Neuroscience Letters 370 (2004) 171–174
Mitochondrial polymporphisms in Parkinson’s Disease D. Otaeguia,∗ , C. Pais´ana,b , A. S´aenza,c , I. Mart´ıd , M. Ribatea , J.F. Mart´ı-Mass´od , J. P´erez-Turb , A. L´opez de Munainc,d a
b
Experimental Unit, Hospital Donostia, Spain Unitat de genetica Molecular, Institut Biomedicina de Valencia, CSIC, Spain c Ilundain Fundazioa, San Sebasti´ an, Spain d Neurology Department, Hospital Donostia, Spain
Received 23 April 2004; received in revised form 5 August 2004; accepted 9 August 2004
Abstract The mtDNA polymorphisms A4336G, A10398G and T4216C have been associated with PD. While A4336G is thought to be a genetic risk factor, A10398G appears to be a protective factor and T4216C is only weakly associated with the disease. In this work we analyzed the association between these three genetic polymorphisms and PD in a Spanish-PD population. The samples were classified by ethnic origin in Basques or other origin. Our analysis confirm the association between A4336G and PD. Our results with A10398G polymorphism highlight the importance of performing the association studies in ethnically homogeneous populations. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Parkinson disease; mtDNA; Polymorphisms; A4336G; Basque population
Parkinson’s disease (PD) is a neurodegenerative disorder characterized by severe neuronal loss in the substantia nigra pars compacta, which provokes the loss of projections from this area to the striatum together with the appearance of Lewy bodies. The cause of PD is unknown although it is now widely accepted that genetic susceptibility factors exist that through interactions with environmental factors, may lead to the development of the disease [7]. Indeed, several genes are directly related with Mendelian forms of PD: SNCA, PRKN, UCHL-1, DJ-1, or PINK1 [15]. Oxidative damage is at the center of one of the hypothesis that attempts to explain the molecular mechanisms underlying PD. Accordingly, the excessive production of reactive oxygen species results in damage to DNA and structural damage in the mitochondria, which in turn provokes neuronal death. Indeed, in the substantia nigra and platelets of PD patients, the biochemical activity in the electron transport chain of complex I is impaired [6]. There is indirect evidence that mitochondrial DNA (mtDNA) may be involved ∗
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in the pathogenesis of PD. Firstly, the mitochondrial toxin MPTP inhibits complex I and reproduces many of the clinical and pathological features of PD. Secondly, some studies of PD families have identified a matrilineal pattern of inheritance [12]. Thirdly, when the mitochondria from platelets of PD patients were used to repopulate mtDNA-less human 0 cells, it became apparent that the defect in complex I activity is transmitted through mtDNA [2,11]. Finally, in hybrid models of sporadic PD in neuroblastoma cells fibrillar and vesicular inclusions are generated that essentially reproduce the antigenic and structural features of Lewy bodies in PD brain tissue [14]. Interestingly, the mtDNA polymorphisms A4336G, A10398G, and T4216C have been associated with PD [13,16]. While A4336G is thought to be a genetic risk factor, A10398G appears to be a protective genetic factor while T4216C is only weakly associated with the disease. In order to further investigate the involvement of mitochondrial proteins in the etiology of PD, we analyzed the association between these three genetic polymorphisms and the risk of developing the disease in a population of Spanish PD patients.
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Table 1 Characteristics of the samples
Age (years) Men Basque origin Other origin
Patients (%, n) (n = 102)
Controls (%, n) (n = 112)
66.8 ± 12.6 54.9 56.9 (58) 13.1 (44)
64.6 ± 5.87 48.6 47.7 (53) 52.3 (58)
Table 2 Allelic distribution of each polymorphism in control and affected subjects Polymorphism
Affected (N, %)
Controls (N, %)
P
4216 T 4216 C
88 (86.3) 14 (13.7)
96 (85.6) 16 (14.4)
0.885
4336 A 4336 G
97 (95.1) 5 (4.9)
112 (100.0) 0 (0.0)
0.018
10398 A 10398 G
75 (79.8) 19 (20.2)
99 (88.6) 13 (11.4)
0.088
Patients were diagnosed according to the Gelb criteria by well-trained neurologists (M, MM, LM) in the Neurology Department at the Hospital Donostia (Spain) [1]. The control subjects were selected from the DNA bank at the Neurology department of the hospital that includes DNA samples from the partners of patients in the hospital that are free of any neurological condition. The demographic characteristics of the PD (n = 102) and controls subjects (n = 112) are shown in Table 1. Both groups were indistinguishable regarding age and sex distribution. DNA was isolated by standard methods from the blood of subjects who had given their informed consent [4]. The subjects were classified by ethnic origin. As we could clearly differentiate Basque surnames from Romanic-derived names, we considered as autochthonous or Basque natives those patients and controls whose maternal grandmother carried both surnames (parental and maternal) with linguistic Basque roots (three mitochondrial generations). The remaining patients and controls were all from Caucasian origin coming from different areas of Spain. Genotyping was performed in two individual PCR amplifications. In one, both the C4216T and A4336G polymorphisms were amplified using the primers and conditions described elsewhere [5]. After PCR, the amplification
products were digested with the restriction enzyme NlaIII and the genotypes of each polymorphism were unequivocally identified: 4216T|4336A yielded a fragment of 313 bp; 4216T|4336G yielded two fragments of 244 and 69 bp; 4216C|4336A also yielded two fragments but of 123 and 190 bp; and finally, 4216C|4336G produced three fragments of 123, 121, and 69 bp. In contrast, the A10398G polymorphism was amplified using the primers and conditions reported in Van der Walt et al. (2003) and the products were digested with the restriction enzyme DdeI. This digestion yields several common fragments (486, 198, 141, 130, 121, 99, and 30 bp), together with a 223 bp fragment in the presence of the A allele, which is transformed into two fragments of 185 and 38 bp when the G allele is present. This polymorphism was only studied in 94 patients because we did not have sufficient DNA to carry out this analysis in all subjects. All the genotypes were resolved on 3–4% agarose gels to unequivocally determine the size of the DNA fragments. Pearson’s χ2 and Fisher analyses were used to compare the frequencies of the mutations in patients and control subjects. Using a multivariate logistic regression model, we assessed the statistical association of the variables: being Basque, suffering PD, and having any one of the polymorphisms. The significance level (Type I error) was established at 0.05 (SYSTAT v9.0, Chicago, IL, USA). When the distribution of each of the polymorphisms was analyzed, we did not detect any significant differences between PD patients and controls for the T4216C (p = 0.885) and the A10398G alleles (p = 0.088). However, the distribution of the A4336G polymorphism was more prevalent in patients than in controls (p = 0.018; Table 2). However, in the group of Basque origin, the distribution of A10398G differed between the affected subjects (18.5%) and the control group (4.8%; p = 0.0221). In the remaining subjects of different origins, a trend was detected between affected subjects (22.5%) and the control group (15.6%), but the difference was not significant (p = 0.3173). Moreover, this polymorphism was less frequent in the normal Basque population (4.8%) than in controls of other ethnic origin (15.6%; p = 0.043) (see Table 3). In order to analyze possible population differences, a second control sample from the same bank of DNA was analyzed (234 samples, mean age: 54.4 years, S.D.: 12.7, range:
Table 3 Allelic distribution of the polymorphisms in affected individuals and controls from a population of Basque origin and a non-defined population Polymorphism
Basque Affected (N, %)
Other origin Controls (N, %)
Affected (N, %)
Controls (N, %)
4216 T 4216 C
52 (89.7) 6 (10.3)
42 (91.3) 4 (8.7)
36 (81.8) 8 (18.2)
54 (81.8) 12 (18.2)
4336 A 4336 G
55 (94.8) 3 (5.2)
46 (100.0) 0 (0.0)
42 (95.45) 2 (4.55)
66 (100.0) 0 (0.0)
10398 A 10398 G
44 (81.5) 10 (18.5)
40 (95.2) 2 (4.8)
31 (77.5) 9 (22.5)
54 (84.4) 10 (15.6)
D. Otaegui et al. / Neuroscience Letters 370 (2004) 171–174
14–86). In this case, the DNA was from individuals with no history of neurological disease, but they were not matched by age or sex with the patient group. Statistically significant differences were observed in the proportions in which this polymorphism was represented in this sample, in the group of Basque (7.1% alelle G) origin and the other mixed group (15.4% Allele G) (p = 0.0266). Our analysis confirms previous findings regarding the association between the A4336G mtDNA polymorphism and PD [13]. The nucleotide involved in this polymorphism connects the amino acid acceptor stem with the TC stem of tRNAGln , and it is conserved in human, cow and chicken mtDNAs but not in mouse and Xenopus [8]. While a difference was observed in the distribution of this polymorphism between the populations analyzed, the relationship with ethnic origin (Basque/Other origin) is not outstanding (data not shown). Despite the lack of functional studies there are some indirect evidences about the effects of this kind of changes. In agreement with an existing structural model of mitochondrial tRNA [10], this change could induce secondarily structural modifications that may have functional consequences (alter the efficiency of the respiratory chain), as seen with homologous changes at the same structural area in cytoplasmic tRNA’s [9]. Indeed, it has been demonstrated that the performance of distinct mitochondrial haplotypes in the respiratory chain does vary [4]. The low predominance of this polymorphism indicates that it is not an important factor in the disease but it could, however, be a risk factor in conjunction with other environmental and/or genetic factors. The A10398G polymorphism is differentially distributed in the Basque population with respect to another population of less well-defined origin. More precisely, the G allele is on the whole less predominant in the population of Basque origin. Indeed, when considering the population of Basque origin alone, the presence of this polymorphism in PD patients is significantly greater than in the control population (p = 0.0221) and as such, it could be associated with the disease. A10398G induces a change in the ND3 protein from threonine to alanine, which represents an alteration in a subunit of the respiratory chain complex affected in PD, complex I. In our subjects, we found this polymorphism more often in the patient group than in the control subjects, contrasting with the protective effect proposed by Van der Walt et al. (2003). Hence, since the A10398G polymorphism is over-represented in the Basque population with Parkinson’s disease, we believe it could represent a risk factor for this disease. However, it should be highlighted that this association is not seen in the general population, illustrating that importance of performing this type of study in more ethnically homogeneous populations. In turn, this also stresses the need to use adequate criteria when selecting the control populations [3]. In conclusion, although there is a growing body of evidence to support the role of mtDNA in PD, biochemical
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studies will be needed to confirm the functional significance of these associations.
Acknowledgments We want to thank Dr. Marina for his helpful advice and Dr. Sefton for his help with the English writing. This work was supported by a Grant from the ‘Fundaci´on Ram´on Areces’ (DI-TER Parkinson). D.O. is supported by a Grant from Department of Education, Gobierno Vasco. C.P. is the recipient of a fellowship from the ‘Ministerio de Ciencia y Tecnolog´ıa FPI’. The authors want to thank M.A. Iribarren for her excellent technical support.
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