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Association of the MTHFR rs1801131 and rs1801133 variants in sporadic Parkinson’s disease patients
Accepted Manuscript Title: Association of the MTHFR rs1801131 and rs1801133 variants in sporadic Parkinson’s disease patients Author: Lamei Yuan Zhi Song Xiong Deng Wei Xiong Zhijian Yang Hao Deng PII: DOI: Reference:
S0304-3940(16)30030-1 http://dx.doi.org/doi:10.1016/j.neulet.2016.01.031 NSL 31789
To appear in:
Neuroscience Letters
Received date: Revised date: Accepted date:
23-12-2015 15-1-2016 18-1-2016
Please cite this article as: Lamei Yuan, Zhi Song, Xiong Deng, Wei Xiong, Zhijian Yang, Hao Deng, Association of the MTHFR rs1801131 and rs1801133 variants in sporadic Parkinson’s disease patients, Neuroscience Letters http://dx.doi.org/10.1016/j.neulet.2016.01.031 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. 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.
Association of the MTHFR rs1801131 and rs1801133 variants in sporadic Parkinson’s disease patients
Lamei Yuan a,b, Zhi Song b, Xiong Deng a, Wei Xiong c, Zhijian Yang a, Hao Deng a,b,*
a
Center for Experimental Medicine, the Third Xiangya Hospital, Central South
University, Changsha, China b
Department of Neurology, the Third Xiangya Hospital, Central South University,
Changsha, China c
Cancer Research Institute, Xiangya School of Medicine, Central South University,
Changsha, China
*
Address correspondence to:
Hao Deng, M.D., Ph.D. Professor of Center for Experimental Medicine and Professor of Neurology Deputy Director of Center for Experimental Medicine The Third Xiangya Hospital, Central South University 138 Tongzipo Road, Changsha, Hunan 410013, P.R. China Tel: 86-731-88618372 Fax: 86-731-88618339 Email: [email protected]
1
HIGHLIGHTS The association of rs1801131 and rs1801133 with Parkinson’s disease was studied. 512 Chinese Han patients with Parkinson’s disease and 512 controls were genotyped. The T allele of rs1801133 may decrease the risk of PD in Chinese Han population. The A-T haplotype of rs1801131-rs1801133 may decrease the risk of PD in Chinese.
ABSTRACT Parkinson’s disease (PD) is a common age-dependent neurodegenerative movement disorder related to multiple factors, and genetic factors play an important role in the pathogenesis of PD. Variants in the methylenetetrahydrofolate reductase gene (MTHFR), a gene encoding a folate-dependent enzyme that is involved in homocysteine metabolism, have been reported to be associated with PD. To explore the role of the MTHFR gene in the development of PD in Chinese Han population, we analyzed two MTHFR variants (rs1801131 and rs1801133) in a patient cohort consisting of 512 patients with PD from mainland China and a control cohort consisting of 512 age, gender and ethnicity matched normal subjects. Statistically significant differences in genotypic and allelic frequencies were detected in the MTHFR variant rs1801133 (P = 0.022 and 0.007, respectively; odds ratio = 0.780, 95% confidence interval = 0.651-0.934). In addition, the A-T haplotype of rs1801131-rs1801133 showed a protective role against PD development (P = 0.007, odds ratio = 0.779, 95% confidence interval = 0.650-0.933). Our results suggested that the T allele of rs1801133 variant and A-T haplotype of rs1801131-rs1801133 in the MTHFR gene may decrease the risk of developing PD in Chinese Han population from mainland China. Abbreviations: PD, Parkinson’s disease; MAPT, the microtubule-associated protein tau gene; GBA, the glucocerebrosidase gene; SMPD1, the sphingomyelin phosphodiesterase 1, acid lysosomal gene; MTHFR, the methylenetetrahydrofolate reductase gene; dbSNP, database of single nucleotide polymorphisms; SIFT, Sorting Intolerant from Tolerant; Polyphen-2, Polymorphism Phenotyping version 2; MALDI-TOF MS, Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry; PCR, polymerase chain reaction; PASW, Predictive Analytics Software; OR, odds ratio; CI, confidence interval. Keywords: Parkinson’s disease, MTHFR gene, Variant, rs1801131, rs1801133
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1. Introduction Parkinson’s disease (PD, MIM 168600) is the second most common age-dependent progressive neurodegenerative disorder following Alzheimer’s disease. PD affects approximately 1% of individuals over 60 years of age worldwide, and affects 4-5% of people over 85 years of age [1-4]. It is pathologically characterized by selective and progressive loss of dopaminergic neurons, which may be associated with neurotoxic effects related to changes in homocysteine levels [4-7]. The neuropathological hallmarks are aggregation of α-synuclein in remaining neurons and the presence of intracytoplasmic inclusions, known as Lewy bodies and neurites [1,2,4,5]. PD is clinically characterized by cardinal motor symptoms, including resting tremor, bradykinesia, rigidity, and postural instability, as well as a favorable response to levodopa [2,8,9]. Meanwhile, non-motor features, such as autonomic dysregulation, depression, sleep disturbances, cognitive impairment and dementia, present in some patients with PD [1,2]. Since 1997, through classic genetic linkage analysis, genome sequencing and genetic association studies, at least 21 genetic loci (designated as PARK1 to PARK21) and 16 disease-associated genes have been identified to be involved in familial and sporadic parkinsonism [2,4,10,11]. Only a small number of PD cases are directly related to rare single monogenic mutations, and the underlying pathogenesis of most patients with PD is likely multifactorial [2,10]. Other susceptibility genes, such as the microtubule-associated protein tau gene (MAPT), the glucocerebrosidase gene (GBA), and the sphingomyelin phosphodiesterase 1, acid lysosomal gene (SMPD1), are reported to be associated with PD [9,10,12]. Many mechanisms, including mitochondrial and lysosomal dysfunctions, oxidative stress damage, misfolded protein accumulation and impairment of cellular clearance system by ubiquitin-proteasome and autophagy pathways, are speculated to have a role in pathogenesis of PD [10,13]. Current dopamine replacement therapy provides only partial symptomatic relief for patients with PD, but is incapable of slowing or halting disease progression [2,12]. Some studies also suggest that changes in homocysteine levels may be neurotoxic to dopaminergic neurons [6,7]. Two common variants in the methylenetetrahydrofolate 3
reductase gene (MTHFR, MIM 607093), encoding a folate-dependent enzyme involved in homocysteine metabolism, have been reported to be associated with susceptibility of PD, possibly related to changes in the homocysteine level [7,14,15]. In order to determine whether variants rs1801131 and rs1801133 of the MTHFR gene are associated with PD in Chinese Han population, we conducted a genotyping assay of these two variants in a patient cohort consisting of 512 Chinese Han patients with PD and in a control cohort consisting of 512 age, gender and ethnicity matched normal subjects from mainland China. 2. Methods 2.1. Subjects In this study, we enrolled 1,024 unrelated Chinese Han subjects, including a PD cohort consisting of 512 patients with sporadic PD (308 males and 204 females) and a control cohort consisting of 512 age, gender and ethnicity matched controls (308 males and 204 females), from the southern part of mainland China. The patients in the PD cohort (age 65.8 ± 10.3 years, and age at onset 62.4 ± 7.8 years) were recruited from Department of Neurology, the Third Xiangya Hospital of Central South University, China. The disease condition was ascertained by two independent movement specialists. PD was clinically diagnosed following published diagnostic criteria, and secondary forms of parkinsonism by other known causes were excluded [16,17]. Among the 512 patients with sporadic PD, some had been previously studied and found to be negative for causal mutations in known genes presumably associated with PD [8,18,19]. The controls (age 65.9 ± 10.5 years) were volunteers without symptoms or history of neurological disorders, and were matched with PD cases for age, gender, ethnicity and residence. Peripheral blood was drawn from all the participants after obtaining a signed informed consent. The research was conducted in accordance with guidelines of Helsinki Declaration and with the approval from the Institutional Review Board, China. 2.2. Variants selection and genotyping We filtered variants in the database of single nucleotide polymorphisms (version 144, dbSNP144) for the MTHFR gene with a minor allele frequency above 0.05. Three 4
bioinformatics prediction software programs, including Sorting Intolerant from Tolerant
(SIFT,
(Polyphen-2,
http://sift.jcvi.org/),
Polymorphism
http://genetics.bwh.harvard.edu/pph2/),
Phenotyping and
version
2
MutationTaster
(http://www.mutationtaster.org/), were applied to evaluate whether variants affect protein structure and function [20-22]. Two variants, rs1801131 (c.1286A>C, p.E429A) and rs1801133 (c.665C>T, p.A222V), in the MTHFR gene (NCBI Reference Sequence: NM_005957.4, NP_005948.3) were selected for evaluating the potential association between MTHFR variants and risk of PD. Isolation of genomic DNA from peripheral blood leucocytes was conducted via standard phenol-chloroform extraction method [8]. Genotyping of the two MTHFR gene variants was conducted in all study subjects by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) using the Sequenom MassARRAY system following the iPLEXTM Gold Application Guide [23]. The primer pairs for allele-specific amplification and single-base extension were designed by Sequenom Assay Design 3.1 software (Sequenom, San Diego, CA, USA), then synthesized and diluted as required, which were listed in Table 1. Allele-specific amplification for targeted regions by polymerase chain reaction (PCR), treatment of PCR products by shrimp alkaline phosphatase, single-base extension with extending primer, and purification of reaction products by resin, were conducted following the manufacturers’ protocols (Bioyong Technologies, Beijing, China) and as previously described [24]. The final extended purified products were transferred to the standard 384-well SpectroCHIP by MassARRAY Nanodispenser RS1000 system (Sequenom Inc.). Mass spectrometric analyses were performed by MALDI-TOF MS system, and then MassARRAY Typer 4.0 software (Sequenom Inc.) was applied to analyze the spectrometric peak charts and to generate the genotype data [25]. The genotyping assay in this study was performed using the Sequenom MassARRAY iPLEX Gold platform by investigators blinded to the sample status (case or control subjects). Quality control was conducted by genotyping duplicate samples, positive and negative controls [26]. In addition, Sanger sequencing was performed in 8% randomly selected samples on an ABI 3500 DNA sequencer to check the reliability and 5
accuracy of the method, following the manufacturer’s instructions (Applied Biosystems Inc., Foster City, CA, US) [27]. 2.3. Statistical analysis Statistical analysis was performed using Predictive Analytics Software (PASW) Statistics 18.0 (SPSS Inc., Chicago, IL, USA). Hardy-Weinberg equilibrium for genotype frequencies was examined to evaluate the normal heterogeneity of the case and control groups. Pearson’s 2 test was used to analyze the differences in the genotypic and allelic frequencies of two variants [19]. Haplotype construction and association
analysis
were
performed
using
the
SHEsis
Online
Version
(http://analysis.bio-x.cn) [28]. Results were expressed as P values, odds ratios (ORs), and 95% confidence intervals (CIs). Two-sided P value less than 0.05 was considered to be statistically significant, as described in previous studies [26,29]. 3. Results Genotypes from 1,024 subjects were obtained by MALDI-TOF MS analysis and confirmed by random Sanger sequencing. Both genotypic and allelic frequencies of 512 patients and 512 controls are summarized in Table 2. For both variants (rs1801131 and rs1801133), no evidence of deviation from Hardy-Weinberg equilibrium was observed in either PD cohort or control cohort (all P > 0.05). Between the two cohorts, statistically significant differences in genotypic and allelic frequencies were identified in the MTHFR rs1801133 (P = 0.022 and 0.007, respectively; OR = 0.780, 95% CI: 0.651-0.934), and the frequency of T allele was 0.338 in patients and 0.396 in controls. However, there was no significant difference in the MTHFR rs1801131 (P = 0.213 and 0.098, respectively; OR = 1.201, 95% CI: 0.967-1.492) (Table 2). For haplotypes composed of rs1801131 and rs1801133, only the A-T haplotype showed an association with sporadic PD (2 = 7.378, P = 0.007, OR = 0.779, 95% CI: 0.650-0.933) (Table 3). 4. Discussion PD is largely a late-onset sporadic neurodegenerative disorder with an age-dependent prevalence, and only about 5-10% of PD cases are familial and are transmitted in a Mendelian fashion [2,30]. With the advent of sequencing technologies, an increasing 6
number of genetic loci and disease-causing or susceptibility genes have been identified to be involved in the pathogenesis of PD [2,4,8,10]. There are increasing numbers of evidence suggesting that genetic variants either exert a protective role against PD development or increase the risk of PD development [2,10,13]. Recently, the MTHFR gene variants have been reported to be associated with PD, though there are some discrepancies among different studies (Table 4) [15,31-35]. In this study, we found that the T allele of rs1801133 variant and the A-T haplotype of rs1801131-rs1801133 are able to decrease the risk of developing PD in Chinese Han individuals from mainland China. The finding that T allele of rs1801133 decreases the risk of PD development is consistent with that in a previously reported case-control study in Chinese Han population [31]. Additionally, our finding that rs1801131 is not associated with development of PD is consistent with previous reports [15,32,35]. The MTHFR gene is localized to chromosome 1p36.3 spanning over 20 kb and contains a noncoding exon (exon 1) and 11 coding exons. The MTHFR gene encodes a ~74.5 kDa catalytically active protein with 656 amino acids [36,37]. The encoded enzyme catalyzes the conversion of 5,10-methylenetetrahydrofolate, a carbon donor in nucleotide biosynthesis, to 5-methyltetrahydrofolate, the main form of circulatory folate and a methyl donor for homocysteine remethylation to methionine [15,36,37]. The enzyme regulates the proportional distribution of one-carbon moieties and homocysteine levels, and plays a pivotal role in folate metabolism [15,37]. Genetic mutations in the MTHFR gene can cause autosomal recessive homocystinuria due to MTHFR deficiency, and gene variants are described to be related to vascular diseases, thromboembolism, neural tube defects, schizophrenia, cancers, as well as neurodegenerative diseases, such as Alzheimer’s disease and PD [37-39]. Increased homocysteine elicits neurotoxic effects and might accelerate dopaminergic neurons death [40-42]. Elevated homocysteine levels caused by dietary folate deficiency or focal infusion of homocysteine may enhance dysfunction and death of dopaminergic neurons in a mouse model of PD, revealing a potential role in PD [43]. The two common variants genotyped in this study have been reported to affect enzymatic activity, particularly in folate deficient state, and change plasma homocysteine levels, 7
influencing the risk of PD development [15,37,44]. The inconsistent findings of rs1801133 may be explained by different genetic background, epigenetics or environmental factors, such as folate, vitamin B6 and vitamin B12 dietary levels, etc. [34,38,39,45]. As noted, this is the first study to concurrently explore the potential association between two variants (rs1801131 and rs1801133), rs1801131-rs1801133 haplotypes and the risk of PD in Chinese Han population from mainland China. Our data may provide additional support for the association between the MTHFR variant (rs1801133) and sporadic PD in Chinese Han population. However, further genetic studies involving larger sample size, and different ethnic or geographic populations, accompanied by functional analysis are necessary to firmly establish the role of these variants in PD development.
Conflict of interest The authors declare no conflict of interest.
Acknowledgments We give our sincere appreciation to all the participators and investigators enrolled in this study. This work was supported by grants from New Xiangya Talents Project, China (H.D.).
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Association of seven functional polymorphisms of one-carbon metabolic pathway with total plasma homocysteine levels and susceptibility to Parkinson’s disease among South Indians, Neurosci. Lett. 568 (2014) 1-5. [47]C.S. Fong, H.Y. Shyu, J.C. Shieh, Y.P. Fu, T.Y. Chin, H.W. Wang, C.W. Cheng, Association of MTHFR, MTR, and MTRR polymorphisms with Parkinson’s disease among ethnic Chinese in Taiwan, Clin. Chim. Acta 412 (2011) 332-338. [48]R.Y. Yuan, J.J. Sheu, J.M. Yu, C.J. Hu, I.J. Tseng, C.S. Ho, C.Y. Yeh, Y.L. Hung, T.R. Chiang, Methylenetetrahydrofolate reductase polymorphisms and plasma homocysteine in levodopa-treated and non-treated Parkinson’s disease patients, J. Neurol. Sci. 287 (2009) 64-68. [49]M.C. Rodriguez-Oroz, P.M. Lage, J. Sanchez-Mut, I. Lamet, J. Pagonabarraga, J.B. Toledo, D. Garcia-Garcia, P. Clavero, L. Samaranch, C. Irurzun, J.M. Matsubara, J. Irigoien, E. Bescos, J. Kulisevsky, J. Perez-Tur, J.A. Obeso, Homocysteine and cognitive impairment in Parkinson’s disease: a biochemical, neuroimaging, and genetic study, Mov. Disord. 24 (2009) 1437-1444. [50]R.M. Camicioli, T.P. Bouchard, M.J. Somerville, Homocysteine is not associated with global motor or cognitive measures in nondemented older Parkinson’s disease patients, Mov. Disord. 24 (2009) 176-182. [51]D. Caccamo, G. Gorgone, M. Curro, G. Parisi, W. Di Iorio, C. Menichetti, V. Belcastro, L. Parnetti, A. Rossi, F. Pisani, R. Ientile, P. Calabresi, Effect of MTHFR
polymorphisms
on
hyperhomocysteinemia
in
levodopa-treated
Parkinsonian patients, Neuromolecular Med. 9 (2007) 249-254. [52]J. Dorszewska, J. Florczak, A. Rozycka, B. Kempisty, J. Jaroszewska-Kolecka, K. Chojnacka, W.H. Trzeciak, W. Kozubski, Oxidative DNA damage and level of thiols as related to polymorphisms of MTHFR, MTR, MTHFD1 in Alzheimer’s and Parkinson’s diseases, Acta Neurobiol. Exp. (Wars) 67 (2007) 113-129. [53]Z. Todorovic, E. Dzoljic, I. Novakovic, D. Mirkovic, R. Stojanovic, Z. Nesic, M. Krajinovic, M. Prostran, V. Kostic, Homocysteine serum levels and MTHFR C677T genotype in patients with Parkinson’s disease, with and without levodopa therapy, J. Neurol. Sci. 248 (2006) 56-61. 14
[54]D. Religa, K. Czyzewski, M. Styczynska, B. Peplonska, J. Lokk, M. Chodakowska-Zebrowska,
K.
Stepien,
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Winblad,
M.
Barcikowska,
Hyperhomocysteinemia and methylenetetrahydrofolate reductase polymorphism in patients with Parkinson’s disease, Neurosci. Lett. 404 (2006) 56-60. [55]U. Wullner, H. Kolsch, M. Linnebank, Methylenetetrahydrofolate reductase in Parkinson’s disease, Ann. Neurol. 58 (2005) 972-973. [56]Y. Momose, M. Murata, K. Kobayashi, M. Tachikawa, Y. Nakabayashi, I. Kanazawa, T. Toda, Association studies of multiple candidate genes for Parkinson’s disease using single nucleotide polymorphisms, Ann. Neurol. 51 (2002) 133-136. [57]D.L. Harmon, D. Ramsbottom, A.S. Whitehead, Y. Ben-Shlomo, G. Davey-Smith, The thermolabile variant of 5,10-methylenetetrahydrofolate reductase is not associated with Parkinson’s disease, J. Neurol. Neurosurg. Psychiatry 62 (1997) 671.
15
Table 1: Primer sequences for the MTHFR gene variants. dbSNP ID
Bioinformatics predictiona
rs1801131
Damaging/Benign/Polymorphism acgttggatgTCTACCTGA acgttggatgTCTCCCGA
rs1801133
Forward primerb (5'3')
Reverse primerb (5'3') Product length
AGAGCAAGTCC
GAGGTAAAGAAC
Damaging/Probably
acgttggatgCACTTGAA
acgttggatgAAGTGATG
damaging/Polymorphism
GGAGAAGGTGTC
CCCATGTCGGTG
109 bp
Extending primerb (5'3') gggatGAGCTGACCAGT GAAG
112 bp
gagaTGCGTGATGATGA AATCG
a
Sorting Intolerant from Tolerant/Polymorphism Phenotyping version 2/MutationTaster prediction
b
The lowercase letters in the primer sequences are 5'-end tags (forward and reverse primers) or nonhomologous sequences (extending primers)
to increase the molecular weights. MTHFR, methylenetetrahydrofolate reductase; dbSNP, database of single nucleotide polymorphisms.
16
Table 2: Genotypic and allelic frequencies of the MTHFR gene variants (rs1801131 and rs1801133) in Chinese Han sporadic PD patients and controls. dbSNP ID
Subject
rs1801131
P value (2)
Genotype (n = 512, %)
Allele (n = 1024, %)
AA
AC
CC
A
C
PD
315 (61.5)
174 (34.0)
23 (4.5)
804 (78.5)
220 (21.5)
Control
342 (66.8)
150 (29.3)
20 (3.9)
834 (81.4)
190 (18.6)
CC
CT
TT
C
T
PD
222 (43.4)
234 (45.7)
56 (10.9)
678 (66.2)
346 (33.8)
Control
182 (35.5)
255 (49.8)
75 (14.6)
619 (60.4)
405 (39.6)
rs1801133
0.213 (3.097)
0.022 (7.618)
P value (2)
OR (95% CI)
0.098 (2.745)
1.201 (0.967-1.492)
0.007 (7.319)
0.780 (0.651-0.934)
Statistically significant P values are marked in bold. MTHFR, methylenetetrahydrofolate reductase; PD, Parkinson’s disease; dbSNP, database of single nucleotide polymorphisms; OR, odds ratio; CI, confidence interval.
17
Table 3: Haplotype analysis of rs1801131 and rs1801133 in the MTHFR gene comparing between PD patients and matched controls. Haplotype
Patients (%) Controls (%)
2 value
P value
OR (95% CI)
A-C
44.9
42.1
1.712
0.191
1.124 (0.943-1.339)
A-T
33.6
39.4
7.378
0.007
0.779 (0.650-0.933)
C-C
21.3
18.4
2.729
0.099
1.201 (0.966-1.493)
C-T
0.2
0.2
-
-
-
Statistically significant P value is marked in bold. MTHFR, methylenetetrahydrofolate reductase; PD, Parkinson’s disease; OR, odds ratio; CI, confidence interval.
18
Table 4: Genetic analysis of the MTHFR gene variants in PD patients. Reference
Location/Ethnicity
PD
Controls
Varianta
P value
OR/RR (95% CI)
Association
Garcia et al., 2015 [34]
Mexican
140
216
rs1801133
0.024
2.06 (1.101-3.873)
Yesb (Increased risk)
Liao et al., 2014 [31]
Chinese Han
765
717
rs1801133
0.003
0.80 (0.688-0.926)
Yes (Decreased risk)
Kumudini et al., 2014 [46]
South Indian
151
416
rs1801133
0.15
1.45 (0.87-2.45)
No
Vallelunga et al., 2014 [32]
Italian
120
-
rs1801131
1.0
-
No
rs1801133
0.024
c
Wu et al., 2013 [15] c
Caucasian and Asian
Yes (Age at onset)
907
820
rs1801131
0.733
1.03 (0.88-1.20)
No
1606
7168
rs1801133
<0.001
1.24 (1.11-1.38)
Yes (Increased risk)
Zhu et al., 2013 [33]
European and Asian
1820
7530
rs1801133
0.0002
1.212 (1.097-1.340)
Yes (Increased risk)
Bialecka et al., 2012 [35]
Poland
320
254
rs1801131
0.304
0.70 (0.39-1.29)
No
rs1801133
0.122
1.62 (0.91-2.90)
No
Gorgone et al., 2012 [6]
Italy
60
82
rs1801133
0.01
N.A.
Yes (Increased risk)
Fong et al., 2011 [47]
Chinese
211
218
rs1801133
>0.05
1.11 (0.81-1.53)
No
Yuan et al., 2009 [48]
Chinese
76
110
rs1801131
>0.05
N.A.
No
rs1801131
0.3277
N.A.
No
rs1801133
0.0239 (>0.0125)
rs1801131
0.50
N.A.
No
rs1801133
0.47
rs1801131
0.9
N.A.
No
rs1801133
0.2
rs1801133 Rodriguez-Oroz et al., 2009
Spain
89
30
[49] Camicioli et al., 2009 [50] Caccamo et al., 2007 [51]
Canada Italy
51 49
50 86
Lin et al., 2007 [39]
Chinese
94
146
rs1801133
0.120
N.A.
No
Dorszewska et al., 2007 [52]
Poland
98
50
rs1801131
>0.05
N.A.
No
rs1801133
19
G1793A
a
Todorovic et al., 2006 [53]
Serbia
113
53
rs1801133
0.2
N.A.
No
Religa et al., 2006 [54]
Poland
114
100
rs1801133
>0.05
N.A.
No
Wullner et al., 2005 [55]
Germany
342
342
rs1801131
0.24
N.A.
No
rs1801133
0.79
de Lau et al., 2005 [7]
Netherlands
65
5855
rs1801133
0.09
1.74 (0.91-3.32)
Yesd (Increased risk)
Momose et al., 2002 [56]
Japanese
232
249
rs1801131
0.458
N.A.
No
Harmon et al., 1997 [57]
Ireland
188
184
rs1801133
0.53
1.39 (0.63-3.12)
No
Variant: rs1801131 and rs1801133, commonly recorded as A1298C and C677T;
b
Risk variant: C allele;
c
Meta-analysis;
d
Borderline
significant. Statistically significant P values are marked in bold. MTHFR, methylenetetrahydrofolate reductase; PD, Parkinson’s disease; OR, odds ratio; RR, relative risk; CI, confidence interval; N.A., not available.
20
S0304-3940(16)30030-1 http://dx.doi.org/doi:10.1016/j.neulet.2016.01.031 NSL 31789
To appear in:
Neuroscience Letters
Received date: Revised date: Accepted date:
23-12-2015 15-1-2016 18-1-2016
Please cite this article as: Lamei Yuan, Zhi Song, Xiong Deng, Wei Xiong, Zhijian Yang, Hao Deng, Association of the MTHFR rs1801131 and rs1801133 variants in sporadic Parkinson’s disease patients, Neuroscience Letters http://dx.doi.org/10.1016/j.neulet.2016.01.031 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. 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.
Association of the MTHFR rs1801131 and rs1801133 variants in sporadic Parkinson’s disease patients
Lamei Yuan a,b, Zhi Song b, Xiong Deng a, Wei Xiong c, Zhijian Yang a, Hao Deng a,b,*
a
Center for Experimental Medicine, the Third Xiangya Hospital, Central South
University, Changsha, China b
Department of Neurology, the Third Xiangya Hospital, Central South University,
Changsha, China c
Cancer Research Institute, Xiangya School of Medicine, Central South University,
Changsha, China
*
Address correspondence to:
Hao Deng, M.D., Ph.D. Professor of Center for Experimental Medicine and Professor of Neurology Deputy Director of Center for Experimental Medicine The Third Xiangya Hospital, Central South University 138 Tongzipo Road, Changsha, Hunan 410013, P.R. China Tel: 86-731-88618372 Fax: 86-731-88618339 Email: [email protected]
1
HIGHLIGHTS The association of rs1801131 and rs1801133 with Parkinson’s disease was studied. 512 Chinese Han patients with Parkinson’s disease and 512 controls were genotyped. The T allele of rs1801133 may decrease the risk of PD in Chinese Han population. The A-T haplotype of rs1801131-rs1801133 may decrease the risk of PD in Chinese.
ABSTRACT Parkinson’s disease (PD) is a common age-dependent neurodegenerative movement disorder related to multiple factors, and genetic factors play an important role in the pathogenesis of PD. Variants in the methylenetetrahydrofolate reductase gene (MTHFR), a gene encoding a folate-dependent enzyme that is involved in homocysteine metabolism, have been reported to be associated with PD. To explore the role of the MTHFR gene in the development of PD in Chinese Han population, we analyzed two MTHFR variants (rs1801131 and rs1801133) in a patient cohort consisting of 512 patients with PD from mainland China and a control cohort consisting of 512 age, gender and ethnicity matched normal subjects. Statistically significant differences in genotypic and allelic frequencies were detected in the MTHFR variant rs1801133 (P = 0.022 and 0.007, respectively; odds ratio = 0.780, 95% confidence interval = 0.651-0.934). In addition, the A-T haplotype of rs1801131-rs1801133 showed a protective role against PD development (P = 0.007, odds ratio = 0.779, 95% confidence interval = 0.650-0.933). Our results suggested that the T allele of rs1801133 variant and A-T haplotype of rs1801131-rs1801133 in the MTHFR gene may decrease the risk of developing PD in Chinese Han population from mainland China. Abbreviations: PD, Parkinson’s disease; MAPT, the microtubule-associated protein tau gene; GBA, the glucocerebrosidase gene; SMPD1, the sphingomyelin phosphodiesterase 1, acid lysosomal gene; MTHFR, the methylenetetrahydrofolate reductase gene; dbSNP, database of single nucleotide polymorphisms; SIFT, Sorting Intolerant from Tolerant; Polyphen-2, Polymorphism Phenotyping version 2; MALDI-TOF MS, Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry; PCR, polymerase chain reaction; PASW, Predictive Analytics Software; OR, odds ratio; CI, confidence interval. Keywords: Parkinson’s disease, MTHFR gene, Variant, rs1801131, rs1801133
2
1. Introduction Parkinson’s disease (PD, MIM 168600) is the second most common age-dependent progressive neurodegenerative disorder following Alzheimer’s disease. PD affects approximately 1% of individuals over 60 years of age worldwide, and affects 4-5% of people over 85 years of age [1-4]. It is pathologically characterized by selective and progressive loss of dopaminergic neurons, which may be associated with neurotoxic effects related to changes in homocysteine levels [4-7]. The neuropathological hallmarks are aggregation of α-synuclein in remaining neurons and the presence of intracytoplasmic inclusions, known as Lewy bodies and neurites [1,2,4,5]. PD is clinically characterized by cardinal motor symptoms, including resting tremor, bradykinesia, rigidity, and postural instability, as well as a favorable response to levodopa [2,8,9]. Meanwhile, non-motor features, such as autonomic dysregulation, depression, sleep disturbances, cognitive impairment and dementia, present in some patients with PD [1,2]. Since 1997, through classic genetic linkage analysis, genome sequencing and genetic association studies, at least 21 genetic loci (designated as PARK1 to PARK21) and 16 disease-associated genes have been identified to be involved in familial and sporadic parkinsonism [2,4,10,11]. Only a small number of PD cases are directly related to rare single monogenic mutations, and the underlying pathogenesis of most patients with PD is likely multifactorial [2,10]. Other susceptibility genes, such as the microtubule-associated protein tau gene (MAPT), the glucocerebrosidase gene (GBA), and the sphingomyelin phosphodiesterase 1, acid lysosomal gene (SMPD1), are reported to be associated with PD [9,10,12]. Many mechanisms, including mitochondrial and lysosomal dysfunctions, oxidative stress damage, misfolded protein accumulation and impairment of cellular clearance system by ubiquitin-proteasome and autophagy pathways, are speculated to have a role in pathogenesis of PD [10,13]. Current dopamine replacement therapy provides only partial symptomatic relief for patients with PD, but is incapable of slowing or halting disease progression [2,12]. Some studies also suggest that changes in homocysteine levels may be neurotoxic to dopaminergic neurons [6,7]. Two common variants in the methylenetetrahydrofolate 3
reductase gene (MTHFR, MIM 607093), encoding a folate-dependent enzyme involved in homocysteine metabolism, have been reported to be associated with susceptibility of PD, possibly related to changes in the homocysteine level [7,14,15]. In order to determine whether variants rs1801131 and rs1801133 of the MTHFR gene are associated with PD in Chinese Han population, we conducted a genotyping assay of these two variants in a patient cohort consisting of 512 Chinese Han patients with PD and in a control cohort consisting of 512 age, gender and ethnicity matched normal subjects from mainland China. 2. Methods 2.1. Subjects In this study, we enrolled 1,024 unrelated Chinese Han subjects, including a PD cohort consisting of 512 patients with sporadic PD (308 males and 204 females) and a control cohort consisting of 512 age, gender and ethnicity matched controls (308 males and 204 females), from the southern part of mainland China. The patients in the PD cohort (age 65.8 ± 10.3 years, and age at onset 62.4 ± 7.8 years) were recruited from Department of Neurology, the Third Xiangya Hospital of Central South University, China. The disease condition was ascertained by two independent movement specialists. PD was clinically diagnosed following published diagnostic criteria, and secondary forms of parkinsonism by other known causes were excluded [16,17]. Among the 512 patients with sporadic PD, some had been previously studied and found to be negative for causal mutations in known genes presumably associated with PD [8,18,19]. The controls (age 65.9 ± 10.5 years) were volunteers without symptoms or history of neurological disorders, and were matched with PD cases for age, gender, ethnicity and residence. Peripheral blood was drawn from all the participants after obtaining a signed informed consent. The research was conducted in accordance with guidelines of Helsinki Declaration and with the approval from the Institutional Review Board, China. 2.2. Variants selection and genotyping We filtered variants in the database of single nucleotide polymorphisms (version 144, dbSNP144) for the MTHFR gene with a minor allele frequency above 0.05. Three 4
bioinformatics prediction software programs, including Sorting Intolerant from Tolerant
(SIFT,
(Polyphen-2,
http://sift.jcvi.org/),
Polymorphism
http://genetics.bwh.harvard.edu/pph2/),
Phenotyping and
version
2
MutationTaster
(http://www.mutationtaster.org/), were applied to evaluate whether variants affect protein structure and function [20-22]. Two variants, rs1801131 (c.1286A>C, p.E429A) and rs1801133 (c.665C>T, p.A222V), in the MTHFR gene (NCBI Reference Sequence: NM_005957.4, NP_005948.3) were selected for evaluating the potential association between MTHFR variants and risk of PD. Isolation of genomic DNA from peripheral blood leucocytes was conducted via standard phenol-chloroform extraction method [8]. Genotyping of the two MTHFR gene variants was conducted in all study subjects by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) using the Sequenom MassARRAY system following the iPLEXTM Gold Application Guide [23]. The primer pairs for allele-specific amplification and single-base extension were designed by Sequenom Assay Design 3.1 software (Sequenom, San Diego, CA, USA), then synthesized and diluted as required, which were listed in Table 1. Allele-specific amplification for targeted regions by polymerase chain reaction (PCR), treatment of PCR products by shrimp alkaline phosphatase, single-base extension with extending primer, and purification of reaction products by resin, were conducted following the manufacturers’ protocols (Bioyong Technologies, Beijing, China) and as previously described [24]. The final extended purified products were transferred to the standard 384-well SpectroCHIP by MassARRAY Nanodispenser RS1000 system (Sequenom Inc.). Mass spectrometric analyses were performed by MALDI-TOF MS system, and then MassARRAY Typer 4.0 software (Sequenom Inc.) was applied to analyze the spectrometric peak charts and to generate the genotype data [25]. The genotyping assay in this study was performed using the Sequenom MassARRAY iPLEX Gold platform by investigators blinded to the sample status (case or control subjects). Quality control was conducted by genotyping duplicate samples, positive and negative controls [26]. In addition, Sanger sequencing was performed in 8% randomly selected samples on an ABI 3500 DNA sequencer to check the reliability and 5
accuracy of the method, following the manufacturer’s instructions (Applied Biosystems Inc., Foster City, CA, US) [27]. 2.3. Statistical analysis Statistical analysis was performed using Predictive Analytics Software (PASW) Statistics 18.0 (SPSS Inc., Chicago, IL, USA). Hardy-Weinberg equilibrium for genotype frequencies was examined to evaluate the normal heterogeneity of the case and control groups. Pearson’s 2 test was used to analyze the differences in the genotypic and allelic frequencies of two variants [19]. Haplotype construction and association
analysis
were
performed
using
the
SHEsis
Online
Version
(http://analysis.bio-x.cn) [28]. Results were expressed as P values, odds ratios (ORs), and 95% confidence intervals (CIs). Two-sided P value less than 0.05 was considered to be statistically significant, as described in previous studies [26,29]. 3. Results Genotypes from 1,024 subjects were obtained by MALDI-TOF MS analysis and confirmed by random Sanger sequencing. Both genotypic and allelic frequencies of 512 patients and 512 controls are summarized in Table 2. For both variants (rs1801131 and rs1801133), no evidence of deviation from Hardy-Weinberg equilibrium was observed in either PD cohort or control cohort (all P > 0.05). Between the two cohorts, statistically significant differences in genotypic and allelic frequencies were identified in the MTHFR rs1801133 (P = 0.022 and 0.007, respectively; OR = 0.780, 95% CI: 0.651-0.934), and the frequency of T allele was 0.338 in patients and 0.396 in controls. However, there was no significant difference in the MTHFR rs1801131 (P = 0.213 and 0.098, respectively; OR = 1.201, 95% CI: 0.967-1.492) (Table 2). For haplotypes composed of rs1801131 and rs1801133, only the A-T haplotype showed an association with sporadic PD (2 = 7.378, P = 0.007, OR = 0.779, 95% CI: 0.650-0.933) (Table 3). 4. Discussion PD is largely a late-onset sporadic neurodegenerative disorder with an age-dependent prevalence, and only about 5-10% of PD cases are familial and are transmitted in a Mendelian fashion [2,30]. With the advent of sequencing technologies, an increasing 6
number of genetic loci and disease-causing or susceptibility genes have been identified to be involved in the pathogenesis of PD [2,4,8,10]. There are increasing numbers of evidence suggesting that genetic variants either exert a protective role against PD development or increase the risk of PD development [2,10,13]. Recently, the MTHFR gene variants have been reported to be associated with PD, though there are some discrepancies among different studies (Table 4) [15,31-35]. In this study, we found that the T allele of rs1801133 variant and the A-T haplotype of rs1801131-rs1801133 are able to decrease the risk of developing PD in Chinese Han individuals from mainland China. The finding that T allele of rs1801133 decreases the risk of PD development is consistent with that in a previously reported case-control study in Chinese Han population [31]. Additionally, our finding that rs1801131 is not associated with development of PD is consistent with previous reports [15,32,35]. The MTHFR gene is localized to chromosome 1p36.3 spanning over 20 kb and contains a noncoding exon (exon 1) and 11 coding exons. The MTHFR gene encodes a ~74.5 kDa catalytically active protein with 656 amino acids [36,37]. The encoded enzyme catalyzes the conversion of 5,10-methylenetetrahydrofolate, a carbon donor in nucleotide biosynthesis, to 5-methyltetrahydrofolate, the main form of circulatory folate and a methyl donor for homocysteine remethylation to methionine [15,36,37]. The enzyme regulates the proportional distribution of one-carbon moieties and homocysteine levels, and plays a pivotal role in folate metabolism [15,37]. Genetic mutations in the MTHFR gene can cause autosomal recessive homocystinuria due to MTHFR deficiency, and gene variants are described to be related to vascular diseases, thromboembolism, neural tube defects, schizophrenia, cancers, as well as neurodegenerative diseases, such as Alzheimer’s disease and PD [37-39]. Increased homocysteine elicits neurotoxic effects and might accelerate dopaminergic neurons death [40-42]. Elevated homocysteine levels caused by dietary folate deficiency or focal infusion of homocysteine may enhance dysfunction and death of dopaminergic neurons in a mouse model of PD, revealing a potential role in PD [43]. The two common variants genotyped in this study have been reported to affect enzymatic activity, particularly in folate deficient state, and change plasma homocysteine levels, 7
influencing the risk of PD development [15,37,44]. The inconsistent findings of rs1801133 may be explained by different genetic background, epigenetics or environmental factors, such as folate, vitamin B6 and vitamin B12 dietary levels, etc. [34,38,39,45]. As noted, this is the first study to concurrently explore the potential association between two variants (rs1801131 and rs1801133), rs1801131-rs1801133 haplotypes and the risk of PD in Chinese Han population from mainland China. Our data may provide additional support for the association between the MTHFR variant (rs1801133) and sporadic PD in Chinese Han population. However, further genetic studies involving larger sample size, and different ethnic or geographic populations, accompanied by functional analysis are necessary to firmly establish the role of these variants in PD development.
Conflict of interest The authors declare no conflict of interest.
Acknowledgments We give our sincere appreciation to all the participators and investigators enrolled in this study. This work was supported by grants from New Xiangya Talents Project, China (H.D.).
8
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15
Table 1: Primer sequences for the MTHFR gene variants. dbSNP ID
Bioinformatics predictiona
rs1801131
Damaging/Benign/Polymorphism acgttggatgTCTACCTGA acgttggatgTCTCCCGA
rs1801133
Forward primerb (5'3')
Reverse primerb (5'3') Product length
AGAGCAAGTCC
GAGGTAAAGAAC
Damaging/Probably
acgttggatgCACTTGAA
acgttggatgAAGTGATG
damaging/Polymorphism
GGAGAAGGTGTC
CCCATGTCGGTG
109 bp
Extending primerb (5'3') gggatGAGCTGACCAGT GAAG
112 bp
gagaTGCGTGATGATGA AATCG
a
Sorting Intolerant from Tolerant/Polymorphism Phenotyping version 2/MutationTaster prediction
b
The lowercase letters in the primer sequences are 5'-end tags (forward and reverse primers) or nonhomologous sequences (extending primers)
to increase the molecular weights. MTHFR, methylenetetrahydrofolate reductase; dbSNP, database of single nucleotide polymorphisms.
16
Table 2: Genotypic and allelic frequencies of the MTHFR gene variants (rs1801131 and rs1801133) in Chinese Han sporadic PD patients and controls. dbSNP ID
Subject
rs1801131
P value (2)
Genotype (n = 512, %)
Allele (n = 1024, %)
AA
AC
CC
A
C
PD
315 (61.5)
174 (34.0)
23 (4.5)
804 (78.5)
220 (21.5)
Control
342 (66.8)
150 (29.3)
20 (3.9)
834 (81.4)
190 (18.6)
CC
CT
TT
C
T
PD
222 (43.4)
234 (45.7)
56 (10.9)
678 (66.2)
346 (33.8)
Control
182 (35.5)
255 (49.8)
75 (14.6)
619 (60.4)
405 (39.6)
rs1801133
0.213 (3.097)
0.022 (7.618)
P value (2)
OR (95% CI)
0.098 (2.745)
1.201 (0.967-1.492)
0.007 (7.319)
0.780 (0.651-0.934)
Statistically significant P values are marked in bold. MTHFR, methylenetetrahydrofolate reductase; PD, Parkinson’s disease; dbSNP, database of single nucleotide polymorphisms; OR, odds ratio; CI, confidence interval.
17
Table 3: Haplotype analysis of rs1801131 and rs1801133 in the MTHFR gene comparing between PD patients and matched controls. Haplotype
Patients (%) Controls (%)
2 value
P value
OR (95% CI)
A-C
44.9
42.1
1.712
0.191
1.124 (0.943-1.339)
A-T
33.6
39.4
7.378
0.007
0.779 (0.650-0.933)
C-C
21.3
18.4
2.729
0.099
1.201 (0.966-1.493)
C-T
0.2
0.2
-
-
-
Statistically significant P value is marked in bold. MTHFR, methylenetetrahydrofolate reductase; PD, Parkinson’s disease; OR, odds ratio; CI, confidence interval.
18
Table 4: Genetic analysis of the MTHFR gene variants in PD patients. Reference
Location/Ethnicity
PD
Controls
Varianta
P value
OR/RR (95% CI)
Association
Garcia et al., 2015 [34]
Mexican
140
216
rs1801133
0.024
2.06 (1.101-3.873)
Yesb (Increased risk)
Liao et al., 2014 [31]
Chinese Han
765
717
rs1801133
0.003
0.80 (0.688-0.926)
Yes (Decreased risk)
Kumudini et al., 2014 [46]
South Indian
151
416
rs1801133
0.15
1.45 (0.87-2.45)
No
Vallelunga et al., 2014 [32]
Italian
120
-
rs1801131
1.0
-
No
rs1801133
0.024
c
Wu et al., 2013 [15] c
Caucasian and Asian
Yes (Age at onset)
907
820
rs1801131
0.733
1.03 (0.88-1.20)
No
1606
7168
rs1801133
<0.001
1.24 (1.11-1.38)
Yes (Increased risk)
Zhu et al., 2013 [33]
European and Asian
1820
7530
rs1801133
0.0002
1.212 (1.097-1.340)
Yes (Increased risk)
Bialecka et al., 2012 [35]
Poland
320
254
rs1801131
0.304
0.70 (0.39-1.29)
No
rs1801133
0.122
1.62 (0.91-2.90)
No
Gorgone et al., 2012 [6]
Italy
60
82
rs1801133
0.01
N.A.
Yes (Increased risk)
Fong et al., 2011 [47]
Chinese
211
218
rs1801133
>0.05
1.11 (0.81-1.53)
No
Yuan et al., 2009 [48]
Chinese
76
110
rs1801131
>0.05
N.A.
No
rs1801131
0.3277
N.A.
No
rs1801133
0.0239 (>0.0125)
rs1801131
0.50
N.A.
No
rs1801133
0.47
rs1801131
0.9
N.A.
No
rs1801133
0.2
rs1801133 Rodriguez-Oroz et al., 2009
Spain
89
30
[49] Camicioli et al., 2009 [50] Caccamo et al., 2007 [51]
Canada Italy
51 49
50 86
Lin et al., 2007 [39]
Chinese
94
146
rs1801133
0.120
N.A.
No
Dorszewska et al., 2007 [52]
Poland
98
50
rs1801131
>0.05
N.A.
No
rs1801133
19
G1793A
a
Todorovic et al., 2006 [53]
Serbia
113
53
rs1801133
0.2
N.A.
No
Religa et al., 2006 [54]
Poland
114
100
rs1801133
>0.05
N.A.
No
Wullner et al., 2005 [55]
Germany
342
342
rs1801131
0.24
N.A.
No
rs1801133
0.79
de Lau et al., 2005 [7]
Netherlands
65
5855
rs1801133
0.09
1.74 (0.91-3.32)
Yesd (Increased risk)
Momose et al., 2002 [56]
Japanese
232
249
rs1801131
0.458
N.A.
No
Harmon et al., 1997 [57]
Ireland
188
184
rs1801133
0.53
1.39 (0.63-3.12)
No
Variant: rs1801131 and rs1801133, commonly recorded as A1298C and C677T;
b
Risk variant: C allele;
c
Meta-analysis;
d
Borderline
significant. Statistically significant P values are marked in bold. MTHFR, methylenetetrahydrofolate reductase; PD, Parkinson’s disease; OR, odds ratio; RR, relative risk; CI, confidence interval; N.A., not available.
20