 22(20) 0.7 0.43-1.39

A/A 0(0) 0(0) 0 0

G* 380(86) 152(68) 1 (ref) 0.6 1 0.42

A 44(10) 22(10) 0.8 0.46-1.38

AD Alzheimer Disease; LOD late-onset depression; *Ancestral Allele; df-degree of freedom, df=1 between 2 alleles, Df=2 between 3 genotypes; a significant
association is indicated in bold and italic (<0,05); Adjusted p-value from 1,000 permutation test (multiple testing-Unphased): 0.2 (Allele) Adjusted p-value
from 1,000 permutation test (multiple testing): 0.1 (Genotypic).

Notwithstanding the involvement of orexin in depression,
the role of orexin-related genes on the susceptibility to this
disorder has been barely explored. Polymorphisms in OX2R
gene seem to be associated with LOD but, as said before,
these results did not persist after 1,000 permutations test.
Despite these findings, some limitations must be ad-
dressed. This is one exploratory study with a small sample
size, and the difference between the numbers of cases com-
pared to comparison group, allied to the fact that they were
very heterogeneous must be taken in account. Women have
been living more years and with a healthier life, so there is a
difficulty to obtain elderly men for all groups. However
these samples were well characterized and the study has 99%
power for the identification of a large or moderate effect
size, as 45% to detect a small effect size.
Given the high prevalence of Alzheimer's disease, it
would be interesting to continue the study of possible genes
associated with the disorder, in order to better understand its
pathophysiology with diagnostic/ therapeutic implications.
CONCLUSION
In conclusion, our results suggest that the OX2R gene poly-
morphism exhibited no association with development of
LOD and AD in a Brazilian population sample. More com-
prehensive studies with larger samples are necessary to de-
termine conclusively the association of OX2R gene with
LOD and AD.
CONFLICT OF INTEREST
The author(s) confirm that this article content has no con-
flict of interest.
Lack of Association between Genetic Polymorphism
ACKNOWLEDGEMENTS
This work was supported by CAPES, FAPEMIG and
CNPq (FAPEMIG: CBB-APQ-00075-09 / CNPq
573646/2008-2).
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