Parkinson disease - genes - SNCA - genetics
doença de Parkinson - genes SNCA - genética
Parkinson’s disease (PD) is characterized by an accelerated loss of dopaminergic neurons
of the substantia nigra pars compacta, which clinically expresses tremor, rigidity,
bradykinesia, postural instability, and progressive impairment of cognitive function[1]. The SNCA gene codifies a small protein called α-synuclein, which has been largely involved
in neurodegeneration processes; nevertheless, its function in health and disease remains
unclear. Various hypotheses have been proposed regarding the pathogenicity of SNCA
misfolding, because it aggregates as a key component of Lewy bodies found in PD. In
the substantia nigra, neuronal cell loss occurs before symptoms develop, and accelerated
cell loss may not always converge with α-synuclein deposition. This raises the possibility
of a functional, rather than anatomic, disturbance due to the abnormal expression
of SNCA protein that occurs in neurodegeneration[2]. Multiple system atrophy (MSA) and PD share the feature of deposition of abnormally
phosphorylated α-synuclein. A genome-wide association study of 1,713 white PD cases
and 3,974 white control subjects also aimed to find significant associations with
MSA. They identified an association with the SNCA locus in both diseases. The odds ratio (OR) associated with the heterozygous combination
of the rs3857059 variant was 1.3 in both diseases (95% confidence interval (CI) in
PD: 1.2 to 1.5; 95%CI in MSA: 1.1 to 1.6), whereas the OR for homozygous carriers
was 3.8 (95%CI: 2.4 to 5.9) in PD and 5.9 (95%CI: 3.2 to 10.9) in MSA[3]. The rs3857059 variant of the SNCA gene has also been associated with an elevated mRNA expression level in the temporal
cortex biopsies of patients with Alzheimer’s disease with Lewy body pathology[4]. There is increasing evidence that genetic variants in the SNCA locus demonstrate
associations with PD in several studies conducted in different populations[5],[6],[7]. Moreover, triplication of SNCA is related to early onset of PD, whereas, when duplicated,
it associates with classical presentation of PD. This suggests that gene dosage affects
the onset and progression of the disease. A recent study in a cellular model carrying
a SNCA gene triplication demonstrated decreased developmental fitness, accelerated
aging, and increased neuronal cell loss[8]. Besides copy number changes in the SNCA gene, three missense mutations are the
most common pathogenic changes: A53T, A30P, and E46K[9]. Nevertheless, a previous work from our group demonstrated the absence of the A30P
change in the subset of patients analyzed in our group. Hence, different genetic variants
in the SNCA gene may represent a risk factor for PD development in our population.
The aim of the present work was to assess the possible association of the rs3857059
variant of the SNCA gene and PD in Mexican Mestizo patients.
METHOD
In total, 241 Mexican Mestizos were included in the study. Allele frequencies were
compared between 106 patients diagnosed with PD and 135 controls. Controls were clinical
healthy participants that were referred for neurological evaluation by a certified
neurologist (García S). Only participants with no family history of PD or other neurodegenerative
disorders were included. Samples were obtained from participants between February
2009 and June 2010, from four tertiary-care level hospitals in Mexico (Neurology Departments
from Centro Médico Nacional “20 de Noviembre”-ISSSTE, Instituto de Ciencias Médicas
y de la Nutrición “Salvador Zubirán,” Mexico City; and División de Genética, Centro
de Investigación Biomédica de Occidente-IMSS, Jalisco, Mexico). Diagnosis was performed
according to the Queen Square Brain Bank criteria[10]. Cognitive impairment was assessed using the Folstein Mini Mental State Examination
Test. Institutional Committees approved the study and informed written consent was
obtained from the participants.
Genomic DNA was isolated from peripheral blood leukocytes according to the method
described by Gustincich et al.[11]; this technique allows high-quality DNA extraction for multiple applications due
to the use of cationic detergents. Genotyping was performed by real time PCR using
TaqMan probes (hydrolysis probes) specific for the rs3857059 (C_8933273_10 assay,
Applied Biosystems, Foster City, CA, USA). Real-time PCR was performed on a LightCycler
480 II (Roche Diagnostics GmbH, Switzerland); PCR reactions were prepared according
to the manufacturer’s instructions. Statistical analysis was performed using SPSS
software v. 18.0 (SPSS Inc., Chicago, IL, USA) and p < 0.05 were considered to indicate
statistically significant results. Expected proportions of genotypes within each group
were tested using the Hardy–Weinberg equilibrium (HWE), this was estimated using the
χ2 test (available online http://ihg.gsf.de/cgi-bin/hw/hwa1.pl). Allele and genotype
frequencies were compared between groups using the χ2 test and Fisher’s exact test.
To test for an association between the rs3857059 and PD, the χ2 test was performed.
To estimate the proportion of the G allele of the rs3857059 variant in each study
group, OR were calculated from 2 × 2 contingency tables. The association of this variant
was also tested by gender using the 2 × 2 χ2 test. To test for differences in the
age at onset and the genotype frequencies of the variant of interest, we performed
one-way ANOVA. Then a Student’s t-test was performed to test for differences in the
mean age of onset between GG/AG and AA genotype groups. A χ2 test was also performed
to test for an association between the variant and cognitive impairment.
RESULTS
Allele frequencies in our study were similar to those reported by the MEX group in
the HAPMAP database (http://hapmap.ncbi.nlm.nih.gov/). Alleles and genotypes were
distributed according to HWE equilibrium in both groups, consistent with unequivocal
genotyping, which is the main factor affecting HWE deviations[12] ([Table 1]). The χ2 test revealed that the G allele in the homozygous state (recessive model)
was associated with PD (OR = 2.40, CI, 1.12 to 5.13, p = 0.02) ([Table 1]). Correction by regression was not necessary because no confounding factors were
found as per the definition of Clarke et al.[13]. To test for an association of the variant by gender, we performed χ2 tests to compare
the GG, AG, and AA genotypes in males and females separately, and a difference was
found in the female group (p < 0.01) ([Table 2]). The OR was estimated in females according to genotype in the following combinations:
GG vsAA + AG using an allele positivity test table; association with PD was found only
in females with the GG genotype (OR = 1.31, CI, 1.01 to 1.7, p = 0.037). We further
analyzed the possible involvement of the variant in cognitive impairment, age at onset,
and early onset in the youngest group of patients (< 40 years old); no association
was found with any of these features (p > 0.05) ([Table 3]).
Table 1
Genotypes of the study groups.
|
Groups
|
Genotypes (rs3857059)
|
Association
|
Significance
|
|
|
|
|
|
|
SNCA analysis
|
AA
|
AG
|
GG
|
OR, (CI)
|
p-value
|
|
Controls n = 135
|
48 (35.6)
|
67 (49.6)
|
20 (14.8)
|
2.4 (1.121–5.138)*
|
0.02
|
|
Patients n = 106
|
25 (23.6)
|
56 (52.8)
|
25 (23.6)
|
OR: Odds ratio; CI: (95% Confidence Interval; *correction by regression was not necessary
as no confounder factors were found according to the definition of Clarke et al.12
Table 2
Genotype frequencies in female and male groups of cases and controls.
|
Genotype / Gender
|
Case
|
Control
|
p-value (Fisher exact)
|
|
|
|
|
AA
|
AG
|
GG
|
AA
|
AG
|
GG
|
|
Female
|
3
|
18
|
10
|
18
|
23
|
5
|
p = 0.004
|
|
Male
|
22
|
38
|
15
|
30
|
44
|
15
|
p = 0.815
|
|
Total
|
n = 106
|
n = 135
|
n = 241
|
Table 3
Descriptive data for the study groups.
|
Descriptive data of the groups of study
|
P-value (Fisher exact)
|
|
Age
|
Mean
|
Min
|
Max
|
p = 0.02
|
|
Controls
|
65.73 ± 9.4
|
40
|
88
|
|
Patients
|
62.52 ± 12.1
|
29
|
93
|
|
Gender
|
Male
|
Female
|
|
p = 0.48
|
|
Controls
|
89
|
46
|
-
|
|
Patients
|
75
|
31
|
-
|
|
Smoking
|
Yes
|
No
|
-
|
p = 0.43
|
|
Controls
|
61
|
74
|
-
|
|
Patients
|
42
|
64
|
-
|
|
Early onset*
|
Yes
|
No
|
|
|
|
Number of Patients
|
18
|
88
|
-
|
-
|
|
Age at onset
|
Mean ± (SD)
|
Min
|
Max
|
Range
|
|
56.26 ± 14.4
|
25
|
87
|
62
|
SD: standard deviation; *Early onset was considered as symptoms < 40 years old; Percentage
of early onset SPD 16.98%.
DISCUSSION
Allelic variants in various genes (PARK genes) have been linked to PD; for instance,
the G2019S in the LRRK2 (PARK 8) gene confers major susceptibility to PD in Ashkenazi
Jews and individuals from the north of Africa; whereas in Han Chinese populations,
the rs7684318 variant of the SNCA gene (PARK1/4) was associated with PD but not with
disease onset[14]. Therefore, major genes and specific variants associated with PD may be different
among populations of distinct ethnic origin. Mexican Mestizos (most present-day Mexicans)
are the result of the admixture of Spaniards and Amerindians[15]; therefore, genetic variants associated with PD may be not the same as those frequently
reported in other populations. Some studies conducted in Mexican Mestizos have gained
insights into population-specific genetic variants associated with PD, for example,
the epsilon4 allele of APOE[16] for late-onset PD and variants in GBA and PARK2 in early-onset PD[17]. Nonetheless, major susceptibility genes remain unknown[18],[19]. Unlike in other ethnic groups, the A30P, IVS4 + 66A-G in SNCA, and other known
point mutations in the PARK2 gene are uncommon in Mexican Mestizos[18],[20],[21]. The rs3857059 variant of the SNCA gene was associated with expression levels of
SNCA mRNA in the temporal cortex of brain biopsies of patients with Alzheimer’s &
Lewy Body pathology; homozygotes for the minor allele (G) demonstrated significantly
higher expression. Hence, we hypothesized that this genetic variant is associated
with PD. To the best of our knowledge, this is the first study to find a positive
association of the GG genotype of the rs3857059 variant and PD and also the first
to find a gender effect, females with the GG genotype being more affected than males.
Gender-based differences in gene expression in human dopaminergic neurons of substantia
nigra pars compacta exist[22]; however, SNCAupregulation was associated with PD in males[22]. In addition, methylation of the SNCAgene and its abnormal expression have been linked to behavioral disorders in females[23],[24]; therefore, further studies are mandatory to gain insights into the gender effect
of the GG genotype of the rs3857059 variant and PD reported herein.
A recent report from our group demonstrated that the rs1801133 variant in the MTHFR
gene was associated with PD[25]. MTHFR is involved in many different biochemical pathways in humans, such as DNA
methylation. The expression of the SNCA gene is regulated in part by methylation of
intron 1, because it was demonstrated in DNA derived from biopsies of substantia nigra,
putamen, and cortex from PD patients[26]. Taken together, these findings suggest that, at least in Mexican Mestizos, allelic
variants of the rs1801133 and rs3857059 in MTHFR and SNCA genes account for susceptibility
to PD and the GG genotype of the rs3857059 variant is particularly associated with
PD in females of this population. Further studies aimed to explore potential interactions
between genetic and epigenetic changes involved in PD pathology may add to the complexity
of this common neurodegenerative disorder.
