Journal of the Neurological Sciences 379 (2017) 58–63

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Journal of the Neurological Sciences

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

Association of polymorphisms and reduced expression levels of the

NR4A2 gene with Parkinson's disease in a Mexican population
Elizabeth Ruiz-Sánchez a, Petra Yescas b, Mayela Rodríguez-Violante c, Nancy Martínez-Rodríguez d,
Jesica N. Díaz-López a, Adriana Ochoa b, Sergio S. Valdes-Rojas e, Daniel Magos-Rodríguez a,
Julio C. Rojas-Castañeda f, Amin Cervantes-Arriaga c, Samuel Canizales-Quinteros g, Patricia Rojas a,⁎
a
Laboratory of Neurotoxicology, Instituto Nacional de Neurología y Neurocirugía, “Manuel Velasco Suárez”, SS, Mexico City, Mexico
b
Department of Genetics, Instituto Nacional de Neurología y Neurocirugía, “Manuel Velasco Suárez”, SS, Mexico City, Mexico
c
Clinical Neurodegenerative Research Unit, Instituto Nacional de Neurología y Neurocirugía, “Manuel Velasco Suárez”, SS, Mexico City, Mexico
d
Community Health Research Department, Hospital Infantil de México, Mexico City, Mexico
e
Direction of Geriatric Attention, Instituto Nacional de las Personas Adultas Mayores (INAPAM), Mexico City, Mexico
f
Laboratory of Reproductive Biology, Instituto Nacional de Pediatria, Mexico City, Mexico
g
Unidad de Genómica de Poblaciones Aplicada a la Salud, Facultad de Química, UNAM-INMEGEN, Mexico

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

Article history: Introduction: The NR4A2 transcription factor is important in the development, survival and phenotype of dopami-
Received 6 December 2016 nergic neurons and it is postulated as a possible biomarker for Parkinson's disease (PD). Therefore, our aim was to
Received in revised form 3 April 2017 analyze in a sample of a Mexican population with idiopathic PD, mutations (in two hotspot mutation regions) and
Accepted 15 May 2017 two polymorphisms (rs34884856 in promotor and rs35479735 intronic regions) of the NR4A2 gene. We also eval-
Available online 17 May 2017
uate the levels of NR4A2 gene expression in peripheral blood for a Mexican population, and identify whether they
are associated with NR4A2 gene polymorphisms.
Keywords:
NR4A2 gene
Methods: We conducted a case-control study, which included 227 idiopathic PD cases and 454 unrelated controls.
Polymorphisms Genetic variants of the NR4A2 gene were genotyped by high-resolution melting (HRM) and validated by an auto-
Parkinson's disease mated sequencing method. The gene expression was performed in peripheral blood using a real-time polymerase
Genetics chain reaction.
Association Results: The rs35479735 polymorphism was associated with a higher risk of developing PD. In addition, NR4A2
gene expression was significantly decreased in patients with PD. Linkage disequilibrium analysis showed a haplo-
type H4 (3C-3G) that showed lower levels of expression, and contained the risk alleles for both polymorphisms.
Conclusions: In summary, this is the first study in a Mexican population that considers the analysis of NR4A2 in pa-
tients with PD. An association was identified between genotype and mRNA expression levels of NR4A2 in patients
with PD. These results suggest that polymorphisms and expression of the NR4A2 gene could play an important role
in the risk of developing PD in Mexican populations.
© 2017 Elsevier B.V. All rights reserved.

1. Introduction

Parkinson's disease (PD) is characterized by degeneration of dopa-

Abbreviations: ACD, Acid citrate dextrose; AIC, Akaike information criteria; AIM,
ancestry-informative markers; ASM, automated sequencing method; CI, confidence
interval; H, Haplotype; HWE, Hardy-Weinberg equilibrium; HRM, High-Resolution
Melting; Indel, Insertion or deletion of bases in the DNA; LD, Linked disequilibrium;
NR4A2, Nuclear Receptor Subfamily 4 Group A Member 2; OR, odd ratio; PD, Parkinson's
disease; PBMC, Peripheral blood mononuclear cells; PCR, Polymerase chain reaction;
SNP, Single nucleotide polymorphism; UPDRS, Unified Parkinson's Disease Rating Scale;
3G, Insertion G; 2G, Deletion G; 3C, Insertion C; 2C, Deletion C.
⁎ Corresponding author at: Laboratory of Neurotoxicology, Instituto Nacional de
Neurología y Neurocirugía, Av. Insurgentes Sur No. 3877, Col. La Fama, C.P. 14269, México
D.F., Mexico.
E-mail address: prcastane@hotmail.com (P. Rojas).
minergic neurons in the substantia nigra pars compacta of the midbrain
and subsequent dopamine depletion in the striatum [1]. Identification of
genes controlling the maintenance of dopaminergic neurons, such as
NR4A2 (also known as Nurr1), have provided new insight into the
mechanisms leading to neuronal death in PD, which so far is not well
understood.
The NR4A2 transcription factor is important in the development,
survival and phenotype of dopaminergic neurons (for a review see
Decressac et al., 2013 [2]). This orphan nuclear receptor is encoded by
the NR4A2 gene that is located on chromosome 2q22-23 and consists
of eight exons [3]. It is an immediate early gene, and highly expressed

http://dx.doi.org/10.1016/j.jns.2017.05.029
0022-510X/© 2017 Elsevier B.V. All rights reserved.
 E. Ruiz-Sánchez et al. / Journal of the Neurological Sciences 379 (2017) 58–63
in dopaminergic neurons [2] and other cells, including peripheral blood
leukocytes [4].
Mutations in the NR4A2 gene are associated with familial PD and are
mainly identified in exon 1 and 3. These mutations are linked with a sig-
nificant decrease in N4R42 mRNA [5,6]. Furthermore, different polymor-
phisms of NR4A2 are described in diverse populations, for example,
rs34884856 in the promoter and rs35479735 in the intron 6 regions
are polymorphisms present in Caucasian and Asian populations but
with different frequencies [7–10]. In particular, the rs35479735 poly-
morphism (insertion/deletion of a G) in NR4A2 is associated with familial
and sporadic PD in Caucasian and Asian populations [11–13]. In addition,
rs34884856 polymorphism (insertion/deletion of a C) has been linked
with disease related to the dopamine system [14,15].
In various studies, NR4A2 gene expression in peripheral blood has
been suggested as a possible biomarker for sporadic PD. In these studies,
NR4A2 gene expression was reduced in human peripheral blood mono-
nuclear cells (PBMC) of Caucasian and Asian patients with sporadic PD
[16–18]. This is consistent with studies of PBMC in PD patients that
showed changes in gene expression that may be related with neurode-
generation [19,20]. Therefore, gene expression of NR4A2 could help in
the early diagnosis of the disease and in the evaluation its progression
from a clinical perspective.
Because of the relationship of NR4A2 in the dopamine system and its
association with PD, our aim was to analyze in a sample of a Mexican pop-
ulation with idiopathic PD, mutations (in two regions with a high muta-
tion rate) and two polymorphisms of the NR4A2 gene (rs34884856 and
rs35479735). In addition, the levels of NR4A2 gene expression were ana-
lyzed in PBMC for a Mexican population. We also evaluated the possible
association between levels of NR4A2 gene expression and the polymor-
phisms analyzed for this gene.
2. Materials and methods
2.1. Participants
A total 227 sporadic PD cases and 454 unrelated controls were exam-
ined. Patients with idiopathic PD from the movement disorder clinic at
the National Institute of Neurology and Neurosurgery of Mexico were in-
cluded. A movement disorder specialist examined these patients, all of
whom fulfilled the UK PD Society Brain Bank Clinical Diagnostic Criteria
of PD [21]. Control subjects were recruited under the following inclusion
criteria: without a diagnosis of neurodegenerative disease, with no fam-
ily history of movement disorders and without severe, uncontrolled
physical disease (e.g., cancer, hypertension and diabetes). The institu-
tional ethics committee approved this study and all the patients gave
their informed written consent. Our study was submitted according to
the principles of the Helsinki Declaration. Patients and controls were
Mexican mestizo, that is, subjects who were born in Mexico, had a Span-
ish-derived last name and a family of Mexican ancestry that could be
traced back to the third generation [22]. To restrict the possible con-
founding effects of age and gender, frequency and individual matchings
were carried out, and a ratio of 1 case per 2 controls was applied.
Whole blood was collected in 6 ml Acid Citrate Dextrose (ACD) tubes
from patients and controls for genotyping analysis and measurement
of gene expression levels of NR4A2.
2.2. Genotyping of NR4A2
Genomic DNA was extracted from PBMC by standard procedures
[23]. Mutations and polymorphisms (Indel) were selected from the pro-
moter, exon and intronic regions of the NR4A2 gene using the Single Nu-
cleotide Polymorphism (SNP) database, and referenced sequences were
taken from the dbSNP public database [Website 1]. Mutations were
scanned in two region-high mutation rates (hotspot mutations).
Genetics variants were studied using high-resolution melting (HRM),
and 20% of samples taken at random were sequenced as a quality control
59
using an ABI3730 sequencing equipment (Applied Biosystems, Carlsbad
CA, USA), and were genotyped with a 99% concordance for validation.
Polymerase chain reaction (PCR) amplification for automated sequenc-
ing and the HRM for analysis of the genetics variants was performed
using the Rotor-Gene 6000 instrument (Corbett Research Pty Ltd., Syd-
ney, Australia). The primers used are listed in Table S1.
2.3. Quantitative reverse transcription PCR (qRT-PCR)
We evaluated NR4A2 mRNA levels in the first 156 patients with PD
and 102 control individuals. The gene expression was performed in
PBMC from patients with PD and control participants using a method de-
scribed previously [24]. Total RNA was extracted from PBCM using TRIzol
reagent (Invitrogen Life Technologies, Carlsbad, CA, USA), following the
manufacturer's instructions. RNA integrity was determined by agarose
gel electrophoresis and the concentration and purity were measured
spectrophotometrically. The cDNA was generated from 1000 ng of total
RNA using Moloney Murine Leukemia Virus Reverse Transcriptase (M-
MLV RT) (Invitrogen Life Technologies, Carlsbad, CA, USA) in a total vol-
ume of 20 μl.
GAPDH was used as an internal reference gene to normalize target
gene transcript levels. The quantitative PCR reactions were performed
using predesigned primer and probes from Applied Biosystems (Foster
City, CA, USA). The relative fold changes were determined by the method
of 2−ΔΔCt as described previously [25].
2.4. Statistical analysis
Demographic and clinical variables between PD and control groups
were analyzed using SPSS software v.12.0 (SPSS Inc., Chicago, IL, USA)
and STATA software v.11 (StataCorp, College station, Tx, USA). Numeric
variables not normally distributed are presented as median and range
and were compared using the Mann-Whitney U test. Comparison be-
tween categorical variables and deviations from Hardy-Weinberg equi-
librium (HWE) were analyzed with chi-square tests, and are presented
as absolute frequencies and proportions. The inheritance hypothesis
was tested according to models: dominant, recessive, and heterozygous.
Logistic regression analysis was used in a bivariate way to estimate the
risk of polymorphisms with Parkinsons's disease. Multiple logistic
models were constructed in order to identify the variables that explain
better the risk of developing Parkinson's between the studied groups.
Models were constructed including variables with biological relevance
or with statistical significance, or both. Confounding bias was accepted
when changes in estimated odds ratios (ORs) were equal or larger than
10%. When a principal effects model was reached, effect modification
was also tested and interaction terms were constructed between the
polymorphism, age and sex; the terms were included in the model
when the significance of the p-value was larger or equal to 0.20. A
Hosmer-Lemeshow Goodness of Fit test was performed for each multiple
logistic model, based on Akaike information criteria (AIC). Pairwise link-
age disequilibrium (LD, D´) estimations between polymorphisms and
haplotype reconstruction were performed with Haploview version 4:1
[26] (Broad Institute of Massachusetts Institute of Technology and Har-
vard University, Cambridge, MA, USA). Statistical power to detect an as-
sociation of rs34884856 and rs35479735 with PD was 0.28 and 0.93,
respectively, as estimated with QUANTO software [Website 2].
Correlations between NR4A2 gene expression levels, genotype and
inheritance models were tested using Kruskal-Wallis or Mann-Whitney
U tests. Statistical significance was set at p ≤ 0.05.
3. Results
Demographic and clinical characteristics of PD patients and controls
included in the study are shown in Table 1. Similar age and gender distri-
bution were observed among individuals.
60 E. Ruiz-Sánchez et al. / Journal of the Neurological Sciences 379 (2017) 58–63

Table 1 frequency between cases and controls (Table 3). A haplotype was H1
Characteristics of PD patients and controls. (3C-2G, insertion C in the promoter polymorphism and deletion G in in-
Characteristics Patients (n = 227) Controls (n = 454) p tron 6) with a frequency of 0.46 in controls and 0.40 for patients. The sec-
Age, mean (SD) 62.01 (±10.6) 62.63 (±11.2) 0.43a
ond haplotype identified was H2 (2C-3G, deletion C for the promoter
Gender, n (%) polymorphism and insertion G in intron 6) with a frequency of 0.419
Men 123 (54.2) 246 (54.2) 1b and 0.481 in controls and patients, respectively. The haplotype H1 (3C-
Woman 104 (45.8) 208 (45.8) 2G) showed an OR = 0.78 (0.62–0.98) (p = 0.037) and H2 (2C-3G)
Age at onset in years (±SD) 55.48 ± 10.37 – –
with an OR = 1.28 (1.02–1.6) (p = 0.030) for PD patients.
PD duration in years (±SD) 5.9 ± 3.79 – –
Hoehn and Yahr stage 2.24 ± 0.79 – –
UPDRS 31.07 ± 20.53 – – 3.3. Gene expression
Therapy –
Levodopa, n (%) 99 (43.6) – –
DA-A + Levodopa, n (%) 61 (26.9) – – The levels of NR4A2 gene expression in 102 patients with PD (medi-
DA-A, n (%) 28 (12.3) – – an (p25, 75); 0.56 (0.25–1.02)) were significantly lower than for 156
Other, n (%) 39 (17.2) – – healthy controls (median (p25, 75); 0.98 (0.45–1.59)) (p = 0.001)
Dyskinesia, n (%) 54 (23.8) – – (Fig. 1A). Our results did not reveal any correlation between expression
Antidepressant therapy 52 (22.9) – –
levels of the NR4A2 gene and age, sex, age of onset and treatment (data
PD, Parkinson's disease; n, total population; DA-A, dopamine agonist; UPDRS, Unified not shown). After analyzed differences in NR4A2 mRNA expression
Parkinson's Disease Rating Scale; SD, standard deviation.
a levels between cases and controls, we proceeded to investigate the pos-
Mann-Whitney U test.
b
Chi2 of the Mantel-Hassen test. sible association between genotypes of the polymorphisms studied
with expression levels of the NR4A2 gene.

3.1. Screening mutation and genotyping results

Mutations identified in other populations for exon 1 and 3 of NR4A2
were analyzed [5,6], and no mutations were found in the cohort of spo-
radic PD in this Mexican population.
We analyzed two polymorphisms of the NR4A2 gene (rs34884856 in
the promoter and rs35479735 in the intron 6 region). Observed and ex-
pected frequencies in all polymorphism sites were in Hardy-Weinberg
equilibrium for the control group. A similar genotype and allele frequen-
cies distribution of the rs34884856 polymorphism was observed in PD
patients and controls (p = 0.258, Table 2). Bivariate logistic regression
analysis for a different model of heredity was not significantly associat-
ed with PD. In contrast, for the rs35479735 polymorphism in the intron
6 region a significant difference was observed in genotype and allele fre-
quencies between cases and controls (p = 0.021, Table 2). The 3G-allele
(as homozygote or heterozygote) carriage rates were 54.6% and 46.4% in
the PD and control groups, respectively. In the construction of inherence
models, only the recessive model of the rs35479735 polymorphism was
associated with an increased risk of developing PD (OR = 1.53, 95%IC =
1.08 to 2.19, p = 0.018).
3.4. Association between genotypes and levels of NR4A2 gene expression
In the group of patients, for the polymorphism of the promoter region
a statistically significant difference among the expression levels of the
three genotypes of this polymorphism was observed (2C/2C, 3C/2C and
3C/3C, Fig. 1B) (p = 0.045). In particular, in the homozygous genotype
for the risk allele (insertion C, 3C/3C) lowest levels of NR4A2 gene ex-
pression were found compared to other genotypes (2C/2C and 3C/2C)
(p = 0.021) (Fig. 1 C). In the control group, the expression levels were
not statistically significant between genotypes (p = 0.21). However, sta-
tistically significant differences in NR4A2 gene expression between cases
and controls were observed independently of genotype.
For the region intronic polymorphism, no association between geno-
type and expression levels of the gene NR4A2 was found.
This study showed a haplotype (H4, 3C-3G) with lower levels of ex-
pression compared to other haplotypes for all participants (p = 0.046)
(Table 3); however, the OR value was not significant (4.85 (0.69 to
33.97); p = 0.112). Interestingly, this haplotype contains risk alleles in
both polymorphisms.

3.2. Haplotype analysis
When we analyzed the linked disequilibrium (LD) among the two
polymorphisms rs34884856 and rs35479735, we observed that the
polymorphisms were in LD (D′ = 0.787, r2 = 0.5). Four haplotypes
were identified, and two of them showed significant differences in
4. Discussion
In the present study, genetic variants and expression levels of the
NR4A2 gene were analyzed in order to establish its role as a susceptibility
biomarker for PD in a Mexican population. The genetic variants were an-
alyzed in 227 PD patients and 454 controls with similar ethnic origin and
matching on factors such as sex and age. The analysis did not include

Table 2
Allele and genotype distribution of NR4A2 polymorphisms and risk analysis as a function of the inheritance model in Parkinson's disease patients and controls.

Polymorphisms Allele frequency n(%) Genotype frequency n(%) Model genetic

Promoter rs34884856 2C 3C 2C/2C 3C/2C 3C/3C Model OR (95%IC) p value

Control (n = 454) 459 (50.5) 449 (49.5) 120 (26.4) 219 (48.2) 115 (25.3) Dominant 1.34 (0.94–1.90) 0.105
PD (n = 227) 242 (53.3) 212 (46.7) 71 (31.3) 100 (44.1) 56 (24.7) Recessive 0.92 (0.64–1.33) 0.657
Heterozygous 0.84 (0.61–1.16) 0.288

Intron 6 2G 3G 2G/2G 3G/2G 3G/3G

rs35479735
Control (n = 454) 487 (53.6) 421 (46.4) 138 (30.4) 211 (46.5) 105 (23.1) Dominant 0.67 (0.46–0.97) 0.035
PD (n = 227) 206 (45.4) 248 (54.6)⁎ 51 (22.5) 104 (45.8) 72 (31.7)⁎ Recessive 1.53 (1.08–2.19) 0.018
Heterozygous 0.97 (0.70–1.33) 0.846

Underlined allele denotes the minor allele.

In bold is shown the recessive model instead of associations that were tested using logistic regression adjusting for sex and age.
OR, odd ratio; 95%CI, confidence interval. Chi2 of Mantel-Hassen.
3G, insertion G; 2G deletion G; 3C, insertion C; 2C, deletion C.
⁎ p b 0.05.
 E. Ruiz-Sánchez et al. / Journal of the Neurological Sciences 379 (2017) 58–63 61

Table 3
NR4A2 haplotypes in PD patients and controls.

Frequency
Haplotypes OR (IC 95%) p value
All Controls Patients

H1 3C-2G 0.441 0.461 0.402 0.78 (0.62–0.98) 0.037

H2 2C-3G 0.440 0.419 0.481 1.28 (1.02–1.60) 0.030
H3 2C-2G 0.068 0.075 0.052 0.69 (0.44–1.11) 0.106
H4 3C-3G 0.051 0.045 0.065 1.53 (0.95–2.50) 0.102

Risk assessment according to haplotypes and NR4A2 expression levels

mRNA NR4A2 expression levels median (p25-p75)

Haplotypes OR (IC 95%)a p value
All Control Patients
H3 2C-2G 1.06 (0.16–1.50) 0.76 (0.12–1.63) 1.06 (0.63–1.14) 1.0 (Ref.) –
H1 3C-2G 0.64 (0.22–1.38) 0.87 (0.49–1.96) 0.27 (0.16–0.67) 1.51 (0.35–6.44) 0.579
H2 2C-3G 0.79 (0.32–1.51) 0.99 (0.55–1.58) 0.74 (0.27–1.40) 3.25 (0.75–14.15) 0.116
H4 3C-3G 0.49 (0.17–0.69) 0.56 (0.10–1.00) 0.42 (0.17–0.69) 4.85 (0.69–33.97) 0.112

The order of the polymorphisms in the haplotypes is according to the positions in the chromosome (promoter rs34884856 and Intron 6 rs35479735). Result with p value b0.05 are shown
(bold).
OR, odd ratio; CI95%, confidence interval; p25-p75.
H1, haplotype1; H2, haplotype1; H3, haplotype 3; H4, haplotype 4.
a
The models were adjusted for mRNA expression levels and age.

ancestry-informative markers (AIM). However, we used standard
criteria to define the Mexican Mestizo, and minimized effects of stratifi-
cation [22].
Fig. 1. Analysis of NR4A2 gene expression. A) Levels of NR4A2 mRNA expression were
significantly lower in the PBMC of PD patients versus the control group (*p = 0.001). B)
Effect of rs34884856 genotype on NR4A2 mRNA expression in PD patients. The three
genotypes showed significant statistical differences in gene expression levels (p = 0.045).
C) In patients, the individuals carrying the 2C alleles were compared with individuals
homozygous for the 3C allele. Genotype 3C/3C was significantly less expressed versus 2C/
2C + 3C/2C in the PD patients (*p = 0.021). The graphs show relative expression of
NR4A2 normalized to GAPDH (logarithmic scale expressed) in PBMC of 102 PD patients
and 156 healthy controls. The values are expressed as medians (—) and percentiles
(————). PBMC, peripheral blood mononuclear cells.
NR4A2 mutations have been identified in a limited number of PD pa-
tients in some populations, but they were an uncommon cause of PD [5,
6,27]. We did not find mutations in exon 1 and 3 of the NR4A2 gene in our
cohort of Mexican sporadic PD cases. This coincides with reports of Mex-
ican Americans that did not have mutations in these exons of NR4A2 [14].
The rs34884856 polymorphism of the promotor region was not as-
sociated with PD in our Mexican population. The lack of association ob-
served for this polymorphism may be due to insufficient statistical
power (28% for rs34884856). Further studies in larger samples are nec-
essary to confirm whether this indel contributes to the risk of PD in
Mexicans. However, we found an association of the rs35479735 poly-
morphism in the intronic region with PD. This polymorphism showed
an acceptable level of statistical power for the sample size analyzed in
our population. In particular, the 3G/3G genotype of the rs35479735
polymorphism showed a significantly increased risk of PD. This associa-
tion was identified in three previous studies, one was in an Asian popu-
lation and two were mixed populations of an American studies [11–13].
Further studies are required to clarify these associations with PD in
other populations, especially in Asian and Latin American populations.
Interestingly, we found that the allele frequency for these polymor-
phisms in the Mexican population was different from that in the Cauca-
sian population [11,12], but closer to the Asian population [13]. Since
studies of genomic medicine have been done mostly in specific popula-
tions (European), other ethnic groups should be studied to identify
which variants are important for each population [28]. Thus, studies per-
formed in Mexican populations are important because it is not possible to
extrapolate data from other populations to be used in our population. It
has been reported that some polymorphisms with high frequency in
Mexican populations, are very different from other populations, and
this could result in our population in an increased susceptibility to com-
plex diseases [28]. The study of NR4A2 in Mexican populations is also nec-
essary because NR4A2 has been postulated as a possible target in therapy
for PD, as well as a potential biomarker [29]. Since PD is a complex dis-
ease, it is also important to study the interaction of NR4A2 with other
components of the dopamine-signaling pathway that contribute to PD.
This study showed that NR4A2 gene expression was significantly de-
creased in patients with PD, which may suggest that the change in NR4A2
mRNA expression levels in PBMC is associated with susceptibility for PD.
In support of our result, NR4A2 gene expression was also found to be re-
duced in PBMC of Caucasian and Asian patients with PD [16–18]. This
study did not shown correlations between the expression of the NR4A2
gene and age, sex, age of onset and treatment (data not shown). Our
data are consistent with reports for Caucasian and Asian populations
[17,18].
62 E. Ruiz-Sánchez et al. / Journal of the Neurological Sciences 379 (2017) 58–63
In addition to being a source of easy accessibility, PMBC of PD patients
have been shown to recapitulate transcriptional changes occurring in
neurodegeneration [19,20]. These changes in the expression of several
genes related with nucleic metabolism, mitochondrial function and im-
mune response were also found in in vitro cells and animal models of
PD, as well as in PBMC and brains of PD patients [20,30]. The deregulation
of these genes has been associated with dopaminergic degeneration [31,
32]. In particular, nuclear receptor NR4A2 has been proposed as a poten-
tial target for DNA repair mechanisms, mitochondrial dysfunction and
anti-inflammatory response [33], which in turn, may be also related
with mechanisms of dopaminergic neurodegeneration. Together, these
findings supports the hypothesis that NR4A2 mRNA levels may be a po-
tential peripheral biomarker of PD.
We found a relationship between the rs34884856 polymorphism in
the promoter region and NR4A2 expression levels in the PD patients.
The genotype 3C/3C showed a lower NR4A2 gene expression than
other genotypes with 2C allele carriers (as homozygote or heterozygote)
in PD patients. This is the first study that associates a genotype and
NR4A2 expression levels in patients with PD. Since a genetic association
of the rs34884856 polymorphism with PD was not found, it is important
to perform functional studies of this variant. For example, functional in
silico analysis could identify whether the rs34884856 polymorphism is
related to the decreased expression of the NR4A2 gene or if this polymor-
phism is in disequilibrium with another polymorphism that changes
NR4A2 gene expression and that is also associated with PD.
An association between levels of NR4A2 gene expression and the
polymorphism in the intronic region was not identified; this is not sur-
prising because it is postulated that polymorphisms in the promoter re-
gions could influence changes in levels of gene expression, and that the
polymorphisms in intronic regions could change or influence the mes-
senger RNA editing and translation processes [34]. One limitation of
the present study is the lack of measurement of protein levels of
NR4A2, to explore the possible relationship with the polymorphisms
studied, especially for the intronic polymorphism.
Haplotype analysis identified the haplotypes H1 (3C-2G) and H2 (2C-
3G) to be associated with PD. In addition, the haplotype H4 (3C-3G)
showed the lowest levels of NR4A2 gene expression compared to other
haplotypes in all participants. Although we could not verify the associa-
tion between the risk haplotype and expression levels, a trend could be
identified. A limitation of this study is the rather number of patients
and controls analyzed for gene expression, and it would be important
to increase the sample size to verify this possible relationship.
The apparent discrepancy between the genotype and phenotype for
the polymorphisms analyzed, could be due to the fact that the suscepti-
bility to complex diseases such as PD, may be due to different factors (ge-
netics and environmental) [35,36]. For this reason, it is necessary to
identify other genetic factors and epigenetic factors that interact with
the polymorphisms of the NR4A2 gene studied and that could be associ-
ated with PD. For example, it is interesting the identification of haplo-
types together with other polymorphisms of this gene, which have
been also associated with dopaminergic dysfunctions [37,38]. Another
possibility is the study of a genetic interaction between polymorphisms
of NR4A2 and of other genes such as PITX3 and EN1, which play a role
in the development of the dopaminergic neuron phenotype and are as-
sociated with susceptibility to PD [39]. Finally, epigenetics changes of
NR4A2 related with cognitive processes [40], and its possible association
with both gene expression and the polymorphisms analyzed is an impor-
tant field to explore in future studies.
In our population, the rs35479735 polymorphism and expression
levels of the NR4A2 gene were associated with a risk of PD using a
case-control study. NR4A2 gene expression was significantly decreased
in Mexican patients with PD as compared with the control group. An as-
sociation was identified between genotype and mRNA expression levels
of NR4A2 in patients with PD in our Mexican population. These results
suggest that polymorphisms and expression of the NR4A2 gene could
play a role in PD susceptibility in Mexican populations.
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.jns.2017.05.029.
Declaration of interest
The authors confirm that they have no conflict of interest.
Funding sources
This research was funded partially by a National Council of Science
and Technology, Mexico (CONACyT) grant, Project number SALUD-
2011-01-162087. The funders had no involvement in the preparation
or writing up of this research. The authors confirm that they have no con-
flict of interest.
Acknowledgements
This work was submitted in partial fulfillment of the requirements
for the PhD degree by Elizabeth Ruiz Sánchez, who is a doctoral student
from the Programa de Doctorado en Ciencias Biomédicas, Universidad
Nacional Autónoma de México (UNAM). We thank to the Instituto
Nacional de las Personas Adultas Mayores (INAPAM) for the support
to recruit participants in this study. In particular, to Director de Atención
Gerontológica-INAPAM. The authors thank Dr. Robyn Elizabeth Hudson
for her valuable comments. We also thank Diana Alvarado and Haydee
N. León for their technical support.
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