- About this Journal ·
- Abstracting and Indexing ·
- Advance Access ·
- Aims and Scope ·
- Article Processing Charges ·
- Articles in Press ·
- Author Guidelines ·
- Bibliographic Information ·
- Citations to this Journal ·
- Contact Information ·
- Editorial Board ·
- Editorial Workflow ·
- Free eTOC Alerts ·
- Publication Ethics ·
- Reviewers Acknowledgment ·
- Submit a Manuscript ·
- Subscription Information ·
- Table of Contents
Volume 35 (2013), Issue 3, Pages 181–185
Genetic Analysis of PARK2 and PINK1 Genes in Brazilian Patients with Early-Onset Parkinson's Disease
1Departamento de Genética, Instituto de Biologia Roberto Alcantara Gomes, Universidade do Estado do Rio de Janeiro, Rua São Francisco Xavier 524, PHLC, Sala 501F, Maracanã, 20550-013 Rio de Janeiro, RJ, Brazil
2Laboratório de Genética Humana, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, RJ, Brazil
3Hospital Universitário Clementino Fraga Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil
4Santa Casa da Misericórdia do Rio de Janeiro, Rio de Janeiro, RJ, Brazil
5Faculdade de Ciências Médicas, Centro Biomédico, Universidade do Estado do Rio de Janeiro, Rio de Janeiro, RJ, Brazil
6Núcleo Neurociências, Hospital das Clínicas, Universidade Federal de Goiás, Goiânia, GO, Brazil
7Instituto Integrado de Neurociências, Goiânia, GO, Brazil
Received 18 June 2013; Accepted 5 August 2013
Academic Editor: Yi-Chia Huang
Copyright © 2013 Karla Cristina Vasconcelos Moura et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Parkinson's disease is the second most frequent neurodegenerative disorder in the world, affecting 1-2% of individuals over the age of 65. The etiology of Parkinson's disease is complex, with the involvement of gene-environment interactions. Although it is considered a disease of late manifestation, early-onset forms of parkinsonism contribute to 5–10% of all cases. In the present study, we screened mutations in coding regions of PARK2 and PINK1 genes in 136 unrelated Brazilian patients with early-onset Parkinson's disease through automatic sequencing. We identified six missense variants in PARK2 gene: one known pathogenic mutation, two variants of uncertain role, and three nonpathogenic changes. No pathogenic mutation was identified in PINK1 gene, only benign polymorphisms. All putative pathogenic variants found in this study were in heterozygous state. Our data show that PARK2 point mutations are more common in Brazilian early-onset Parkinson's disease patients (2.9%) than PINK1 missense variants (0%), corroborating other studies worldwide.
Parkinson’s disease (PD) is a complex neurodegenerative disorder that affects different regions of the nervous system, although it is clinically recognized by typical motor manifestations . Even though much of the etiology of PD remains unknown, early-onset PD (EOPD) (onset before 50 years old), which accounts for approximately 5%–10% of all PD cases, can be explained by monogenic causes .
Mutations in coding regions of PARK2 gene are often implicated as the most common cause of EOPD, followed by PINK1 gene variants. These two genes are associated with the autosomal recessive forms of parkinsonism and may act in the same pathway, controlling mitochondrial homeostasis [2–4].
PARKIN is an ubiquitin E3 ligase and, therefore, participates in the ubiquitin-mediated proteasomal degradation pathway , whose activity seems to be compromised by pathogenic mutations. Since the first description of PARK2 mutations in Japanese patients with juvenile EOPD , more than 170 different mutations have been described throughout its sequence, including large deletions or amplifications, small deletions/insertions as well as missense mutations. As a common molecular consequence, such mutations are known to cause loss of PARKIN function, leading to impaired mitochondrial integrity .
PINK1 (PARK6) encodes a mitochondrial serine/threonine kinase, which is expressed ubiquitously in the human brain. Recent evidence suggests that the physiological role of PINK1 comprises the phosphorylation of mitochondrial proteins in response to cellular stress and the protection of mitochondria against various stressors [8, 9]. Up to date, homozygous and compound heterozygous losses of function mutations affecting the kinase domain of PINK1 gene were observed, all of them reducing its enzymatic activity [10, 11]. The frequency of these mutations varies according to the geographic region from 0 to 15% all over the world [7, 12].
PARKIN and PINK1 are thought to participate in the same pathway concerning controlling mitochondrial integrity and function, with PINK1 functioning upstream from PARKIN [13, 14]. In the present study, we investigated the presence of PARK2 and PINK1 sequence mutations in patients with EOPD from the Southeast and Midwest regions of Brazil.
2. Materials and Methods
We analyzed 136 unrelated Brazilian patients (86 men and 50 women; mean age years; mean age at onset years) with idiopathic PD manifesting before 51 years old and 200 healthy Brazilian controls. All patients and healthy volunteers were from the same geographic area, both with similar age and socioeconomic status. Amongst the patients included in this study, 31 cases had familial history of PD in relatives of 1st and 2nd degrees and 105 represented isolated cases of the disease. The Ethics Committee of State University of Rio de Janeiro approved this study and a written informed consent was obtained from all subjects.
The DNA of patients was obtained from peripheral blood. The presence of exon rearrangements in PARK2 and PINK1 genes was formerly screened in 102 probands of our sample through the MLPA analysis . The coding regions of PARK2 and PINK1 genes were sequenced using primers previously described . The sequencing reactions were prepared according to the manufacturer, using the Big Dye Terminator v3.1 Kit (Applied Biosystems). Control DNA samples were screened for p.P437L, p.A339V, and p.K220R substitutions. All reactions were processed on an automated sequencer ABI Prism 3130 (Applied Biosystems), and the sequence analysis was performed using the softwares Chromas Lite 2.0 (Technelysium) and BioEdit Sequence Alignment Editor Version 6.0.6 (Isis Pharmaceuticals, Inc.). DNA samples which showed sequence variations underwent PCR-RFLP analysis or Taqman SNP genotyping to confirm the alteration found. The prediction analysis of the effects of changes which cause amino acid substitutions in proteins was performed using electronic tools PolyPhen and Pmut. To exclude the possible effect of silent mutations on splicing, we have used SpliceView and NNsplice softwares.
We identified twelve sequence variants in PARK2 gene: three silent variants and nine missense mutations (Table 1). Between the alterations found, five are nonpathogenic polymorphisms: c.500G > A (p.S167N), c.1138G > C (p.V380L), c.783A > G (p.L261L), c.1180G > A (p.D394N), and c.111G > A (p.P37P) . The already-known c.245C > A (p.A82E) and c.719C > T (p.T240M) variants were found in one patient each, both in heterozygous state. The p.A82E substitution had also been found in control subjects worldwide [17, 18]. We also identified the heterozygous c.1310C > T (p.P437L) variant in two probands and one healthy individual. This well-known alteration had already been reported in PD patients and controls in some populations [18, 19]. The patient found with the heterozygous c.434G > A (p.S145N) substitution also harbors the p.S167N polymorphism in PARK2 exon 4. This variant has an uncertain pathogenic nature, and it has never been identified in controls worldwide.
Three changes found in this study have never been described: c.659A > G (p.K220R), c.1016C > T (p.A339V), and c.1021C > T (p.L341L) (Table 1). The new variant recognized in exon 9 of PARK2 gene, c.1021C > T, was identified in two patients and resulted in a silent mutation (p.L341L).
Among the 200 healthy controls, the c.1310C > T (p.P437L) substitution in PARK2 gene was found just in one individual in heterozygous state. Besides, we investigated the presence of c.1016C > T (p.A339V) and c.659A > G (p.K220R) variants in the same population, and we were unable to detect these mutations in any of the control individuals analyzed.
In PINK1 gene we found only known benign polymorphisms, including the exon 5 variant c.1018G > A (p.A340T) in seven patients, the silent variant c.1173T > C (p.D391D) in one patient (exon 6), the exon 7 variant c.1426G > A (p.E476K) in two patients, and exon 8 variant c.1562A > C (p.N521T) that was found in half of our patients (66/132) (Table 2).
The quantitative analysis, previously realized by our group, identified 4 patients with dosage mutations: one proband with exon 1 heterozygous deletion of PINK1 gene; an index case with heterozygous deletion of PARK2 exon 4; one patient with heterozygous duplication of PARK2 exon 4, and a compound heterozygous patient that harbors two PARK2 mutations, a deletion of exons 5-6, and a duplication of exon 3 . Among PD patients with heterozygous pathogenic or probably pathogenic PARK2 variants, the quantitative analysis with MLPA has not revealed dosage alterations.
So far, PARK2 and PINK1 are the genes most frequently associated with autosomal recessive EOPD, and both coding products participate in the same metabolic pathway centered on maintenance of the morphological integrity of mitochondria. Studies of PARK2 and PINK1 genes in Latin American populations are very scarce. In our study, we found twelve substitutions in PARK2 gene, from which nine are nonpathogenic variants and three putative pathogenic mutations. Among the heterozygous variants identified, three are silent mutations, including one that is novel (p.L341L).
The exon 3 c.245C > A (p.A82E) variant was identified in one proband and was previously described by other authors in PD patients and controls [17, 18], suggesting that the p.A82E variant is probably nonpathogenic. Besides, the in silico analysis through the PolyPhen and Pmut softwares showed that this alteration is benign. We also identified the c.719C > T (p.T240M) substitution in one patient. This change had already been described, and it had been previously classified as a known pathogenic variant [18, 20]. Our in silico analysis corroborated these data and considered that this alteration affects the PARKIN function, being probably pathogenic.
Another mutation, c.1310C > T (p.P437L), was found in two patients and one healthy subject. Other groups have reported this variant in a similar frequency between cases and controls in North American and European populations [18, 19]. Although our in silico predictions have classified this variant as probably pathogenic, this mutation was present in a Brazilian control individual (59 years old), which leads us to believe that it is a nonpathogenic polymorphism. However, the evaluation of pathogenicity of this variant must be done with caution. We have identified this mutation in three asymptomatic daughters (35, 33, and 31 years old) and a granddaughter (12 years old) of a carrier patient. Functional analyses should help to clarify if the p.P437L substitution is a risk factor for PD.
The c.434G > A (p.S145N) variant was found in a patient that also harbors the polymorphism c.500G > A (p.S167N) in the same exon of PARK2 gene. The in silico analysis had contradictory results, and this mutation has its pathogenic nature unclear in the literature . Although the pathogenicity is not yet confirmed, we believe that functional analyses are important to establish if the p.S145N variant is related with a high risk of developing PD.
Three changes identified in this study have never been described: c.659A > G (p.K220R), c.1016C > T (p.A339V), and c.1021C > T (p.L341L). The first one (p.K220R) was found in only one patient, in heterozygous state. We were unable to detect this mutation in any of the 200 healthy subjects. All in silico evidence points this variant as nondeleterious for PARKIN protein. In addition, this mutation does not encode any part of the catalytic domain of the protein. We believe that it might be a rare polymorphism, but functional studies and segregation analysis would be valuable tools to determine the pathogenicity of this substitution.
Two novel mutations were found in exon 9 of PARK2: p.A339V in one patient with familial history of PD and p.L341L in two sporadic cases. We did not observe the p.A339V variant in 400 control chromosomes, reinforcing the rarity of this variant or its potential association with the patient’s phenotype. On the other hand, the in silico analysis showed contradictory results, which did not help to clarify the pathogenicity of this mutation. Another known missense mutation in the same amino acid residue had already been identified and does not cause phenotypic changes . We cannot classify the pathogenicity of this variant until functional studies are performed.
The second new alteration found in PARK2 exon 9 (c.1021C > T) results in a silent mutation, p.L341L. The in silico analysis using SpliceView and NNsplice programs showed that this variant does not affect the recognition of donor and receptor sites of splicing, and, because of its silent nature, this change is considered nonpathogenic.
No pathogenic mutation was identified in PINK1 gene. We found the variant c.1426G > A (p.E476K) in two patients. As this substitution has already been found in some healthy control subjects by other studies [1, 25], it was predicted to be benign by bioinformatic programs and is poorly conserved even within mammals , and we suggest it is a nonpathogenic variant.
All putative pathogenic point mutations identified in this study in PARK2 gene were in heterozygous state (Table 1). Until now, the role of homozygous and compound heterozygous variants was already established as a cause of autosomal recessively inherited EOPD, but the pathogenic significance of heterozygous mutations is still uncertain, particularly for missense substitutions in the context of recessive inheritance. Different mechanisms have been suggested to explain the effects of single heterozygous variants like loss-of-function mutations by lowering the biological activity of the encoded protein (haploinsufficiency), dominant-negative property, or gain-of-function dominant mutations . So, increasing evidence indicates that heterozygous variants are noncausative mutations but rather genetic susceptibility factors which may contribute to the risk of developing PD .
Our findings showed that PARK2 point mutations are more frequent than PINK1 pathogenic variants in our sample of Brazilian EOPD patients. The absence of pathogenic mutations in PINK1 gene in our population is consistent with other studies [19, 23, 27], supporting the hypothesis that mutations in PINK1 may not be a relevant cause of EOPD among Brazilian sporadic and familial patients. Our results of PARK2 gene are in agreement with studies worldwide that have found similar frequencies of PARK2 point mutations in different populations [18–20, 23, 24] (Table 3), although they differ from the frequency identified by other Brazilian groups (5.5% and 11.1%) [21, 22]. One possible explanation for these different results would be the reduced sample sizes tested by them (72 and 45 patients, resp.) (Table 3).
In conclusion, we strengthen that the functional analyses of the missense variants found by us are still missing and would help us to clarify the real pathogenic value of these mutations in our population. Besides, whether heterozygous mutations in recessive genes act as susceptibility factors or as causal agents in the PD process remains to be determined.
Conflict of Interests
The authors have no conflict of interests to declare.
The authors thank the study participants. This work was supported by funds from Ministério da Saúde, Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (E-26/110.765/2010; E-26/103.065/2011), Conselho Nacional de Desenvolvimento Científico e Tecnológico (303256/2009-5), and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior.
- C. H. Hawkes, “The prodromal phase of sporadic Parkinson's disease: does it exist and if so how long is it?” Movement Disorders, vol. 23, no. 13, pp. 1799–1807, 2008.
- I. Martin, V. L. Dawson, and T. M. Dawson, “Recent advances in the genetics of parkinson's disease,” Annual Review of Genomics and Human Genetics, vol. 12, pp. 301–325, 2011.
- J. J. Palacino, D. Sagi, M. S. Goldberg et al., “Mitochondrial dysfunction and oxidative damage in parkin-deficient mice,” The Journal of Biological Chemistry, vol. 279, no. 18, pp. 18614–18622, 2004.
- E. M. Valente, S. Salvi, T. Ialongo et al., “PINK1 mutations are associated with sporadic early-onset parkinsonism,” Annals of Neurology, vol. 56, no. 3, pp. 336–341, 2004.
- H. Shimura, N. Hattori, S. Kubo et al., “Familial Parkinson disease gene product, parkin, is a ubiquitin-protein ligase,” Nature Genetics, vol. 25, no. 3, pp. 302–305, 2000.
- T. Kitada, S. Asakawa, N. Hattori et al., “Mutations in the parkin gene cause autosomal recessive juvenile parkinsonism,” Nature, vol. 392, no. 6676, pp. 605–608, 1998.
- K. Nuytemans, J. Theuns, M. Cruts, and C. van Broeckhoven, “Genetic etiology of Parkinson disease associated with mutations in the SNCA, PARK2, PINK1, PARK7, and LRRK2 genes: a mutation update,” Human Mutation, vol. 31, no. 7, pp. 763–780, 2010.
- L. F. Burbulla and R. Krüger, “Converging environmental and genetic pathways in the pathogenesis of Parkinson's disease,” Journal of the Neurological Sciences, vol. 306, no. 1-2, pp. 1–8, 2011.
- A. Rakovic, A. Grünewald, P. Seibler et al., “Effect of endogenous mutant and wild-type PINK1 on Parkin in fibroblasts from Parkinson disease patients,” Human Molecular Genetics, vol. 19, no. 16, Article ID ddq215, pp. 3124–3137, 2010.
- M. Atsumi, Y. Li, H. Tomiyama, K. Sato, and N. Hattori, “A 62-year-old woman with early-onset Parkinson's disease associated with the PINK1 gene deletion,” Clinical Neurology, vol. 46, no. 3, pp. 199–202, 2006.
- N. Exner, B. Treske, D. Paquet et al., “Loss-of-function of human PINK1 results in mitochondrial pathology and can be rescued by parkin,” Journal of Neuroscience, vol. 27, no. 45, pp. 12413–12418, 2007.
- E. Deas, H. Plun-Faureau, and N. W. Wood, “PINK1 function in health and disease,” EMBO Molecular Medicine, vol. 1, no. 3, pp. 152–165, 2009.
- J. Park, S. B. Lee, S. Lee et al., “Mitochondrial dysfunction in Drosophila PINK1 mutants is complemented by parkin,” Nature, vol. 441, no. 7097, pp. 1157–1161, 2006.
- J. W. Um, C. Stichel-Gunkel, H. Lübbert, G. Lee, and K. C. Chung, “Molecular interaction between parkin and PINK1 in mammalian neuronal cells,” Molecular and Cellular Neuroscience, vol. 40, no. 4, pp. 421–432, 2009.
- K. C. V. Moura, M. C. Junior, A. L. Z. de Rosso et al., “Exon dosage variations in Brazilian patients with Parkinson's disease: analysis of SNCA, PARKIN, PINK1 and DJ-1 genes,” Disease Markers, vol. 32, no. 3, pp. 173–178, 2012.
- J. M. Choi, M. S. Woo, H. Ma et al., “Analysis of PARK genes in a Korean cohort of early-onset Parkinson disease,” Neurogenetics, vol. 9, no. 4, pp. 263–269, 2008.
- J. Brooks, J. Ding, J. Simon-Sanchez, C. Paisan-Ruiz, A. B. Singleton, and S. W. Scholz, “Parkin and PINK1 mutations in early-onset Parkinson's disease: comprehensive screening in publicly available cases and control,” Journal of Medical Genetics, vol. 46, no. 6, pp. 375–381, 2009.
- N. Pankratz, D. K. Kissell, M. W. Pauciulo et al., “Parkin dosage mutations have greater pathogenicity in familial PD than simple sequence mutations,” Neurology, vol. 73, no. 4, pp. 279–286, 2009.
- K. Nuytemans, B. Meeus, D. Crosiers et al., “Relative contribution of simple mutations versus copy number variations in five Parkinson disease genes in the Belgian population,” Human Mutation, vol. 30, no. 7, pp. 1054–1061, 2009.
- K. S. Marder, M. X. Tang, H. Mejia-Santana et al., “Predictors of Parkin mutations in early-onset Parkinson disease: the consortium on risk for early-onset Parkinson disease study,” Archives of Neurology, vol. 67, no. 6, pp. 731–738, 2010.
- P. D. C. Aguiar, P. S. Lessa, C. Godeiro Jr. et al., “Genetic and environmental findings in early-onset Parkinson's disease Brazilian patients,” Movement Disorders, vol. 23, no. 9, pp. 1228–1233, 2008.
- S. T. Camargos, L. O. Dornas, P. Momeni et al., “Familial parkinsonism and early onset Parkinson's disease in a Brazilian movement disorders clinic: phenotypic characterization and frequency of SNCA, PRKN, PINK1, and LRRK2 mutations,” Movement Disorders, vol. 24, no. 5, pp. 662–666, 2009.
- B. R. Zhang, Z. X. Hu, X. Z. Yin et al., “Mutation analysis of parkin and PINK1 genes in early-onset Parkinson's disease in China,” Neuroscience Letters, vol. 477, no. 1, pp. 19–22, 2010.
- C. Klein, A. Djarmati, K. Hedrich et al., “PINK1, Parkin, and DJ-1 mutations in Italian patients with early-onset parkinsonism,” European Journal of Human Genetics, vol. 13, no. 9, pp. 1086–1093, 2005.
- R. Marongiu, A. Ferraris, T. Ialongo et al., “PINK1 heterozygous rare variants: prevalence, significance and phenotypic spectrum,” Human Mutation, vol. 29, no. 4, p. 565, 2008.
- C. Klein, K. Lohmann-Hedrich, E. Rogaeva, M. G. Schlossmacher, and A. E. Lang, “Deciphering the role of heterozygous mutations in genes associated with parkinsonism,” The Lancet Neurology, vol. 6, no. 7, pp. 652–662, 2007.
- C. Godeiro-Junior, P. M. de Carvalho-Aguiar, A. C. Felício et al., “PINK1 mutations in a Brazilian cohort of early-onset Parkinson's disease patients,” Movement Disorders, vol. 24, no. 11, pp. 1693–1696, 2009.