Neurogenetics

, Volume 6, Issue 4, pp 171–177

Lrrk2 pathogenic substitutions in Parkinson's disease

Authors

  • Ignacio F. Mata
    • Departments of Neurology and NeuroscienceMayo Clinic
  • Jennifer M. Kachergus
    • Departments of Neurology and NeuroscienceMayo Clinic
  • Julie P. Taylor
    • Departments of Neurology and NeuroscienceMayo Clinic
  • Sarah Lincoln
    • Departments of Neurology and NeuroscienceMayo Clinic
  • Jan Aasly
    • Department of NeurologySt. Olav's Hospital
  • Timothy Lynch
    • Department of NeurologyMater Misericordiae University Hospital
    • Conway Institute of Biomolecular and Biomedical Research
  • Mary M. Hulihan
    • Departments of Neurology and NeuroscienceMayo Clinic
  • Stephanie A. Cobb
    • Departments of Neurology and NeuroscienceMayo Clinic
  • Ruey-Meei Wu
    • Department of Neurology, National Taiwan University Hospital, College of MedicineNational Taiwan University
  • Chin-Song Lu
    • Department of NeurologyChang Gung Memorial Hospital
  • Carlos Lahoz
    • Servicio de NeurologíaHospital Universitario Central de Asturias
  • Zbigniew K. Wszolek
    • Departments of Neurology and NeuroscienceMayo Clinic
    • Departments of Neurology and NeuroscienceMayo Clinic
    • Department of NeuroscienceMolecular Genetics Laboratory and Core, Morris K. Udall Parkinson's Disease Research Center of Excellence
Original Article

DOI: 10.1007/s10048-005-0005-1

Cite this article as:
Mata, I.F., Kachergus, J.M., Taylor, J.P. et al. Neurogenetics (2005) 6: 171. doi:10.1007/s10048-005-0005-1
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Abstract

Leucine-rich repeat kinase 2 (LRRK2) mutations have been implicated in autosomal dominant parkinsonism, consistent with typical levodopa-responsive Parkinson's disease. The gene maps to chromosome 12q12 and encodes a large, multifunctional protein. To identify novel LRRK2 mutations, we have sequenced 100 affected probands with family history of parkinsonism. Semiquantitative analysis was also performed in all probands to identify LRRK2 genomic multiplication or deletion. In these kindreds, referred from movement disorder clinics in many parts of Europe, Asia, and North America, parkinsonism segregates as an autosomal dominant trait. All 51 exons of the LRRK2 gene were analyzed and the frequency of all novel sequence variants was assessed within controls. The segregation of mutations with disease has been examined in larger, multiplex families. Our study identified 26 coding variants, including 15 nonsynonymous amino acid substitutions of which three affect the same codon (R1441C, R1441G, and R1441H). Seven of these coding changes seem to be pathogenic, as they segregate with disease and were not identified within controls. No multiplications or deletions were identified.

Keywords

LRRK2KinaseIdiopathic Parkinson's diseaseMutationPolymorphism

Introduction

Parkinson's disease (PD) is a prevalent, age-associated movement disorder affecting 1.8% of the population more than 65 years old. The disease is progressive and characterized clinically by resting tremor, bradykinesia, and rigidity, typically with an asymmetric onset and a positive response to dopamine replacement therapy [1]. Pathological findings include loss of pigmented neurons within the substantia nigra, with proteinaceous Lewy body inclusions in surviving cells [2]. The causes of disease have remained obscure and controversial for more than a century. Although parkinsonism may aggregate within families, most twin studies have not supported a genetic etiology and several environmental agents have been proposed [35]. Nevertheless, in the last 7 years mutations in six genes (SNCA, PRKN, PINK1, DJ-1, MAPT, and UCH-L1) have been linked to familial parkinsonism [6].

Mutations in a seventh gene, Leucine-rich repeat kinase 2 (LRRK2), have recently been identified in autosomal dominant PD [712]. LRRK2 (PARK8; OMIM*609007) is located at chromosome 12q12 (GenBank accession no. AY792511). The locus was mapped originally in the Japanese “Sagamihara family” by Funayama and colleagues [13]. LRRK2 is encoded by 51 exons and seems to be expressed in most brain regions; the protein has a predicted molecular weight of 286 kDa. It is highly conserved among vertebrates and shares homology to the ROCO protein family [14]. In silico analysis predicts the presence of five conserved domains: a leucine-rich repeat (LRR), a Roc (Ras in complex proteins; Rab GTPase), a COR (domain C-terminal of Roc), a catalytic core common to both tyrosine and serine/threonine kinases (mixed-lineage MAPKKK), and a WD40 domain [7]. All of these regions may be involved in multiple functions, including substrate binding, protein phosphorylation, and protein–protein interactions.

This study provides a comprehensive summary of LRRK2 coding mutations and their frequency within appropriately matched controls. We report synonymous, nonsynonymous, and pathogenic amino acid substitutions within the Lrrk2 protein, in familial parkinsonism.

Results

To determine the frequency of LRRK2 mutations as a cause of familial parkinsonism and to identify novel mutations in the gene, including whole gene multiplications and deletions, all 51 exons were sequenced and semiquantitative multiplex PCR was performed in affected probands from 100 multiplex families. In these pedigrees, parkinsonism is consistent with an autosomal dominant trait. We identified 26 variants within the exonic sequence. Fifteen lead to nonsynonymous amino acid substitutions, three of these have been described previously as pathogenic (R1441C, R1441G, and G2019S) [712], two were also observed in our previous study (R1514Q and M1646T) [7], and five are documented as single nucleotide polymorphisms (SNPs) within public databases (N551K, I723V, R1398H, S1647T, and T2397M). The remaining five nonsynonymous amino acid substitutions are novel (R1441H, P1542S, R1628P, M1869T, and G2385R) (Table 1). One homozygous LRRK2 149G>A transition (R50H) was identified in all exon 1 sequences, suggesting an error within the consensus genomic sequence (accession NT_029419, contig position 2762388, dbSNP rs2256408). No whole gene multiplications or deletions were found.
Table 1

Twenty-six exonic variants and their frequencies

Exon

Accession no.

Nucleotide

Amino acid

Frequency

5

rs10878245

457C>T

L153L

0.46

5

ss48398554

546A>G

K182K

<0.01

12

ss48398555

1383T>C

S461S

<0.01

14

rs7308720

1653C>G

N551K

0.07

18

rs10878307

2167A>G

I723V

0.05

22

rs7966550

2857T>C

L953L

0.14

30

rs7133914

4193G>A

R1398H

0.07

30

rs11175964

4269G>A

K1423K

0.07

31

ss48398556

4321C>T

R1441C [7]

<0.01

31

ss48398557

4321C>G

R1441G [8]

<0.01

31

ss48398558

4322G>A

R1441H

<0.01

32

ss48398559

4541G>A

R1514Q [7]

<0.01

32

ss48398560

4624C>T

P1542S

0.01

34

rs1427263

4872A>C

G1624G

0.35

34

ss48398561

4883G>C

R1628P

0.01

34

rs11176013

4911G>A

K1637K

0.45

34

ss48398562

4937T>C

M1646T [7]

0.02

34

rs11564148

4939T>A

S1647T

0.29

37

rs10878371

5457C>T

G1819G

0.41

38

ss48398563

5606T>C

M1869T

<0.01

41

ss48398564

6055G>A

G2019S [9–12]

<0.01

43

rs10878405

6324G>A

E2108E

0.30

44

ss48398566

6510C>A

G2170G

<0.01

48

ss48398568

7153G>A

G2385R

<0.01

48

ss48398567

7155A>G

G2385G

0.12

49

rs3761863

7190C>T

T2397M

0.35

Major and minor alleles are shown at each nucleotide variant position. Minor allele frequencies for all variants are calculated based on genotyping 300 US Caucasian controls; rs7133914 and rs11175964 are in complete linkage disequilibrium. Ten variants are mutations (frequency <0.01) of which seven lead to nonsynonomous amino acid substitutions (highlighted in bold). Of the latter, five coding substitutions have been previously described, R1441C, R1441G, and G2019S as pathogenic mutations and R1514Q and M1646T as SNPs [712]

For the 26 exonic variants observed in this study, we assessed genotype frequencies in more than 300 North American and European control samples. Sixteen variants correspond to SNPs with minor allele frequencies 1% or more in controls, whereas 10 are mutations with frequencies of less than 1%. Of these 10 mutations, seven lead to nonsynonymous amino acid substitutions (Table 1).

To assess genetic evidence for pathogenicity we examined whether these seven LRRK2 mutations segregate with disease. Family probands have clinical parkinsonism consistent with typical, asymmetric, levodopa-responsive late-onset PD (Table 2). Their kindreds originate from Ireland (n=1), North America (n=2), Norway (n=1), Spain (n=1), and Taiwan (n=2) (Fig. 1). For LRRK2 4321C>G (R1441G) and 5606T>C (M1869T) mutations, there was one affected individual who was not a mutation carrier. LRRK2 4541G>A (R1514Q) was found in a pair of identical twins, with similar features and ages of onset (50 and 52 years), which precludes segregation analysis. Similarly, the LRRK2 7153G>A (G2385R) has only been observed once in a parent and their offspring. Subsequently, we genotyped all seven variants in a larger, ethnically diverse set of 1,000 control samples (600 North American, 200 Taiwanese, 200 Norwegians, 200 Irish, and 200 Spanish) and no further mutations were observed.
Table 2

Clinical information for probands with nonsynonymous coding substitutions in Lrrk2

Mutation

Gender

No. affected

Age at onset

Ethnicity

Initial symptoms

RT

R

B

PI

AS

l-Dopa response

MMSE scores

Other features

R1441C

M

3

55

Caucasian

B

+

+

+

+

+

na

R1441G

M

7

61

Caucasian

B and RT

+

+

+

+

+

+

na

R1441H

M

2

58

Asian

LtH RT

+

+

+

+

26

R1514Q

F

3

50

Caucasian

na

+

+

+

+

+

30

M1869T

F

3

70

Caucasian

RtH and arm RT

+

+

+

+

+

na

G2019S

M

3

40

Caucasian

LtH RT

+

+

+

+

+

30

G2385R

F

2

50

Asian

LtL B

+

+

+

+

29

Hyperreflexia

RT resting tremor, R rigidity, B bradykinesia, PI postural instability, AS asymmetry, MMSE Mini-Mental State Examination, LtH left hand, RtH right hand, LtL left leg, na not available

https://static-content.springer.com/image/art%3A10.1007%2Fs10048-005-0005-1/MediaObjects/10048_2005_5_Fig1_HTML.gif
Fig. 1

Pedigrees of seven families identified with LRRK2 mutations. Blackened symbols denote subjects with parkinsonism, + implies that DNA was available. m Mutated, wt wild-type alleles. To protect confidentiality, genotypes and genders of some unaffected individuals are not shown

Several additional amino acid substitutions were not observed in our sequence analysis of affected probands including five within public SNP databases (P1262A, V1598E, R1725STOP, P2119L, and N2261I), one recently identified (I2012T) [15] and seven documented in our prior publication (L119P, R793M, L1114L, I1122V, Y1699C, I2020T, and N2081D) [7]. Herein, we provide a comprehensive summary of all exonic variants identified and their position within Lrrk2 protein domains. This list includes SNPs within public databases as denoted by their accession numbers (Table 3).
Table 3

All LRRK2 exonic nucleotide variants

https://static-content.springer.com/image/art%3A10.1007%2Fs10048-005-0005-1/MediaObjects/10048_2005_5_Tab3_HTML.gif

The table highlights all coding variants within 51 exons of the LRRK2 gene. Several variants are documented in dbSNP (http://snp.cshl.org/) as indicated by “rs” nomenclature; “ss” numbers are recent submissions from this study. Nucleotide positions are labeled from the start ATG. The shading corresponds to the location of predicted protein domains: LRR leucine-rich repeat, Roc Rab GTPase, COR C-terminal of Roc, MAPKKK mixed-lineage protein kinase, and WD40 [7]. An ideogram of LRRK2 exons 1–51 and the protein is shown to the left. Coding substitutions referenced were not identified in this sequence analysis but were documented in prior publications [7, 15]. Putative pathogenic substitutions are highlighted in bold

Thus, of the coding substitutions observed in this study, Lrrk2 R1441C, R1441H, and G2019S meet criteria for pathogenic amino acid substitutions. Lrrk2 R1441G is most likely pathogenic given past reports [8, 16], whereas the evidence for Lrrk2 R1514Q, M1869T, and G2385R remains equivocal [7]. It is notable that three mutations cluster within the same codon (R1441) of the Roc domain in three different populations; two of these affect the same nucleotide (4321C>G (R1441G) and 4321C>T (R1441C)) and have been previously observed in Family D (Western Nebraska, USA) and Basque (Spain) families [7, 8] and the same codon is changed within a Taiwanese family with 4322G>A (R1441H). Adjacent codons of the MAPKKK domain have been described with neighboring pathogenic LRRK2 6055G>A (G2019S) and 6059T>C (I2020T) mutations. More recently 6035T>C (I2012T) was also reported within this domain [7, 15]. To date, Lrrk2 G2019S has been the most frequently reported amino acid substitution in sporadic and familial PD [912].

Discussion

In this study, 7% (7/100) of autosomal dominant, late-onset parkinsonism may be attributed to point mutations in the LRRK2 gene. The patients' clinical diagnoses and mean ages of onset (58±15 years, mean±SD; range 40–79 years) are typical for levodopa-responsive PD. Our families were referred from many North American, European, and Asian centers and a rigorous epidemiological assessment of mutation frequency in incident PD has yet to be made. Evidently, there are many coding mutations, resulting in synonymous and nonsynonymous amino acid substitutions, within the LRRK2 gene. We did not observe any whole gene multiplications or deletions. However, the resolution of our study precludes exclusion of all exonic rearrangements. Presently only Lrrk2 R1441C, R1441G, Y1699C, I2012T, G2019S, and I2020T meet statistical genetic evidence for disease causality (mLOD >3, θ=0) within extended families [7, 11, 15, 17, 18].

The COR domain harbors the majority of coding variability, including nine amino acid substitutions of which some might be expected to have an effect on protein structure (R1514Q, P1542S, V1598E, R1725STOP). We previously described a pathogenic mutation 5096A>G (Y1699C) in this region in Family A associated with PD, amyotrophy, and dementia [7]. Pathogenicity for the R1514Q and M1869T substitutions has yet to be confirmed. Of interest, the N-terminus harbors the fewest nonsynonymous amino acid substitutions with only four amino acid changes between exons 1 and 24. Given this region's lack of homology to other proteins, its amino acid sequence conservation may highlight its functional importance and that of the leucine-rich repeat domain. The C-terminal WD40 domain has three amino acid substitutions of which Lrrk2 G2385R may be pathogenic; the frequency and function of coding changes in this domain must be further assessed. The many polymorphic amino acid substitutions throughout Lrrk2 may contribute to its function and possibly to the population-attributable risk of PD. Thus, case–control studies of LRRK2 polymorphic variability are warranted.

Three deleterious mutations were identified within the Roc domain, all affecting arginine at position 1441, albeit in Spanish, North American, and Taiwanese families. Our Spanish family originates from Asturias, Northwest Spain, not from the Basque region [8]; however, a common haplotype has been shown between this family, other affected R1441G carriers from the same region, and in affected carriers from the Basque region [16]. Members of the Rab GTPase superfamily are generally small proteins associated with the cytoskeleton and the regulation of intracellular traffic [19]. Whether the GTPase domain in Lrrk2 is functional also remains to be determined. Alignment of this region against Rab proteins indicates that residue 1441 falls into the variable “RabSF3” region. RabSF regions are located on the surface of Rab GTPases and are thought to be involved in molecular interactions and protein binding [20]. In addition, this motif has recently been implicated in the membrane targeting of some Rab GTPases to specific organelles [21]. Alternate pathologies, synucleinopathy and tauopathy, were previously associated with Lrrk2 R1441C within a single family [7]. Whether the pathology observed is a direct consequence or secondary manifestation of Lrrk2 substitutions in this domain remains to be tested within animal models of disease. The neuronal pathology of additional patients with Lrrk2 R1441-associated disease may also clarify this issue.

The Lrrk2 G2019S and I2020T substitutions fall within a putative activation segment of the MAPKKK domain, a region that would normally protect the active site. In kinases, this region begins and ends with highly conserved amino acid triplets, DRG and APE [20]. Within this activation segment is an activation loop responsible for kinase regulation. In many kinases, the loop covers the catalytic site maintaining the enzyme in an inactive state by preventing substrate access. The loop undergoes a critical conformational change to activate the kinase; its movement exposes the catalytic region and, in addition, may provide a substrate platform. The Lrrk2 G2019S and I2020T substitutions change the highly conserved glycine and isoleucine at the start of the activation segment (the hinge to the activation loop). Of interest, substitutions within the activation segment of other kinases have an activating and oncogenic effect [22]. However, two alternate hypotheses can be invoked. The DRG of the kinase sequence also overlaps with the adjacent, structurally conserved magnesium-binding loop of the kinase, within which the I2012T substitution is also located. It is therefore, equally possible that I2012T, G2019S, and I2020T will cause a reduction in enzymatic activity. Interestingly, all three of these substitutions may introduce new phosphorylation sites (serine or threonine), which could also influence the enzymatic activity or substrate-binding properties of the protein. Kinase activity of recombinant Lrrk2 will be required to directly address the pathogenic effect of these amino acid substitutions.

In conclusion, LRRK2 genetic findings explain a proportion of familial parkinsonism, consistent with a diagnosis of idiopathic PD. This belies most twin studies that suggest the heritability of late-onset PD is negligible. LRRK2 point mutations are relatively frequent in familial parkinsonism, and we postulate common coding variability may contribute to disease risk for some neurodegenerative disorders. Interestingly, the mechanism whereby mutations in different Lrrk2 domains cause clinical PD may be different, and this, in part, could be responsible for the pleomorphic pathology observed.

Thus, genetic insights in PD may now facilitate accurate/earlier patient diagnosis, better models of disease as well as the rationale for novel drug development. Therapeutic interventions might now be tailored to specific patient groups, and perhaps offer both symptomatic benefit and a halt to disease progression.

Subjects, materials, and methods

Gene sequencing

To identify novel LRRK2 mutations and determine their frequency, we have sequenced 100 affected probands with family history of parkinsonism. The average age of onset was 53±12 years (range 23–79 years, 65 men/35 women). Families were referred from movement disorders clinics throughout Europe, North America, and Asia (85% Caucasians and 15% Asians). For each kindred, DNA was available from at least two affected family members (72% of the families had 2–5 known affected members, 20% had 6–10, and 8% had more than 10 affected members). Parkinsonism in each pedigree was consistent with an autosomal dominant pattern of inheritance and each patient was assessed by neurologists specialized in movement disorders. The examination included a full medical history, including family history, and a standard neurological assessment. The Folstein Mini-Mental State Examination (MMSE) was used to assess cognitive function. A clinical diagnosis of PD required the presence of at least two of three cardinal signs (resting tremor, bradykinesia, and rigidity), improvement from adequate dopaminergic therapy, and the absence of atypical features or other causes of parkinsonism [1, 23]. Appropriate institutional review and informed consent was obtained for clinicogenetic research.

Genomic DNA was extracted from peripheral blood lymphocytes using standard protocols. Primer pairs for all LRRK2 exons were designed with Gene Runner version 3.05 software, and were used to amplify all 51 exons by polymerase chain reaction (PCR) (see online Supplement). PCR products were purified from unincorporated nucleotides using a Millipore PCR purification plate. A total volume of 6 μl, containing 15–90 ng of the clean product and 1.6 pM of one of the primers (forward or reverse), was used for sequencing. Electropherograms were analyzed with SeqScape v2.1.1 (ABI, Applied Biosystems, Foster City, CA, USA). ABI “assay-by-design” probes were designed for all novel variants and screened in ethnically matched control samples using SDS 2.1 software on an ABI 7900. For mutation 7153G>A (G2385R), the presence of a frequent SNP 2-bp 3′ prevented the design of a specific ABI probe; thus, additional genotyping was performed by direct sequencing. Similarly, for novel SNP 4883G>C (R1628P), a restriction enzyme digest was needed to genotype control samples. Variant frequencies were assessed in appropriately matched control chromosomes. In addition, segregation analysis was performed in families with specific LRRK2 mutations by testing all available DNA from affected and nonaffected family members.

Gene multiplication and deletion analysis

Semiquantitative multiplex PCR was performed for the detection of whole gene LRRK2 multiplications and deletions in affected probands from 100 families in which parkinsonism segregates as an autosomal dominant trait. Four multialleleic repeat markers (D12S2514, D12S2515, D12S2521, D12S2523) and five exons (3, 25, 33, 41, and 48) within the LRRK2 locus were optimized for multiplexing along with an internal control [forward control primer 5′-(Hex)ACGTTCCTGATAATGGGATC-3′ and reverse 5′-CCTCTCTCTACCAAGTGAGG-3′]. One primer of each pair was labeled with a fluorescent tag. Semiquantitative PCR was performed inside the log-linear amplification range, approximately 18–23 cycles using multiplex panels of three or more markers spanning the LRRK2 locus. The PCR reaction included 80 ng of genomic DNA, 1 U Taq polymerase, 5 μl Q solution (Qiagen, Valencia, CA, USA), 2.5 μl 10× buffer, and 5 mM of each dNTP. Initial 94°C denaturing (1 min) was followed by about 18–23 cycles of 94°C (30 s), 55°C (30 s), and 72°C (2 min), with a final extension at 72°C for 5 min. PCR products were run on an ABI 3100 genetic analyzer and results were analyzed using Genotyper 3.7 software. Each sample was analyzed independently at least twice.

Acknowledgements

We would like to acknowledge patients and family members for their participation. In addition, we thank all collaborating investigators and physicians of the Udall Center, Mayo Clinic (Jacksonville, FL) for their continued effort. Minnie Schreiber is thanked for laboratory support. NINDS P01 NS40256 funded the Udall Clinical and Genetic Cores. The Asociacion Parkinson Asturias helped fund I.F.M.

Copyright information

© Springer-Verlag 2005