Mitochondrial DNA polymorphisms and risk of Parkinson's disease in Spanish population

Journal of the Neurological Sciences 236 (2005) 49 – 54 www.elsevier.com/locate/jns

Mitochondrial DNA polymorphisms and risk of Parkinson’s disease in Spanish population Cecilia Huerta a, Mo´nica G. Castro a, Eliecer Coto a, Marta Bla´zquez b, Rene´ Ribacoba b, Luis M. Guisasola b, Carlos Salvador b, Carmen Martı´nez b, Carlos H. Lahoz b, Victoria Alvarez a,* a

Gene´tica Molecular-Instituto de Estudios Nefrolo´gicos, Hospital Central de Asturias, Maternidad, 33006 Oviedo, Spain b Neurologı´a, Hospitales Central Asturias, Cabuen˜es-Gijo´n, and Alvarez Buylla-Mieres, Asturias, Spain Received 4 February 2005; received in revised form 26 April 2005; accepted 29 April 2005 Available online 21 June 2005

Abstract Mutations in mitochondrial DNA (mtDNA) have been implicated in the development of Parkinson’s disease (PD). Mitochondrial function is necessary to supply the energy required for cell metabolism, and mutations in mitochondrial genes should have a deleterious effect in neuronal function. An association between several common mtDNA-polymorphisms and the risk of PD has been described. To test this association among Spanish patients, we genotyped 271 PD-patients and 230 healthy controls for 13 single-nucleotide polymorphisms (SNPs) through polymerase chain reaction (PCR) followed by digestion with a restriction enzyme. Alleles at eight of these SNPs define nine common European haplotypes, the mitochondrial haplogroups. In our population, no haplogroup showed significantly different frequencies between patients and controls. A significant association was found for the 4336T/C SNP (a polymorphism in the tRNA gln gene), with allele 4336C having a significantly increased frequency in PD-women compared to controls (OR = 4.45; 95%CI = 1.23 – 15.96; p = 0.011). We also sequenced five of the complex I genes (ND1 to ND5) in the patients who were 4336C, and no mutation in these genes was found. We also found a significantly reduced frequency of 10398G in patients ( p = 0.009; OR = 0.53), confirming a previously described protective effect for this allele in PD. In conclusion, we provided further evidence of the involvement of mitochondrial DNA variation in PD. In agreement with previous reports, we described a higher risk for PD among women with the mitochondrial 4336C allele in our population, and a protective effect for 10398G. D 2005 Elsevier B.V. All rights reserved. Keywords: Mitochondria; Parkinson’s disease; DNA-polymorphisms; Mitochondrial haplogroups

1. Introduction Mitochondria are the cellular organelles that perform the metabolic reactions necessary to generate energy as adenosine triphosphate (ATP). Mitochondria contain their own DNA in a single circular chromosome (mtDNA), and their own machinery for RNA and protein synthesis. The mitochondrial genome has only 37 intronless genes that encode 13 subunits of the electron-transfer chain, 2 ribosomal RNAs, and 22 transfer RNAs [1]. * Corresponding author. Tel.: +34 985 10 79 68. E-mail address: [email protected] (V. Alvarez). 0022-510X/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jns.2005.04.016

Several reports have suggested that a mitochondrial dysfunction could be involved in Parkinson’s disease (PD) [2 –4]. A moderate impairment of complex I (the first system in the electron transport chain) has been demonstrated in patients with PD, and the substantia nigra and platelets of PD-patients should have a reduced activity of this complex [5 – 8]. In animal models, inhibition of complex I by several substances leads to the selective degeneration of dopaminergic neurons, a hallmark of PD [9]. The mtDNA encodes seven of the protein subunits of complex I, and the genetic variation at the mitochondrial genome could contribute to the risk of developing PD [10 – 12]. To test this hypothesis, some authors have previously

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C. Huerta et al. / Journal of the Neurological Sciences 236 (2005) 49 – 54

Table 1 Main characteristics of patients with Parkinson’s disease (PD) and healthy controls

Mean age (years) Age range (years) Male/female

PD

Controls

Controls < 55 years

Controls  55 years

N = 271

N = 230

N = 126

N = 104

63 T 11

51 T18

38 T 6

77 T 7

26 – 88

19 – 92

19 – 54

55 – 92

125/146

132/98

78/48

54/50

men, 146 women). They were recruited through the Neurology Departments of three hospitals from the region of Asturias (Northern Spain, total population 1 million). The main characteristics of these patients and controls are summarised in Table 1. A total of 230 healthy individuals, aged 19 to 92 years (132 men, 98 women), were used as population controls. They were blood bank donors and healthy spouses of patients, and did not have a history of PD or any other neurological disease. All the individuals participating in the study gave their informed consent to participate, and the study was approved by the Ethical Committee of Hospital Central Asturias.

genotyped several mitochondrial single-nucleotide polymorphisms (SNPs) in PD-patients and controls [13 – 16]. According to their data, some of these mtSNPs could significantly contribute to the risk of developing PD. In this work, we genotyped a group of Spanish PDpatients and healthy controls for 13 mitochondrial SNPs. Allele and haplogroup frequencies were compared between patients and controls.

2.2. mtDNA genotyping DNA was obtained from 10 ml of blood following a salting-out method [17]. A total of 13 mitochondrial DNA fragments were polymerase chain reaction (PCR) amplified. Each reaction contained approximately 100 ng of genomic DNA, 10 pmol of each primer-pair, and 2 mM of each dNTP, in a final vol of 20 Al. PCR consisted on a initial denaturation of 95 -C – 5 min, followed by 35 cycles of 95 -C – 30 s, the annealing temperature for 1 min (Table 2), and 72 -C – 1 min, followed by a final extension at 72 -C –5 min. The genotype corresponding to each polymorphism was determined through digestion of the PCR-fragment with a restriction enzyme which target sequence was affected by the nucleotide change (RFLP) (Table 2). Ten microliters of

2. Patients and methods 2.1. Patients and controls The study included a total of 271 patients diagnosed as having PD (mean age 63 T 11 years; range 26 –88 years; 125

Table 2 Mitochondrial polymorphisms analysed through digestion with a restriction enzyme Polymorphism

Primers

Annealing (-C)

Restriction enzyme

Allele-sizes (base pairs)

G1719A*

TCACCCTCCTCAAGTATACTTCA ATTTGGGTAAATGGTTTGGC AAACTTCCTACCACTCACCCTAGCATT TCGGGGTAGGGCCCGA AAACTTCCTACCACTCACCCTAGCATT TCGGGGTAGGGCCCGA ACCTATCACACCCCATCCTAAA AAGGATTATGGATGCGGTT AATGATCTGCTGCAGTGCTC TCCGGATAGGCCGAGAA ATGCAATTCCCGGACGTCT TTCACTGTAAAGAGGTGTTGGTTCT CCCATACTAGTTATTATCGA TAGGGGTCATGGGCGGGTT CCCATACTAGTTATTATCGA TAGGGGTCATGGGCGGGTT AGCCCTACAAACAACTAACCTGC AGTAGGGAGGATATGAGGTGTGAG ATTTACCGAGAAAGCTCACAAG TTTTATTTGGAGTTGCACCAAGAT TAGCCTTCTCCACTTCAAGTC AGAAACCTGTAGGAAAGGTATT CCTCACAGGTTTCTACTCCAAA AAGTCCTAGGAAAGTGACAGC AACCTACCCACCCTTAACAG CATCGTGATGTCTTATTTAAGGG

58

Dde I

62

Nla III

62

Nla III

60

Nhe I

60

Alu I

54

Hae III

58

Hae III

58

Hha I

53

Dde I

60

Hinf I

60

Sau96 I

57

Fnu4 HI

60

Sau96 I

G: 148 + 79 + 23 A: 148 + 102 T: 265 C: 227 + 38 T: 265 C: 158 + 107 G: 200 + 100 A: 300 C: 156 + 152 T: 152 + 126 + 30 G: 149 + 114 A: 263 G: 215 + 55 + 30 A: 215 + 85 G: 185 + 115 A: 300 A: 205 + 68 G: 167 + 68 + 38 A: 134 G: 109 + 25 G: 137 + 127 A: 264 G: 197 + 121 A: 318 G: 114 + 104 + 65 A: 172 + 114

T4216C T4336C G4580A* C7028T* G8251A G8994A G9055A* A10398G A12308G* G13368A* G13708A* G16391A*

The PCR-annealing temperature for each primer pair, the restriction enzymes used to analyse each SNP, an the size of the alleles are also indicated. Asterisks denote the eight polymorphisms used to define the nine mitochondrial haplogroups.

C. Huerta et al. / Journal of the Neurological Sciences 236 (2005) 49 – 54

A

B

Table 4 Frequencies of the nine mitochondrial haplogroups defined by eight mitochondrial polymorphisms in patients with Parkinson’s disease (PD) and healthy controls SNP

1

2

3

4

5

L

1

51

2

3

L

Fig. 1. A. Genotypes for the T4336C and the T4216C polymorphisms. Lanes 1, 4 (4336T, 4216T); lanes 2, 3, 4336C; lane 5,4216C. B. Genotypes for A10398G. Lane 1, 10398G; lanes 2, 3, 10398A. Lane L contains the DNA size marker (1kb ladder)

G1719A G4580A C7028T G9055A A12308G G13368A G13708A G16391A

Haplogroups H

I

J

K

T

U

V

W

X

G G C G A G G G

A G T G A G G A

G G T G A G A G

G G T A G G G G

G G T G A A G G

G G T G G G G G

G A T G A G G G

G G T G A G G G

A G T G A G G G

H

each PCR product were digested with the appropriated restriction enzyme (New England Biolabs), digestions were electrophoresed on 3% agarose gels, and fragments visualised after ethidium bromide staining (Fig. 1). To determine the accuracy of this genotyping method, DNAs representative of each RFLP-genotype were amplified, the PCR-fragments were purified, and both strands were sequenced in an automated system (ABI310), using Big-Dye terminator v3.1 cycle sequencing kit chemistry (Applied Biosystems). To determine if some mutation was linked to the 4336C allele, seven mitochondrial DNA fragments corresponding to the NADH dehydrogenase subunits ND1 to ND5 genes were PCR-amplified with the primers described in Table 3 in the 22 patients who were 4336C, and sequenced as described above. Nucleotides in the mtDNA were numbered according to the mitomap database (www.mitomap.org).

PD male N = 125 PE Female N = 146 Controls 55 years N = 104 Controls <55 years N = 126

I

J

K

T

U

V

W X

Other

46% 0% 9% 4% 9% 11% 10% 6% 2% 3% 47% 0% 8% 5% 9% 11% 10% 7% 2% 3% 45% 1% 10% 9% 8% 10% 9% 6% 1% 1% 44% 1% 13% 9% 8% 11%

9% 4% 1% 0%

analysis we followed a previously described approach, comparing each haplogroup with all other haplogroups pooled into one group [14]. Because multiple comparisons were taken into account (9 haplogroups and 13 SNPs), we used the Bonferroni’s correction and a p < 0.01 was considered as the level of statistical significance. All statistical analyses were performed using the BMDP new systems software (BMDP, Cork, Ireland).

3. Results 2.3. Statistical analysis Allele and haplogroup frequencies between patients and controls were compared through a v 2 test. Odds ratios (ORs) and their 95% confidence intervals (CIs) were also calculated, to assess the odds of carrying each allele in patients compared with controls. For the haplogroups

In this study we analysed 13 mitochondrial SNPs in 271 PD-patients and 230 healthy controls. Genotypes for each SNP were statistically compared between the two groups. Eight of these SNPs define the nine common European mitochondrial haplogroups, and we did not find significant differences between the frequencies of these haplogroups

Table 3 Primers used to amplify the genes of the complex I mitochondrial chain, which were sequenced in the 22 patients who were 4336C Mitochondrial region (nucleotides)

Mitochondrial genes

PCR-primers

PCR-annealing (-C)

3130 – 3720

ND1

63

3661 – 4441

ND1

4667 – 5523

ND2

10,055 – 10,410

ND3

10,465 – 10,778

ND4L

12,469 – 13,250

ND5

13,490 – 14,159

ND5

GGACAAGAGAAATAAGGCCTACTTCAC TTTGGGCTACTGCTCGCAGT CAGGGTGAGCATCAAACTCAAACTACG TTCGGGGTATGGGCCCGATAG TCCATAATCCTTCTAATAGC AACCTAAATTTCTATAAGATTATTA GTAATAAACTTCGCCTTAATTTT TATACCAATTCGGTTCAGTC CCAAATGCCCCTCATTTACA GGGACGATTAGTTTTAGCATTGG TTATCAGTCTCTTCCCCACAAC GGAGAAGGCTACGATTTTTTT CCTCACAGGTTTCTACTCCAAA CGGGGGAATAGGTTATGTGATT

71 53 55 62 58 62

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C. Huerta et al. / Journal of the Neurological Sciences 236 (2005) 49 – 54

Table 5 Frequencies for the alleles of the SNPs not used to define the mitochondrial haplogroups Polymorphism

Alelles

Female PD N = 146

Male PD N = 125

Total female controls N = 98

Total male controls N = 132

Total controls  55 years N = 104

T4216C

T C T C G A G A A G

118 (81%) 28 (19%) 128 (88%) 18 (12%) 139 (95%) 7 (5%) 139 (95%) 7 (5%) 128 (88%) 18 (12%)

101 (81%) 24 (19%) 121 (97%) 4 (3%) 120 (96%) 5 (4%) 115 (92%) 10 (8%) 109 (87%) 16 (13%)

83 15 95 3 95 3 95 3 78 20

111 (84%) 21 (16%) 130 (98%) 2 (2%) 127 (96%) 5 (4%) 126 (95%) 6 (5%) 103 (79%) 29 (21%)

89 15 102 2 100 4 100 4 82 22

T4336C# G8251A G8994A A10398G&

(85%) (15%) (97%) (3%) (97%) (3%) (97%) (3%) (80%) (20%)

(85%) (15%) (98%) (2%) (96%) (4%) (96%) (4%) (79%) (21%)

#p = 0.003; OR = 3.98 (95%CI = 1.46 – 10.80); Total PD vs. total controls (4336C). #p = 0.011; OR = 4.45 (95% CI = 1.23 – 15.96); Female PD vs. female controls (4336C). &p = 0.009; OR = 0.53 (95% CI = 0.33 – 0.86); Total PD vs. total controls (10398G).

between patients and controls (Table 4). These frequencies did not differ between controls <55 (n = 126) and 55 (n = 104) years, suggesting that they were not related with survival in our population. In addition, frequencies in patients were similar between men and women, indicating the lack of a gender effect (Table 4). Table 5 summarises the genotype frequencies for the five SNPs that were not used to define the haplogroups. We found a significantly reduced frequency of 10398G in patients compared to controls ( p = 0.009; OR = 0.53; 95%CI = 0.33– 0.86). The 10398G causes a nonconservative threonine to alanine change within the NADH dehydrogenase 3 (ND3) gene. We also found a significantly increased frequency of the 4336C allele in patients compared to controls ( p = 0.003), but when divided according to gender this difference was only observed in women ( p = 0.011; OR = 4.45; 95%CI = 1.23 –15.96). We also analysed the influence of these mitochondrial SNPs on the age at the onset of PD. Mean onset-ages were similar between patients 10398G (73.7 years) and 10398A (72.9 years), or between 4336C (67.5 years) and 4336T (71.3 years). The 4336C polymorphism is in the tRNA gln gene and could be directly responsible for the observed association, or the responsible could be a different nucleotide change linked to 4336C. To clarify this issue, we sequenced the five NADH dehydrogenase genes ND1 to ND5 in the 22 patients who were 4336C. In these cases, we only found nucleotide changes corresponding to some of the SNPs included in the study, but no mutation in any of them.

4. Discussion Mitochondria are essential to provide the energy necessary for cellular function. Mammalian mitochondria have their own DNA, in a single chromosome that contains 37 genes. Mutations in mtDNA could reduce the capacity to produce ATP, and this impairment in energy supply could affect neurons and other cell types. It has been well

established that mutations in mtDNA are responsible for some syndromes, such as MELAS (mitochondrial encephalomyopathy, lactic acidosis, and strokelike episodes), MERFF (myoclonic episodes with ragged-red fibers), Kearns– Sayre syndrome, or Leber’s neuropathy, among others [18,19]. The mtDNA mutations involved in these severe syndromes affect dramatically the mitochondrial function, and frequently involve several genes (large deletions), or mitochondrial protein synthesis (mutations in mitochondrial rRNAs and tRNAs) [18,20,21]. These are commonly seen as familial diseases, with non-mendelian maternal transmission [19,20]. In addition, common mtDNA polymorphisms could influence the risk of developing some multifactorial neurodegenerative disorders, such as Parkinson and Alzheimer diseases [14,15,22]. Sporadic PD is likely a consequence of the interaction between genetic susceptibility and environmental exposures [2,8,23]. Because mitochondrial dysfunction has been involved in the expression of PD, mtDNA polymorphisms could influence the risk of developing PD, either directly or through interaction with other genes or with some environmental toxins [13,14]. To test this hypothesis we, and others before, genotyped patients and controls for several mtDNA SNPs. The 4216C variant was found at an increased frequency in Irish PD cases, but in our population the frequencies for alleles at this SNP did not differ between patients and controls [15]. The mtDNA SNP T4336C has been previously linked to PD [16,24,25]. Interestingly, we found a significantly increased frequency for 4336C in women with PD compared to controls, but this effect was not observed in male patients. Nucleotide 4336 is in the tRNA gln gene, and connects the amino acid acceptor domain with the TCC domain [26]. This SNP could determine some structural change with functional consequences, such as an alteration of the respiratory chain. However, it is also possible that 4336C is in some haplotype with other changes that are responsible for the association. As an initial approach to clarify this, we sequenced five genes encoding peptides of the complex I respiratory chain in 22 patients who were 4336C, and no mutation was found

C. Huerta et al. / Journal of the Neurological Sciences 236 (2005) 49 – 54

in these cases. This supports that 4336C could be directly involved in the risk for PD among women, but a complete sequencing of the mtDNA in these cases should be necessary to confirm or refute this finding. There is no clear explanation for the gender difference in the association between 4336C and PD. The incidence of PD among men is higher than that among women (male/ female ratio 1.5– 2.5:1.0) [27,28]. Although the reason for this gender difference is unknown, several studies have found that estrogens have neuroprotective effects, and postmenopausal estrogen therapy may be associated with a reduced risk of PD in women [29,30]. Moreover, mitochondria would play a central role in estrogen-mediated neuroprotection [31,32]. In addition, previous studies have also found associations between SNPs in nuclear genes and PD in women [33]. Van der Walt et al. described a reduced frequency of allele 10398G in PD, and the protective effect of this allele should be stronger in women than in men [14]. We also found a significantly reduced frequency of 10398G among our patients, but the protective effect in PD in our population was similar in men and women. This allele causes a nonconservative change from Thr to Ala within the ND3 gene, one of the sububits of complex I, and it is thus possible that 10398G may increase the performance of complex I. If so, carriers of this allele could have an enhanced capability for energy metabolism under cellular stress, thus being at a lower risk for developing PD. Finally, a negative association between haplogroups J and K and PD has been described in Irish [14]. We found non-significantly reduced frequencies for these haplogroups in PD compared to controls, and this likely reflects the fact that 10398G is in haplogroups J and K [34]. In conclusion, in agreement with some previous studies from other populations, we confirmed the increased risk for PD among carriers of 4336C. However, in our population only women who were 4336C had an increased risk for PD. In addition, the 10398G allele had a protective effect for the risk of developing this common neurodegenerative disorder.

Acknowledgements Authors wish to thank all the patients and controls participating in the study. This work was supported by two grants from the Spanish Fondo de Investigaciones Sanitarias (FIS 02/0022 and Red Tema´tica Centros-C03/07). CH is the recipient of a predoctoral fellowship from Asociacio´n Parkinson Asturias.

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