Genetic Variants of LRRK2 in Taiwanese Parkinson’s Disease

Genetic variants of leucine-rich repeat kinase 2 (LRRK2) were reported to alter the risk for Parkinson’s disease (PD). However, the genetic spectrum of LRRK2 variants has not been clearly disclosed yet in Taiwanese population. Herein, we sequenced LRRK2 coding region in 70 Taiwanese early onset PD patients (age at onset ≤ 50), and found six amino acid-changing single nucleotide polymorphisms (SNPs, N551K, R1398H, R1628P, S1647T, G2385R and M2397T), one reported (R1441H) and 2 novel missense (R767H and S885N) mutations. We examined the frequency of identified LRRK2 variants by genotyping 573 Taiwanese patients with PD and 503 age-matched control subjects. The results showed that PD patients demonstrated a higher frequency of G2385R A allele (4.6%) than control subjects (2.1%; odds ratio = 2.27, 95% confidence interval: 1.38–3.88, P = 0.0017). Fewer PD patients (27.7%) carried the 1647T-2397T haplotype as compared with the control subjects (33.0%; odds ratio = 0.80, 95% confidence interval: 0.65–0.97, P = 0.0215). However, the frequency of 1647T-2385R-2397T haplotype (4.3%) in PD patients was still higher than in control subjects (1.9%, odds ratio: 2.15, 95% confidence interval: 1.27–3.78, P = 0.0058). While no additional subject was found to carry R767H and R1441H, one more patient was observed to carry the S885N variant. Our results indicate a robust risk association regarding G2385R and a new possible protective haplotype (1647T-2397T). Gene-environmental interaction and a larger cohort study are warranted to validate our findings. Additionally, two new missense mutations (R767H and S885N) regarding LRRK2 in PD patients were identified. Functional studies are needed to elucidate the effects of these LRRK2 variants on protein function.


Introduction
Parkinson's disease (PD) is the second most common neurodegenerative disorder in the world [1]. It affects 1% of the population aged over sixty, and is characterized by a slowness of movement (bradykinesia) and a difficulty in initiating movement (akinesia) [1]. The pathogenesis of PD is associated with progressive degeneration of dopaminergic (DA) neurons and the presence of eosinophilic cytoplasmic inclusion bodies (Lewy bodies) with enrichment of α-synuclein in the ventral midbrain [2].
The etiology of PD remains to be explored. Mutations in the gene for leucine-rich repeat kinase 2 (LRRK2) account for some patients with autosomal dominantly inherited PD [3,4].
In Taiwan, the LRRK2 G2385R and R1628P variants may play significant roles in susceptibility to PD [7][8][9][10]. In contrast, LRRK2 G2019S, a common mutation amongst PD patients in North America, Europe and North Africa [3,4,[11][12][13][14], has not been found in Taiwanese PD patients [15]. The disease penetrance for G2019S carriers is age dependent, increasing from less than 20% at age 50 years or younger to 80~85% at age 70 years [16,17]. Age at onset (AAO) of mutation carriers is broad, ranged from 28 to 73 years, and mutation carriers were clinically indistinguishable from idiopathic PD [18,19]. To further examine the genetic variations of LRRK2 in Taiwanese PD, we sequenced the LRRK2 coding region in 70 Taiwanese PD patients and assessed the association of identified SNPs with the risk of PD by utilizing a large case-control cohort of patients and controls, to provide more insight into LRRK2 variants in Taiwanese PD patients.

G2385R and M2397T
A case-control study in a cohort of PD (n = 573) and ethnically matched controls (n = 503) was conducted to assess the association of the six amino acid-changing variants with risk of PD. The genotype distributions in PD and controls did not deviate significantly from Hardy-Weinberg equilibrium for any of the six variants examined (data not shown). The SNPSpD method was employed for correction of multiple SNP testing. SNPSpD output of six λs was shown in Table 2. As described by Cheverud [24], high correlation among variables leads to high λs. In this case, the first λ (2.37) was less than 6 (the number of variables in the correlation matrix), suggesting that not all variables are completely correlated. The magnitude of pair-wise LD was quantified by the metrics D' and Δ 2 . The D' and Δ 2 coefficients of 551 and 1398 sites were 0.94 and 0.77, respectively, suggesting less historical recombination and more LD between 551 and 1398 sites. This was also true for 1647 and 2397 sites, with a D' coefficient of 0.93 and a Δ 2 coefficient of 0.59.
The genotype and allele distributions of the six variants for both patients and controls are outlined in Table 3. A statistically significant difference in G2385R A allele (4.6% vs. 2.1%, P = 0.0013) distribution between patients and controls was observed. When odds ratios of the at-risk genotype/allele were calculated, an increase in risk of developing PD was demonstrated for G2385R A allele (odds ratio: 2.27, 95% confidence interval: 1.38 -3.88, P = 0.0017). The allele distribution of G2385R was further analyzed after being stratified by age. In the early onset PD (EOPD) group (AAO ≤ 50), a significant difference in G2385R A allele (5.1% vs. 0.8%, P = 0.0063) distribution between patients and controls was observed. EOPD patients with A allele have odds ratio 6.61 (95% confidence interval: 1.72 -43.35, P = 0.0155) as compared with controls. In the late onset PD (LOPD) group (AAO > 50), a significant difference in G2385R A allele (4.5% vs. 2.5%, P = 0.0295) distribution between patients and controls was also observed. The LOPD patients with A allele has an odds ratio of 1.84 (95% confidence interval: 1.08 -3.26, P = 0.0288) as compared with controls. The difference in A allele distribution between EOPD and LOPD groups were not significant (5.1% vs. 4.5%, P = 0.7329). The allele distribution of other variants did not show a significant difference between early and late onset PD patients groups as well as controls.

Discussion
The present study consolidates the role of LRRK2 G2385R as a risk factor of PD, and supports that S1647T-M2397T haplotype may lower the susceptibility of PD among Taiwanese population. We also identify one reported (R1441H) and two novel missense mutations (R767H and S885N) of LRRK2. Although the genome-wide association studies (GWAS) reported a strong association between LRRK2 genetic variations and PD [25,26], the GWAS association signal has not been driven by identified missense variant as the G2385R, which may be due to this risk variant is ethnic specific.
The G2835R variant on the WD40 domain was first reported in a PD patient from Taiwan, with less than 1% frequency in Caucasian controls [20]. This variant is more common in Asia and is associated with an increased risk of PD in Japan, Singapore and Mainland China [27][28][29][30], in addition to Taiwan [7][8][9]. When over-expressed in human HEK cells, the G2835R variant was more toxic and associated with a higher rate of apoptosis under condition of oxidative stress [27]. Acting differently from the common LRRK2 kinase-activating G2019S mutation [31], the G2385R mutant causes a partial loss of the kinase function of LRRK2 [32]. In M17 neuroblastoma cell line, G2019S mutation decreased the average length of neurites and G2019S/G2385R double mutants counteract the neurite shortening effect of G2019S, suggesting that the impact of G2385R is strong enough to overcome the kinase-activating effect of the G2019S [32]. Since both loss and gain of kinase function variants are pathogenic, it is likely that the kinase activity of LRRK2 can be tolerated over only a narrow range. It is also possible that the G2385R mutation leads to pathogenic effects via other mechanism, which raises another therapeutic aspect for PD.
The protective LRRK2 variants and haplotypes have been reported in PD patients. For example, R1398H and N551K reduce the risk of PD in Han-Chinese population [33]. Individuals carrying haplotype 551K-1398H-1423K have a significant reduction of PD risk in the white, Asian, and Arab-Berber populations [34]. Herein we identified a new LRRK2 haplotype 1647T-2397T related to the reduced risk for PD, although results seen in single variant disease-association analysis does not find risk alterations in these two polymorphisms. S1647T is located at the highly evolutionconserved COR domain, which is thought to be a regulator of ROC GTPase activity [35]. In a Taiwanese study, S1647T is associated with increased PD risk, after considering the interaction effects with pesticide exposure [36]. These results contrast with the effect of 1647T-2397T to reduce PD risk, suggesting that other, yet unknown, molecular mechanisms are involved. Located on WD40 domain, M2397T is a riskassociated polymorphism in inflammatory bowel disease [37]. This variant decreases the amount of LRRK2 by altering the protein stability when expressed in HEK-293 cells [38]. This mechanism may contribute to its protective role in PD. As the risk of developing PD with 1647T-2385R-2397T haplotype is similar to that with 2385R allele alone, the protective effect of 1647T-2397T haplotype may be absent in the population carrying G2385R risk variant. Alternatively, the protect effect of 1647T-2397T may be attributable to the absence of G2385R variant. A larger cohort study will be needed to delineate the genetic effect of 1647T-2397T haplotype on PD risk reduction.
Two novel (R767H and S885N) and one reported (R1441H) missense mutations were identified in this population study. R767H is located in the ANK domain [6], which may play a role in protein folding [39]. Although the substitution of arginine with histidine would not dramatically affect the protein polarity, the newly added guanidine group may affect the protein stability by modifying the folding structure of LRRK2. S885N mutation substitutes serine with asparagine at the hinge between the ANK and LRR domains. The molecular mechanism of this mutation remains elusive. R1441H lies within the ROC GTPase domain, and more recently identified mutations affecting the same amino acid (R1441C, R1441G) have been described in affected PD patients [4,20]. R1441C mutation has been shown to increase LRRK2 kinase activity [31]. Both R1441C and R1441G mutations affect the GTPase activity of LRRK2 [40]. Lymphoblastoid cell lines carrying R1441H mutation showed increased apoptosis following exposure to proteasome inhibitor [41]. Thus, these mutations act dominantly and most likely cause enzymatic or structural gain-of-function that leads to neuronal toxicity. Although our results are significant, there are limitations in this study. The role of gene-environmental interaction has not been evaluated. The sample size in our study may not be able to identify an association when the genetic effect of the allele is weak. This may explain the lack of protective effects of R1398H and N551K and increased risk of R1628P seen in a Chinese multicenter study [33]. Additionally, there is insufficient segregation to prove the pathogenicity of the two novel mutations (R767H and S885N). Nevertheless, our population study provides more information about the genetic variant of LRRK2 in Taiwanese PD patients, and discovers two novel LRRK2 mutations. Further study is needed to identify the functional implications of these genetic variants, which may shed light on developing new therapeutic strategies for PD.

Ethics statement
This study was performed according to a protocol approved by the Institutional Review Board of Chang Gung Memorial Hospital (ethical license No: 97-2476A3), and all examinations were performed after obtaining written informed consents.

Patient population
A total of 573 unrelated Taiwanese PD subjects (44.7% females) were recruited from the neurology clinics of Chang

Statistical analysis
The genotype frequency data and the expected genotypic frequency under random mating were computed and Chisquare tested for Hardy-Weinberg equilibrium using a standardized formula. The genotype and allele association analysis was carried out using the Chi-square test. The SNPSpD method [43] was used to generate an adjusted significance threshold for correction of multiple SNP testing (http://genepi.qimr.edu.au/general/daleN/SNPSpD/). The experiment-wide significance threshold of 0.0092 was required to keep the type I error rate at 5%. Measures of pairwise linkage disequilibrium (LD) between SNPs, including Lewontin's standardized disequilibrium coefficients (D'), the squared pairwise correlations (Δ 2 ), and eigenvalues (λs) were computed with the LDMAX software-part of the GOLD Table 5. Primers and conditions for PCR amplification of LRRK2 cDNA and genomic DNA.

Test (amplified region)
Command Line Tools package [44]. PHASE version 2.1 was used to infer the LRRK2 gene haplotypes [45]. The LRRK2 pairwise haplotype frequencies were computed and Chi-square tested for significance. Odds ratios with 95% confidence intervals (95% CI) were calculated to test association between genotype/allele/haplotype and disease.