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Identification of novel variants in LRRK2 gene in patients with Parkinson's disease in Serbian population
Journal of the Neurological Sciences 353 (2015) 59–62
Contents lists available at ScienceDirect
Journal of the Neurological Sciences journal homepage: www.elsevier.com/locate/jns
Identification of novel variants in LRRK2 gene in patients with Parkinson's disease in Serbian population Milena Z. Janković a, Nikola D. Kresojević a, Valerija S. Dobričić a, Vladana V. Marković a, Igor N. Petrović a, Ivana V. Novaković b,1, Vladimir S. Kostić a,⁎,1 a b
Neurology Clinic, School of Medicine, University of Belgrade, Dr. Subotica 6, Belgrade, Serbia Institute for Human Genetics, School of Medicine, University of Belgrade, Visegradska 26, Belgrade, Serbia
a r t i c l e
i n f o
Article history: Received 15 December 2014 Received in revised form 10 March 2015 Accepted 2 April 2015 Available online 12 April 2015 Keywords: Parkinson's disease Genetics LRRK2 PARK8 Late onset Mutation
a b s t r a c t Background: Mutations in LRRK2 (leucine-rich repeat kinase 2) are the most common cause of autosomal dominant Parkinson's disease (PD). Large international studies have revealed that pathogenic mutations are clustered in several exons coding for functional domains of LRRK2 protein, but the mutation frequency differs among populations. Systematic study of LRRK2 mutation prevalence and phenotype in Serbian population has not been performed. Methods: Comprehensive mutation screening of selected exons of LRRK2 was performed in 486 Serbian PD patients. Results: Previously reported mutations I1371V and G2019S were identified in a single patient each, and c.4536+3ANG substitution in two patients. G2019S is the most common, pathogenic mutation, while pathogenic roles for recurrent variants I1371V and c.4536+3ANG are not confirmed yet. Two novel variants S1508G and I1991V were discovered in 2 unrelated patients. These variants are considered as disease causing according to several software predictions, but additional segregation and functional analyses are required. Conclusions: Mutation frequency in our study (1.23%) was similar to other European populations, although the most common mutations were underestimated and novel variants were detected. In most cases, symptoms of LRRK2-PD are similar to sporadic PD, so estimation of frequency and penetrance of mutations in different populations is important for efficient genetic testing strategy and counseling. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Parkinson's disease (PD) is a progressive neurodegenerative disorder that affects about 1% of the population above the age of 60 and 4–5% above the age of 85 [1]. So far, several genes have been found to be involved in PD, and among them the PARK8 locus that encodes for leucine rich repeat kinase 2 (LRRK2) is the most frequent genetic cause associated with autosomal dominant PD, accounting for about 6% of familial cases and 2% of sporadic PD cases [2–6]. The LRRK2 gene has 51 exons, encoding a very large protein (LRRK2 or dardarin), which contains two predicted enzymatic domains (GTPase and kinase) and multiple protein–protein interaction domains [7]. LRRK2-associated PD is clinically indistinguishable from idiopathic PD, most commonly with late-onset of the disease, although several early-onset patients with LRRK2 mutations have also been reported [3]. At least seven mutations clustered in several exons can be considered as definitely ⁎ Corresponding author at: Neurology Clinic CCS, Dr. Subotica 6, Belgrade, Serbia. Tel.: +381 112685596; fax: +381 112684577. E-mail address: [email protected] (V.S. Kostić). 1 These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.jns.2015.04.002 0022-510X/© 2015 Elsevier B.V. All rights reserved.
disease-causing, on the basis of co-segregation with disease in families and absence in controls [8], affecting central catalytic ROC–COR-kinase domain of LRRK2 [7]. It has been shown that mutation frequency in LRRK2 gene is different across populations [9], so it is important to establish relative frequency in different ethnic groups, in order to create international guidelines for genetic testing strategy for PD. Herein, we used a selective approach in the analysis of this gene in a large cohort of Serbian PD patients in order to estimate the prevalence and phenotype of mutations in LRRK2 gene. 2. Patients and methods 2.1. Patients The study was approved by the Ethics Committee of Clinical Center of Serbia, and written informed consent was obtained from each participant or authorized family member. The consecutive patients with PD were recruited from a tertiary referral center (Institute of Neurology, Clinical Center of Serbia) without regard to age at onset and family history status of the disease. All patients and controls were examined by at least one movement disorder
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M.Z. Janković et al. / Journal of the Neurological Sciences 353 (2015) 59–62
Table 1 Sequence variations found both in coding and non-coding regions of the LRRK2 gene in Serbian patients with Parkinson's disease. Detected variant
Position
g.DNA
c.DNA
Amino acid change
Protein domain
Ref. no.
No. of patients
c.4111ANG heta c.4193GNA het c.4193GNA homb c.4269GNA het c.4269GNA hom c.4290CNT het c.4522ANG het c.4536+3ANG het c.5170+21TNC het c.5170+23TNA het c.5170+23TNA hom c.5318-9ANG het c.5948+34TNC het c.5948+48CNT het c.5971ANG het c.6055GNA het
Exon 29 Exon 30 Exon 30 Exon 30 Exon 30 Exon 30 Exon 31 Intron 31 Intron 35 Intron 35 Intron 35 Intron 37 Intron 40 Intron 40 Exon 41 Exon 41
g.111875ANG g.112366GNA g.112366GNA g.112442GNA g.112442GNA g.112463CNT g.113892ANG g.113909ANG g.124466TNC g.124468TNA g.124468TNA g.126408ANG g.138448TNC g.138462CNT g.143573ANG g.143657GNA
4169ANG 4251GNA 4251GNA 4327GNA 4327GNA 4348CNT 4580ANG N/Ac N/A N/A N/A N/A N/A N/A 6029ANG 6113GNA
Ile1371Val Arg1398His Arg1398His Lys1423= Lys1423= Ala1430= Ser1508Gly N/A N/A N/A N/A N/A N/A N/A Ile1991Val Gly2019Ser
ROC ROC ROC ROC ROC ROC ROC N/A N/A N/A N/A N/A N/A N/A Kinase Kinase
rs17466213 rs7133914 rs7133914 rs11175964 rs11175964 rs111435410 novel rs41286476 novel rs7307276 rs7307276 rs41286460 rs17444152 rs2404834 novel rs34637584
1 50 2 50 2 3 1 2 1 36 65 1 2 41 1 1
Novel variants are typed in bold. a Heterozygote. b Homozygote. c N/A — not applicable.
specialists (VSK, IP, VM) when detailed interview on demographic and clinical features was applied. PD was diagnosed according to the UK Brain Bank criteria [10]. Familial PD was defined if one or more, first- or second-degree relatives of the patient, were reported as PD cases. In total, 486 PD patients (301 males, 185 females) and 143 healthy control subjects older than 50 years (77 males, 66 females) of Serbian ethnic background, were recruited for this study. 2.2. Methods All patients were analyzed for the presence of mutations in the exons 30, 31, 35 and 41 and their flanking intronic sequences, known to harbor recurrent, proven pathogenic mutations [8]. In the subgroup of 232 patients with the age of onset N 45 years or positive family history for PD, the analysis was expanded to include exons 24, 25, 29, 38 and 40, reported to harbor mutations with no clear clinical importance [3,11]. Peripheral blood was obtained from all individuals and genomic DNA was isolated using standard methods. Selected exons and their flanking intronic sequences were amplified and directly sequenced in forward and reverse directions using the ABI Big Dye terminator chemistry and an ABI 3500 instrument (Applied Biosystems, Foster City, CA). Primer sequences and PCR conditions are available on request. Sequences were analyzed using the Sequencher software (Gene Codes Corporation, Ann Arbor, MI). Sequence variants are numbered according to the references NM_198578.3/NP_940980.3. Identified mutations were confirmed by analyzing the blood sample from the proband in duplicate. Impact of detected non-synonymous coding variants on protein function was predicted by MutationTaster [12], PolyPhen-2 [13], PROVEAN [14], SIFT [15], SNPs&GO [16] and MutPred [17] software, while the effect on splicing efficiency was determined using the NNsplice [18], NetGene2 [19] and Human Splicing Finder [20] programs. 3. Results Thirty-nine PD patients had positive family history and 148 were defined as early-onset cases (age at onset ≤45 years). The mean age of PD onset in the whole cohort was 53.1 years (range: 22 to 79 years). Sequence analysis of selected exons revealed four heterozygous missense substitutions in the coding regions in a single patient each (Table 1): c.6055GNA (p.Gly2019Ser), c.4111ANG (p.Ile1371Val), c.4522ANG (p.Ser1508Gly) and c.5971ANG (p.Ile1991Val). The first two are known pathogenic mutations. The latter two variants are the variants of unknown pathogenic significance, and are absent from the Exome Variant Server [21], dbSNP141 [22], 1000 Genomes [23] and
Exome Aggregation Consortium [24] databases. Pathogenicity of all novel variants was predicted using in silico analysis (Table 2). Unfortunately, family members were not available for co-segregation analysis. Variants in exon 30 c.4193GNA (p.Arg1398His) and c.4269GNA (p.Lys1423=) were detected in 52 patients (50 heterozygous and 2 homozygous). In the same exon another silent change, c.4290CNT (p.Ala1430=) was detected in 3 patients. Besides coding variants, six nucleotide changes in the non-coding regions of LRRK2 were detected: c.4536+3ANG, c.5170+21TNC, c.5170+23TNA, c.5318-9ANG, c.5948+34TNC and c.5948+48CNT (Table 1). Variant c.5318-9ANG was detected in one patient, .4536+3ANG and c.5948+34TNC in two unrelated patients each, and c.5948+48CNT in 41 patients. c.5170+23TNA was detected in the heterozygous state in 36 patients, and in the homozygous state in 65 patients. Substitution c.5170+21TNC, detected in a single patient, has not been described in the literature yet, and according to prediction software, it is unlikely to cause PD (Table 2). None of the novel, potentially disease causing mutations was detected in 286 ethnically matched control chromosomes. Substitution c.4536+3ANG is detected in three healthy controls. The phenotypes of patients with LRRK2 mutations are given in Table 3. 4 . Discussion Our study is the first comprehensive analysis of the LRRK2 variants associated with PD in Serbian population. Previously, 14 Serbian PD were included in an international study of LRRK2 gene, but no mutations were revealed in our patients [25]. Table 2 Results of in silico analysis for novel variants found in the LRRK2 gene. Variant
p.Ser1508Gly
MutationTaster PolyPhen-2 PROVEAN SNPs&GO SIFT MutPred
Disease causing Probably damaging Neutral Neutral Damaging Probability of deleterious mutation 0.662 NN splice No effect on splicing NetGene2 No effect on splicing Humane Splicing Probably no impact Finder on splicing
a
N/A — not applicable.
c.5170+21TNC Polymorphism N/Aa N/A N/A N/A N/A
p.Ile1991Val
Disease causing Possibly damaging Neutral Neutral Neutral Probability of deleterious mutation 0.629 No effect on splicing No effect on splicing No effect on splicing No effect on splicing Probably no impact Potential alteration on splicing of splicing
M.Z. Janković et al. / Journal of the Neurological Sciences 353 (2015) 59–62
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Table 3 Demographic and clinical characteristics of patients with LRRK2 mutations. Patient
Mutation
Gender
Age at onset (years)
Family history
Presenting symptom(s)
1 2 3 4 5 6
I1371V S1508G c.4536+3ANG c.4536+3ANG I1991E G2019S
Female Female Female Female Male Female
57 58 59 62 41 56
Negative Negative Negative Negative Negative Positive
Orthostatic tremor, dragging of left leg Mild hypomimia, unilateral, left-sided bradykinesia and rigidity Unknown Tremor at rest and rigidity, more pronounced on the right Tremor at rest (left arm) Hypokinetic-rigid syndrome, more pronounced on the left
The most common proven LRRK2 pathogenic mutation, p.G2019S, was detected in a heterozygous state in a female patient whose first symptoms appeared at the age of 56. Patient's mother also had PD, but she was not available for the genetic testing. Multicentric international studies showed that 0.58% of PD patients carried this mutation [26], and that in Europe it has a decreasing gradient from South to North [10]. With the frequency of 0.21% (1/486), this mutation seems to be underrepresented in our population. Another known mutation, p.I1371V, was detected in one patient with the late onset of first symptoms (57 years), negative family history and typical presentation of the disease. This mutation was previously reported in several unrelated PD patients, but it was also detected in one healthy control [27,28]. Although this mutation, located in the ROC domain, affects highly conserved residue among the LRRK2 protein homologues [28] and a neuropathological study in one PD case carrying this mutation showed classical Lewy body-positive pathology [29], its pathogenic role remains uncertain. In addition to known mutations, two novel LRRK2 missense variants were detected. Variant p.S1508G was found in a patient with the late onset and no affected family members. This variant is affecting one of the most conserved functional domains of LRRK2, ROC domain, and several in silico analyses predicted this variant to be pathogenic. However, it is important to emphasize that this patient also harbors the heterozygous mutation in the glucocerebrosidase gene (N370S), which is a wellknown risk factor for PD [30]. Another novel variant, p.I1991V, was detected in a patient with young-onset (41 years) and negative family history. This sequence change is affecting conserved amino acid in kinase domain, and, although isoleucine and valine are both aliphatic amino acids, four performed software predictions consider this variant as pathogenic. None of these novel variants were found in 286 control chromosomes, but further functional and co-segregation analyses are required to confirm their possible pathogenicity. Intronic variation c.4536+3ANG, found in two late-onset patients, was reported in several studies, in both familial and sporadic PD cases [31,32]. However, it was previously detected in unaffected family member of one patient [32], and we detected three mutation carriers among healthy controls. In silico analysis predicted that this change affects splicing, resulting in a truncated protein without functional GTPase and kinase domains [31]. Other detected intronic variants, one novel (c.5170+21TNC) and 4 known (c.5170+23TNA, c.5318-9ANG, c.5948+34TNC and c.5948+48CNT) are considered as benign polymorphisms because they are not predicted to affect splicing. In our study, the mean age of onset in patients with mutations was 55.5 years (range: 41–62 years). Five patients had age of onset N55 years, which are in accordance with previous studies showing that LRRK2-associated PD was clinically similar to sporadic PD. In one patient PD symptoms started at the age of 41, indicating that LRRK2 gene should be also considered in genetic testing of earlyonset PD. Frequency of female mutation carriers in our study was 83% (5 of 6), although we included 1.63 times more male than female PD patients. These finding is in line with the previous studies in Ashkenazi Jews [33,34], and Italian PD patients [35] and may suggest that genetic contribution is greater for women then for man with PD.
Similar with other European populations [4], the frequency of LRRK2 mutations in our study is 1.23% (6 of 486). A potential caveat of our study is that we did not analyze the entire coding region of the LRRK2 gene. However, we still believe that our data are valuable since pathogenic LRRK2 mutations cluster in exons coding for functional domains of LRRK2 protein, thus partially justifying our selective genetic testing approach [36]. Disclosure of conflicts of interest The authors declare that they have no conflict of interest. Ethical standard This study has been approved by the appropriate ethics committee and has therefore been performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki. Acknowledgment We thank all the participating subjects for their contribution. This study was supported by grants from the Ministry of Education and Science of Republic of Serbia (ON175090 and ON175091). The authors would like to thank the Exome Aggregation Consortium and the groups that provided exome variant data for comparison. A full list of contributing groups can be found at http://exac.broadinstitute.org/about. References [1] Lees AJ, Hardy J, Revesz T. Parkinson's disease. Lancet 2009;373:2055–66. http://dx. doi.org/10.1016/S0140-6736(09)60492-X. [2] Paisán-Ruíz C, Jain S, Evans EW, et al. Cloning of the gene containing mutations that cause PARK8-linked Parkinson's disease. Neuron 2004;44:595–600. http://dx.doi. org/10.1016/j.neuron.2004.10.023. [3] Zimprich A, Biskup S, Leitner P, et al. Mutations in LRRK2 cause autosomal-dominant parkinsonism with pleomorphic pathology. Neuron 2004;44:601–7. http://dx.doi. org/10.1016/j.neuron.2004.11.005. [4] Healy DG, Falchi M, O'Sullivan SS, et al. Phenotype, genotype, and worldwide genetic penetrance of LRRK2-associated Parkinson's disease: a case-control study. Lancet Neurol 2008;7:583–90. http://dx.doi.org/10.1016/S1474-4422(08)70117-0. [5] Bekris LM, Mata IF, Zabetian CP. The genetics of Parkinson disease. J Geriatr Psychiatry Neurol 2010;23:228–42. http://dx.doi.org/10.1177/0891988710383572. [6] Aasly JO, Vilariño-Güell C, Dachsel JC, et al. Novel pathogenic LRRK2 p.Asn1437His substitution in familial Parkinson's disease. Mov Disord 2010;25(13):2156–63. http://dx.doi.org/10.1002/mds.23265. [7] Gasser T. Molecular pathogenesis of Parkinson disease: insights from genetic studies. Expert Rev Mol Med 2009;11:e22. http://dx.doi.org/10.1017/S1462399409001148. [8] Cookson MR. Cellular effects of LRRK2 mutations. Biochem Soc Trans 2012;40: 1070–3. http://dx.doi.org/10.1042/BST20120165. [9] Ross OA, Soto-Ortolaza AI, Heckman MG, et al. Association of LRRK2 exonic variants with susceptibility to Parkinson's disease: a case–control study. Lancet Neurol 2011; 10:898–908. http://dx.doi.org/10.1016/S1474-4422(11)70175-2. [10] Hughes AJ, Daniel SE, Kilford L, et al. Accuracy of clinical diagnosis of idiopathic Parkinson's disease: a clinico-pathological study of 100 cases. J Neurol Neurosurg Psychiatry 1992;55:181–4. http://dx.doi.org/10.1136/jnnp.55.3.181. [11] Lesage S, Brice A. Parkinson's disease: from monogenic forms to genetic susceptibility factors. Hum Mol Genet 2009;18(R1):48–59. http://dx.doi.org/10.1093/hmg/ ddp012. [12] Schwarz JM, Rodelsperger C, Schuelke M, Seelow D. MutationTaster evaluates disease-causing potential of sequence alterations. Nat Methods 2010;7:575–6. http://dx.doi.org/10.1038/nmeth0810-575. [13] Adzhubei IA, Schmidt S, Peshkin L, et al. A method and server for predicting damaging missense mutations. Nat Methods 2010;7:248–9. http://dx.doi.org/10.1038/ nmeth0410-248.
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[14] Choi Y, Sims GE, Murphy S, Miller JR, Chan AP. Predicting the functional effect of amino acid substitutions and indels. PLoS One 2012;7(10):e46688. http://dx.doi. org/10.1371/journal.pone.0046688. [15] Ng PC, Henikoff S. Predicting deleterious amino acid substitutions. Genome Res 2001;11(5):863–74. [16] Calabrese R, Capriotti E, Fariselli P, Martelli PL, Casadio R. Functional annotations improve the predictive score of human disease-related mutations in proteins. Hum Mutat 2009;30(8):1237–44. http://dx.doi.org/10.1002/humu.21047. [17] Li B, Krishnan VG, Mort ME, et al. Automated inference of molecular mechanisms of disease from amino acid substitutions. Bioinformatics 2009;25(21):2744–50. http:// dx.doi.org/10.1093/bioinformatics/btp528. [18] Reese MG, Eeckman FH, Kulp D, Haussler D. Improved splice site detection in Genie. J Comput Biol 1997;4(3):311–23. http://dx.doi.org/10.1089/cmb.1997.4.311. [19] Hebsgaard SM, Korning PG, Tolstrup N, Engelbrecht J, Rouze P, Brunak S. Splice site prediction in Arabidopsis thaliana DNA by combining local and global sequence information. Nucleic Acids Res 1996;24(17):3439–52. http://dx.doi.org/10.1093/ nar/24.17.3439. [20] Desmet FO, Hamroun D, Lalande M, Collod-Béroud G, Claustres M, Béroud C. Human Splicing Finder: an online bioinformatics tool to predict splicing signals. Nucleic Acids Res 2009;37(9):e67. http://dx.doi.org/10.1093/nar/gkp215. [21] NHLBI Exome Sequencing Project (ESP). Exome Variant Server http://evs.gs. washington.edu/EVS/. [accessed 01/11/2014]. [22] Sherry ST, Ward MH, Kholodov M, et al. dbSNP: the NCBI database of genetic variation. Nucleic Acids Res 2001;29(1):308–11. http://dx.doi.org/10.1093/nar/29.1.308. [23] 1000 Genomes Project Consortium, Abecasis GR, Auton A, et al. An integrated map of genetic variation from 1,092 human genomes. Nature 2012;491:56–65. http://dx. doi.org/10.1038/nature11632. [24] Exome Aggregation Consortium (ExAC), Cambridge, MA. URL: http://exac. broadinstitute.org. [accessed 30/01/2015]. [25] Hedrich K, Winkler S, Hagenah J, et al. Recurrent LRRK2 (Park8) mutations in earlyonset Parkinson's disease. Mov Disord 2006;21(9):1506–10. http://dx.doi.org/10. 1002/mds.20990. [26] Correia Guedes L, Ferreira JJ, Rosa MM, Coelho M, Bonifati V, Sampaio C. Worldwide frequency of G2019S LRRK2 mutation in Parkinson's disease: a systematic review.
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Parkinsonism Relat Disord 2010;16:237–42. http://dx.doi.org/10.1016/j.parkreldis. 2009.11.004. Paisan-Ruiz C, Lang AE, Kawarai T, et al. LRRK2 gene in Parkinson disease: mutation analysis and case control association study. Neurology 2005;65:696–700. http://dx. doi.org/10.1212/01.WNL.0000167552.79769.b3. Di Fonzo A, Tassorelli C, De Mari M, et al. Comprehensive analysis of the LRRK2 gene in sixty families with Parkinson's disease. Eur J Hum Genet 2006;14:322–31. http:// dx.doi.org/10.1038/sj.ejhg.5201539. Giordana MT, D'Agostino C, Albani G, et al. Neuropathology of Parkinson's disease associated with the LRRK2 Ile1371Val mutation. Mov Disord 2006;22(2):275–8. http://dx.doi.org/10.1002/mds.21281. Sidransky E, Nalls M, Aasly J. Multicenter analysis of glucocerebrosidase mutations in Parkinson's disease. N Engl J Med 2009;361:1651–61. http://dx.doi.org/10.1056/ NEJMoa0901281. Zabetian CP, Samii A, Mosley AD, et al. A clinic-based study of the LRRK2 gene in Parkinson disease yields new mutations. Neurology 2005;65:741–4. http://dx.doi. org/10.1212/01.wnl.0000172630.22804.73. Shojaee S, Sina F, Farboodi N, et al. A clinic-based screening of mutations in exons 31, 34, 35, 41, and 48 of LRRK2 in Iranian Parkinson's disease patients. Mov Disord 2009; 24(7):1023–7. http://dx.doi.org/10.1002/mds.22503. Orr-Urtreger A, Shifrin C, Rozovski U, et al. The LRRK2 G2019S mutation in Ashkenazi Jews with Parkinson disease: is there a gender effect? Neurology 2007;69: 1595–602. http://dx.doi.org/10.1212/01.wnl.0000277637.33328.d8. Saunders-Pullman R, Stanley K, San Luciano M, et al. Gender differences in the risk of familial parkinsonism: beyond LRRK2? Neurosci Lett 2011;496:125–8. http://dx.doi. org/10.1016/j.neulet.2011.03.098. Cilia R, Siri C, Rusconi D, et al. LRRK2 mutations in Parkinson's disease: confirmation of a gender effect in the Italian population. Parkinsonism Relat Disord 2014;20: 911–4. http://dx.doi.org/10.1016/j.parkreldis.2014.04.016. Paisán-Ruíz C, Nath P, Washecka N, et al. Comprehensive analysis of LRRK2 in publicly available Parkinson's disease cases and neurologically normal controls. Hum Mutat 2008;29:485–90. http://dx.doi.org/10.1002/humu.20668.
Contents lists available at ScienceDirect
Journal of the Neurological Sciences journal homepage: www.elsevier.com/locate/jns
Identification of novel variants in LRRK2 gene in patients with Parkinson's disease in Serbian population Milena Z. Janković a, Nikola D. Kresojević a, Valerija S. Dobričić a, Vladana V. Marković a, Igor N. Petrović a, Ivana V. Novaković b,1, Vladimir S. Kostić a,⁎,1 a b
Neurology Clinic, School of Medicine, University of Belgrade, Dr. Subotica 6, Belgrade, Serbia Institute for Human Genetics, School of Medicine, University of Belgrade, Visegradska 26, Belgrade, Serbia
a r t i c l e
i n f o
Article history: Received 15 December 2014 Received in revised form 10 March 2015 Accepted 2 April 2015 Available online 12 April 2015 Keywords: Parkinson's disease Genetics LRRK2 PARK8 Late onset Mutation
a b s t r a c t Background: Mutations in LRRK2 (leucine-rich repeat kinase 2) are the most common cause of autosomal dominant Parkinson's disease (PD). Large international studies have revealed that pathogenic mutations are clustered in several exons coding for functional domains of LRRK2 protein, but the mutation frequency differs among populations. Systematic study of LRRK2 mutation prevalence and phenotype in Serbian population has not been performed. Methods: Comprehensive mutation screening of selected exons of LRRK2 was performed in 486 Serbian PD patients. Results: Previously reported mutations I1371V and G2019S were identified in a single patient each, and c.4536+3ANG substitution in two patients. G2019S is the most common, pathogenic mutation, while pathogenic roles for recurrent variants I1371V and c.4536+3ANG are not confirmed yet. Two novel variants S1508G and I1991V were discovered in 2 unrelated patients. These variants are considered as disease causing according to several software predictions, but additional segregation and functional analyses are required. Conclusions: Mutation frequency in our study (1.23%) was similar to other European populations, although the most common mutations were underestimated and novel variants were detected. In most cases, symptoms of LRRK2-PD are similar to sporadic PD, so estimation of frequency and penetrance of mutations in different populations is important for efficient genetic testing strategy and counseling. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Parkinson's disease (PD) is a progressive neurodegenerative disorder that affects about 1% of the population above the age of 60 and 4–5% above the age of 85 [1]. So far, several genes have been found to be involved in PD, and among them the PARK8 locus that encodes for leucine rich repeat kinase 2 (LRRK2) is the most frequent genetic cause associated with autosomal dominant PD, accounting for about 6% of familial cases and 2% of sporadic PD cases [2–6]. The LRRK2 gene has 51 exons, encoding a very large protein (LRRK2 or dardarin), which contains two predicted enzymatic domains (GTPase and kinase) and multiple protein–protein interaction domains [7]. LRRK2-associated PD is clinically indistinguishable from idiopathic PD, most commonly with late-onset of the disease, although several early-onset patients with LRRK2 mutations have also been reported [3]. At least seven mutations clustered in several exons can be considered as definitely ⁎ Corresponding author at: Neurology Clinic CCS, Dr. Subotica 6, Belgrade, Serbia. Tel.: +381 112685596; fax: +381 112684577. E-mail address: [email protected] (V.S. Kostić). 1 These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.jns.2015.04.002 0022-510X/© 2015 Elsevier B.V. All rights reserved.
disease-causing, on the basis of co-segregation with disease in families and absence in controls [8], affecting central catalytic ROC–COR-kinase domain of LRRK2 [7]. It has been shown that mutation frequency in LRRK2 gene is different across populations [9], so it is important to establish relative frequency in different ethnic groups, in order to create international guidelines for genetic testing strategy for PD. Herein, we used a selective approach in the analysis of this gene in a large cohort of Serbian PD patients in order to estimate the prevalence and phenotype of mutations in LRRK2 gene. 2. Patients and methods 2.1. Patients The study was approved by the Ethics Committee of Clinical Center of Serbia, and written informed consent was obtained from each participant or authorized family member. The consecutive patients with PD were recruited from a tertiary referral center (Institute of Neurology, Clinical Center of Serbia) without regard to age at onset and family history status of the disease. All patients and controls were examined by at least one movement disorder
60
M.Z. Janković et al. / Journal of the Neurological Sciences 353 (2015) 59–62
Table 1 Sequence variations found both in coding and non-coding regions of the LRRK2 gene in Serbian patients with Parkinson's disease. Detected variant
Position
g.DNA
c.DNA
Amino acid change
Protein domain
Ref. no.
No. of patients
c.4111ANG heta c.4193GNA het c.4193GNA homb c.4269GNA het c.4269GNA hom c.4290CNT het c.4522ANG het c.4536+3ANG het c.5170+21TNC het c.5170+23TNA het c.5170+23TNA hom c.5318-9ANG het c.5948+34TNC het c.5948+48CNT het c.5971ANG het c.6055GNA het
Exon 29 Exon 30 Exon 30 Exon 30 Exon 30 Exon 30 Exon 31 Intron 31 Intron 35 Intron 35 Intron 35 Intron 37 Intron 40 Intron 40 Exon 41 Exon 41
g.111875ANG g.112366GNA g.112366GNA g.112442GNA g.112442GNA g.112463CNT g.113892ANG g.113909ANG g.124466TNC g.124468TNA g.124468TNA g.126408ANG g.138448TNC g.138462CNT g.143573ANG g.143657GNA
4169ANG 4251GNA 4251GNA 4327GNA 4327GNA 4348CNT 4580ANG N/Ac N/A N/A N/A N/A N/A N/A 6029ANG 6113GNA
Ile1371Val Arg1398His Arg1398His Lys1423= Lys1423= Ala1430= Ser1508Gly N/A N/A N/A N/A N/A N/A N/A Ile1991Val Gly2019Ser
ROC ROC ROC ROC ROC ROC ROC N/A N/A N/A N/A N/A N/A N/A Kinase Kinase
rs17466213 rs7133914 rs7133914 rs11175964 rs11175964 rs111435410 novel rs41286476 novel rs7307276 rs7307276 rs41286460 rs17444152 rs2404834 novel rs34637584
1 50 2 50 2 3 1 2 1 36 65 1 2 41 1 1
Novel variants are typed in bold. a Heterozygote. b Homozygote. c N/A — not applicable.
specialists (VSK, IP, VM) when detailed interview on demographic and clinical features was applied. PD was diagnosed according to the UK Brain Bank criteria [10]. Familial PD was defined if one or more, first- or second-degree relatives of the patient, were reported as PD cases. In total, 486 PD patients (301 males, 185 females) and 143 healthy control subjects older than 50 years (77 males, 66 females) of Serbian ethnic background, were recruited for this study. 2.2. Methods All patients were analyzed for the presence of mutations in the exons 30, 31, 35 and 41 and their flanking intronic sequences, known to harbor recurrent, proven pathogenic mutations [8]. In the subgroup of 232 patients with the age of onset N 45 years or positive family history for PD, the analysis was expanded to include exons 24, 25, 29, 38 and 40, reported to harbor mutations with no clear clinical importance [3,11]. Peripheral blood was obtained from all individuals and genomic DNA was isolated using standard methods. Selected exons and their flanking intronic sequences were amplified and directly sequenced in forward and reverse directions using the ABI Big Dye terminator chemistry and an ABI 3500 instrument (Applied Biosystems, Foster City, CA). Primer sequences and PCR conditions are available on request. Sequences were analyzed using the Sequencher software (Gene Codes Corporation, Ann Arbor, MI). Sequence variants are numbered according to the references NM_198578.3/NP_940980.3. Identified mutations were confirmed by analyzing the blood sample from the proband in duplicate. Impact of detected non-synonymous coding variants on protein function was predicted by MutationTaster [12], PolyPhen-2 [13], PROVEAN [14], SIFT [15], SNPs&GO [16] and MutPred [17] software, while the effect on splicing efficiency was determined using the NNsplice [18], NetGene2 [19] and Human Splicing Finder [20] programs. 3. Results Thirty-nine PD patients had positive family history and 148 were defined as early-onset cases (age at onset ≤45 years). The mean age of PD onset in the whole cohort was 53.1 years (range: 22 to 79 years). Sequence analysis of selected exons revealed four heterozygous missense substitutions in the coding regions in a single patient each (Table 1): c.6055GNA (p.Gly2019Ser), c.4111ANG (p.Ile1371Val), c.4522ANG (p.Ser1508Gly) and c.5971ANG (p.Ile1991Val). The first two are known pathogenic mutations. The latter two variants are the variants of unknown pathogenic significance, and are absent from the Exome Variant Server [21], dbSNP141 [22], 1000 Genomes [23] and
Exome Aggregation Consortium [24] databases. Pathogenicity of all novel variants was predicted using in silico analysis (Table 2). Unfortunately, family members were not available for co-segregation analysis. Variants in exon 30 c.4193GNA (p.Arg1398His) and c.4269GNA (p.Lys1423=) were detected in 52 patients (50 heterozygous and 2 homozygous). In the same exon another silent change, c.4290CNT (p.Ala1430=) was detected in 3 patients. Besides coding variants, six nucleotide changes in the non-coding regions of LRRK2 were detected: c.4536+3ANG, c.5170+21TNC, c.5170+23TNA, c.5318-9ANG, c.5948+34TNC and c.5948+48CNT (Table 1). Variant c.5318-9ANG was detected in one patient, .4536+3ANG and c.5948+34TNC in two unrelated patients each, and c.5948+48CNT in 41 patients. c.5170+23TNA was detected in the heterozygous state in 36 patients, and in the homozygous state in 65 patients. Substitution c.5170+21TNC, detected in a single patient, has not been described in the literature yet, and according to prediction software, it is unlikely to cause PD (Table 2). None of the novel, potentially disease causing mutations was detected in 286 ethnically matched control chromosomes. Substitution c.4536+3ANG is detected in three healthy controls. The phenotypes of patients with LRRK2 mutations are given in Table 3. 4 . Discussion Our study is the first comprehensive analysis of the LRRK2 variants associated with PD in Serbian population. Previously, 14 Serbian PD were included in an international study of LRRK2 gene, but no mutations were revealed in our patients [25]. Table 2 Results of in silico analysis for novel variants found in the LRRK2 gene. Variant
p.Ser1508Gly
MutationTaster PolyPhen-2 PROVEAN SNPs&GO SIFT MutPred
Disease causing Probably damaging Neutral Neutral Damaging Probability of deleterious mutation 0.662 NN splice No effect on splicing NetGene2 No effect on splicing Humane Splicing Probably no impact Finder on splicing
a
N/A — not applicable.
c.5170+21TNC Polymorphism N/Aa N/A N/A N/A N/A
p.Ile1991Val
Disease causing Possibly damaging Neutral Neutral Neutral Probability of deleterious mutation 0.629 No effect on splicing No effect on splicing No effect on splicing No effect on splicing Probably no impact Potential alteration on splicing of splicing
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Table 3 Demographic and clinical characteristics of patients with LRRK2 mutations. Patient
Mutation
Gender
Age at onset (years)
Family history
Presenting symptom(s)
1 2 3 4 5 6
I1371V S1508G c.4536+3ANG c.4536+3ANG I1991E G2019S
Female Female Female Female Male Female
57 58 59 62 41 56
Negative Negative Negative Negative Negative Positive
Orthostatic tremor, dragging of left leg Mild hypomimia, unilateral, left-sided bradykinesia and rigidity Unknown Tremor at rest and rigidity, more pronounced on the right Tremor at rest (left arm) Hypokinetic-rigid syndrome, more pronounced on the left
The most common proven LRRK2 pathogenic mutation, p.G2019S, was detected in a heterozygous state in a female patient whose first symptoms appeared at the age of 56. Patient's mother also had PD, but she was not available for the genetic testing. Multicentric international studies showed that 0.58% of PD patients carried this mutation [26], and that in Europe it has a decreasing gradient from South to North [10]. With the frequency of 0.21% (1/486), this mutation seems to be underrepresented in our population. Another known mutation, p.I1371V, was detected in one patient with the late onset of first symptoms (57 years), negative family history and typical presentation of the disease. This mutation was previously reported in several unrelated PD patients, but it was also detected in one healthy control [27,28]. Although this mutation, located in the ROC domain, affects highly conserved residue among the LRRK2 protein homologues [28] and a neuropathological study in one PD case carrying this mutation showed classical Lewy body-positive pathology [29], its pathogenic role remains uncertain. In addition to known mutations, two novel LRRK2 missense variants were detected. Variant p.S1508G was found in a patient with the late onset and no affected family members. This variant is affecting one of the most conserved functional domains of LRRK2, ROC domain, and several in silico analyses predicted this variant to be pathogenic. However, it is important to emphasize that this patient also harbors the heterozygous mutation in the glucocerebrosidase gene (N370S), which is a wellknown risk factor for PD [30]. Another novel variant, p.I1991V, was detected in a patient with young-onset (41 years) and negative family history. This sequence change is affecting conserved amino acid in kinase domain, and, although isoleucine and valine are both aliphatic amino acids, four performed software predictions consider this variant as pathogenic. None of these novel variants were found in 286 control chromosomes, but further functional and co-segregation analyses are required to confirm their possible pathogenicity. Intronic variation c.4536+3ANG, found in two late-onset patients, was reported in several studies, in both familial and sporadic PD cases [31,32]. However, it was previously detected in unaffected family member of one patient [32], and we detected three mutation carriers among healthy controls. In silico analysis predicted that this change affects splicing, resulting in a truncated protein without functional GTPase and kinase domains [31]. Other detected intronic variants, one novel (c.5170+21TNC) and 4 known (c.5170+23TNA, c.5318-9ANG, c.5948+34TNC and c.5948+48CNT) are considered as benign polymorphisms because they are not predicted to affect splicing. In our study, the mean age of onset in patients with mutations was 55.5 years (range: 41–62 years). Five patients had age of onset N55 years, which are in accordance with previous studies showing that LRRK2-associated PD was clinically similar to sporadic PD. In one patient PD symptoms started at the age of 41, indicating that LRRK2 gene should be also considered in genetic testing of earlyonset PD. Frequency of female mutation carriers in our study was 83% (5 of 6), although we included 1.63 times more male than female PD patients. These finding is in line with the previous studies in Ashkenazi Jews [33,34], and Italian PD patients [35] and may suggest that genetic contribution is greater for women then for man with PD.
Similar with other European populations [4], the frequency of LRRK2 mutations in our study is 1.23% (6 of 486). A potential caveat of our study is that we did not analyze the entire coding region of the LRRK2 gene. However, we still believe that our data are valuable since pathogenic LRRK2 mutations cluster in exons coding for functional domains of LRRK2 protein, thus partially justifying our selective genetic testing approach [36]. Disclosure of conflicts of interest The authors declare that they have no conflict of interest. Ethical standard This study has been approved by the appropriate ethics committee and has therefore been performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki. Acknowledgment We thank all the participating subjects for their contribution. This study was supported by grants from the Ministry of Education and Science of Republic of Serbia (ON175090 and ON175091). The authors would like to thank the Exome Aggregation Consortium and the groups that provided exome variant data for comparison. A full list of contributing groups can be found at http://exac.broadinstitute.org/about. References [1] Lees AJ, Hardy J, Revesz T. Parkinson's disease. Lancet 2009;373:2055–66. http://dx. doi.org/10.1016/S0140-6736(09)60492-X. [2] Paisán-Ruíz C, Jain S, Evans EW, et al. Cloning of the gene containing mutations that cause PARK8-linked Parkinson's disease. Neuron 2004;44:595–600. http://dx.doi. org/10.1016/j.neuron.2004.10.023. [3] Zimprich A, Biskup S, Leitner P, et al. Mutations in LRRK2 cause autosomal-dominant parkinsonism with pleomorphic pathology. Neuron 2004;44:601–7. http://dx.doi. org/10.1016/j.neuron.2004.11.005. [4] Healy DG, Falchi M, O'Sullivan SS, et al. Phenotype, genotype, and worldwide genetic penetrance of LRRK2-associated Parkinson's disease: a case-control study. Lancet Neurol 2008;7:583–90. http://dx.doi.org/10.1016/S1474-4422(08)70117-0. [5] Bekris LM, Mata IF, Zabetian CP. The genetics of Parkinson disease. J Geriatr Psychiatry Neurol 2010;23:228–42. http://dx.doi.org/10.1177/0891988710383572. [6] Aasly JO, Vilariño-Güell C, Dachsel JC, et al. Novel pathogenic LRRK2 p.Asn1437His substitution in familial Parkinson's disease. Mov Disord 2010;25(13):2156–63. http://dx.doi.org/10.1002/mds.23265. [7] Gasser T. Molecular pathogenesis of Parkinson disease: insights from genetic studies. Expert Rev Mol Med 2009;11:e22. http://dx.doi.org/10.1017/S1462399409001148. [8] Cookson MR. Cellular effects of LRRK2 mutations. Biochem Soc Trans 2012;40: 1070–3. http://dx.doi.org/10.1042/BST20120165. [9] Ross OA, Soto-Ortolaza AI, Heckman MG, et al. Association of LRRK2 exonic variants with susceptibility to Parkinson's disease: a case–control study. Lancet Neurol 2011; 10:898–908. http://dx.doi.org/10.1016/S1474-4422(11)70175-2. [10] Hughes AJ, Daniel SE, Kilford L, et al. Accuracy of clinical diagnosis of idiopathic Parkinson's disease: a clinico-pathological study of 100 cases. J Neurol Neurosurg Psychiatry 1992;55:181–4. http://dx.doi.org/10.1136/jnnp.55.3.181. [11] Lesage S, Brice A. Parkinson's disease: from monogenic forms to genetic susceptibility factors. Hum Mol Genet 2009;18(R1):48–59. http://dx.doi.org/10.1093/hmg/ ddp012. [12] Schwarz JM, Rodelsperger C, Schuelke M, Seelow D. MutationTaster evaluates disease-causing potential of sequence alterations. Nat Methods 2010;7:575–6. http://dx.doi.org/10.1038/nmeth0810-575. [13] Adzhubei IA, Schmidt S, Peshkin L, et al. A method and server for predicting damaging missense mutations. Nat Methods 2010;7:248–9. http://dx.doi.org/10.1038/ nmeth0410-248.
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