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Association of genetic variants within HLA-DR region with Parkinson's disease in Taiwan
Journal Pre-proof Association of genetic variants within HLA-DR region with Parkinson’s disease in Taiwan Kuo-Hsuan Chang, MD PhD, Yih-Ru Wu, MD, Yi-Chun Chen, MD, Hon-Chung Fung, MD PhD, Chiung-Mei Chen, MD PhD PII:
S0197-4580(19)30391-4
DOI:
https://doi.org/10.1016/j.neurobiolaging.2019.11.002
Reference:
NBA 10709
To appear in:
Neurobiology of Aging
Received Date: 21 February 2019 Revised Date:
1 November 2019
Accepted Date: 1 November 2019
Please cite this article as: Chang, K.-H., Wu, Y.-R., Chen, Y.-C., Fung, H.-C., Chen, C.-M., Association of genetic variants within HLA-DR region with Parkinson’s disease in Taiwan, Neurobiology of Aging (2019), doi: https://doi.org/10.1016/j.neurobiolaging.2019.11.002. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Elsevier Inc. All rights reserved.
1
Association of genetic variants within HLA-DR region with Parkinson’s
2
disease in Taiwan
3 4
Kuo-Hsuan Chang# MD PhD, Yih-Ru Wu# MD, Yi-Chun Chen MD, Hon-Chung Fung
5
MD PhD, Chiung-Mei Chen* MD PhD
6
a
7
College of Medicine, Taipei, Taiwan
Department of Neurology, Chang Gung Memorial Hospital, Chang Gung University
8 9
#
Contribute to the article equally
10 11
*Corresponding author:
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Chiung-Mei Chen MD PhD, Department of Neurology, Chang Gung Memorial Hospital,
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No.5, Fusing St., Gueishan Township, Taoyuan County 333, Taiwan
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Tel: 886-3-3281200 Ext. 8721
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Fax: +886-3-3287226
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Email: Chiung-Mei [email protected]
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Keywords: HLA-DR, rs4248166, rs9268515, rs2395163, rs75855844, rs660895,
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Parkinson’s disease, Polymorphism, Disease association
20 21
All authors report no financial disclosures.
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1
1
Abstract
2
Previous genome-wide association studies in Caucasians suggest genetic loci of human
3
leucocyte antigen (HLA)-DR region may be associated with Parkinson’s disease (PD).
4
However, these genetic-disease associations were limitedly reported in Asian
5
populations. Herein, we investigated the effects of 5 top PD-associated genetic variants
6
within HLA-DR region in Caucasians, including rs4248166, rs9268515, rs2395163,
7
rs75855844 and rs660895, by genotyping 486 Taiwanese patients with PD and 473
8
age-matched control subjects. Although the association between rs2395163 C allele and
9
PD patients demonstrated marginal significance (odds ratio (OR) = 0.74, 95% CI:
10
0.55~0.99, P = 0.045). The frequency of rs2395163 C allele (8.65%) in male PD patients
11
was significantly lower than in male control subjects (14.02%; OR = 0.58, 95% CI:
12
0.39~0.88, P = 0.009). The genetic associations between PD patients and other tested
13
genetic variants were negative. Although strong linkage disequilibriums
14
(rs4248166-rs9626515-s2395163 and rs9626515-rs660895) were observed, the
15
haplotype analysis did not find any significant risk-associated allelic combinations.
16
These results suggest a distinct genetic background within HLA-DR region between
17
Taiwanese and Caucasian PD patients.
18 19
Keywords: HLA-DR, rs4248166, rs9268515, rs2395163, rs75855844 and rs660895,
20
Parkinson’s disease, Polymorphism, Disease association 2
1 2
Abbreviations: EOPD = early-onset Parkinson’s disease; eQTL = expression
3
quantitative train locus; GWAS = genome wide-association studies; HLA = human
4
leucocyte antigen; LOPD = late-onset Parkinson’s disease; OR = odds ratio; PD =
5
Parkinson’s disease; SNP = single nucleotide polymorphism;
6
3
1
Introduction
2
Parkinson's disease (PD) is an age-related neurodegenerative disease characterized by
3
resting tremor, bradykinesia, rigidity and postural instability, which are caused by the
4
progressive loss of nigrostriatal dopaminergic (DA) neurons with deposition of protein
5
aggregates containing α-synuclein (Lang and Lozano, 1998). To date, the mechanisms of
6
PD neurodegeneration remains elusive. Neuroinflammation has been proposed to be an
7
important contributor to PD pathogenesis (Simon-Sanchez et al., 2011). In the brain of
8
PD patients, large numbers of microglia expressing human leucocyte antigen (HLA)-DR
9
have been detected in the substantia nigra (Simon-Sanchez et al., 2011), suggesting the
10
important role of genetic variants within this region in immunopathogenesis of PD.
11
The HLA-DR antigen, encoded by HLA-DRA and HLA-DRB alleles, acts as an
12
antigen-presenting molecule or regulatory molecule involved in innate immune response
13
(Klein and Sato, 2000). Interestingly, the associations of noncoding single nucleotide
14
polymorphisms (SNPs) in HLA-DR region with PD were discovered in genome-wide
15
association studies (GWAS), which were also found in subsequent other GWAS and
16
SNP-based studies mainly in Caucasian populations (Ahmed et al., 2012; Hamza et al.,
17
2010; Hill-Burns et al., 2011; International Parkinson Disease Genomics et al., 2011;
18
Pankratz et al., 2012; Simon-Sanchez et al., 2011; Wissemann et al., 2013). The first
19
PD-associated SNP, rs3129882, is in intron 1 of HLA-DRA (Hamza et al., 2010), which
20
was further confirmed in a meta-analysis of five PD GWAS (Pankratz et al., 2012), but 4
1
not in a GWAS from the Netherlands and a case-control study from Northern Spain
2
(Mata et al., 2011; Pankratz et al., 2012). The other PD associated SNPs are also
3
noncoding and map intergenicaly near HLA-DRA, HLA-DRB1, HLA-DRB5 and
4
HLA-DQB1 (Ahmed et al., 2012; Hill-Burns et al., 2011; International Parkinson
5
Disease Genomics et al., 2011; Nalls et al., 2014; Pankratz et al., 2012; Simon-Sanchez
6
et al., 2011; Wissemann et al., 2013). However, the HLA complex with different ethnic
7
and geographic origins is highly polymorphic. GWAS study in Japanese PD patients has
8
shown no evidence of HLA association (Satake et al., 2009). Hamza et al. demonstrated
9
rs3129882 minor G allele increases risk of PD (Hamza et al., 2010), but Guo et al. found
10
minor A allele increased PD risk (Guo et al., 2011), and in contrast, Zhao et al. found
11
association of minor A allele with a decreased risk in ethnic Chinese PD patients (Zhao
12
et al., 2013). Previously we and other groups found absence of association between
13
rs3129882 and PD in Taiwanese and Chinese populations (Chiang et al., 2012; Zhao et
14
al., 2013). It is noticed that other reported associations of SNPs in HLA region
15
(rs4248166, rs9268515, rs2395163, rs75855844 and rs660895) with PD have not been
16
studied in Chinese and Asian populations (Ahmed et al., 2012; Hill-Burns et al., 2011;
17
Pankratz et al., 2012; Simon-Sanchez et al., 2011; Wissemann et al., 2013). To provide
18
more insights about HLA-DR region contributing to PD across different populations, we
19
conducted a case-control study by examining the genotypic and allelic frequencies of
20
five SNPs within the HLA-DR region in 486 Taiwanese PD patients and 473 control 5
1
subjects.
2 3
Subjects and Methods
4
Ethics Statement
5
This study was conducted according to the protocol approved by the Institutional
6
Review Boards of Chang Gung Memorial Hospital (Ethical license No: 20171921A3)
7
and all examinations were performed with written informed consents.
8
Patient population
9
We recruited PD patients in the neurological clinics of Chang Gung Memorial
10
Hospital-Linkou Medical Center. PD patients were diagnosed according to the clinical
11
diagnostic criteria of UK PD Society Brain Bank (Hughes et al., 1992). We also
12
recruited unrelated healthy individuals matched with age, gender, and ethnicity as
13
control subjects. PD patients were divided into early-onset PD (EOPD) with an age at
14
onset of < 50 years and late-onset PD (LOPD) with an age at onset of 55 years.
15
Genetic analysis
16
Five genetic loci (rs4248166, rs9268515, rs2395163, rs75855844 and rs660895) within
17
HLA region were selected from the PD risk loci identified in literatures (Ahmed et al.,
18
2012; Hill-Burns et al., 2011; Pankratz et al., 2012; Simon-Sanchez et al., 2011;
19
Wissemann et al., 2013). Intronic genetic variant rs3129882 has been previously
20
examined in Taiwanese PD patients (Chiang et al., 2012) and was not repeatedly tested 6
1
in this study. The SNP genotyping was performed by Agena MassARRAY platform
2
with iPLEX gold chemistry (Agena, San Diego, CA) following the manufacture
3
instruction. The specific PCR primer and extension primer sequences (Supplementary
4
Table S1) were designed with Assay Designer software package (v.4.0). Briefly,
5
genomic DNA sample (10 ng) was loaded to mutiplex PCR reaction in 5 µL containing
6
PCR primers (500 nmol for each), dNTPs (2.5 mM for each) and Taq polymerase (1
7
unit, Agena, PCR accessory and Enzyme kit). Thermocycling was set at 94℃ for 4
8
minutes followed by 45 cycles of 94℃ for 20 seconds, 56℃ for 30 seconds and 72℃
9
for 1 minute, than 72℃ for 3 minutes. Unincorporated dNTPs were deactivated using
10
shrimp alkaline phosphatase (0.3 U). The single base extension reaction was using
11
iPLEX enzyme, terminator mix, and extension primer mix followed by 94℃ for 30
12
seconds followed by 40 cycles of 94℃ for 5 seconds, and 5 inner cycle of 56℃ for 5
13
seconds and 80℃ for 5 seconds, than 72℃ for 3 minutes (Agena, iPLEX gold kit).
14
Purified primer extension reaction (7 nL) was loaded onto a matrix pad of a
15
SpectroCHIP (Agena) and then analyzed by MassARRAY Analyzer 4 (Agena).
16
Statistics
17
The genotypes of each variant in the PD patients and controls did not deviate from the
18
Hardy-Weinberg equilibrium. The Pearson’s χ2 test was used to compare allelic and
19
genotypic frequencies between the PD patients and controls. As this study involved 5
20
independent genetic loci, we made a modest correction using Bonferroni method for 7
1
multiple comparisons with statistical significance defined at P < 0.01, while P < 0.05
2
was considered as marginal significance. Haplotype correlations between each loci were
3
analyzed by SHEsis (http://analysis.bio-x.cn/myAnalysis.php). Only haplotypes with
4
frequencies of ≥ 0.01 in total cases were included. Given the observed allele frequency
5
before stratification, we had power greater than 0.8 to identify an association of the
6
allele with PD susceptibility when the per-allele genetic effect was greater than an odds
7
ratio of 1.5 for rs4248166 and rs660895, and 1.7 for rs9268515 and rs2395163.
8 9
Results
10
A total of 959 subjects, including 486 PD patients (female/male: 249/237) and 473
11
control subjects (female/male: 234/239) were recruited. To minimize the effect by the
12
same SNP within the same family, only one proband with familial PD was recruited.
13
The mean age at onset of PD symptoms was 63.53 ± 10.70 years (range 19 ~ 89), and
14
that of control subjects upon recruitment was 63.73 ± 11.86 years (range 26 ~ 94).
15
Rs2395163 C allele showed a lower frequency in PD patients compared to control
16
subjects with marginally statistical significance (OR = 0.74, 95% CI: 0.55 ~ 0.99, P =
17
0.045, Table 1). All subjects had G allele of rs75855844. The genotype analysis did not
18
find any significant association of all tested loci with PD (Table 2). However, the
19
stratification according to gender showed significant association between rs2395163 C
20
allele and PD in male patients (OR = 0.58, 95% CI: 0.39 ~ 0.88, P = 0.009, Table 3). A 8
1
lower frequency of rs9268585 C allele was detected in male PD patients compared to
2
male control subjects with marginally statistical significance (OR = 0.64, 95% CI: 0.41
3
~ 1.00, P = 0.049). GG genotype of rs660895 showed a lower prevalence in male PD
4
patients compared to male control subjects with marginally statistical significance (OR =
5
0.45, 95% CI: 0.21 ~ 0.97, P = 0.037). There were no statistically significant differences
6
in allelic and genotypic frequencies between PD patients and controls after being
7
stratified by age at onset (Table 4).
8 9
Linkage disequilibrium analysis showed rs4248166, rs9268515 and rs2395163 were highly correlated with each other, while rs660895 was highly correlated with
10
rs9268515 (Figure). Haplotype analysis did not find any significant association between
11
tested haplotypes and PD (Table 5).
12 13
Discussion
14
Although SNP loci within HLA-DR region are significantly associated with PD in
15
Caucasian populations (Ahmed et al., 2012; Hill-Burns et al., 2011; International
16
Parkinson Disease Genomics et al., 2011; Nalls et al., 2014; Pankratz et al., 2012;
17
Simon-Sanchez et al., 2011; Wissemann et al., 2013), the present study shows that only
18
rs2395163 C allele is associated with PD in male Taiwanese patients, whereas the
19
associations between other tested SNPs within HLA-DR region (rs4248166, rs9268515,
20
rs75855844 and rs660895) and PD are absent. It is important to notice that the minor 9
1
allelic frequency of rs9268515 in our control subjects (9.51%) is similar to that in
2
Japanese (9.6%) but very different from that in Europeans (16.3%) or Americans (18.9%)
3
from 1000 Genome (http://www.1000genomes.org/home). Rs2395163 minor allelic
4
frequency in our control subjects (11.73%) is similar to that in Han-Chinese in Beijing
5
(13.1%) or in Japanese (11.1%), but different from that of Europeans (19.8%) or
6
Americans (22.6%). The rs75855844 are monomorphic (All carrying GG genotype) in
7
our population. These distinct genetic backgrounds may contribute to the differential
8
effects of genetic loci within HLA-DR region on PD risk between Asian and Caucasian
9
populations.
10
While rs2395163 demonstrating significant association with male PD patients is
11
located near the HLA-DR gene, it is also an expression quantitative trait locus (eQTL)
12
that correlated with gene expression levels of HLA-DQA1 and HLA-DQA2, located 217
13
to 321 kb away, in cortical regions (Kleinjan and van Heyningen, 2005), monocytes
14
(Zeller et al., 2010) and lymphoblastoid cell lines (Veyrieras et al., 2008). While most
15
eQTLs lie either within genes or close to genes (cis-eQTL), 5% of eQTLs lie more than
16
20 kb upstream of the transcription start site (trans-eQTL) (Veyrieras et al., 2008). The
17
mechanisms by which trans-eQTLs alter transcription of their linked gene expression
18
are still poorly understood. It is possible that that transcription factors residing close to
19
the corresponding cis-eQTLs could mediate expression of distant gene expression
20
(Veyrieras et al., 2008), allowing trans-eQTLs to remotely affect gene expression. Other 10
1
potential regulatory mechanisms of trans-eQTLs include variation in noncoding RNA,
2
inter-chromosomal interaction, or haplotype variation in nearby sequence. Future studies
3
to determine the gene expression network controlled by rs2395163, particularly in DA
4
neurons, will be warranted to characterize the role of HLA plays in pathogenesis of PD.
5
The differences between male and female PD patients in symptoms, course, and
6
cognitive effects have been recognized. More men than women have PD by a ratio of
7
approximately 2: 1 (Baldereschi et al., 2000). The age at onset in male PD patients are
8
2.1 years earlier compared to female patients (Haaxma et al., 2007). Clinical
9
characteristics that appear in men more often than women include rigidity and rapid eye
10
movement behavior disorder, whereas more women than men exhibit postural instability,
11
dyskinesia and depression (Baba et al., 2005). Genetically, GRN rs5848 and NURR1
12
IVS6 modify the risk of PD particularly in the female populations (Chang et al., 2013;
13
Chen et al., 2007). Our study reveals protective effect of rs2395163 C allele in male PD
14
patients. The factor contributing to these gender differences of PD is still uncertain. In
15
female PD patients, age at onset positively correlates with fertile life span, parity, and
16
age of menopause, suggesting the neuroprotection of estrogen (Haaxma et al., 2007).
17
Environmental factors, such as exposures to toxins or heavy metals and pesticides, may
18
be more present in the male working environment and that may result in the higher
19
prevalence of PD in male populations (Baldereschi et al., 2000). Further, there may also
20
be gender-related differences in expression of genes related to PD pathways 11
1
(Cantuti-Castelvetri et al., 2007; Simunovic et al., 2010). The expression level of SNCA
2
and PINK1 are up-regulated in DA neurons of male patients compared to the male
3
controls (Cantuti-Castelvetri et al., 2007). Genes involved in oxidative phosphorylation,
4
apoptosis, synaptic transmission and transmission of nerve impulse are down-regulated
5
in DA neurons of the male patients compared to the male controls (Simunovic et al.,
6
2010). Expression levels of genes related to PD or DA neuron development show
7
significant difference between males and females in the normal physiological state or
8
under stress conditions (Tao et al., 2012). Furthermore, substantial evidence has shown
9
the interaction between androgen signaling and immune responses may contribute to the
10
gender-related differential disease prevalence and progression (Gubbels Bupp and
11
Jorgensen, 2018). A recent study also shows that SRY, encoding Y chromosome,
12
co-localizes with DA neurons in the substantia nigra and lowering nigral SRY expression
13
with antisense oligonucleotides in male rats diminished motor deficits and nigral DA
14
neuronal loss in 6-hydroxydopamine- and rotenone-induced rat models of PD,
15
suggesting SRY directly contributes to the sex differences in PD (Lee et al., 2019). These
16
studies suggest SRY or androgen may play a crucial role in pathogenesis of PD.
17
Therefore, we propose that the rs2395163 C allele in male PD may interact with
18
male-specific proteins or signaling to achieve a protection effect. A large-scaled study
19
on male PD patients should be carried out to consolidate these gender-specific
20
associations. 12
1
Previous reports have shown that the SNPs rs4248166, rs9268515, rs7585844 and
2
rs660895 are closely linked to PD in Caucasians (Ahmed et al., 2012; Hill-Burns et al.,
3
2011; International Parkinson Disease Genomics et al., 2011; Pankratz et al., 2012;
4
Simon-Sanchez et al., 2011; Wissemann et al., 2013). However, GWAS of PD including
5
East Asians, including China, Hong Kong, Korea, Malaysia, Singapore and Taiwan,
6
cannot recapitulate these genetic-disease associations (Foo et al., 2017). Consistently,
7
GWAS study in Japanese shows absence of the association between HLA region and PD
8
(Satake et al., 2009). In Chinese patients, the linkage between the SNPs surrounding
9
rs3129882 in HLA-DRA and PD is absent (Mo et al., 2015). HLA-DQB1 rs9275326 are
10
identified as a protective genetic variant for PD in Caucasian population (Nalls et al.,
11
2014), while this association has not been described in Taiwanese populations (Chang et
12
al., 2015). These results indicate the potential ethnic divergence within HLA region, as
13
well as suggest a distinct immunogenetic background between Asian and Caucasian PD
14
patients.
15
Our study provides important information about the weak association of HLA-DR
16
region with PD patients in Taiwanese. However, there are limitations. The natural bias
17
of hospital-based study may lead to the false positive result. The small sample size for
18
the EOPD patients may reduce the statistical power. The potential interactions of
19
unknown environmental factors with HLA-DR region were not explored. In future works,
20
larger series in different ethnic populations will be mandatory to further evaluate the 13
1
potential association between SNPs that showed marginally statistical significances in
2
PD. Causal inference studies between identified eQTLs and PD phenotypes will further
3
improve our understanding of PD pathogenesis.
4 5
Author Contribution
6
Conceptualization, Chen CM. and Wu YR.; Methodology, Chen CM. and Wu YR.;
7
Formal Analysis, Chang KH. and Chen CM.; Investigation, Chang KH, Wu YR., Chen
8
YC., Fung HC. and Chen CM.; Resources, Chang KH., Wu YR., Chen YC., Fung HC.
9
and Chen CM.; Data Curation, Chen CM., Wu YR. and Chang KH.; Writing – Original
10
Draft Preparation, Chang KH.; Writing – Review & Editing, Chang KH, Wu YR and
11
Chen CM..; Funding Acquisition, Wu YR and Chen CM.
12 13
Disclosure statement
14
The authors do not have any actual or potential conflicts of interest.
15 16
Data availability
17
The datasets used and/or analysed during the current study are available from the
18
corresponding author upon request.
19 20
Acknowledgment 14
1
This study was sponsored by Chang Gung Medical Foundation (grant no.
2
CMRPG3F0382, CMRPG3H147 and CMRPG3J127). The funders had no role in study
3
design, data collection and analysis, decision to publish, or preparation of the
4
manuscript.
5 6
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18
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19
501-504.
21
Table 1. Comparisons of minor allelic frequencies of single nucleotide polymorphisms (SNPs) between Parkinson's disease (PD) patients and the controls Minor allele of each SNP rs4248166, C rs9268515, C rs2395163, C rs660895, G
PD Allele, N=972 (%)
Controls Allele, N=946 (%)
188 (19.34) 74 (7.61) 87 (8.95) 245 (25.21)
180 (19.03) 90 (9.51) 111 (11.73) 262 (27.70)
OR (95% CI)
1.02 (0.81 ~ 1.28) 0.8614 0.78 (0.57 ~ 1.08) 0.1368 0.74 (0.55 ~ 0.99) 0.04521 0.88 (0.72 ~ 1.08) 0.2168
CI: confidence interval, OR: odds ratio, PD: Parkinson’s disease, SNP: single nucleotide polymorphism.
22
P value
Table 2. Genetic models of single nucleotide polymorphisms (SNPs) in Parkinson's disease (PD) patients and controls SNP
Genotype
PD N=486 (%)
Controls N=473 (%)
TT TC CC CC+TC vs TT
313 (64.40) 158 (32.51) 15 (3.09) 173 (35.60)
307 (64.90) 152 (32.14) 14 (2.96) 166 (35.10)
CC vs TC+CC GG GC
15 (3.09) 413 (84.98) 72 (14.81)
CC CC+GC vs GG CC vs GC+GG TT
OR (95% CI)
P value
1.02 (0.78 ~ 1.34) 1.05 (0.50 ~ 2.21) 1.02 (0.78 ~ 1.33)
0.8893 0.8961 0.8710
14 (2.96) 389 (82.24) 78 (16.49)
1.04 (0.50 ~ 2.19)
0.9089
0.87 (0.61 ~ 1.23)
0.4318
1 (0.21) 73 (15.02) 1 (0.21) 402 (82.72)
6 (1.27) 84 (17.76) 6 (1.27) 371 (78.44)
0.16 (0.02 ~ 1.31) 0.82 (0.58 ~ 1.15) 0.16 (0.02 ~ 1.34)
0.1088 0.2527 0.1150
TC CC CC+TC vs TT CC vs TC+TT AA AG
81 (16.67) 3 (0.62) 84 (17.28) 3 (0.62) 271 (55.76) 185 (38.07)
93 (19.66) 9 (1.90) 102 (21.56) 9 (1.90) 251 (53.07) 182 (38.48)
0.80 (0.58 ~ 1.12) 0.31 (0.08 ~ 1.15) 0.76 (0.55 ~ 1.05) 0.32 (0.09 ~ 1.19)
0.1938 0.1148 0.09373 0.07340
0.94 (0.72 ~ 1.23)
0.6581
Dominant Model
GG GG+AG vs AA
30 (6.17) 215 (44.24)
40 (8.46) 222 (46.93)
0.69 (0.42 ~ 1.15) 0.90 (0.70 ~ 1.16)
0.1547 0.4020
Recessive Model
GG vs AG+AA
30 (6.17)
40 (8.46)
0.71 (0.44 ~ 1.16)
0.1742
rs4248166
Dominant Model Recessive Model
rs9268515
Dominant Model Recessive Model
rs2395163
Dominant Model Recessive Model
rs660895
CI: confidence interval, OR: odds ratio, PD: Parkinson’s disease, SNP: single nucleotide polymorphism.
23
Table 3. Comparisons of minor allelic and genotypic frequencies of single nucleotide polymorphisms (SNPs) between female and male Parkinson's disease (PD) patients and the controls Minor allele of each SNP
PD (%)
Controls (%)
498 101 (20.28) 155 (62.25) 87 (34.94)
468 87 (18.59) 156 (66.67) 69 (29.49)
CC rs9268515, C
7 (2.81) 39 (7.83)
GG GC CC rs2395163, C TT TC CC rs660895, G
OR (95% CI)
P value
1.11 (0.81 ~ 1.53)
0.5070
1.27 (0.86 ~ 1.87)
0.2269
9 (3.85) 37 (7.91)
0.78 (0.28 ~ 2.15) 0.99 (0.62 ~ 1.58)
0.6347 0.9656
211 (84.74) 37 (14.86) 1 (0.40) 46 (9.23)
198 (84.62) 35 (14.96) 1 (0.43) 44 (9.40)
0.99 (0.60 ~ 1.64) 0.94 (0.06 ~ 15.10) 0.98 (0.64 ~ 1.51)
0.9750 0.9642 0.9298
204 (81.93) 44 (17.67) 1 (0.40) 129 (25.90)
192 (82.05) 40 (17.09) 2 (0.85) 125 (26.71)
1.04 (0.65 ~ 1.66) 0.47 (0.04 ~ 5.23) 0.96 (0.72 ~ 1.28)
0.8853 0.9586 0.7762
AA AG
139 (55.82) 91 (36.55)
126 (53.85) 91 (38.89)
0.91 (0.62 ~ 1.32)
0.6102
GG
19 (7.63)
17 (7.26)
1.01 (0.50 ~ 2.04)
0.9708
474 87 (18.35)
478 93 (19.46)
0.93 (0.67 ~ 1.29)
0.6643
158 (66.67) 71 (29.96)
151 (63.18) 83 (34.73)
0.82 (0.55 ~ 1.20)
0.3098
CC rs9268515, C GG GC CC
8 (3.38) 35 (7.38) 202 (85.23) 35 (14.77) 0 (0.00)
5 (2.09) 53 (11.09) 191 (79.92) 43 (17.99) 5 (2.09)
1.53 (0.49 ~ 4.78) 0.64 (0.41 ~ 1.00)
0.6545 0.04852
0.77 (0.47 ~ 1.25)
0.2938
rs2395163, C TT
41 (8.65) 198 (83.54)
67 (14.02) 179 (74.90)
0.58 (0.39 ~ 0.88)
0.009034
Female (allele) rs4248166, C TT TC
Male (allele) rs4248166, C TT TC
24
TC CC rs660895, G AA AG GG
37 (15.61) 2 (0.84) 116 (24.47)
53 (22.18) 7 (2.93) 137 (28.66)
0.63 (0.40 ~ 1.01) 0.26 (0.05 ~ 1.26) 0.81 (0.60 ~ 1.08)
0.05178 0.1418 0.1436
132 (55.70) 94 (39.66) 11 (4.64)
125 (57.30) 91 (38.08) 23 (9.62)
0.98 (0.67 ~ 1.42) 0.45 (0.21 ~ 0.97)
0.9090 0.03720
CI: confidence interval, OR: odds ratio, PD: Parkinson’s disease, SNP: single nucleotide polymorphism.
25
Table 4. Comparisons of minor allelic and genotypic frequencies of single nucleotide polymorphisms (SNPs) between early (EOPD) and late-onset Parkinson's disease (LOPD) patients and the controls Minor allele of each SNP
PD (%)
Controls (%)
OR (95% CI)
P value
86 18 (20.93) 34 (79.07) 9 (20.93)
116 24 (20.69) 36 (62.07) 20 (34.49)
1.015 (0.51 ~ 2.02)
0.9668
0.48 (0.19 ~ 1.19)
0.1092
CC rs9268515, C
0 (0.00) 6 (6.98)
2 (3.45) 17 (14.66)
0.44 (0.16 ~ 1.16)
0.08938
GG GC CC rs2395163, C
37 (86.05) 6 (13.95) 0 (0.00) 9 (10.47)
42 (72.41) 15 (25.86) 1 (1.72) 19 (16.38)
0.45 (0.16 ~ 1.29)
0.1330
0.60 (0.26 ~ 1.39)
0.2296
TT TC CC rs660895, G
34 (79.07) 9 (20.93) 0 (0.00) 19 (22.09)
40 (68.97) 17 (29.31) 1 (1.72) 38 (32.76)
0.62 (0.25 ~ 1.58)
0.3176
0.58 (0.31 ~ 1.10)
0.09586
AA AG
25 (58.14) 17 (39.53)
24 (41.38) 30 (51.72)
0.54 (0.24 ~ 1.23)
0.1427
GG
1 (2.33)
4 (6.90)
0.24 (0.03 ~ 2.30)
0.1863
886 179 (20.20)
830 156 (18.80)
1.09 (0.86 ~ 1.39)
0.4621
279 (62.98) 149 (33.63)
271 (65.30) 132 (31.81)
1.10 (0.82 ~ 1.46)
0.5307
CC rs9268515, C GG GC CC
15 (3.39) 68 (7.67) 376 (84.88) 66 (14.90) 1 (0.23)
12 (2.89) 73 (8.80) 347 (83.61) 63 (15.18) 5 (1.20)
1.21 (0.56 ~ 2.64) 0.86 (0.61 ~ 1.22)
0.6241 0.3984
0.97 (0.66 ~ 1.41) 0.18 (0.02 ~ 1.59)
0.8599 0.08452
rs2395163, C TT
78 (8.80) 368 (83.07)
91 (10.96) 331 (79.76)
0.78 (0.57 ~ 17.08)
0.1335
EOPD (allele) rs4248166, C TT TC
LOPD (allele) rs4248166, C TT TC
26
TC CC rs660895, G AA AG GG
72 (16.25) 3 (0.68) 226 (25.51)
76 (18.31) 8 (1.92) 224 (26.99)
0.85 (0.60 ~ 1.22) 0.33 (0.09 ~ 1.28) 0.93 (0.75 ~ 1.15)
0.3765 0.09460 0.4861
246 (55.53) 168 (37.92) 29 (6.55)
227 (54.70) 152 (36.63) 36 (8.67)
1.02 (0.77 ~ 1.36) 0.74 (0.44 ~ 1.25)
0.8919 0.2645
CI: confidence interval, OR: odds ratio, PD: Parkinson’s disease, SNP: single nucleotide polymorphism.
27
Table 5. Comparisons of sub-haplotypes between Parkinson's disease patients and the controls Sub-haplotypes rs4248166-rs9268515-rs2395163 T-G-T C-G-T T-C-C T-G-C
PD (%)
Control (%)
OR (95% CI)
P value
71.6 19.3
69.0 19.0
1.13 (0.93 ~ 1.37) 1.02 (0.81 ~ 1.28)
0.2257 0.8872
7.4 1.5
9.3 2.4
0.78 (0.56 ~ 1.08) 0.63 (0.33 ~ 1.21)
0.1349 0.1626
74.2 18.2 7.0
71.4 19.1 8.6
1.14 (0.93 ~ 1.40) 0.94 (0.75 ~ 1.18) 0.80 (0.57 ~ 1.11)
0.2019 0.6007 0.1808
rs660895-rs9268515 A-G G-G G-C
CI: confidence interval, OR: odds ratio, PD: Parkinson’s disease.
28
Figure. Linkage disequilibrium among the single nucleotide polymorphisms in HLA-DR region. Linkage disequilibrium was measured as D’. Numbers in cells are % D’.
29
30
1. Genetic loci within HLA-DR region may be associated with PD in Caucasian. 2. Five top PD risk loci within HLA-DR region were analyzed in Taiwanese PD patients. 3. Only rs2395163 C allele is associated with male PD patients in Taiwan. 4. PD patients display ethnic variation of genetic background within HLA-DR region.
Author Contribution: Conceptualization, Chen CM. and Wu YR.; Methodology, Chen CM. and Wu YR.; Formal Analysis, Chang KH. and Chen CM.; Investigation, Chang KH, Wu YR., Chen YC., Fung HC. and Chen CM.; Resources, Chang KH., Wu YR., Chen YC., Fung HC. and Chen CM.; Data Curation, Chen CM., Wu YR. and Chang KH.; Writing – Original Draft Preparation, Chang KH.; Writing – Review & Editing, Chang KH, Wu YR and Chen CM..; Funding Acquisition, Wu YR and Chen CM.
S0197-4580(19)30391-4
DOI:
https://doi.org/10.1016/j.neurobiolaging.2019.11.002
Reference:
NBA 10709
To appear in:
Neurobiology of Aging
Received Date: 21 February 2019 Revised Date:
1 November 2019
Accepted Date: 1 November 2019
Please cite this article as: Chang, K.-H., Wu, Y.-R., Chen, Y.-C., Fung, H.-C., Chen, C.-M., Association of genetic variants within HLA-DR region with Parkinson’s disease in Taiwan, Neurobiology of Aging (2019), doi: https://doi.org/10.1016/j.neurobiolaging.2019.11.002. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Elsevier Inc. All rights reserved.
1
Association of genetic variants within HLA-DR region with Parkinson’s
2
disease in Taiwan
3 4
Kuo-Hsuan Chang# MD PhD, Yih-Ru Wu# MD, Yi-Chun Chen MD, Hon-Chung Fung
5
MD PhD, Chiung-Mei Chen* MD PhD
6
a
7
College of Medicine, Taipei, Taiwan
Department of Neurology, Chang Gung Memorial Hospital, Chang Gung University
8 9
#
Contribute to the article equally
10 11
*Corresponding author:
12
Chiung-Mei Chen MD PhD, Department of Neurology, Chang Gung Memorial Hospital,
13
No.5, Fusing St., Gueishan Township, Taoyuan County 333, Taiwan
14
Tel: 886-3-3281200 Ext. 8721
15
Fax: +886-3-3287226
16
Email: Chiung-Mei [email protected]
17 18
Keywords: HLA-DR, rs4248166, rs9268515, rs2395163, rs75855844, rs660895,
19
Parkinson’s disease, Polymorphism, Disease association
20 21
All authors report no financial disclosures.
22
1
1
Abstract
2
Previous genome-wide association studies in Caucasians suggest genetic loci of human
3
leucocyte antigen (HLA)-DR region may be associated with Parkinson’s disease (PD).
4
However, these genetic-disease associations were limitedly reported in Asian
5
populations. Herein, we investigated the effects of 5 top PD-associated genetic variants
6
within HLA-DR region in Caucasians, including rs4248166, rs9268515, rs2395163,
7
rs75855844 and rs660895, by genotyping 486 Taiwanese patients with PD and 473
8
age-matched control subjects. Although the association between rs2395163 C allele and
9
PD patients demonstrated marginal significance (odds ratio (OR) = 0.74, 95% CI:
10
0.55~0.99, P = 0.045). The frequency of rs2395163 C allele (8.65%) in male PD patients
11
was significantly lower than in male control subjects (14.02%; OR = 0.58, 95% CI:
12
0.39~0.88, P = 0.009). The genetic associations between PD patients and other tested
13
genetic variants were negative. Although strong linkage disequilibriums
14
(rs4248166-rs9626515-s2395163 and rs9626515-rs660895) were observed, the
15
haplotype analysis did not find any significant risk-associated allelic combinations.
16
These results suggest a distinct genetic background within HLA-DR region between
17
Taiwanese and Caucasian PD patients.
18 19
Keywords: HLA-DR, rs4248166, rs9268515, rs2395163, rs75855844 and rs660895,
20
Parkinson’s disease, Polymorphism, Disease association 2
1 2
Abbreviations: EOPD = early-onset Parkinson’s disease; eQTL = expression
3
quantitative train locus; GWAS = genome wide-association studies; HLA = human
4
leucocyte antigen; LOPD = late-onset Parkinson’s disease; OR = odds ratio; PD =
5
Parkinson’s disease; SNP = single nucleotide polymorphism;
6
3
1
Introduction
2
Parkinson's disease (PD) is an age-related neurodegenerative disease characterized by
3
resting tremor, bradykinesia, rigidity and postural instability, which are caused by the
4
progressive loss of nigrostriatal dopaminergic (DA) neurons with deposition of protein
5
aggregates containing α-synuclein (Lang and Lozano, 1998). To date, the mechanisms of
6
PD neurodegeneration remains elusive. Neuroinflammation has been proposed to be an
7
important contributor to PD pathogenesis (Simon-Sanchez et al., 2011). In the brain of
8
PD patients, large numbers of microglia expressing human leucocyte antigen (HLA)-DR
9
have been detected in the substantia nigra (Simon-Sanchez et al., 2011), suggesting the
10
important role of genetic variants within this region in immunopathogenesis of PD.
11
The HLA-DR antigen, encoded by HLA-DRA and HLA-DRB alleles, acts as an
12
antigen-presenting molecule or regulatory molecule involved in innate immune response
13
(Klein and Sato, 2000). Interestingly, the associations of noncoding single nucleotide
14
polymorphisms (SNPs) in HLA-DR region with PD were discovered in genome-wide
15
association studies (GWAS), which were also found in subsequent other GWAS and
16
SNP-based studies mainly in Caucasian populations (Ahmed et al., 2012; Hamza et al.,
17
2010; Hill-Burns et al., 2011; International Parkinson Disease Genomics et al., 2011;
18
Pankratz et al., 2012; Simon-Sanchez et al., 2011; Wissemann et al., 2013). The first
19
PD-associated SNP, rs3129882, is in intron 1 of HLA-DRA (Hamza et al., 2010), which
20
was further confirmed in a meta-analysis of five PD GWAS (Pankratz et al., 2012), but 4
1
not in a GWAS from the Netherlands and a case-control study from Northern Spain
2
(Mata et al., 2011; Pankratz et al., 2012). The other PD associated SNPs are also
3
noncoding and map intergenicaly near HLA-DRA, HLA-DRB1, HLA-DRB5 and
4
HLA-DQB1 (Ahmed et al., 2012; Hill-Burns et al., 2011; International Parkinson
5
Disease Genomics et al., 2011; Nalls et al., 2014; Pankratz et al., 2012; Simon-Sanchez
6
et al., 2011; Wissemann et al., 2013). However, the HLA complex with different ethnic
7
and geographic origins is highly polymorphic. GWAS study in Japanese PD patients has
8
shown no evidence of HLA association (Satake et al., 2009). Hamza et al. demonstrated
9
rs3129882 minor G allele increases risk of PD (Hamza et al., 2010), but Guo et al. found
10
minor A allele increased PD risk (Guo et al., 2011), and in contrast, Zhao et al. found
11
association of minor A allele with a decreased risk in ethnic Chinese PD patients (Zhao
12
et al., 2013). Previously we and other groups found absence of association between
13
rs3129882 and PD in Taiwanese and Chinese populations (Chiang et al., 2012; Zhao et
14
al., 2013). It is noticed that other reported associations of SNPs in HLA region
15
(rs4248166, rs9268515, rs2395163, rs75855844 and rs660895) with PD have not been
16
studied in Chinese and Asian populations (Ahmed et al., 2012; Hill-Burns et al., 2011;
17
Pankratz et al., 2012; Simon-Sanchez et al., 2011; Wissemann et al., 2013). To provide
18
more insights about HLA-DR region contributing to PD across different populations, we
19
conducted a case-control study by examining the genotypic and allelic frequencies of
20
five SNPs within the HLA-DR region in 486 Taiwanese PD patients and 473 control 5
1
subjects.
2 3
Subjects and Methods
4
Ethics Statement
5
This study was conducted according to the protocol approved by the Institutional
6
Review Boards of Chang Gung Memorial Hospital (Ethical license No: 20171921A3)
7
and all examinations were performed with written informed consents.
8
Patient population
9
We recruited PD patients in the neurological clinics of Chang Gung Memorial
10
Hospital-Linkou Medical Center. PD patients were diagnosed according to the clinical
11
diagnostic criteria of UK PD Society Brain Bank (Hughes et al., 1992). We also
12
recruited unrelated healthy individuals matched with age, gender, and ethnicity as
13
control subjects. PD patients were divided into early-onset PD (EOPD) with an age at
14
onset of < 50 years and late-onset PD (LOPD) with an age at onset of 55 years.
15
Genetic analysis
16
Five genetic loci (rs4248166, rs9268515, rs2395163, rs75855844 and rs660895) within
17
HLA region were selected from the PD risk loci identified in literatures (Ahmed et al.,
18
2012; Hill-Burns et al., 2011; Pankratz et al., 2012; Simon-Sanchez et al., 2011;
19
Wissemann et al., 2013). Intronic genetic variant rs3129882 has been previously
20
examined in Taiwanese PD patients (Chiang et al., 2012) and was not repeatedly tested 6
1
in this study. The SNP genotyping was performed by Agena MassARRAY platform
2
with iPLEX gold chemistry (Agena, San Diego, CA) following the manufacture
3
instruction. The specific PCR primer and extension primer sequences (Supplementary
4
Table S1) were designed with Assay Designer software package (v.4.0). Briefly,
5
genomic DNA sample (10 ng) was loaded to mutiplex PCR reaction in 5 µL containing
6
PCR primers (500 nmol for each), dNTPs (2.5 mM for each) and Taq polymerase (1
7
unit, Agena, PCR accessory and Enzyme kit). Thermocycling was set at 94℃ for 4
8
minutes followed by 45 cycles of 94℃ for 20 seconds, 56℃ for 30 seconds and 72℃
9
for 1 minute, than 72℃ for 3 minutes. Unincorporated dNTPs were deactivated using
10
shrimp alkaline phosphatase (0.3 U). The single base extension reaction was using
11
iPLEX enzyme, terminator mix, and extension primer mix followed by 94℃ for 30
12
seconds followed by 40 cycles of 94℃ for 5 seconds, and 5 inner cycle of 56℃ for 5
13
seconds and 80℃ for 5 seconds, than 72℃ for 3 minutes (Agena, iPLEX gold kit).
14
Purified primer extension reaction (7 nL) was loaded onto a matrix pad of a
15
SpectroCHIP (Agena) and then analyzed by MassARRAY Analyzer 4 (Agena).
16
Statistics
17
The genotypes of each variant in the PD patients and controls did not deviate from the
18
Hardy-Weinberg equilibrium. The Pearson’s χ2 test was used to compare allelic and
19
genotypic frequencies between the PD patients and controls. As this study involved 5
20
independent genetic loci, we made a modest correction using Bonferroni method for 7
1
multiple comparisons with statistical significance defined at P < 0.01, while P < 0.05
2
was considered as marginal significance. Haplotype correlations between each loci were
3
analyzed by SHEsis (http://analysis.bio-x.cn/myAnalysis.php). Only haplotypes with
4
frequencies of ≥ 0.01 in total cases were included. Given the observed allele frequency
5
before stratification, we had power greater than 0.8 to identify an association of the
6
allele with PD susceptibility when the per-allele genetic effect was greater than an odds
7
ratio of 1.5 for rs4248166 and rs660895, and 1.7 for rs9268515 and rs2395163.
8 9
Results
10
A total of 959 subjects, including 486 PD patients (female/male: 249/237) and 473
11
control subjects (female/male: 234/239) were recruited. To minimize the effect by the
12
same SNP within the same family, only one proband with familial PD was recruited.
13
The mean age at onset of PD symptoms was 63.53 ± 10.70 years (range 19 ~ 89), and
14
that of control subjects upon recruitment was 63.73 ± 11.86 years (range 26 ~ 94).
15
Rs2395163 C allele showed a lower frequency in PD patients compared to control
16
subjects with marginally statistical significance (OR = 0.74, 95% CI: 0.55 ~ 0.99, P =
17
0.045, Table 1). All subjects had G allele of rs75855844. The genotype analysis did not
18
find any significant association of all tested loci with PD (Table 2). However, the
19
stratification according to gender showed significant association between rs2395163 C
20
allele and PD in male patients (OR = 0.58, 95% CI: 0.39 ~ 0.88, P = 0.009, Table 3). A 8
1
lower frequency of rs9268585 C allele was detected in male PD patients compared to
2
male control subjects with marginally statistical significance (OR = 0.64, 95% CI: 0.41
3
~ 1.00, P = 0.049). GG genotype of rs660895 showed a lower prevalence in male PD
4
patients compared to male control subjects with marginally statistical significance (OR =
5
0.45, 95% CI: 0.21 ~ 0.97, P = 0.037). There were no statistically significant differences
6
in allelic and genotypic frequencies between PD patients and controls after being
7
stratified by age at onset (Table 4).
8 9
Linkage disequilibrium analysis showed rs4248166, rs9268515 and rs2395163 were highly correlated with each other, while rs660895 was highly correlated with
10
rs9268515 (Figure). Haplotype analysis did not find any significant association between
11
tested haplotypes and PD (Table 5).
12 13
Discussion
14
Although SNP loci within HLA-DR region are significantly associated with PD in
15
Caucasian populations (Ahmed et al., 2012; Hill-Burns et al., 2011; International
16
Parkinson Disease Genomics et al., 2011; Nalls et al., 2014; Pankratz et al., 2012;
17
Simon-Sanchez et al., 2011; Wissemann et al., 2013), the present study shows that only
18
rs2395163 C allele is associated with PD in male Taiwanese patients, whereas the
19
associations between other tested SNPs within HLA-DR region (rs4248166, rs9268515,
20
rs75855844 and rs660895) and PD are absent. It is important to notice that the minor 9
1
allelic frequency of rs9268515 in our control subjects (9.51%) is similar to that in
2
Japanese (9.6%) but very different from that in Europeans (16.3%) or Americans (18.9%)
3
from 1000 Genome (http://www.1000genomes.org/home). Rs2395163 minor allelic
4
frequency in our control subjects (11.73%) is similar to that in Han-Chinese in Beijing
5
(13.1%) or in Japanese (11.1%), but different from that of Europeans (19.8%) or
6
Americans (22.6%). The rs75855844 are monomorphic (All carrying GG genotype) in
7
our population. These distinct genetic backgrounds may contribute to the differential
8
effects of genetic loci within HLA-DR region on PD risk between Asian and Caucasian
9
populations.
10
While rs2395163 demonstrating significant association with male PD patients is
11
located near the HLA-DR gene, it is also an expression quantitative trait locus (eQTL)
12
that correlated with gene expression levels of HLA-DQA1 and HLA-DQA2, located 217
13
to 321 kb away, in cortical regions (Kleinjan and van Heyningen, 2005), monocytes
14
(Zeller et al., 2010) and lymphoblastoid cell lines (Veyrieras et al., 2008). While most
15
eQTLs lie either within genes or close to genes (cis-eQTL), 5% of eQTLs lie more than
16
20 kb upstream of the transcription start site (trans-eQTL) (Veyrieras et al., 2008). The
17
mechanisms by which trans-eQTLs alter transcription of their linked gene expression
18
are still poorly understood. It is possible that that transcription factors residing close to
19
the corresponding cis-eQTLs could mediate expression of distant gene expression
20
(Veyrieras et al., 2008), allowing trans-eQTLs to remotely affect gene expression. Other 10
1
potential regulatory mechanisms of trans-eQTLs include variation in noncoding RNA,
2
inter-chromosomal interaction, or haplotype variation in nearby sequence. Future studies
3
to determine the gene expression network controlled by rs2395163, particularly in DA
4
neurons, will be warranted to characterize the role of HLA plays in pathogenesis of PD.
5
The differences between male and female PD patients in symptoms, course, and
6
cognitive effects have been recognized. More men than women have PD by a ratio of
7
approximately 2: 1 (Baldereschi et al., 2000). The age at onset in male PD patients are
8
2.1 years earlier compared to female patients (Haaxma et al., 2007). Clinical
9
characteristics that appear in men more often than women include rigidity and rapid eye
10
movement behavior disorder, whereas more women than men exhibit postural instability,
11
dyskinesia and depression (Baba et al., 2005). Genetically, GRN rs5848 and NURR1
12
IVS6 modify the risk of PD particularly in the female populations (Chang et al., 2013;
13
Chen et al., 2007). Our study reveals protective effect of rs2395163 C allele in male PD
14
patients. The factor contributing to these gender differences of PD is still uncertain. In
15
female PD patients, age at onset positively correlates with fertile life span, parity, and
16
age of menopause, suggesting the neuroprotection of estrogen (Haaxma et al., 2007).
17
Environmental factors, such as exposures to toxins or heavy metals and pesticides, may
18
be more present in the male working environment and that may result in the higher
19
prevalence of PD in male populations (Baldereschi et al., 2000). Further, there may also
20
be gender-related differences in expression of genes related to PD pathways 11
1
(Cantuti-Castelvetri et al., 2007; Simunovic et al., 2010). The expression level of SNCA
2
and PINK1 are up-regulated in DA neurons of male patients compared to the male
3
controls (Cantuti-Castelvetri et al., 2007). Genes involved in oxidative phosphorylation,
4
apoptosis, synaptic transmission and transmission of nerve impulse are down-regulated
5
in DA neurons of the male patients compared to the male controls (Simunovic et al.,
6
2010). Expression levels of genes related to PD or DA neuron development show
7
significant difference between males and females in the normal physiological state or
8
under stress conditions (Tao et al., 2012). Furthermore, substantial evidence has shown
9
the interaction between androgen signaling and immune responses may contribute to the
10
gender-related differential disease prevalence and progression (Gubbels Bupp and
11
Jorgensen, 2018). A recent study also shows that SRY, encoding Y chromosome,
12
co-localizes with DA neurons in the substantia nigra and lowering nigral SRY expression
13
with antisense oligonucleotides in male rats diminished motor deficits and nigral DA
14
neuronal loss in 6-hydroxydopamine- and rotenone-induced rat models of PD,
15
suggesting SRY directly contributes to the sex differences in PD (Lee et al., 2019). These
16
studies suggest SRY or androgen may play a crucial role in pathogenesis of PD.
17
Therefore, we propose that the rs2395163 C allele in male PD may interact with
18
male-specific proteins or signaling to achieve a protection effect. A large-scaled study
19
on male PD patients should be carried out to consolidate these gender-specific
20
associations. 12
1
Previous reports have shown that the SNPs rs4248166, rs9268515, rs7585844 and
2
rs660895 are closely linked to PD in Caucasians (Ahmed et al., 2012; Hill-Burns et al.,
3
2011; International Parkinson Disease Genomics et al., 2011; Pankratz et al., 2012;
4
Simon-Sanchez et al., 2011; Wissemann et al., 2013). However, GWAS of PD including
5
East Asians, including China, Hong Kong, Korea, Malaysia, Singapore and Taiwan,
6
cannot recapitulate these genetic-disease associations (Foo et al., 2017). Consistently,
7
GWAS study in Japanese shows absence of the association between HLA region and PD
8
(Satake et al., 2009). In Chinese patients, the linkage between the SNPs surrounding
9
rs3129882 in HLA-DRA and PD is absent (Mo et al., 2015). HLA-DQB1 rs9275326 are
10
identified as a protective genetic variant for PD in Caucasian population (Nalls et al.,
11
2014), while this association has not been described in Taiwanese populations (Chang et
12
al., 2015). These results indicate the potential ethnic divergence within HLA region, as
13
well as suggest a distinct immunogenetic background between Asian and Caucasian PD
14
patients.
15
Our study provides important information about the weak association of HLA-DR
16
region with PD patients in Taiwanese. However, there are limitations. The natural bias
17
of hospital-based study may lead to the false positive result. The small sample size for
18
the EOPD patients may reduce the statistical power. The potential interactions of
19
unknown environmental factors with HLA-DR region were not explored. In future works,
20
larger series in different ethnic populations will be mandatory to further evaluate the 13
1
potential association between SNPs that showed marginally statistical significances in
2
PD. Causal inference studies between identified eQTLs and PD phenotypes will further
3
improve our understanding of PD pathogenesis.
4 5
Author Contribution
6
Conceptualization, Chen CM. and Wu YR.; Methodology, Chen CM. and Wu YR.;
7
Formal Analysis, Chang KH. and Chen CM.; Investigation, Chang KH, Wu YR., Chen
8
YC., Fung HC. and Chen CM.; Resources, Chang KH., Wu YR., Chen YC., Fung HC.
9
and Chen CM.; Data Curation, Chen CM., Wu YR. and Chang KH.; Writing – Original
10
Draft Preparation, Chang KH.; Writing – Review & Editing, Chang KH, Wu YR and
11
Chen CM..; Funding Acquisition, Wu YR and Chen CM.
12 13
Disclosure statement
14
The authors do not have any actual or potential conflicts of interest.
15 16
Data availability
17
The datasets used and/or analysed during the current study are available from the
18
corresponding author upon request.
19 20
Acknowledgment 14
1
This study was sponsored by Chang Gung Medical Foundation (grant no.
2
CMRPG3F0382, CMRPG3H147 and CMRPG3J127). The funders had no role in study
3
design, data collection and analysis, decision to publish, or preparation of the
4
manuscript.
5 6
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Zeller, T., Wild, P., Szymczak, S., Rotival, M., Schillert, A., Castagne, R., Maouche, S.,
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Germain, M., Lackner, K., Rossmann, H., Eleftheriadis, M., Sinning, C.R., Schnabel,
12
R.B., Lubos, E., Mennerich, D., Rust, W., Perret, C., Proust, C., Nicaud, V., Loscalzo,
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J., Hübner, N., Tregouet, D., Münzel, T., Ziegler, A., Tiret, L., Blankenberg, S.,
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Cambien, F., 2010. Genetics and beyond--the transcriptome of human monocytes and
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disease susceptibility. PloS One 5, e10693.
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Zhao, Y., Gopalai, A.A., Ahmad-Annuar, A., Teng, E.W., Prakash, K.M., Tan, L.C., Au,
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2013. Association of HLA locus variant in Parkinson's disease. Clin. Genet. 84,
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501-504.
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Table 1. Comparisons of minor allelic frequencies of single nucleotide polymorphisms (SNPs) between Parkinson's disease (PD) patients and the controls Minor allele of each SNP rs4248166, C rs9268515, C rs2395163, C rs660895, G
PD Allele, N=972 (%)
Controls Allele, N=946 (%)
188 (19.34) 74 (7.61) 87 (8.95) 245 (25.21)
180 (19.03) 90 (9.51) 111 (11.73) 262 (27.70)
OR (95% CI)
1.02 (0.81 ~ 1.28) 0.8614 0.78 (0.57 ~ 1.08) 0.1368 0.74 (0.55 ~ 0.99) 0.04521 0.88 (0.72 ~ 1.08) 0.2168
CI: confidence interval, OR: odds ratio, PD: Parkinson’s disease, SNP: single nucleotide polymorphism.
22
P value
Table 2. Genetic models of single nucleotide polymorphisms (SNPs) in Parkinson's disease (PD) patients and controls SNP
Genotype
PD N=486 (%)
Controls N=473 (%)
TT TC CC CC+TC vs TT
313 (64.40) 158 (32.51) 15 (3.09) 173 (35.60)
307 (64.90) 152 (32.14) 14 (2.96) 166 (35.10)
CC vs TC+CC GG GC
15 (3.09) 413 (84.98) 72 (14.81)
CC CC+GC vs GG CC vs GC+GG TT
OR (95% CI)
P value
1.02 (0.78 ~ 1.34) 1.05 (0.50 ~ 2.21) 1.02 (0.78 ~ 1.33)
0.8893 0.8961 0.8710
14 (2.96) 389 (82.24) 78 (16.49)
1.04 (0.50 ~ 2.19)
0.9089
0.87 (0.61 ~ 1.23)
0.4318
1 (0.21) 73 (15.02) 1 (0.21) 402 (82.72)
6 (1.27) 84 (17.76) 6 (1.27) 371 (78.44)
0.16 (0.02 ~ 1.31) 0.82 (0.58 ~ 1.15) 0.16 (0.02 ~ 1.34)
0.1088 0.2527 0.1150
TC CC CC+TC vs TT CC vs TC+TT AA AG
81 (16.67) 3 (0.62) 84 (17.28) 3 (0.62) 271 (55.76) 185 (38.07)
93 (19.66) 9 (1.90) 102 (21.56) 9 (1.90) 251 (53.07) 182 (38.48)
0.80 (0.58 ~ 1.12) 0.31 (0.08 ~ 1.15) 0.76 (0.55 ~ 1.05) 0.32 (0.09 ~ 1.19)
0.1938 0.1148 0.09373 0.07340
0.94 (0.72 ~ 1.23)
0.6581
Dominant Model
GG GG+AG vs AA
30 (6.17) 215 (44.24)
40 (8.46) 222 (46.93)
0.69 (0.42 ~ 1.15) 0.90 (0.70 ~ 1.16)
0.1547 0.4020
Recessive Model
GG vs AG+AA
30 (6.17)
40 (8.46)
0.71 (0.44 ~ 1.16)
0.1742
rs4248166
Dominant Model Recessive Model
rs9268515
Dominant Model Recessive Model
rs2395163
Dominant Model Recessive Model
rs660895
CI: confidence interval, OR: odds ratio, PD: Parkinson’s disease, SNP: single nucleotide polymorphism.
23
Table 3. Comparisons of minor allelic and genotypic frequencies of single nucleotide polymorphisms (SNPs) between female and male Parkinson's disease (PD) patients and the controls Minor allele of each SNP
PD (%)
Controls (%)
498 101 (20.28) 155 (62.25) 87 (34.94)
468 87 (18.59) 156 (66.67) 69 (29.49)
CC rs9268515, C
7 (2.81) 39 (7.83)
GG GC CC rs2395163, C TT TC CC rs660895, G
OR (95% CI)
P value
1.11 (0.81 ~ 1.53)
0.5070
1.27 (0.86 ~ 1.87)
0.2269
9 (3.85) 37 (7.91)
0.78 (0.28 ~ 2.15) 0.99 (0.62 ~ 1.58)
0.6347 0.9656
211 (84.74) 37 (14.86) 1 (0.40) 46 (9.23)
198 (84.62) 35 (14.96) 1 (0.43) 44 (9.40)
0.99 (0.60 ~ 1.64) 0.94 (0.06 ~ 15.10) 0.98 (0.64 ~ 1.51)
0.9750 0.9642 0.9298
204 (81.93) 44 (17.67) 1 (0.40) 129 (25.90)
192 (82.05) 40 (17.09) 2 (0.85) 125 (26.71)
1.04 (0.65 ~ 1.66) 0.47 (0.04 ~ 5.23) 0.96 (0.72 ~ 1.28)
0.8853 0.9586 0.7762
AA AG
139 (55.82) 91 (36.55)
126 (53.85) 91 (38.89)
0.91 (0.62 ~ 1.32)
0.6102
GG
19 (7.63)
17 (7.26)
1.01 (0.50 ~ 2.04)
0.9708
474 87 (18.35)
478 93 (19.46)
0.93 (0.67 ~ 1.29)
0.6643
158 (66.67) 71 (29.96)
151 (63.18) 83 (34.73)
0.82 (0.55 ~ 1.20)
0.3098
CC rs9268515, C GG GC CC
8 (3.38) 35 (7.38) 202 (85.23) 35 (14.77) 0 (0.00)
5 (2.09) 53 (11.09) 191 (79.92) 43 (17.99) 5 (2.09)
1.53 (0.49 ~ 4.78) 0.64 (0.41 ~ 1.00)
0.6545 0.04852
0.77 (0.47 ~ 1.25)
0.2938
rs2395163, C TT
41 (8.65) 198 (83.54)
67 (14.02) 179 (74.90)
0.58 (0.39 ~ 0.88)
0.009034
Female (allele) rs4248166, C TT TC
Male (allele) rs4248166, C TT TC
24
TC CC rs660895, G AA AG GG
37 (15.61) 2 (0.84) 116 (24.47)
53 (22.18) 7 (2.93) 137 (28.66)
0.63 (0.40 ~ 1.01) 0.26 (0.05 ~ 1.26) 0.81 (0.60 ~ 1.08)
0.05178 0.1418 0.1436
132 (55.70) 94 (39.66) 11 (4.64)
125 (57.30) 91 (38.08) 23 (9.62)
0.98 (0.67 ~ 1.42) 0.45 (0.21 ~ 0.97)
0.9090 0.03720
CI: confidence interval, OR: odds ratio, PD: Parkinson’s disease, SNP: single nucleotide polymorphism.
25
Table 4. Comparisons of minor allelic and genotypic frequencies of single nucleotide polymorphisms (SNPs) between early (EOPD) and late-onset Parkinson's disease (LOPD) patients and the controls Minor allele of each SNP
PD (%)
Controls (%)
OR (95% CI)
P value
86 18 (20.93) 34 (79.07) 9 (20.93)
116 24 (20.69) 36 (62.07) 20 (34.49)
1.015 (0.51 ~ 2.02)
0.9668
0.48 (0.19 ~ 1.19)
0.1092
CC rs9268515, C
0 (0.00) 6 (6.98)
2 (3.45) 17 (14.66)
0.44 (0.16 ~ 1.16)
0.08938
GG GC CC rs2395163, C
37 (86.05) 6 (13.95) 0 (0.00) 9 (10.47)
42 (72.41) 15 (25.86) 1 (1.72) 19 (16.38)
0.45 (0.16 ~ 1.29)
0.1330
0.60 (0.26 ~ 1.39)
0.2296
TT TC CC rs660895, G
34 (79.07) 9 (20.93) 0 (0.00) 19 (22.09)
40 (68.97) 17 (29.31) 1 (1.72) 38 (32.76)
0.62 (0.25 ~ 1.58)
0.3176
0.58 (0.31 ~ 1.10)
0.09586
AA AG
25 (58.14) 17 (39.53)
24 (41.38) 30 (51.72)
0.54 (0.24 ~ 1.23)
0.1427
GG
1 (2.33)
4 (6.90)
0.24 (0.03 ~ 2.30)
0.1863
886 179 (20.20)
830 156 (18.80)
1.09 (0.86 ~ 1.39)
0.4621
279 (62.98) 149 (33.63)
271 (65.30) 132 (31.81)
1.10 (0.82 ~ 1.46)
0.5307
CC rs9268515, C GG GC CC
15 (3.39) 68 (7.67) 376 (84.88) 66 (14.90) 1 (0.23)
12 (2.89) 73 (8.80) 347 (83.61) 63 (15.18) 5 (1.20)
1.21 (0.56 ~ 2.64) 0.86 (0.61 ~ 1.22)
0.6241 0.3984
0.97 (0.66 ~ 1.41) 0.18 (0.02 ~ 1.59)
0.8599 0.08452
rs2395163, C TT
78 (8.80) 368 (83.07)
91 (10.96) 331 (79.76)
0.78 (0.57 ~ 17.08)
0.1335
EOPD (allele) rs4248166, C TT TC
LOPD (allele) rs4248166, C TT TC
26
TC CC rs660895, G AA AG GG
72 (16.25) 3 (0.68) 226 (25.51)
76 (18.31) 8 (1.92) 224 (26.99)
0.85 (0.60 ~ 1.22) 0.33 (0.09 ~ 1.28) 0.93 (0.75 ~ 1.15)
0.3765 0.09460 0.4861
246 (55.53) 168 (37.92) 29 (6.55)
227 (54.70) 152 (36.63) 36 (8.67)
1.02 (0.77 ~ 1.36) 0.74 (0.44 ~ 1.25)
0.8919 0.2645
CI: confidence interval, OR: odds ratio, PD: Parkinson’s disease, SNP: single nucleotide polymorphism.
27
Table 5. Comparisons of sub-haplotypes between Parkinson's disease patients and the controls Sub-haplotypes rs4248166-rs9268515-rs2395163 T-G-T C-G-T T-C-C T-G-C
PD (%)
Control (%)
OR (95% CI)
P value
71.6 19.3
69.0 19.0
1.13 (0.93 ~ 1.37) 1.02 (0.81 ~ 1.28)
0.2257 0.8872
7.4 1.5
9.3 2.4
0.78 (0.56 ~ 1.08) 0.63 (0.33 ~ 1.21)
0.1349 0.1626
74.2 18.2 7.0
71.4 19.1 8.6
1.14 (0.93 ~ 1.40) 0.94 (0.75 ~ 1.18) 0.80 (0.57 ~ 1.11)
0.2019 0.6007 0.1808
rs660895-rs9268515 A-G G-G G-C
CI: confidence interval, OR: odds ratio, PD: Parkinson’s disease.
28
Figure. Linkage disequilibrium among the single nucleotide polymorphisms in HLA-DR region. Linkage disequilibrium was measured as D’. Numbers in cells are % D’.
29
30
1. Genetic loci within HLA-DR region may be associated with PD in Caucasian. 2. Five top PD risk loci within HLA-DR region were analyzed in Taiwanese PD patients. 3. Only rs2395163 C allele is associated with male PD patients in Taiwan. 4. PD patients display ethnic variation of genetic background within HLA-DR region.
Author Contribution: Conceptualization, Chen CM. and Wu YR.; Methodology, Chen CM. and Wu YR.; Formal Analysis, Chang KH. and Chen CM.; Investigation, Chang KH, Wu YR., Chen YC., Fung HC. and Chen CM.; Resources, Chang KH., Wu YR., Chen YC., Fung HC. and Chen CM.; Data Curation, Chen CM., Wu YR. and Chang KH.; Writing – Original Draft Preparation, Chang KH.; Writing – Review & Editing, Chang KH, Wu YR and Chen CM..; Funding Acquisition, Wu YR and Chen CM.