Neurogenetics

, Volume 7, Issue 3, pp 133–138

A common missense variant in the LRRK2 gene, Gly2385Arg, associated with Parkinson’s disease risk in Taiwan

Authors

  • Alessio Di Fonzo
    • Department of Clinical GeneticsErasmus MC Rotterdam
    • Centro Dino Ferrari, Department of Neurological SciencesUniversity of Milan, and Foundation “Ospedale Maggiore Policlinico
  • Yah-Huei Wu-Chou
    • Neuroscience Research CenterChang Gung Memorial Hospital, Chang Gung University College of Medicine
    • Human Molecular Genetics LaboratoryChang Gung Memorial Hospital, Chang Gung University College of Medicine
  • Chin-Song Lu
    • Neuroscience Research CenterChang Gung Memorial Hospital, Chang Gung University College of Medicine
    • Department of NeurologyChang Gung Memorial Hospital, Chang Gung University College of Medicine
  • Marina van Doeselaar
    • Department of Clinical GeneticsErasmus MC Rotterdam
  • Erik J. Simons
    • Department of Clinical GeneticsErasmus MC Rotterdam
  • Christan F. Rohé
    • Department of Clinical GeneticsErasmus MC Rotterdam
  • Hsiu-Chen Chang
    • Neuroscience Research CenterChang Gung Memorial Hospital, Chang Gung University College of Medicine
  • Rou-Shayn Chen
    • Neuroscience Research CenterChang Gung Memorial Hospital, Chang Gung University College of Medicine
    • Department of NeurologyChang Gung Memorial Hospital, Chang Gung University College of Medicine
  • Yi-Hsin Weng
    • Neuroscience Research CenterChang Gung Memorial Hospital, Chang Gung University College of Medicine
    • Department of NeurologyChang Gung Memorial Hospital, Chang Gung University College of Medicine
  • Nicola Vanacore
    • National Centre of EpidemiologyNational Institute for Health
  • Guido J. Breedveld
    • Department of Clinical GeneticsErasmus MC Rotterdam
  • Ben A. Oostra
    • Department of Clinical GeneticsErasmus MC Rotterdam
    • Department of Clinical GeneticsErasmus MC Rotterdam
Original Article

DOI: 10.1007/s10048-006-0041-5

Cite this article as:
Di Fonzo, A., Wu-Chou, Y., Lu, C. et al. Neurogenetics (2006) 7: 133. doi:10.1007/s10048-006-0041-5

Abstract

Mutations in the LRRK2 gene are a cause of autosomal dominant Parkinson’s disease (PD). Whether LRRK2 variants influence susceptibility to the commoner, sporadic forms of PD remains largely unknown. Data are particularly limited concerning the Asian population. In search for novel, biologically relevant variants, we sequenced the LRRK2 coding region in Taiwanese patients with PD. Four newly identified variants and another variant recently found in a Taiwanese PD family were tested for association with the disease in a sample of 608 PD cases and 373 ethnically matched controls. Heterozygosity for the Gly2385Arg variant was significantly more frequent among PD patients than controls (nominal p value=0.004, corrected for multiple comparisons=0.012, gender- and age-adjusted odds ratio=2.24, 95% C.I.: 1.29–3.88); this variant was uniformly distributed across genders and age strata. Two novel variants, Met1869Val and Glu1874Stop, were found in one PD case each; their pathogenic role remains, therefore, uncertain. The remaining two novel variants (Ala419Val and Pro755Leu) were present with similar frequency in cases and controls, and were therefore, interpreted as disease-unrelated polymorphisms. Our findings suggest that the LRRK2 Gly2385Arg is the first identified, functionally relevant variant, which acts as common risk factor for sporadic PD in the population of Chinese ethnicity.

Keywords

Parkinson’s diseaseLRRK2MutationPolymorphismRisk factorTaiwan

Introduction

Mutations in the gene leucine-rich repeat kinase 2(LRRK2, PARK8) are the most frequent known cause of familial autosomal dominant Parkinson’s disease (PD) [1, 2]. Moreover, due to incomplete penetrance or missing genealogical information, LRRK2 mutations are also found in apparently sporadic PD cases. The available data suggest that the prevalence of the LRRK2 mutation varies markedly across populations. A first founder mutation, Arg1441Gly, is frequent but is limited to the Basque population, while a second founder mutation, Gly2019Ser, is common in countries of Southern Europe, being found in ~3–7% of familial PD and in ~1–2% of sporadic cases, and it might be much more frequent in populations from North Africa and the Middle East [39]. Very limited data are available on the nature and frequency of LRRK2 mutations and on the polymorphism content of this large gene in patients of Asian and other ethnicities.

Three known PD-causing mutations (Ile2012Thr, Gly2019Ser, and Ile2020Thr) appear to be very rare or absent in Asian PD patients [1012], while novel variants of uncertain pathogenic role were recently identified in single Taiwanese families with PD (Arg1441His and Gly2385Arg) [13]. Whether LRRK2 variants influence the susceptibility to the commoner, sporadic forms of PD also remains largely unknown. No evidence for a major role of common LRRK2 variants was found in the German population [14], while evidence for a PD-associated haplotype was identified in the population of Chinese ethnicity from Singapore [15]. However, the biologically relevant variant within this haplotype remains unknown. A third smaller study found no association between PD and four coding LRRK2 variants in Caucasians [16]. In search for novel, biologically relevant variants in the Chinese population, we sequenced the LRRK2 coding region in Taiwanese PD patients. Five variants were then studied for association with PD in a large sample of cases and ethnically matched controls.

Patients and methods

We studied 608 sporadic patients (246 females, 362 males) of Chinese ethnicity with a clinical diagnosis of idiopathic PD, ascertained at a single referral center in Taiwan. The average age at the last examination and at disease onset were 66±12 years (range 24–97) and 54.9±11.9 years (range 12–94), respectively. The clinical diagnosis of PD was established according to published criteria [17]. The whole PD sample was previously screened by us and found negative for three LRRK2 mutations (Ile2012Thr, Gly2019Ser, and Ile2020Thr) [12]. In cases with disease onset before the age of 40 years [n=68], the parkin and PINK1 genes were also screened by single-strand conformation polymorphism analysis (SSCP), and no mutations were found.

The control sample included 373 individuals free from PD, matched to the patients by ethnicity, collected at the same center, and originating from the same geographical areas (212 females and 161 males, mean age at sampling 54.7±18.2 years, range 11–87). More exactly, the controls were spouses of PD patients (67.5%), spouses of patients with unrelated diseases (16.5%), and healthy volunteers (16%). The project was approved from the local Ethical authorities and written informed consent was obtained from all subjects. Genomic DNA was isolated from peripheral blood using standard protocols.

In 15 randomly chosen PD patients, the whole LRRK2 coding sequence (51 exons) and exon–intron boundaries were studied by polymerase chain reaction (PCR) using previously described primers and protocols [18]. Direct sequencing of both strands was performed using Big Dye Terminator chemistry ver.3.1 (Applied Biosystems). Fragments were loaded on an ABI3100 and analyzed with DNA Sequencing Analysis (ver.3.7) and SeqScape (ver.2.1) software (Applied Biosystems). Mutations were named according to the LRRK2 cDNA sequence deposited in Genbank (accession n. AY792511). Novel sequence variants identified were submitted to Genbank.

Variants identified in these 15 patients were prioritized for further study and tested by direct sequencing in the whole sample of 608 sporadic PD patients and 373 controls. Positive and negative controls were included in each PCR plate. Statistical analyses included contingency tables, multiple logistic regression, and Student’s t-test as appropriate, and were performed using the SPSS ver. 13.0 package. Permutation test was performed using the program Haploview ver. 3.2.

Results

By sequencing the whole LRRK2 coding region and splice sites in 15 patients with sporadic PD, we identified 17 exonic (Table 1 and Fig. 1) and 22 intronic variants (not shown). Similar to what was previously found by us and others in Caucasians [13, 18], one variant (rs2256408, predicted amino acid effect Arg50His) was present in all samples in homozygous state. This is likely due to an error in the genomic reference sequence. Among the remaining 16 variants, we initially prioritized four for further analysis: the three novel missense variants (Ala419Val, Pro755Leu, and Met1869Val) and one additional variant (Gly2385Arg), which was recently identified in one Taiwanese family with PD and considered as a putative pathogenic mutation [13]. The prioritized variants were tested for association with PD in our entire sample of 608 cases and 373 ethnically matched controls. While sequencing the large sample, a further, novel heterozygous coding variant, Glu1874Stop, was found in one PD case.
Table 1

Coding variants identified in 15 Taiwanese patients with PD

Exon

Accession number

Amino acid effect

MAF

Genotypes

1

rs2256408

Arg50His

1.00

15 hom

5

rs10878245

Leu153Leu

0.3

2 hom and 5 het

11

ss49853007

Ala419Val

0.03

1 het

14

rs7308720

Asn551Lys

0.1

3 het

19

ss49853008

Pro755Leu

0.03

1 het

22

rs7966550

Leu953Leu

0.23

1 hom and 5 het

30

rs7133914

Arg1398His

0.06

2 het

30

rs11175964

Lys1423Lys

0.06

2 het

34

rs1427263

Gly1624Gly

0.47

4 hom and 6 het

34

ss48398561

Arg1628Pro

0.06

2 het

34

rs11176013

Lys1637Lys

0.47

4 hom and 6 het

34

rs11564148

Ser1647Thr

0.4

3 hom and 6 het

37

rs10878371

Gly1819Gly

0.47

4 hom and 6 het

38

ss pending

Met1869Val

0.03

1 het

43

rs10878405

Glu2108Glu

0.4

3 hom and 6 het

48

ss48398568

Gly2385Arg

0.06

2 het

49

rs3761863

Met2397Thr

0.5

4 hom and 7 het

MAF Minor allele frequency, hom homozygous, het heterozygous

https://static-content.springer.com/image/art%3A10.1007%2Fs10048-006-0041-5/MediaObjects/10048_2006_41_Fig1_HTML.gif
Fig. 1

a Aligment of LRRK2 protein homologues at positions of two missense mutations analysed in this study. The alignment was generated using T-COFFEE. The conserved amino acids are highlighted. hsHomo sapiens (accession number NP_940980), mmMus Musculus (NP_080006.2), rnRattus norvegicus (XP_235581.3), tn Tetraodon nigroviridans (Q4S189), dmDrosophila melanogaster (Q9VDJ9), ceCaenorhabditis elegans (NP_492839.3), ddDictyostelium discoideum (AAO12857.1). b Protein domain structure of LRRK2. The position of the variants studied is indicated. ANK-like Ankyrin-like repeat region, LRR leucine-rich repeat region, Roc Ras in complex proteins, COR C-terminal of Roc, WD40 WD40-like repeats domain, MKL-like mixed-lineage kinase-like consensus region

The results of the association analyses are shown in Table 2. All variants were in Hardy–Weinberg equilibrium in controls and cases. The allelic or genotypic frequencies were not statistically different in cases and controls for two novel variants (Ala419Val, and Pro755Leu). The remaining novel variants, Met1869Val and Glu1874Stop were not observed in other PD or control individuals tested (Table 2). The heterozygosity for the Gly2385Arg variant was significantly higher among PD cases (61 heterozygous carriers out of 608 tested cases) than controls (18 heterozygous carriers out of 373 tested controls [nominal p value=0.004, corrected for multiple comparisons=0.012; permutation test, p=0.02 (5,000 replicates); gender- and age-adjusted odds ratio 2.24, 95% C.I.: 1.29–3.88]. The frequency of this variant was uniform across gender and age strata (Table 3).
Table 2

Allelic and genotypic frequencies in cases and controls

 

Cases

Controls

Genotypes

Alleles

Genotypes

Alleles

Exon

Nucleotide change

Amino acid change

WT/WT

WT/Var

Var/Var

WT

Var

WT/WT

WT/Var

Var/Var

WT

Var

11

C1256T

Ala419Val

582 (0.98)

10 (0.02)

0 (0.00)

1174 (0.992)

10 (0.008)

341 (0.99)

3 (0.01)

0 (0.00)

685 (0.996)

3 (0.004)

19

C2264T

Pro755Leu

578 (0.99)

7 (0.01)

0 (0.00)

1163 (0.994)

7 (0.006)

339 (0.97)

10 (0.03)

0 (0.00)

688 (0.985)

10 (0.015)

38

A5605G

Met1869Val

592 (0.999)

1 (0.001)

0 (0.00)

1185 (0.999)

1 (0.001)

370 (1.00)

0 (0.00)

0 (0.00)

740 (1.00)

0 (0.00)

38

G5620T

Glu1874Stop

592 (0.999)

1 (0.001)

0 (0.00)

1185 (0.999)

1 (0.001)

370 (1.00)

0 (0.00)

0 (0.00)

740 (1.00)

0 (0.00)

48

G7153A

Gly2385Arg

547 (0.9)

61 (0.1)

0 (0.00)

1155 (0.950)

61 (0.05)

355 (0.95)

18 (0.05)

0 (0.00)

728 (0.975)

18 (0.025)

Nominal p value =0.004, corrected p value=0.012—comparison of Gly2385Arg genotypic and allelic frequency in PD cases vs controls (chi square test), permutation test: p=0.02 (5,000 replicates); Nominal p value=0.065, corrected p value=0.195—comparison of Pro755Leu genotypic and allelic frequency in PD cases vs controls (chi square test); differences between genotypic and allelic frequency of the Ala419Val polymorphism in PD cases vs controls are not significant

Table 3

Frequency of the Gly2385Arg variant in different age strata and genders

Age at PD onset

Total cases

Gly2385Arg carriers

Percentage (%)

Females Carrier

Males Carrier

Cases

 <50

215

23

0.107

9

14

 50<60

152

15

0.099

7

8

 60<70

184

20

0.109

9

11

 70 and >70

57

3

0.053

2

1

 <60

367

38

0.104

  

 60 and >60

241

23

0.095

  

Total females

246

27

0.110

  

Total males

362

34

0.094

  

Total

608

61

0.100

  

Controls

     

 Age at examination

Total controls

Gly2385Arg carriers

Percentage

Females carrier

Males carrier

 <50

109

7

0.064

4

3

 50<60

78

2

0.026

2

0

 60<70

111

6

0.054

3

3

 70 and >70

75

3

0.040

1

2

 <60

187

9

0.048

  

 60 and >60

186

9

0.048

  

Total females

212

10

0.047

  

Total males

161

8

0.050

  

Total

373

18

0.048

  

The age at examination was similar in PD cases that carried the Gly2385Arg variant and those who did not carry it (carriers: 66.5±12 years, non-carriers: 66.2±11.6 years, p=0.84, Student’s t-test). The average disease onset in the 61 carriers of the Gly2385Arg variant was 53.2±12.5 years, range 12–82, which is earlier though not significantly different from the average onset age in non-carriers (55±11.9 years, range 20–94, p=0.24). In the Gly2385Arg carrier with the earliest onset, foot dystonia started at the age of 12 years, followed by bradykinesia and rigidity. The clinical phenotype was otherwise typical of juvenile PD, including good levodopa response, slow course and levodopa-induced dyskinesias. Parkin mutations were not identified.

The average disease duration in carriers of the Gly2385Arg variant was slightly longer than that in non-carriers (average: 13.4±6.2 years in carriers and 11.2±6 in non-carriers, p=0.007, Student’s t-test). Male patients accounted for much of the difference in disease duration [average: 15.1±5.9 years in male carriers (n=34) and 11.6±6.2 in non-carriers, p=0.002], while disease duration was not significantly different among the female patients [average 11.3±6 years in female carriers (n=27) and 10.5±5.8 in non-carriers, p=0.53]. Additional relatives of the cases carrying the novel variants were not available for study. Alignment of LRRK2 protein homologues at the level of the Met1869Val and Gly2385Arg variants is shown in Fig. 1.

Discussion

In this association study, we focused on coding variants, which are more likely to be biologically relevant, identified by direct scanning of the LRRK2 gene in the target population [19]. Four of the five variants investigated are reported here for the first time (Tables 1 and 2).

Two novel variants (Ala419Val, and Pro755Leu) were present with similar frequencies in cases and controls, and therefore, are likely to represent neutral, disease-unrelated changes. The remaining two novel variants, Met1869Val and Glu1874Stop, were found each in only one PD case and were not observed in other PD cases (n=592) or controls (n=370). Due to their rarity, these are, therefore, to be considered as mutations, rather than polymorphisms. However, their pathogenic significance remains uncertain. The patients carrying these mutations are sporadic, and relatives were dead or not available for testing. Hence, we cannot determine if these are “de novo” mutations. The fact that the same codon targeted by the Met1869Val mutation was also affected by another mutation found in PD cases, Met1869Thr [13, 20], supports the contention that these changes are pathogenic. The Met1869 residue is close to one of the known LRRK2 functional domains (Cor, C-terminal of Roc), but has not been highly conserved in evolution, and some species even have a threonine at this position (Fig. 1). However, the conservation of the residue is not an absolute requisite of pathogenic mutations.

In addition to be a novel variant, the Glu1874Stop is, to our knowledge, the first LRRK2 nonsense mutation identified in a PD case and is predicted to encode a protein lacking the entire kinase and WD domains (Fig. 1). The identification of this allele could be a coincidental finding due to the large number of individuals sequenced for this exon. However, the Glu1874Stop could also represent a true disease-causing de novo mutation or an inherited mutation displaying reduced penetrance. In this patient, PD symptoms started at age 69. Inspection of the pedigree of this patient shows no other PD cases among first-degree relatives (parents, five siblings and two children). Both parents died at around 65 years of age during World War II. Only one brother of the patient remains alive and is, today, aged 90, but he could not be sampled for gene testing. This is potentially a very important finding for understanding the mechanisms of the disease caused by LRRK2 mutations. Recent studies found evidence that some of the LRRK2 mutations (Gly2019Ser, Ile2020Thr, and Arg1441Cys) increase the kinase activity of the encoded protein, thus, suggesting a gain-of-function mechanism [21, 22]. If carrying a heterozygous truncating allele, which lacks the whole kinase domain, was the cause of PD in our patient, we would have to postulate that an increased kinase activity is not the invariable mechanism of LRRK2-related neurodegeneration. Functional studies of the Glu1874Stop allele are now warranted.

The last variant studied (Gly2385Arg) was recently reported in a PD family from Taiwan [13]. Evidence for co-segregation with PD in that family was limited due to the small pedigree size; however, the mutation was reported to be absent in 200 ethnically matched controls, and therefore, interpreted as putatively pathogenic. We identified this variant in several PD patients and controls, but its frequency was significantly higher among PD cases, also after adjustment for possible confounders such as gender and age and after correction for multiple testing. This suggests that the Gly2385Arg variant, or another variant in linkage disequilibrium, is associated with PD in the Taiwanese population. The residue targeted (Gly2385) lies in the WD domain and is not conserved in evolution, but amino acids with a net positive charge (such as arginine) are not observed at this codon in the known LRRK2 protein homologues (Fig. 1). Functional studies are needed to investigate whether the presence of the arginine residue influences folding or stability of the LRRK2 protein, or the strength of binding to other, still unknown interactor(s).

The average disease onset in carriers of Gly2385Arg was 2 years earlier but was not significantly different than that of non-carriers. The longer disease duration in male carriers is likely to be a chance finding, in part, as a consequence of the earlier onset; however, it might also suggest, albeit indirectly, a slower disease course among the male carriers. Further, prospective clinical analyses are warranted to further characterize the clinical phenotype associated with the Gly2385Arg variant. It will also be interesting to study the prevalence of the Gly2385Arg variant in Chinese patients with familial forms of PD.

Genetic risk factors might be population specific, and therefore, it is not surprising that evidence for LRRK2 common variants associated with PD was not found, so far, in Caucasians [14, 16] but was found in Chinese (this study and [15]). It will be interesting to explore if Gly2385Arg is the biologically relevant variant contained on the PD-associated haplotype reported recently in Chinese patients from Singapore [15].

Allelic association studies are prone to false-positive results, mainly due to small sample sizes, population stratification, and genotyping errors. Our study was based on a large sample; cases and controls were matched for ethnicity; odds ratios were adjusted for age and gender; furthermore, samples from cases and controls were processed using high quality standards and identical genotyping protocols; these considerations argue against the existence of any of the aforementioned causes of false-positive results. However, it will be important to replicate this association in independent, large samples.

In conclusion, we identified novel LRRK2 variants in the Chinese population, including two novel, potentially pathogenic mutations and the first truncating mutation reported in a PD case. Furthermore, we suggest that the LRRK2 Gly2385Arg is the first identified, functionally relevant variant, acting as a common risk factor for sporadic PD in the Chinese population. These findings have potential important implications for the dissection of the causes and mechanisms of the common forms of PD.

Acknowledgements

This study was supported by grants from the Internationaal Parkinson Fonds (The Netherlands) to V. Bonifati and from the Chang Gung Medical Research Council (CMRPG 1015) (Taiwan) to C-S. Lu.

We declare that the experiments reported in this paper comply with the current laws of the country in which they were performed.

Copyright information

© Springer-Verlag 2006