Download 10. Wang T, Liang ZH, Sun SG, Cao XB, Peng H, Liu HJ, et al

Lack of association between three SNPs in the PARK9, PARK15, and BST1 genes and
Parkinson’s disease in the northern Han Chinese population
ZHU Lan-hui, LUO Xiao-guang, ZHOU Yi-shu, LI Feng-rui, YANG Yi-chun, REN Yan and PANG Hao
School of Forensic Medicine, China Medical University, Shenyang 110001, P.R. China (Zhu LH, Zhou YS, Li FR
and Pang H)
Department of Neurology, the First Hospital of China Medical University, Shenyang 110001, P.R. China (Luo XG
and Ren Y)
Department of Forensic Medicine, Baotou Medical College, Baotou 014060, P.R. China (Li FR)
Department of Neurosurgery, Dalian University Affiliated Xinhua Hospital, Dalian 116021, P.R China (Yang YC)
Corresponding to:
Dr. PANG Hao, School of Forensic Medicine, China Medical University, No. 92 Beier Road, Heping district,
Shenyang 110001, P.R. China (Tel: 86-24-23256666 ext 5417. Fax: 86-24-23267698. Email: [email protected])
This work was supported by grant from National Natural Science Foundation of China (30771833).
Keywords: Parkinson’s disease; PARK9; PARK15; BST1; Association study
Background
Parkinson’s disease is an autosomally inherited neurodegenerative disease in elderly
people. The etiology of Parkinson’s disease has long been thought to be associated with both genetic and
environmental factors. To explore potential genetic risk factors for Parkinson’s disease in the northern
Han Chinese population, we investigated three SNPs (rs4538475, rs11107 and rs12564040) in the BST1,
PARK15 and PARK9 genes.
Genomic DNA from 215 Parkinson’s disease patients and 212 matched controls was
Methods
amplified in two independent PCR systems and subsequently genotyped by digestion with the
endonuclease PstI. Genetic parameter and association studies were carried out with SPSS 13.0 and
PLINK 1.07 software.
Results
We could accurately detect all genotypes in the three loci with the PCR-RFLP or mismatched
PCR-RFLP techniques. The observed heterozygosities of the rs4538475 and rs11107 loci in Parkinson’s
disease and control groups ranged from 0.460–0.481 and 0.410–0.441, in BST1, PARK15 respectively,
while we detected no heterozygosity at the rs12564040 locus in PARK9. The similar distributions of
genotypic frequency between both groups suggest that the three SNPs investigated in this study are
unlikely to play roles as common risk factors or pathogenic mutations for Parkinson’s disease in northern
Han Chinese.
Conclusions
The SNPs investigated in the BST1, PARK15 and PARK9 genes associated with
Parkinson’s disease susceptibility are not associated with Parkinson’s disease in the northern Han Chinese
population.
中文摘要：
研究背景
帕金森病（Parkinson’s disease, PD）是多发于老年人群中的常染色体神经退行性疾病。
尽管准确的病因不清，但目前的研究显示 PD 的发病原因与环境因素和遗传因素密切相关。在已经
鉴定的约 16 种 PD 易感基因中存在着大量的与 PD 相关的单核苷酸多态性（SNP），然而，中国 PD
人群中相关的数据报道甚少。为了调查 PD 潜在的遗传危险因素，本研究首次以中国北方人群为样
本，对 BST1, PARK15 和 PARK9 三种 PD 易感基因的三个 SNP 位点（rs4538475，rs11107 和
rs12564040）的遗传多态性进行了调查。
研究方法 常规分离提取 215 名 PD 患者及 212 名平均年龄相仿的正常个体的静脉血基因组 DNA，
应用 PCR-RFLP 和错配 PCR-RFLP 技术，在两个独立的 PCR 反应体系和一个接续的单一限制性内
切酶 Pst I 的消化反应体系中，经单一常规的聚丙烯酰胺凝胶电泳分离，鉴定三个位点的基因型。
所得基因型数据采用 SPSS 13.0 和 PLINK-1.07 软件进行遗传参数及相关性分析。
实验结果 应用本研究设计的方法可以准确地鉴定所有三个位点的基因型。实验结果显示，
rs4538475 和 rs11107 两个位点观察的杂合度在 PD 病人组和对照组中排列范围分别为 0.460 ~ 0.481
和 0.410 ~ 0.441；而 rs12564040 位点上疾病和对照组均没有发现杂合子。疾病和对照组之间相似的
基因型频率分布经相关性分析显示，本研究所调查的三个 SNP 位点至少在中国北方汉族人中不构
成常见的 PD 危险因素，也不考虑其为 PD 的致病性突变位点。
结论 本研究所调查的三个 SNP 位点在中国北方汉族人群中与 PD 缺乏相关性。
Parkinson’s disease (PD), the second most common neurodegenerative diseases after Alzheimer’s disease,
affects over 1% of individuals over 65 years of age. The main features of PD are bradykinesia, rigidity
and tremor of variable severity, caused by the loss of dopaminergic neurons in the substantia nigra. The
clinical definition of the disease is based on the core features listed above and includes initial
responsiveness to treatment with levodopa.1 The etiology of PD is complex and multifactorial, likely
involving a combination of environmental exposures, polygenic inheritance, and gene-environment
interactions. Until now, sixteen PD susceptible genes, PARK1 to PARK16,2 have been reported. Mutations
in two of these genes (PARK1 and PARK8) exhibit an autosomal dominant inheritance pattern, while
mutations in PARK2 (PARKIN), PARK6 (PINK1), PARK7 (DJ-I) and PARK9 are inherited in an autosomal
recessive fashion.3 Additionally, genome-wide association studies (GWAS) for Parkinson’s disease have
linked more loci to risk of Parkinson’s disease.4,5
Increasing data on the underlying genetics of PD gathered in the last two decades have provided
researchers with incredible amounts of information regarding the pathogenesis of PD. Among the genes
associated with PD susceptibility, the ATP13A2 gene is mapped to the PARK9 locus and is responsible for
Kufor-Rakeb syndrome (KRS), a recessive, juvenile-onset, atypical parkinsonism with pyramidal
degeneration and cognitive dysfunction.6 Recently, mutations in this gene have been found in patients
with early-onset Parkinsonism.7 PARK15 (FBXO7), an autosomal recessive inheritance factor for PD,
encodes F-box protein 7 (Fbxo7). Fbxo7 is characterized by a 40-amino acid domain (the F-box) in its
C-terminal half. Mutations in this gene produce clinically similar phenotypes to other PD-associated
mutations: the patients present with rapidly progressive Parkinsonism, initially responsive to levodopa
treatment.7 From recent GWAS reports, BST1 (bone marrow stromal cell antigen 1) on chromosome 4p15
is recognized as a new risk locus related to PD in the Japanese population,8 but a US/UK/German study
failed to replicate the association, which shows it to be a locus with population differences.5
Ethnic and regional backgrounds of the patients affect the mutation rate of the loci mentioned above.2,9
Association with PD of a single mutation in different genes has been analyzed in many populations
including Asian,10 European and American, and increasing data indicate the significance of these
mutations. However, data about the frequency of mutations in the BST1, ATP13A2 and FBXO7 genes
have not been reported in the northern Han Chinese population. Some of them have not been reported in
mainland China so far either. Additionally, methods using multiple SNP detection in PD-related loci are
very limited. In this study, we conducted a multiplexed PCR-RFLP assay with PstI endonuclease
digestion to identify three SNPs simultaneously and to explore their possible association with PD in the
northern Han Chinese population.
METHODS
Patients and Controls
A total of 215 ethnic Han Chinese patients (mean age, 62.59±10.72 years ranging from 24–85 years old;
53.02% male) were recruited from northern China. All patients fulfilled the criteria for clinical diagnosis
of PD with at least 2 of 3 cardinal signs (bradykinesia, tremor, rigidity) and a positive response to
levodopa therapy. They were diagnosed with idiopathic PD by movement disorder neurologists at the
First Affiliated Hospital of China Medical University in Liaoning province in P.R. China. Control subjects
matching the PD patients in ethnicity, age, gender (mean age, 62.96±10.73 years ranging from 34–87
years old; 59.43% male) were healthy, including no diagnoses of neurodegenerative diseases. There was
no statistical difference in age or sex between these two groups (P > 0.05). Informed consent was
obtained from all subjects and approval was obtained from the local ethics committee.
Genotyping
All genomic DNA was isolated from leukocytes collected from the subjects’ peripheral blood samples,
and extracted using the SDS-proteinase K-phenol- chloroform method. According to the published
sequences of SNPs rs4538475 in BST1 (NT_006316.16: g.6919734A>G; located downstream of the BST1
gene on 4p15), rs11107 in PARK15 (c.345C>T, p.M115I; located in exon 3 of FBXO7 gene on 22q12-13),
we synthetically generated a PstI restriction endonuclease site in each amplified product using
mismatched PCR primers (Sangon, Shanghai, China). A natural Pst I endonuclease site exists in the
sequence around rs12564040 in PARK9 (c.1754G>T, p.585A>D; located in exon 17 of ATP13A2 gene on
1p36). Sequences of these primers are shown in Table 1.
PCR-RFLP was used to identify polymorphisms in the SNPs mentioned above. Two different reaction
mixes for PCR amplification were prepared. The reaction for rs4538475 was carried out in a final volume
of 20 l PCR buffer containing 1X Es Taq MasterMix (CWBIO, Beijing, China), 50–100 ng genomic
DNA, and primer concentrations shown in Table 1. The reaction for the remaining two SNPs was carried
out in a final volume of 20 l PCR buffer containing 1X LA PCR Buffer II (TaKaRa, Dalian, China), 1.6
M dNTP Mixture, 0.05 U/l TaKaRa LA Taq, 50–100 ng genomic DNA and primer concentrations
shown in Table 1. PCR reactions were performed under the following conditions: denaturation at 94°C for
1 minute; 94°C for 30 seconds, 65°C (primer sets 2, 3) or 67°C (primer set 1) for 30 seconds, 72°C for 30
s. The last 3 steps were repeated for 30 cycles (primer sets 2, 3) or 35 cycles (primer set 1). For restriction
enzyme digestion, about 3l PCR product from each reaction and 2.5 U Pst I (TaKaRa, Dalian, China)
were added to 10 l 1X H Buffer. The PCR products were incubated at 37°C for more than 2 hours. The
digested products were separated by polyacrylamide gel electrophoresis (T = 6%, C = 5%). Gels were
dyed with 10 X genefinder (Bio-V, Xiamen, China) and observed under UV. To confirm the formation of
synthetically mismatched sequences, direct DNA sequencing of each PCR product fragment was
performed with a BigDye Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, US). The
non-mismatched sequencing primers are shown in Table 1.
Statistical analysis
All data were analyzed with the Statistical Package for the Social Sciences version 13.0 (SPSS, version
13.0) (SPSS Inc., US) and PLINK for Windows version1.07 (Free Software Foundation, Inc., US).11 A
two-tailed P value < 0.05 was considered statistically significant.
RESULTS
Mismatched sequences containing the Pst I recognition site were correctly generated for all loci and
confirmed by DNA sequencing. The amplified PCR products are of 257 bp (rs4538475), 212 bp (rs11107)
and 128 bp (rs12564040) (Figure 1). The PCR products were digested with the restriction enzyme Pst I
and subsequently separated by electrophoresis. The fragments that remain undigested represent
g.6919734A, c.345T and c.1754T alleles, while the fragments of 234, 189, and 85 bp, represent
g.6919734G, c.345C and c.1754G alleles, respectively. Fragments shorter than 43 bp ran off the gel under
the electrophoresis conditions used. These results indicate that the mismatched multiplex PCR-RFLP
assay was successful and the genotypes of the three SNPs can be correctly identified using this method.
Genotypic frequencies of the 3 SNPs located on the three genes studied were investigated using 215 PD
patients and 212 controls. The genetic parameters for each SNP site are shown in Table 2. The observed
genotypes were in Hardy-Weinberg equilibrium. In the rs4538475 locus, the frequency of the g.6919734
AA genotype was similar to the frequency of the g.6919734 GG genotype in both patients and controls.
The frequency of the c.345 TT plus CT genotype in the rs11107 locus was over 90% in both patients and
controls. However, we detected only the c.1754G allele in the investigated cohorts in this study. We
subsequently performed association analyses of the three SNPs (Table 2) and found that the OR (odds
ratio) values from the three SNPs are 1.12, showing a lack of association between rs11107, rs4538475 and
rs12564040 genotypes and Parkinson’s disease in the northern Han Chinese population.
DISCUSSION
In previous studies, we analyzed gene polymorphisms in four human erythrocytic isoenzymes by
mismatch and mismatched multiplex PCR-RFLP techniques. We could successfully, rapidly and
accurately determine all genotypes in the systems investigated.12,13 In this study, to explore the association
between PD and associated susceptibility genes, we applied the same strategies to investigate the allelic
frequency of three SNPs located on three PD-associated genes. As expected, the genotyping technique we
used effectively detected the SNPs in investigated PD patients and normal subjects. Advances in
genotyping technology at present have allowed rapid genome-wide screening (GWAS) of common
variants in large populations, launching a new era in the investigation of the genetic basis of
neurodegenerative diseases. In most GWAS studies, investigations in stage II or III usually continue to
use previously employed robust and resource-intensive approaches to systematic genome-wide
association analysis.4,5,8 However, we must consider that high-powered large-scale studies may not be
undertaken in the majority of laboratories due to low resources. Thus, genotyping methods different from
those generally used in stage I of GWAS may be employed to replicate results that have demonstrated
significant differences in SNPs from various loci. The multiplexed PCR-RFLP technique used in the
present study is an efficient, easy, timesaving and economic method. This straightforward assay will
allow for more SNPs in more PD-associated genes to be detected as easily as possible in further disease
association research by using this strategy.
Influences of ethnicity on SNP frequencies as well as founder effects have been documented for several
PD genes. Consequently, only a complete mutation analysis of these genes will allow the identification of
all relevant mutations, both in individual patients and in populations of interest. Additionally, it is
important to continue the genetic characterization of patients even if they have been shown to carry
certain SNPs in known genes, because unexpected digenic combinations might explain atypical clinical
presentations in individual PD patients. In this study, based on previous data on six other SNP analyses in
genes associated with PD susceptibility, we selected three additional SNPs as targets for a PD association
analysis in the northern Han Chinese population. We eventually aim to accumulate data on SNPs in all
PD susceptibility genes. BST1 is a newly identified PD-associated gene and its products, with different
SNPs in certain regions, may modify ADP-ribosylcyclase activity and lead to calcium dyshomeostasis in
dopaminergic neurons. Satake et al. firstly identified common variants at the BST1 locus as genetic risk
factors for PD through a genome-wide association study and indicated that four SNPs (rs11931532,
rs12645693, rs4698412 and rs4538475) showed strong disease association with almost the same
significance levels.14 Among them, rs4538475 was the most strongly associated. Another GWAS study in
Japanese samples, reported more recently, similarly described 4p15 (BST1) as a risk locus for PD. Strong
evidence of association for rs4698412 was found in the Japanese genome scan, but a much weaker signal
was obtained in the data from a US/UK/German study.5 In addition, the Japanese group found that the
most associated SNP (rs4698412) maps to a 15 kb linkage disequilibrium (LD) block and is in high LD
with the next top two BST1 variants (rs4698412 and rs4538475). Thus, we selected to investigate the
genotypic frequency of the rs4538475 SNP in the BST1 locus for the first time in a Chinese population.
Our data show that the frequency of the AA genotype in the rs4538475 locus in BST1 is similar to the
frequency of the GG genotype in both PD and control groups, ranging from 0.236 - 0.283. However, we
could not find a difference in the frequency distribution between PD and control groups, indicating that
the rs4538475 locus may not be a genetic risk factor for PD in north Chinese. Interestingly, the rs4538475
locus was reported to have a significant association with PD in the Japanese population, 10 but none in the
French population.5 According to previously reported and our own data, genetic heterogeneity on the
rs4538475 locus not only appears between populations of Asian and European descent, but also occurs
between different populations of Asian descent. Racial and regional differences may be responsible for
this phenomenon.
Two other genes, ATP13A2 and FBXO7, have been officially designated as PARK9 and PARK15.
Pathogenic mutations of FBXO7 are likely to induce dysfunction of the Fbxo7 protein and then cause
abnormal ubiquitination. A disease associated missense mutation (c.1132C→G), resulting in the
nonconservative amino acid substitution of glycine for arginine at position 378 (R378G) in F-box protein
7 has been found.14 In 2009, Di Fonzo et al. found a FBXO7 homozygous truncating mutation (R498Stop)
in an Italian autosomal recessive early-onset parkinsonism pedigree,15 while compound heterozygous
mutations (a splice-site IVS7+1G/T mutation and a missense T22M mutation) presented in a Dutch family.
However, many polymorphisms in FBXO7 are detected in normal, healthy people. In a study of the
southern Chinese population, 10 polymorphisms, but no pathogenic mutations in the FBXO7 gene were
detected. Their result suggests that FBXO7 mutations may be rare in Chinese early-onset parkinsonism
patients.16 The rs11107 locus is located in the coding region of PARK15. Association analysis from the
present study indicates no evidence for the variant PARK15 allele as a genetic risk factor for PD in the
northern Chinese population, consistent with the previous report in the southern Chinese population.
Clinically, PARK9 was identified as a genetic marker for early onset levodopa-responsive
dystonia-parkinsonism with pyramidal signs and eye movement abnormalities.17 Homozygous and
compound heterozygous ATP13A2 mutations were first described in patients of Jordanian and Chilean
ancestry. Multiple mutations and variants have been reported in the ATP13A2 gene so far,18 but the
frequencies of different mutations in the ATP13A2 gene are still not very clear among different
geographic origins.19,20 Lin et al. sequenced all 29 coding exons of the ATP13A2 gene. The ATP13A2
A746T variant was observed in three unrelated PD patients (1.7%), but not in the controls. Lin’s group
concluded that the ATP13A2 gene variant was associated with an increased risk of PD among ethnic
Chinese.21Another study, conducted by Mao et al., did not find any PD patients harboring the A746T
variant among 173 subjects with early onset PD (EOPD) and 47 subjects with familial PD (FPD).22 This
suggests that the A746T mutation in ATP13A2 is rare in EOPD and FPD patients from mainland China. In
the present study, we failed to detect the ATP13A2 A746T variant in either the patient or control group.
We infer that the rs12564040 SNP is rare in the northern Han Chinese population. We did not detect any
suggestive evidence that variation in ATP13A2 can cause PD, or significant evidence that the allelic
variant can increase the risk of PD. Our data also contribute to a growing line of evidence that ATP13A2
is not strongly associated with Parkinson’s disease in the Han Chinese population.
In summary, we obtained genetic parameters and performed association analysis on the three SNPs
mentioned above in northern Chinese PD and normal subjects for the first time. The three SNPs
investigated in this study are unlikely to play roles as common risk factors or pathogenic mutations for
PD in the northern Han Chinese population.
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Table 1. Information about the three SNPs and parameters for mismatched multiplex PCR amplification
SNP
rs4538475
Nucleotide
changes
Amino acid
changes
A>G
Primer
sets
Primer
direction
Set 1
Forward
Reversed
Fragment
Size (bp)
Primer concentration
TACCCTTGGCAAGACAAAcTgCA*
ACCTCTGTGTCCTTGGTTCAAGCAG
257 (234, 23)
0.2
0.2
Sequence (5’→3’)
(mol/L)
rs11107
C345T
M115I
Set 2
Forward
Reversed
ggATAAAATGTGCAATCTATCGATGATAA*
CACCAGCTCCAATCAGACTAGCcT*
212 (189, 23)
0.15
0.15
rs12564040
G1754T
A585D
Set 3
Forward
Reversed
AGCTCAAATCTCTCTGTGcGCCAAA*
GGGGTCTCATCACTGCgAAGACCT*
128 (85, 43)
0.075
0.075
*The small letters indicate mismatched bases.
Table 2. Genotypes of the three SNPs and statistical parameters
Locus
rs4538475
rs11107
rs12564040
Genotypes
AA
AG
GG
CC
CT
TT
TT
TG
GG
Case
Control
Observed
Freq
Observed
Freq
59
99
57
20
95
100
0
0
215
0.274
0.460
0.265
0.093
0.441
0.465
0
0
1
50
102
60
20
87
105
0
0
212
0.236
0.481
0.283
0.094
0.410
0.495
0
0
1
Association with PD
OR=1.12
95%CI (0.853,1.472)
P = 0.412
OR=1.085
95%CI (0.807,1.457)
P = 0.590
OR=NA
95%CI (NA)
P = NA
Parameters were calculated by PLINK version 1.07. All SNP genotypes were in
Hardy-Weinberg equilibrium. NA: not applicable.
Figure 1. Genotyping of the three SNPs by PCR-RFLP
The figure shows all the genotypes in this study. The lane marked M indicates a DNA
ladder ranging from 80 bp to 300 bp, used as a molecular size marker. The lanes
numbered 1–9 represent nine band patterns, which demonstrate all of the genotypes
found.