# Analysis ofLRRK2,SNCA, andITGA8 Gene Variants with Sporadic Parkinson’s Disease Susceptibility in Chinese Han Population

Authors: Jie Fang; Kehui Yi; Mingwei Guo; Xingkai An; Hongli Qu; Qing Lin; Min Bi; Qilin Ma
Journal: Parkinson’s Disease
 (2016)

---

## Abstract

Background. Parkinson’s disease (PD) is an age-related neurodegenerative disease affected by multiple genetic and environmental factors. We performed a case-control study on candidate gene to scrutinize whether genetic variants inLRRK2,SNCA, andITGA8 genes could be associated with sporadic PD in Chinese Han population.Methods. Five single-nucleotide polymorphisms (SNPs) ofLRRK2 (rs1491942),SNCA (rs2301134, rs2301135, and rs356221), andITGA8 (rs7077361) were selected and genotyped among 583 unrelated PD patients and 558 healthy controls.Results. Rs1491942 ofLRRK2 gene had a significantly higher genotype frequency (P
=
3.543
E
-
09) and allelic G/C frequencies (P
=
2.601
E
-
10) in PD patients than controls. Rs2301135 ofSNCA gene also showed an obvious difference in genotype frequency (P
=
4.394
E
-
07) and allelic G/C frequencies (P
=
9.11
6
E
-
13) between PD patients and controls. SNPs rs2301134 and rs356221 ofSNCA gene and rs7077361 ofITGA8 gene lacked the significant association with the susceptibility of PD in Chinese Han population.Conclusions. Our study firstly expresses that rs1491942 ofLRRK2 and rs2301135 ofSNCA gene are substantially associated with sporadic Parkinson’s disease in Chinese Han population.


---

## Body

## 1. Introduction

Parkinson’s disease (PD), the second most common neurodegenerative disease after Alzheimer’s disease, consists of two major pathological hallmarks: loss of dopaminergic neurons and the presence of Lewy bodies (LB). The classic manifestations of PD are characterized by resting tremor, rigidity, bradykinesia, and impairment of postural reflexes. In addition, some untypical nonmotor features, such as sleep disturbances, mood disorders, autonomic dysfunction, sensory problems, and cognitive impairment, are highly concerned recently. Even when treated with effective therapies, PD is progressive and somehow leads to disability or even mortality.Increasing evidence supports that complex factors contribute a lot to the susceptibility of PD, which includes genetic and environmental factors [1–3]. In the past decades, a large number of Genome-Wide Association Studies (GWAS), Candidate Gene Replication Study (CGRS), and subsequent meta-analysis studies have found that a number of potential genes and single-nucleotide polymorphisms (SNPs) associated with PD, including both risk variants and protective variants [4]. In addition, the previous candidate genetic studies provided conclusive evidence showing SNPs inLRRK2,SNCA, andITGA8 genes significantly impact PD susceptibility and disease characteristics.Several variations ofLRRK2 gene were identified as risk factors for PD. For example, rs34778348 (G2385R, c.7153G>A) and rs33949390 (R1628P, c.4883G>C) were seen to associate with PD in Asian population [5, 6]. Another novel SNP withinLRRK2, rs1491942, was found to be responsible for PD in Caucasian populations [7, 8]. However, it was never reported in Chinese Han population before.SNCA, as the first pathogenic gene identified in PD, encodes α-synuclein, the primary component of LB, the pathological hallmark of PD. From then on, several SNPs ofSNCA were highly considered as the genetic risk factors for sporadic PD. Located in the promoter region ofSNCA, two SNPs (rs2301134 and rs2301135) with high allele frequency were reported in some studies as PD-related SNPs in European and Taiwanese cohorts [9, 10]. One SNP (rs356221) in the 3′UTR region ofSNCA gene showed association with susceptibility to sporadic PD in Japanese and Taiwanese cohorts [10, 11]. All these three SNPs ofSNCA (rs2301134, rs2301135, and rs356221) have not been investigated in Han population on the Mainland of China.WhileITGA8 (encoding integrin alpha 8, a type-I transmembrane protein) gene was firstly proved to connect with idiopathic PD in Caucasian population in Simón-Sánchez’s study [4], it was not featured as a PD relevant gene until Lill’s study revealed its potential association with PD [8]. Additional studies are needed to screen the potential pathogenic variants within this gene and assess the potential role of these variants in PD pathogenesis.There are no study that explores the association of the three genes and their SNPs with Parkinson’s disease in Chinese Han population. Here, we perform the first SNP replication study on previously published SNPs withinSNCA (rs356221, rs2301134, and rs2301135),LRRK2 (rs1491942), andITGA8 (rs7077361) gene in Chinese Han population to explore the ethnic differences and recognize predictive factors for the diagnosis of PD.


## 2. Methods

### 2.1. Subjects

This study recruits 1136 cases in the Neurology Department of the First Affiliated Hospital of Xiamen University, which includes 583 Chinese Han sporadic PD patients and 553 matched healthy controls. PD diagnosis coincided well with the diagnostic criteria of UK Parkinson’s Disease Society Brain Bank [12]. Among all the PD patients, the mean age is 65.10 ± 8.90 and the ratio of male to female patients is 320 : 263. The group of controls consists of healthy volunteers from the Medical Center of the First Affiliated Hospital of Xiamen University, the mean age of which is 65.37 ± 9.03, and the ratio of male to female patients is 286 : 267 (Table 1). All subjects are Han population, and the two groups are matched for age, gender, ethnicity, and area of residence. Moreover, this study has gained approval of the local ethics committees, and all patients and controls signed informed consents.Table 1
Demographic characteristics of Parkinson’s disease (PD) cases and controls.
Characteristics
Cases = 583
Controls = 553
Gender (n, %)∗
Female
320
54.9
286
51.7
Male
263
45.1
267
48.3
Age at collection (mean, SD)†
65.10
8.90
65.37
9.03
Age at onset (n, %)
<50
131
22.5
n.a.
n.a.
≧50
452
77.5
n.a.
n.a.
All the subjects were ethnic Hans;∗PD compared with controls by gender: P
>
0.05; †PD compared with controls by age: P
>
0.05; n.a.: not applicable.


### 2.2. Genetic Analysis

Venous blood specimens are collected directly from all PD patients and the healthy controls with ethylene diamine tetraacetic acid (EDTA) anticoagulant. Genomic DNA is extracted from the blood samples with the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) under the manufacturer’s instructions and stored at −20°C. The five SNPs are genotyped by Multiplex Snapshot technique (Applied Biosystems by Life Technologies, Foster City, CA, USA). The primers are designed for each SNP locus using Primer 5 and listed in Table2. Multiplex PCR reactions are performed to amplify target regions containing the selected SNPs. All products are analyzed by the ABI PRISM 3730 DNA Sequence, of which the sequence analyses are conducted by DNA Sequencing Analysis software, GeneMapper4.0. To confirm the results, 10% patients and 10% controls are randomly selected for Sanger sequencing approaches. The concordance rate for replicate approaches was 100%.Table 2
PCR and Snapshot probe primer sequences.
Polymorphisms
Primers
Sequence5
′
→
3
′
LRRK2
rs1491942
Forward
CAGGCTTGGGCAATTTCTAA
Reverse
GCCTATTGTGCTTCCTGCTC
Probe
40Ts+CAGGCTCCCCTGGGTT
SNCA
rs2301134
Forward
ATCACGCTGGATTTGTCTCC
Reverse
CACGGTCACAGGTTACAACG
Probe
41Ts+GACTCTTCCTTAGTAGTCTCCC
rs356221
Forward
TGCCATAGAAACAACGAGGA
Reverse
TTGAAGAACCCAAAATGCAA
Probe
24Ts+AAGAGAAGCCATCCTAGT
rs2301135
Forward
ACTTAACGTGAGGCGCAAAA
Reverse
CGTCCTCCTCCTCCTAGTCC
Probe
54Ts+CCGGGAGAGGGGCGGG
IGTA8
rs7077361
Forward
TGCGAAAACTATTTGGTGAAA
Reverse
CCCACCCACCAAATCTCTAA
Probe
31Ts+GAAATCATCTAGGGGATA


### 2.3. Statistical Analysis

The statistical analyses are processed with SPSS, version 20.0 (IBM, Armonk, NY, USA). The clinical data are expressed as the means± standard deviation (SD) for the continuous variables and as numbers (percentage) for the quantitative variables. Student’st-test is used to compare the age variables between the patients and controls. The gender variables are assessed by the chi-square test. Differences in frequencies of the alleles and genotypes between cases and controls are tested for each SNP through Pearson’s chi-square test and Fisher’s exact test. The criterion for significance is set at P
<
0.05 based on two sides for all of the tests. The statistical power is calculated by Power and Sample Size Calculations version 3.1.2. The Hardy-Weinberg equilibrium (HWE) is tested by adopting the public statistics web tool (http://ihg.gsf.de/cgi-bin/hw/hwa1.pl). The Haploview program [13] is used for the calculation of linkage disequilibrium (LD) among the three SNPs inSNCA.


## 2.1. Subjects

This study recruits 1136 cases in the Neurology Department of the First Affiliated Hospital of Xiamen University, which includes 583 Chinese Han sporadic PD patients and 553 matched healthy controls. PD diagnosis coincided well with the diagnostic criteria of UK Parkinson’s Disease Society Brain Bank [12]. Among all the PD patients, the mean age is 65.10 ± 8.90 and the ratio of male to female patients is 320 : 263. The group of controls consists of healthy volunteers from the Medical Center of the First Affiliated Hospital of Xiamen University, the mean age of which is 65.37 ± 9.03, and the ratio of male to female patients is 286 : 267 (Table 1). All subjects are Han population, and the two groups are matched for age, gender, ethnicity, and area of residence. Moreover, this study has gained approval of the local ethics committees, and all patients and controls signed informed consents.Table 1
Demographic characteristics of Parkinson’s disease (PD) cases and controls.
Characteristics
Cases = 583
Controls = 553
Gender (n, %)∗
Female
320
54.9
286
51.7
Male
263
45.1
267
48.3
Age at collection (mean, SD)†
65.10
8.90
65.37
9.03
Age at onset (n, %)
<50
131
22.5
n.a.
n.a.
≧50
452
77.5
n.a.
n.a.
All the subjects were ethnic Hans;∗PD compared with controls by gender: P
>
0.05; †PD compared with controls by age: P
>
0.05; n.a.: not applicable.


## 2.2. Genetic Analysis

Venous blood specimens are collected directly from all PD patients and the healthy controls with ethylene diamine tetraacetic acid (EDTA) anticoagulant. Genomic DNA is extracted from the blood samples with the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) under the manufacturer’s instructions and stored at −20°C. The five SNPs are genotyped by Multiplex Snapshot technique (Applied Biosystems by Life Technologies, Foster City, CA, USA). The primers are designed for each SNP locus using Primer 5 and listed in Table2. Multiplex PCR reactions are performed to amplify target regions containing the selected SNPs. All products are analyzed by the ABI PRISM 3730 DNA Sequence, of which the sequence analyses are conducted by DNA Sequencing Analysis software, GeneMapper4.0. To confirm the results, 10% patients and 10% controls are randomly selected for Sanger sequencing approaches. The concordance rate for replicate approaches was 100%.Table 2
PCR and Snapshot probe primer sequences.
Polymorphisms
Primers
Sequence5
′
→
3
′
LRRK2
rs1491942
Forward
CAGGCTTGGGCAATTTCTAA
Reverse
GCCTATTGTGCTTCCTGCTC
Probe
40Ts+CAGGCTCCCCTGGGTT
SNCA
rs2301134
Forward
ATCACGCTGGATTTGTCTCC
Reverse
CACGGTCACAGGTTACAACG
Probe
41Ts+GACTCTTCCTTAGTAGTCTCCC
rs356221
Forward
TGCCATAGAAACAACGAGGA
Reverse
TTGAAGAACCCAAAATGCAA
Probe
24Ts+AAGAGAAGCCATCCTAGT
rs2301135
Forward
ACTTAACGTGAGGCGCAAAA
Reverse
CGTCCTCCTCCTCCTAGTCC
Probe
54Ts+CCGGGAGAGGGGCGGG
IGTA8
rs7077361
Forward
TGCGAAAACTATTTGGTGAAA
Reverse
CCCACCCACCAAATCTCTAA
Probe
31Ts+GAAATCATCTAGGGGATA


## 2.3. Statistical Analysis

The statistical analyses are processed with SPSS, version 20.0 (IBM, Armonk, NY, USA). The clinical data are expressed as the means± standard deviation (SD) for the continuous variables and as numbers (percentage) for the quantitative variables. Student’st-test is used to compare the age variables between the patients and controls. The gender variables are assessed by the chi-square test. Differences in frequencies of the alleles and genotypes between cases and controls are tested for each SNP through Pearson’s chi-square test and Fisher’s exact test. The criterion for significance is set at P
<
0.05 based on two sides for all of the tests. The statistical power is calculated by Power and Sample Size Calculations version 3.1.2. The Hardy-Weinberg equilibrium (HWE) is tested by adopting the public statistics web tool (http://ihg.gsf.de/cgi-bin/hw/hwa1.pl). The Haploview program [13] is used for the calculation of linkage disequilibrium (LD) among the three SNPs inSNCA.


## 3. Results

Genotype and allele frequencies of each SNP of all 1136 subjects (583 patients and 553 healthy controls) are shown in Table3. Among PD patients, 131 (22%) had an early age of onset (<50 years) and around 77% of the patients were LOPD (≧50 years). The age (P
=
0.860) and gender (P
=
0.284) show no statistical difference between the PD patients and the controls in our study. All the subjects were ethnic Hans.Table 3
Comparison of the genotype frequencies and the allele frequencies ofLRRK2, SNCA, and ITGA8 polymorphisms.
Gene SNP
Group
Genotypen%
P
H
W
E
Allele Min/Maj
MAFa
MAFb
OR (95% CI)
P
Powerc
CC
GC
GG
G/C
LRRK2
rs1491942
Patients total
291
49.9
240
41.2
52
8.9
0.84
344/822
0.30
0.36
1.884 (1.546–2.297)
2.60E − 10∗
1.00
Controls total
372
67.3
161
29.1
20
3.6
0.67
201/905
0.18
EOPD
65
49.6
54
41.2
12
9.2
0.84
78/184
1.924 (1.244–2.976)
3.03E − 03∗
Controls < 50 y
74
68.5
29
26.9
5
4.6
0.33
39/177
LOPD
226
50.0
186
41.2
40
8.8
0.82
266/638
1.874 (1.500–2.340)
2.46E − 08∗
Controls ≧ 50 y
298
67.0
132
29.7
15
3.4
0.87
162/728
GG
AG
AA
A/G
SNCA
rs2301134
Patients total
437
75.0
132
22.6
14
2.4
0.29
160/1006
0.14
0.20
1.237 (0.964–1.588)
9.43E – 02
0.42
Controls total
436
78.8
108
19.5
9
1.6
0.40
126/980
0.11
EOPD
97
74.0
32
24.4
2
1.5
1.00
36/226
1.561 (0.875–2.786)
1.29E − 01
Controls < 50 y
88
81.5
20
18.5
0
0.0
0.59
20/196
LOPD
340
75.2
100
22.1
12
2.7
0.16
124/780
1.176 (0.891–1.552)
2.52E − 01
Controls ≧ 50 y
348
78.2
88
19.8
9
2.0
0.25
106/784
AA
TA
TT
T/A
rs356221
Patients total
210
36.0
282
48.4
91
15.6
0.86
464/702
0.40
0.40
1.167 (1.985–1.382)
7.50E − 02
0.45
Controls total
231
41.8
244
44.1
78
14.1
0.31
400/706
0.36
EOPD
43
32.8
67
51.1
21
16.0
0.59
109/153
1.286 (0.887–1.864)
1.84E − 01
Controls < 50 y
46
42.6
47
43.5
15
13.9
0.67
77/139
LOPD
167
36.9
215
47.6
70
15.5
1.00
355/549
1.135 (0.938–1.374)
1.93E − 01
Controls ≧ 50 y
185
41.6
197
44.3
63
14.2
0.36
323/567
CC
GG
G/C
rs2301135
Patients total
544
93.3
39
6.7
<0.05
78/1088
0.07
0.19
7.857 (4.046–15.258)
9.12E − 13∗
1.00
Controls total
548
99.1
5
0.9
<0.05
10/1096
0.01
EOPD
129
98.5
2
1.5
<0.05
4/258
1.016 (1.000–1.031)
1.30E − 01
Controls < 50 y
108
100.0
0
0.0
<0.05
0/216
LOPD
415
91.8
37
8.2
<0.05
74/830
7.846 (4.026–15.288)
1.45E − 12∗
Controls ≧ 50 y
440
98.9
5
1.1
<0.05
10/880
TT
CT
C/T
ITGA8
rs7077361
Patients total
580
99.5
3
0.5
1.00
3/1163
0.00
0.00
2.85 (0.296–27.443)
6.25E − 01
0.34
Controls total
552
99.8
1
0.2
1.00
1/1105
0.00
EOPD
131
100.0
0
0.0
<0.05
0/262
0.995 (0.986–1.004)
4.52E − 01
Controls < 50 y
107
99.1
1
0.9
1.00
0/0
LOPD
449
99.3
3
0.7
1.00
3/449
1.003 (1.000–1.007)
2.50E − 01
Controls ≧ 50 y
445
100.0
0
0.0
<0.05
0/445
EOPD: early onset Parkinson’s disease; LOPD: late-onset Parkinson’s disease;P
H
W
E: P value obtained in the Hardy-Weinberg equilibrium (HWE) test; ∗: significant P value obtained in the case-control analysis; Min: minor; Maj: major; MAF: minor allele frequency; a: this study; b: 1000 genomes (Southern Han Chinese); c: power was calculated by Power and Sample Size Calculations version 3.1.2.Linkage disequilibrium between theSNCA SNPs rs2301134 and rs356221 is r
2
= 0.235, while for rs356221 and rs2301135 it is r
2 = 0.066 and for rs2301134 and rs2301135 it is r
2 = 0.127. This shows weakly correlation with in rs2301134, rs2301135, and rs356221.Single marker analysis showed a number of significantly statistical associations in our study. Two SNPs fromSNCA andLRRK2 genes displayed P values < 0.05 prior to correction, both of which were estimated ORs > 1.4. The best P value SNPs from bothLRRK2 andSNCA genes were further analyzed for age stratification.Between all PD patients and controls,LRRK2 gene showed a significant difference in the genotype frequency of variant rs1491942 (P
=
3.543
E
-
09) and allelic G/C frequencies (P
=
2.601
E
-
10, O
R
=
1.884, and 95% CI: 1.55–2.30). In the three SNPs ofSNCA gene, only variant rs2301135 met the statistics standard in genotype frequencies (P
=
4.39
E
-
07) and the allelic G/C frequencies (P
=
9.116
E
-
13, OR = 7.857, and 95% CI: 4.05–15.26). TheITGA8 variant rs7077361 failed to show significant difference in this group (P
>
0.05).In the subgroup of EOPD patients and the controls (age < 50 years), the differences were still obvious in the genotype frequencies of variant rs1491942 ofLRRK2 gene (P
=
1.200
E
-
02) and allelic G/C frequencies (P
=
3.028
E
-
03, OR = 1.924, and 95% CI: 1.24–2.98). Three SNPs ofSNCA gene (P
>
0.05) and one SNP ofITGA8 gene (P
>
0.05) failed to show significant difference in this group. Our statistic data of the LOPD patients and controls aged ≧ 50 years also surpassed the significance thresholds. The variant rs1491942 ofLRRK2 gene showed a P value of 2.538E-07 in genotype frequencies. And the allelic G/C frequencies have a P value of 2.459E-08 while the OR is 1.874, and the 95% CI ranged from 1.50 to 2.34. The difference is obvious in the variant rs2301135 ofSNCA gene in the genotype frequencies (P
=
5.561
E
-
07) and allelic G/C frequencies (P
=
1.45
E
-
12, OR = 7.846, and 95% CI: 4.03–15.29) in the subgroup. All SNPs in our study met Hardy-Weinberg equilibrium except for the rs2301135 ofSNCA gene, shown in Table 3.


## 4. Discussion

Since the first Genome-Wide Association Study on sporadic Parkinson’s disease was performed in 2005, a new era starts to gain attention in the genetic basis of Parkinson’s disease [14]. Advances in genotyping technology and meta-analysis have allowed researchers to rapidly identify common variants related to PD in different populations [4]. Though some of the previously nominated PD risk genes were firstly reported in familiar Parkinson’s disease (such asSNCA andLRRK2), both of them were successfully replicated in unrelated sporadic PD patients [15]. Additional associated studies and subsequent meta-analysis contributed a lot to identifying the unnoticed variants that can also drive PD risk,ITGA8 as an example [8].LRRK2 gene was firstly featured as a PD-related gene in Zimprich’s study of families with autosomal-dominant, late-onset Parkinsonism in 2004 [16]. Variants in different domains ofLRRK2 have been identified in both familial and sporadic PD in different populations [17–19]. Rs1494942 inLRRK2 was previously found associated with PD in US and European series [8, 20]. Our result, being consistent with previous studies, suggests that polymorphism rs1491942 ofLRRK2 is a risk loci of sporadic PD, and the variant carriers may share a similar pathomechanism in different populations. As it is reported,LRRK2 variant carriers share similar clinical and pathological features, including a wild range of onset ages, typical Parkinsonism presentation, and sensitivity to L-dopa therapy [21]. In our PD patients, the rs1491942 shows significantly higher frequencies in both EOPD and LOPD subgroups compared with matched controls. These suggest that rs1491942 does not influence the onset age but contributes to the pathogenesis of EOPD and LOPD in a similar way.SNCA is the first identified causal gene in familial PD [22]. Our findings are partly consistent with previous reports of the association of polymorphisms inSNCA with the susceptibility of PD in US, Norway, and Italian studies [23–25]. The linkage disequilibrium of three SNPs ofSNCA gene showed that those SNPs are independent. Only one SNP near the promoter region (rs2301135) shows significant differences between PD patients and the controls in Chinese Han population. Concerning the age of onset, rs2301135 is more likely to associate with late-onset PD in our study, while another study in UK suggested thatSNCA risk alleles for PD may associate with earlier onset of PD [26]. Given that genotype frequencies and allelic frequencies of rs2301135 ofSNCA gene do not follow the Hardy-Weinberg equilibrium in our study, this suggests the possibility of inappropriate population stratification and selection or other confounding factors in our study. Therefore, these results should be interpreted carefully.ITGA8 expressing in brain mediates cell-cell interactions and regulates neurite outgrowth of sensory and motor neurons [8].ITGA8 gene was firstly shown associated with PD in Caucasian population in Simón-Sánchez’s study but failed to replicate in other studies of Greece, Irish, and Polish series [4, 19, 20].ITGA8 variant rs7077361 showed no evidence of relation to PD in our population. In patients and controls, the observed MAFs of the SNP rs7077361 were similar to those reported in the 1000 genomes Southern Han Chinese (CHS) population. However, there is insufficient power to detect the association of rs7077361 with PD in the current sample size. The lack of association of the rs7077361 in Chinese Han population could be ascribed to the limited sample size and the rare existence of this SNP in Chinese population. Large-sample trials from multicenter are required to better understand the contribution of rs7077361 inITGA8 to PD susceptibility.In conclusion, the present study provides considerable evidence to support the significant influence of genetic variants on PD risk. It does not replicate the susceptibility of rs2301134 and rs356221 inSNCA and rs7077361 inITGA8 for PD but confirms that single-nucleotide polymorphisms rs1491942 ofLRRK2 and rs2301135 of SNCA gene are susceptible to sporadic PD in Chinese Han population. Certain variants are responsible for the incidence of this disease, while other variants may modify the onset age, which suggests that distinct aspects of PD have a specific genetic architecture. Further studies are required to enrich genetic architecture of PD.



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*Source: 3474751-2016-09-07.xml*