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Genotype in the 24-kDa Subunit Gene (NDUFV2) of Mitochondrial Complex I and Susceptibility to Parkinson Disease
GENOMICS
49, 52–58 (1998) GE975192
ARTICLE NO.
Genotype in the 24-kDa Subunit Gene (NDUFV2) of Mitochondrial Complex I and Susceptibility to Parkinson Disease Nobutaka Hattori,1 Hiroyo Yoshino, Masashi Tanaka,* Hiroshi Suzuki,† and Yoshikuni Mizuno Department of Neurology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo, Tokyo 113-8431, Japan; *Department of Gene Therapy, Gihu International Institute of Biotechnology, Yagi Memorial Park, Mitake, Gihu 505-0116, Japan; and †Department of Bioscience, Faculty of Molecular Biology and Biotechnology, Fukui Prefectural Unniversity, Matsuoka-cho, Yoshida-gun, Fukui 910-1195, Japan Received September 18, 1997; accepted December 18, 1997
cause of PD is still unknown, oxidative stress and mitochondrial respiratory failure appear to be two major contributors to nigral degeneration. It is likely that oxidative stress and mitochondrial respiratory failure occur within the nigral neurons as a consequence of this interaction. The increase of superoxide dismutase (Marttila et al., 1988; Saggu et al., 1989), the accumulation of iron (Riederer et al., 1989; Dexter et al., 1990; Good et al., 1992; Jellinger et al., 1992), the loss of glutathione (Riederer et al., 1989; Jenner et al., 1992a), and the increase of lipid peroxidation (Dexter et al., 1989; Yoritaka et al., 1996) are evidence of oxidative stress, while complex I deficiency (Mizuno et al., 1989; Schapira et al., 1989, 1990; Hattori et al., 1991) and the loss of a-ketoglutarate dehydrogenase complex (Mizuno et al., 1994) are evidence of mitochondrial failure. Genetic susceptibility to PD may reside in a polymorphism of genes for proteins and enzymes expressed within the mitochondria and/or regulating free radical metabolism. Thus, although many genetic association studies have been reported (Kondo and Kanazawa, 1991; Armstrong et al., 1992; Smith et al., 1992; Kurth et al., 1993; Kurth and Kurth, 1993; Tsuneoka et al., 1993; Hotamisligil et al., 1994; Ho et al., 1995; Morimoto et al., 1995; Shimoda-Matsubayashi et al., 1996), the results are controversial. Within the central nervous system at least, complex I deficiency appears to be confined to the substantia nigra (SN). We also demonstrated decreased amounts of the 30-, 25-, and 24-kDa subunits of complex I (Mizuno et al., 1989). However, there is no evidence to define the molecular basis of these defects. Previously, we cloned the gene encoding the 24-kDa subunit of complex I (Hattori et al., 1995), which was first cloned by de Coo et al. (1995). Together with the 75- and 51kDa subunits, the 24-kDa subunit forms a structural and functional unit (Ohnishi et al., 1985). These three subunits are closely related to subunits of a soluble NAD/ hydrogenase of the bacterium Alcaligenes eutrophus (Tran-Betcke et al., 1990; Pilkington et al., 1991).
We analyzed the gene encoding the 24-kDa subunit of mitochondrial complex I, which has been implicated in the pathogenesis of Parkinson disease (PD). We set out to identify a polymorphism in the 24-kDa subunit gene (NDUFV2) in patients with PD and determine whether genetic polymorphism of this gene is associated with a higher risk of PD. The subjects comprised 126 patients with PD, and the control group comprised 113 unrelated individuals without neurodegenerative disorders. A novel polymorphism (Ala29Val) in the mitochondrial targeting sequence of NDUFV2 was found in patients with PD. The distribution of the three genotypes was significantly different between the two groups (x2 Å 7.53, df Å 2, P Å 0.023). The frequency of homozygotes for the mutation was significantly higher in PD patients (23.8%) than in control subjects (11.5%, Fisher’s exact test, P Å 0.0099 õ 0.01). The risk of developing PD associated with homozygosity for this mutation was calculated as 2.40 (95% CI: 1.18–4.88). NDUFV2 constitutes one genetic risk factor for PD, and the mutation may well be a cause of complex I deficiency in this disease. q 1998 Academic Press Key Words: Parkinson disease; genetic predisposition; complex I; 24-kDa subunit; mitochondria
INTRODUCTION
In recent years, growing evidence has indicated that the interaction of environmental factors and genetic predisposition initiates the neurodegenerative process in Parkinson disease (PD) (Calne and Langston, 1983; Rajput et al., 1987; Langston, 1989; Jenner et al., 1992b; Payami et al., 1994; Mizuno et al., 1995; Wilhelmsen and Wszollek, 1995). Although the primary Nucleotide sequence data from this article have been deposited with the DDBJ, EMBL, and GenBank Data Libraries under Accession Nos. D88542–D88548. 1 To whom correspondence should be addressed. Telephone: //81-3-5802-1072. Fax: //81-3-3813-7440. E-mail: nhattori@med. juntendo.ac.jp.
0888-7543/98 $25.00 Copyright q 1998 by Academic Press All rights of reproduction in any form reserved.
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The 24-kDa subunit contains one (2Fe–2S) cluster and plays an important role in a substantial part of the electron transport pathway of complex I. The human 24-kDa subunit of complex I is encoded by a single gene (NDUFV2) located on chromosome 18 at region p11.3, which spans 31.5 kb and consists of eight exons (Hattori et al., 1995). To determine whether genetic polymorphism of this gene is associated with a higher risk of PD, we screened all exons of NDUFV2 for mutations in patients with PD, and we found a new polymorphic mutation in the signal peptide of this 24-kDa precursor protein. This paper reports on the new polymorphic mutation and its frequency in PD and control populations. SUBJECTS AND METHODS Subjects. The subjects consisted of 126 PD patients and 113 controls who were followed by the Neurology Service of Juntendo University Medical School Hospital. The average age { SD was 62.7 { 9.3 years for the PD patients and 62.6 { 13.7 for the control subjects. None of the PD patients had a family history of PD or were related to one another. One of the PD patients had sporadic young-onset disease (onset at 38 years old). The criteria used for the diagnosis of idiopathic PD were based on the recently published consensus statement (Calne et al., 1992). All subjects were of Japanese origin. The control subjects consisted of 113 unrelated Japanese individuals who attended our clinic and were diagnosed as not having neurodegenerative disorders. Informed consent was obtained from all the patients and control subjects. Mutation screening of NDUFV2. Human genomic DNA was isolated as described previously (Miller et al., 1988). Samples were either used immediately or stored at 0207C until analysis. All exons (1 to 8) of NDUFV2 were screened for mutations by direct sequencing polymerase chain reaction (PCR) in 20 PD patients randomly selected from among the PD group. PCR was performed with chimera primers that were specific to the sequence for NDUFV2 and also possessed the sequence of the standard sequencing primers (M13 universal and reverse primers) on their 5* ends. The amplified fragments were sequenced by the dideoxy chain termination method using the fluorescence-based automated dye primer sequencing technique with an Applied Biosystems 373A DNA sequencer according to the manufacturer’s instructions (Applied Biosystems Division of Perkin Elmer, Foster City, CA). PCR products for exons 1, 2, 4, 5, 6, and 7 were obtained by nested PCR using two sets of primers. PCR products for exons 3 and 8 were obtained using a single set of primers. The sequences of the oligonucleotide primers are shown in Table 1. Primers for exR and exU are chimera oligonucleotides that possessed the M13 forward universal sequence and the reverse sequence, respectively, at the 5* end. The sequences of these oligonucleotide primers have been deposited with the DNA Data Bank of Japan (DDBJ) (Accession Nos. D64176 and D88542–D88548). PCR conditions are shown in Table 2. One hundred nanograms of each sample was added to a 50-ml reaction mixture containing 50 mM KCl, 10 mM Tris (ph 8.3), 1.5 mM MgCl2 , 0.01% gelatin with 10 pmol of the paired primers, 10 nmol of each dNTP, and 2.5 units of Taq polymerase. LA Taq DNA polymerase (Takara Co., Kyoto, Japan) was used for the first PCRs except for exons 3 and 8. AmpliTaq DNA polymerase (Applied Biosystems Division of Perkin Elmer, Foster City, CA) was used for all the second PCRs including PCRs for exons 3 and 8. The second PCRs were performed with the same PCR mixture containing 1 ml of the first PCR products. The excess primers and dNTPs were removed from the PCR products by Ultrafree-MC centrifugal filter units (Millipore, Tokyo, Japan). The purified PCR products were used as the direct sequencing templates. RFLP-creating PCR screening for Ala29Val mutation in patients with sporadic PD. As will be shown later, we found a novel Ala29-
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Val polymorphic mutation in NDUFV2. We analyzed the frequency of this polymorphism in 126 PD patients and 113 control subjects by RFLP-creating PCR. The Ala29Val mutation (C-T substitution) found in the present study neither created nor abolished any restriction sites. To develop a simple detection method for the C-T substitution, RFLP was artificially induced at the substitution site by introducing a single base mismatch of an oligonucleotide primer. As shown in Table 1, a mismatched primer (ex2AS-mis) and the second primer (ex2S2) were constructed. After amplification, the PCR products were digested with MaeIII (Boehringer Mannheim GmbH, Mannheim, Germany). The products were electrophoresed on a 3.5% agarose gel and visualized with ethidium bromide to detect the 211and (or) 180-bp bands. DNA sequences, amino acid sequences, and protein secondary structure analysis. The DNA sequences and translated amino acid sequences were analyzed using the genetic information processing software Genetyx-Mac Version 8.0 (Software Development Co., Tokyo, Japan). The protein secondary structure was analyzed by theoretical prediction analysis according to the algorithm of Chou and Fasmann (1974). A hydropathy plot of the amino acid sequence was calculated by the method of Kyte and Doolittle (1982) using a window average of seven residues. The computer program used for this analysis was also Genetyx-Mac Version 8.0. Statistical analysis. Statistical analysis was performed with StatView-J Version 4.02 (Abacus Concept Inc., Barkeley, CA), employing the x2 test and Fisher’s exact probability test.
RESULTS
We found a novel structural mutation (a C-to-T transition) in exon 2, which replaces 29 Ala (GCT) with Val (GTT) in the signal peptide. No other mutations were found in the coding region of NDUFV2 among the 20 randomly selected patients. This alanine is conserved among three mammalian species, i.e., bovine (Pilkington and Walker, 1989), human (Pilkington and Walker, 1989; Toda et al., 1989), and rat (Nishikimi et al., 1988) (Fig. 1). The chemical distance (D) between Ala and Val calculated according to the method of Grantham (1974) was relatively large (D Å 64). Computer analysis suggested that replacement of this amino acid caused a change in the hydropathy profile at the first hydrophilic domain (Fig. 2) and that this replacement also caused changes in the secondary structure of the gene product from an a-helix conformation (residues 24–32) to a bsheet conformation (residues 22–32). Thus, the mutant with a valine residue at position 29 shows disruption of the a-helix conformation and a long b-sheet stretch through residues 24–32 (Fig. 3). To investigate the prevalence of this base substitution in a large population, we developed a simple detection method using PCR with a mismatched primer. This procedure creates an artificial RFLP that is affected by the C-T substitution. The polymorphism can be detected as a MaeIII site in exon 2. The MaeIII digestion distinguishes the respective genotypes by their own distinctive restriction patterns (Fig. 4). The mutant allele was found in 32.7% of the 123 PD patients and 38.9% of the 113 control subjects. The frequencies of the mutant allele were not significantly different between the two groups (x2 Å 1.95, P Å 0.16). However, the distribution of the three genotypes was significantly different (x2 Å 7.53, df Å 2, P Å 0.023)
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TABLE 1 Primers Used for Amplification of 24-kDa Subunit Gene (NDUFV2) Sequence (5* r 3*)
Primersa
Region Exon 1
ext EX1S ext EX2AS int EX1S-R int EX1AS-U ext EX2S ext EX4AS int EX2S-R int EX2AS-U EX3S-R EX3AS-U ext EX4S ext EX5AS int EX4S-R int EX4AS-U int EX5S-R int EX5AS-U ext EX6S ext EX7AS int EX6S-R int EX6AS-U int EX7S-R int EX7AS-U EX8S-R EX8AS-U EX2S2 EX2AS-mis
Exon 2
Exon 3 Exons 4 and 5
Exons 6 and 7
Exon 8 PCR for exon2 using mismatched primer
5*-GCG 5*-GGA 5*-CAG 5*-TGT 5*-AGT 5*-AAC 5*-CAG 5*-TGT 5*-CAG 5*-TGT 5*-TAA 5*-GTG 5*-CAG 5*-TGT 5*-CAG 5*-TGT 5*-ATA 5*-CTA 5*-CAG 5*-TGT 5*-CAG 5*-TGT 5*-CAG 5*-TGT 5*-GAT 5*-ATA
CGG TTT GAA AAA TGA TAA GAA AAA GAA AAA CCC TTA GAA AAA GAA AAA ACC TCC GAA AAA GAA AAA GAA AAA GGA AAG
CTG ACC ACA ACG TTC GGC ACA ACG ACA ACG TGA CAA ACA ACG ACA ACG TGG AAA ACA ACG ACA ACG ACA ACG TAG CTC
GGG CCA GCT ACG CAT AAA GCT ACG GCT ACG TAC ATA GCT ACG GCT ACG TCC GCT GCT ACG GCT ACG GCT ACG GGT CTC
AAG ATT ATG GCC TCC AAT ATG GCC ATG GCC TCC TCA ATG GCC ATG GCC TTA CCT ATG GCC ATG GCC ATG GCC AGA CAG
GTG TCC ACC AGT ATT GAA ACC AGT ACC AGT ATT TGT ACC AGT ACC AGT GAG ACC ACC AGT ACC AGT ACC AGT ATA CTC
AAC AGT GT-3* AAG TC-3* GCG CGG CTG GGG AAG CAG TAT GGC AAC CCT GTT GTA-3* TGC CAG-3* AGT TGA TTC CAT TCC GGA TTT ACC CCA ATT CTT GAG AGA ATT TGG CTG CTG CTT TAT GGC TGA TTT-3* ATT CCC T-3* TAA CCC TGA TAC TCC AAC TAA GGC AAA AAT GCT CCA TTA CTA ACA GTG TTA CAA ATA TCA TGT T-3* AA-3* ATA ACC TGG TCC TTA GAA ACC AAC ATC TAA GCA ACA TAG TGA GAC CTA TCC AAA GCT CCT AAC TGG GCT CTT GTG TTA AAG GCC TGC TTG CCA TAT TCC TTA-3* *TA-3* CAT TTT GCG
GTG AAC AGT GT-3* CCC TCG G-3 *
ATT TCC ATA CTT
GTT AAG AGT CTG
GTA-3* TC-3 * CTG-3* GAT-3 *
ATT GAA CTG TGT
TGA TGC TAA ATT
TTT-3* CAG-3 * TTA T-3* CCC T-3*
GAG GGA CTT ACC AGG TAC
TGT T-3* CAC A-3 * GTC-3 * AA-3 * TAA CCA TTA-3* ACC AAA TCC AG-3*
Note. The M13 forward universal sequence and the reverse sequence are underlined. A single base mismatch is indicated by an asterisk. a ext, external reaction; int, internal reaction.
TABLE 2 Fragment Sizes and Thermal Cycling Protocols Fragment size (bp)
Primers pairs Exon 1 EX1S–EX2AS EX1S-R–EX1AS-U Exon 2 EX2S–EX4S EX2S-R–EXAS-U Exon 3 EX3S-R–EXAS-U Exons 4 and 5 EX4S–EX5AS EX4S-R–EX4AS-U EX5S-R–EX5AS-U Exons 6 and 7 EX6S–EX7AS EX6S-R–EXAS-U EX7S-R–EX7AS-U Exon 8 EX8S-R–EX8AS-U PCR for exon 2 using mismatched primer EX2S–EX2AS-mis
PCR conditions
15K 284a
957C/1 min; 987C/20 s, 657C/12 min (30 cycles); 727C/10 min 957C/5 min; 957C/1 min, 637C/1 min, 727C/1 min (35 cycles); 72/7C/7 min
2K 229a
947C/5 min; 947C/1 min, 557C/2 min, 727C/2 min (30 cycles); 727C/7 min 957C/1 min; 957C/30 s, 587C/30 s, 727C/1 min (35 cycles)
242a
957C/1 min; 957C/30 s, 657C/30 s, 727C/1 min (35 cycles)
4K 271a 249a
947C/5 min; 947C/1 min, 557C/1 min, 727C/2 min (30 cycles); 727C/7 min 957C/1 min; 957C/30 s, 587C/30 s, 727C/1 min (35 cycles) 957C/5 min; 957C/30 s, 587C/30 s, 727C/1 min (35 cycles)
2.3K 239a 280a
947C/5 min; 947C/1 min, 557C/1 min, 727C/2 min (30 cycles); 727C/7 min 957C/1 min; 957C/30 s, 607C/30 s, 727C/1 min (35 cycles) 957C/1 min; 957C/30 s, 607C/30 s, 727C/1 min (35 cycles)
283a
957C/1 min; 957C/30 s, 657C/30 s, 727C/1 min (35 cycles)
211
957C/2 min; 957C/1 min, 607C/1 min, 727C/1 min (35 cycles); 727C/7 min
Note. Fragment size is that of the exon plus flanking intronic sequences. EX-R and EX-U are chimera oligonucleotides that possessed the universal and reverse sequences, respectively, at the 5* end. a Fragment size dose not include the sequences of M13 universal and reverse primers.
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FIG. 1. Homology of the amino acid sequences of the signal peptide for the 24-kDa subunit of human, bovine, and rat complex I. Homologous regions are boxed. The arrow indicates the amino acid replaced by the C-to-T transition in exon 2.
(Table 3). Furthermore, the expected frequencies of the three genotypes, under the assumption of Hardy– Weinberg equilibrium, did not differ from the observed frequencies in the control group (x2 Å 1.95, df Å 2, P Å 0.38). On the other hand, there was extreme deviance from Hardy–Weinberg equilibrium in the PD patients (x2 Å 10.18, df Å 2, P Å 0.006 õ 0.01) (Table 3). The frequency of homozygosity for the mutation was significantly higher in the PD patients (23.8%) than in the control subjects (11.5%, Fisher’s exact probability test, P Å 0.0099 õ 0.01). The risk of developing PD associated with homozygosity for this mutation was calculated as 2.40 (95% CI: 1.18–4.88) (Table 2). The frequency of 029Ala homozygotes did not differ significantly between the PD patients and the controls, while the frequency of 029Ala/Val heterozygotes was significantly less common among the PD patients (30.2%) than among the controls (42.5%) (Fisher’s exact probability test, P Å 0.032 õ 0.05). In other words, homozygous mutants occur more frequently and heterozygotes less frequently than expected in the PD patients. DISCUSSION
In our present study, we found a statistically significant difference in the frequency of the novel polymorphism in NDUFV2 in PD patients compared with control subjects. The presence of homozygosity for the Ala29Val mutation increased the risk of developing PD by 2.40-fold. Our results indicate that homozygosity for
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FIG. 3. Secondary structure of the signal peptide for the 24-kDa subunit. The replacement of 029Ala to Val changes the a-helix (at position 24 to 32) to the b-sheet structure (at position 22 to 32).
the Ala29Val mutation in exon 2 of NDUFV2 confers increased susceptibility to PD. The alanine residue located at the polymorphic site was highly conserved among three mammalian species, suggesting the functional importance of this residue. It is predicted that the Ala29Val mutation causes an alteration in the secondary structure of the gene product from an a-helix conformation located in the C-terminal domain of the signal peptide to a b-sheet structure. In the signal pepetide of the 24-kDa subunit, there are two a-helix structures with one b-sheet structure in between. The N-terminal of the signal peptide, which can form an amphiphilic a-helix, seems to be crucial for targeting–recognition events (von Heijne et al., 1989). On the other hand, the a-helix structure of the C-terminal domain of the signal peptide is essential for processing–recognition events by mitochondrial processing peptidase (MPP) (Sjoling et al., 1994). Furthermore, a study based on theoretical analysis of 37 mitochondrial presequences indicates that the a-helical element of the C-terminal domain participates in helix/helix interactions with the peptidase (von Heijne et al., 1989). Thus, the Ala29Val mutation may affect cleavage of the signal peptide, which in turn influences the level of the mature form of 24-kDa subunit of complex I within the mitochondria. Complex I, consisting of at least 41 subunits, is the most complicated of the electron transfer complexes and is composed of subunits encoded by both mitochondrial and nuclear genomes (Walker et al., 1992). Most of the genetic information required for
FIG. 2. Hydropathy plot of the amino acid sequence of the signal peptide for the 24-k Da subunit of human complex I. Positive values indicate hydrophobic regions, whereas negative values indicate hydrophilic regions. The arrow indicates the change in hydrophobicity that is caused by the replacement of 029Ala with Val in the hydrophilic region.
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FIG. 4. MaeIII RFLP of PCR products with a mismatched primer on agarose gel. w/w, m/m, and w/m indicate wildtype homozygote, mutant homozygote, and heterozygote, respectively. M is a 100-bp ladder marker.
mitochondrial biogenesis in complex I is provided by nuclear DNA (nDNA), while only 7 subunits are encoded by mitochondrial DNA (mtDNA). Complex I deficiency is well known to exist in PD, but the cause of this deficiency is unclear. It may be due to exposure to endogenous or exogenous toxins, but it may well be the result of a genetic predisposition. Both
nuclear and mitochondrial DNA mutations can affect the activity of complex I. We have found several point mutations of mtDNA in PD patients that would cause conformational changes in the structure of the gene products (Ikebe et al., 1995), however, we could not find a predominant mtDNA point mutation associated with PD. Shoffner et al. (1993) found a higher incidence of the A-to-G transition at the tRNA Gln gene in PD; however, we could not reproduce their results (unpublished data). As not all the PD patients have this mutation in NDUFV2, complex I deficiency in PD may be due to distinct clusters of mutations in mtDNA and/or nDNA. Although we have not analyzed the relationship between the activity of complex I and the genotypes of NDUFV2, this mutation may well be one of the genetic factors that leads to premature loss of complex I. Such a possibility needs to be studied further. To our knowledge, this is the first report on the association of PD with the genes encoding subunits of complex I. Our findings may thus open up a new pathway for exploring the molecular basis of complex I deficiency in PD and its role in the pathogenesis of this disease.
TABLE 3 NDUFV2 Allele and Genotype Frequencies in PD Patients and Controls
No. subjects No. chromosomes
Control (%)
PD (%)
Total (%)
113 226
126 252
239 478
154 (67.3) 98 (32.7)
306 (64.0) 172 (36.0)
Allele frequency 152 (61.1) 74 (38.9)
029Ala 029Val
x2 Å 1.95, df Å 1, P Å 0.16a Genotype frequency Control (%)
Ala/Ala Ala/Val Val/Val
PD (%)
Observed
Expected
Observed
Expected
Total observed (%)
52 (46.0) 48 (42.5) 13 (11.5)
42 54 17
58 (46.0)b 38 (30.2)c 30 (23.8)d
57 56 13
110 (46.0) 86 (36.0) 43 (18.0)
x2 Å 1.95, df Å 2, P Å 0.38e
x2 Å 10.18, df Å 2, P Å 0.006 f x2 Å 7.53, df Å 2, P Å 0.023 g
a There was no significant difference in allele frequencies between the PD patients and the controls. Expected values were calculated from the allele frequency in the respective groups. b The frequency of Ala/Val heterozygotes was significantly lower in PD than in other homozygotes combined (Fisher’s exact probability test, P Å 0.032 õ 0.05). c The frequency of Val homozygotes is significantly higher in PD than in other genotypes combined (Fisher’s exact probability test, P Å 0.0099 õ 0.01). Odds ratio Å 2.40, 95% CI: 1.18–4.88. d The frequency of Ala homozygotes did not differ significantly between the two groups compared with other two genotypes combined (x2 Å 0.000005, P Å 0.99). e In the controls, the expected frequencies of the genotype did not differ from the observed frequencies. f There was extreme deviance from Hardy–Weinberg equilibrium in the PD patients. g The distribution of the three genotypes was significantly different between the PD patients and the controls.
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sights into the cause of Parkinson’s disease. Neurology 42: 2241– 2250.
ACKNOWLEDGMENTS This work was supported in part by a Grant-in-Aid for Scientific Research on Priority Areas to Y.M. and by a Grant-in-Aid for Scientific Research (08770474 to N.H.) from the Ministry of Education, Science and Culture, Japan, by a Grant-in-Aid for Neurodegenerative Disorders from the Ministry of Health and Welfare, Japan, and by a ‘‘Center of Excellence’’ Grant from the National Parkinson Foundation, Miami, Florida.
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Kondo, I., and Kanazawa, I. (1991) Association of Xba I allele (Xba I 44kb) of the human cytochrome P-450 (CYP2D6) gene in Japanese patients with idiopathic Parkinson’s disease. In ‘‘Parkinson’s Disease: From Clinical Aspects to Molecular Basis’’ (T. Nagatsu, H. Narabayashi, and M. Yoshida, Eds.), pp. 111–117, Springer-Verlag, Vienna. Kurth, J. H., Kurth, M. C., Poduslo, S. E., and Schwankhaus, J. D. (1993). Association of a monoamine oxidase B allele with Parkinson’s disease. Ann. Neurol. 33: 368–372. Kurth, M. C., and Kurth, J. H. (1993). Variant cytochrome P450 CYP2D6 allelic frequencies in Parkinson’s disease. Am. J. Med. Genet. 48: 166–168. Kyte, J., and Doolittle, R. F. (1982). A simple method for displaying the hydropathic character of a protein. J. Mol. Biol. 157: 105–132. Langston, J. W. (1989). Current theorities on the cause of Parkinson’s disease. J. Neurol. Neurosurg. Psychiatry 52(suppl): 13–17. Marttila, R. J., Lorentz, H., and Rinne, U. K. (1988). Oxygen toxicity protecting enzymes in Parkinson’s disease: Increase of superoxide dismutase-like activity in the substantia nigra and basal nucleus. J. Neurol. Sci. 86: 321–331. Miller, S. A., Dykes, D. D., and Polesky, H. F. (1988). A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res. 16: 1215. Mizuno, Y., Ikebe, S., Hattori, N., Nakagawa, H. Y., Mochizuki, H., Tanaka, M., and Ozawa, T. (1995). Role of mitochondria in the etiology and pathogenesis of Parkinson’s disease. Biochim. Biophys. Acta 1271: 265–274. Mizuno, Y., Matsuda, S., Yoshino, H., Mori, H., Hattori, N., and Ikebe, S. (1994). An immunohistochemical study on a-ketoglutarate dehydrogenase complex in Parkinson’s disease. Ann. Neurol., 35. Mizuno, Y., Ohta, S., Tanaka, M., Takamiya, S., Suzuki, K., Sato, T., Oya, H., et al. (1989). Deficiencies in complex I subunits of the respiratory chain in Parkinson’s disease. Biochem. Biophys. Res. Commun. 163: 1450–1455. Morimoto, Y., Murayama, N., Kuwano, A., Kondo, I., Yamashita, Y., and Mizuno, Y. (1995). Association analysis of a polymorphism of the monoamine oxidase B gene with Parkinson’s disease in a Japanese population. Am. J. Med. Genet. 60: 570–572. Nishikimi, M., Hosokawa, Y., Toda, H., Suzuki, H., and Ozawa, T. (1988). The amino acid sequence of the 24-kDa subunit, an iron– sulfur protein, of rat liver mitochondrial NADH dehydrogenase deduced from cDNA sequence. Biochem. Biophys. Res. Commun. 157: 914–920. Ohnishi, T., Ragan, C. I., and Hatefi, Y. (1985). EPR studies of iron– sulfur clusters in isolated subunits and subfractions NADH-ubiquinone oxidoreductase. J. Biol. Chem. 260: 2782–2788. Payami, H., Larsen, K., Bernard, S., and Nutt, J. (1994). Increased risk of Parkinson’s disease in parents and siblings of patients. Ann. Neurol. 36: 659–661. Pilkington, S. J., Skehel, J. M., Gennis, R. B., and Walker, J. E. (1991). Relationship between mitochondrial NADH-ubiquinone reductase and a bactarial NAD-reducing hydrogenase. Biochemistry 30: 2166–2175. Pilkington, S. J., and Walker, J. E. (1989). Mitochondrial NADHubiquinone reductase: Complementary DNA sequences of import precursors of the bovine and human 24-kDa subunit. Biochemistry 28: 3257–3264. Rajput, A. H., Uitti, R. J., Stern, W., Laverty, W., O’Donnell, K., O’Donnell, D., Yuen, W. K., et al. (1987). Geography, drinking water chemistry, pesticides and herbicides and the etiology of Parkinson’s disease. Can. J. Neurol. Sci. 14: 414–418. Riederer, P., Sofic, E., Rausch, W. D., Schmidt, B., Reynolds, G. P., Jellinger, K., and Youdim, M. B. H. (1989). Transition metals, fer-
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ritin, glutathione, and ascorbic acid in parkinsonian brains. J. Neurochem. 55: 515–520. Saggu, H., Cooksey, J., Dexter, D., Wells, F. R., Lees, A., Jenner, P., and Marsden, C. D. (1989). A selective increase in particulate superoxide dismutase activity in parkinsonian sunstantia nigra. J. Neurochem. 53: 692–697. Schapira, A. H., Cooper, J. M., Dexter, D., Jenner, P., Clark, J. B., and Marsden, C. D. (1989). Mitochondrial complex I deficiency in Parkinson’s disease. Lancet 1: 1269. Schapira, A. H., Mann, V. M., Cooper, J. M., Dexter, D., Daniel, S. E., Jenner, P., Clark, J. B., et al. (1990). Anatomic and disease specificity of NADH CoQ1 reductase (complex I) deficiency in Parkinson’s disease. J. Neurochem. 55: 2142–2145. Shimoda-Matsubayashi, S., Matsumine, H., Kobayashi, T., Nakagawa-Hattori, Y., Shimizu, Y., and Mizuno, Y. (1996). Identification of dimorphic muattion in the mitochondrial targeting sequence in the human MnSOD gene and its allelic association with Parkinson’s disease. Biochem. Biophys. Res. Commun. 226: 561– 565. Shoffner, J. M., Brown, M. D., Torroni, A., Lott, M. T., Cabell, M. F., Mirra, S. S., Beal, M. F., et al. (1993). Mitochondrial DNA variations observed in Alzheimer disease and Parkinson disease patients. Genomics 17: 171–184. Sjoling, S., Eriksson, A. C., and Glaser, E. (1994). A helical element in the C-terminal domain of the N. plumbaginifolia F1 beta presequence is important for recognition by the mitochondrial processing peptidase. J. Biol. Chem. 269: 32059–32062. Smith, C. A., Gough, A. C., Leigh, P. N., Summers, B. A., Harding, A. E., Maranganore, D. M., Sturman, S. G., et al. (1992). Debriso-
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quine hydroxylase gene polymorphism and susceptibility to Parkinson’s disease. Lancet 339: 1375–1377. Toda, H., Hosokawa, Y., Nishikimi, M., Suzuki, H., Kato, K., and Ozawa, T. (1989). Cloning and sequencing of cDNA encoding the precursor to the 24 kDa iron–sulfur protein of human mitochondrial NADH dehydrogenase. Int. J. Biochem. 21: 1161–1168. Tran-Betcke, A., Warnecke, U., Bo¨cker, C., Zaborosch, C., and Friedrich, B. (1990). Cloning and nucleotide sequences of the genes for the subunits of NAD-reducing hydrogenase of Alcaligenes eutrophus. J. Bacteriol. 226: 1051–1072. Tsuneoka, Y., Matsuno, Y., Iwasaki, K., Takeuchi, H., and Ichikawa, Y. (1993). A novel cytochrome P-450IID6 gene associated with Parkinson’s disease. J. Biochem. 114: 263–266. von Heijne, G., Steppuhn, J., and Herrmann, R. G. (1989). Domain structure of mitochondrial and chloroplast targeting peptides. Eur. J. Biochem. 180: 535–545. Walker, J. E., Arizmendi, J. M., Dupuis, A., Fearnley, I. M., Finel, M., Medd, S. M., Pilkington, S. J., et al. (1992). Sequences of 20 subunits of NADH:ubiquinone oxidoreductase from bovine heart mitochondria. Application of a novel strategy for sequencing proteins using the polymerase chain reaction. J. Mol. Biol. 226: 1051– 1072. Wilhelmsen, K. C., and Wszollek, Z. K. (1995). Is there a genetic susceptibility to idiopathic Parkinsonism. Parkinsonism Rel. Disorders 1: 73–84. Yoritaka, A., Hattori, N., Uchida, K., Tanaka, M., Stadtman, E. R., and Mizuno, Y. (1996). Immunohistochemical detection of 4-hydroxynonenal protein adducts in Parkinson disease. Proc. Natl. Acad. Sci. USA 93: 2696–2701.
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ARTICLE NO.
Genotype in the 24-kDa Subunit Gene (NDUFV2) of Mitochondrial Complex I and Susceptibility to Parkinson Disease Nobutaka Hattori,1 Hiroyo Yoshino, Masashi Tanaka,* Hiroshi Suzuki,† and Yoshikuni Mizuno Department of Neurology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo, Tokyo 113-8431, Japan; *Department of Gene Therapy, Gihu International Institute of Biotechnology, Yagi Memorial Park, Mitake, Gihu 505-0116, Japan; and †Department of Bioscience, Faculty of Molecular Biology and Biotechnology, Fukui Prefectural Unniversity, Matsuoka-cho, Yoshida-gun, Fukui 910-1195, Japan Received September 18, 1997; accepted December 18, 1997
cause of PD is still unknown, oxidative stress and mitochondrial respiratory failure appear to be two major contributors to nigral degeneration. It is likely that oxidative stress and mitochondrial respiratory failure occur within the nigral neurons as a consequence of this interaction. The increase of superoxide dismutase (Marttila et al., 1988; Saggu et al., 1989), the accumulation of iron (Riederer et al., 1989; Dexter et al., 1990; Good et al., 1992; Jellinger et al., 1992), the loss of glutathione (Riederer et al., 1989; Jenner et al., 1992a), and the increase of lipid peroxidation (Dexter et al., 1989; Yoritaka et al., 1996) are evidence of oxidative stress, while complex I deficiency (Mizuno et al., 1989; Schapira et al., 1989, 1990; Hattori et al., 1991) and the loss of a-ketoglutarate dehydrogenase complex (Mizuno et al., 1994) are evidence of mitochondrial failure. Genetic susceptibility to PD may reside in a polymorphism of genes for proteins and enzymes expressed within the mitochondria and/or regulating free radical metabolism. Thus, although many genetic association studies have been reported (Kondo and Kanazawa, 1991; Armstrong et al., 1992; Smith et al., 1992; Kurth et al., 1993; Kurth and Kurth, 1993; Tsuneoka et al., 1993; Hotamisligil et al., 1994; Ho et al., 1995; Morimoto et al., 1995; Shimoda-Matsubayashi et al., 1996), the results are controversial. Within the central nervous system at least, complex I deficiency appears to be confined to the substantia nigra (SN). We also demonstrated decreased amounts of the 30-, 25-, and 24-kDa subunits of complex I (Mizuno et al., 1989). However, there is no evidence to define the molecular basis of these defects. Previously, we cloned the gene encoding the 24-kDa subunit of complex I (Hattori et al., 1995), which was first cloned by de Coo et al. (1995). Together with the 75- and 51kDa subunits, the 24-kDa subunit forms a structural and functional unit (Ohnishi et al., 1985). These three subunits are closely related to subunits of a soluble NAD/ hydrogenase of the bacterium Alcaligenes eutrophus (Tran-Betcke et al., 1990; Pilkington et al., 1991).
We analyzed the gene encoding the 24-kDa subunit of mitochondrial complex I, which has been implicated in the pathogenesis of Parkinson disease (PD). We set out to identify a polymorphism in the 24-kDa subunit gene (NDUFV2) in patients with PD and determine whether genetic polymorphism of this gene is associated with a higher risk of PD. The subjects comprised 126 patients with PD, and the control group comprised 113 unrelated individuals without neurodegenerative disorders. A novel polymorphism (Ala29Val) in the mitochondrial targeting sequence of NDUFV2 was found in patients with PD. The distribution of the three genotypes was significantly different between the two groups (x2 Å 7.53, df Å 2, P Å 0.023). The frequency of homozygotes for the mutation was significantly higher in PD patients (23.8%) than in control subjects (11.5%, Fisher’s exact test, P Å 0.0099 õ 0.01). The risk of developing PD associated with homozygosity for this mutation was calculated as 2.40 (95% CI: 1.18–4.88). NDUFV2 constitutes one genetic risk factor for PD, and the mutation may well be a cause of complex I deficiency in this disease. q 1998 Academic Press Key Words: Parkinson disease; genetic predisposition; complex I; 24-kDa subunit; mitochondria
INTRODUCTION
In recent years, growing evidence has indicated that the interaction of environmental factors and genetic predisposition initiates the neurodegenerative process in Parkinson disease (PD) (Calne and Langston, 1983; Rajput et al., 1987; Langston, 1989; Jenner et al., 1992b; Payami et al., 1994; Mizuno et al., 1995; Wilhelmsen and Wszollek, 1995). Although the primary Nucleotide sequence data from this article have been deposited with the DDBJ, EMBL, and GenBank Data Libraries under Accession Nos. D88542–D88548. 1 To whom correspondence should be addressed. Telephone: //81-3-5802-1072. Fax: //81-3-3813-7440. E-mail: nhattori@med. juntendo.ac.jp.
0888-7543/98 $25.00 Copyright q 1998 by Academic Press All rights of reproduction in any form reserved.
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ALLELIC ASSOCIATION OF PARKINSON DISEASE AND NDUFV2
The 24-kDa subunit contains one (2Fe–2S) cluster and plays an important role in a substantial part of the electron transport pathway of complex I. The human 24-kDa subunit of complex I is encoded by a single gene (NDUFV2) located on chromosome 18 at region p11.3, which spans 31.5 kb and consists of eight exons (Hattori et al., 1995). To determine whether genetic polymorphism of this gene is associated with a higher risk of PD, we screened all exons of NDUFV2 for mutations in patients with PD, and we found a new polymorphic mutation in the signal peptide of this 24-kDa precursor protein. This paper reports on the new polymorphic mutation and its frequency in PD and control populations. SUBJECTS AND METHODS Subjects. The subjects consisted of 126 PD patients and 113 controls who were followed by the Neurology Service of Juntendo University Medical School Hospital. The average age { SD was 62.7 { 9.3 years for the PD patients and 62.6 { 13.7 for the control subjects. None of the PD patients had a family history of PD or were related to one another. One of the PD patients had sporadic young-onset disease (onset at 38 years old). The criteria used for the diagnosis of idiopathic PD were based on the recently published consensus statement (Calne et al., 1992). All subjects were of Japanese origin. The control subjects consisted of 113 unrelated Japanese individuals who attended our clinic and were diagnosed as not having neurodegenerative disorders. Informed consent was obtained from all the patients and control subjects. Mutation screening of NDUFV2. Human genomic DNA was isolated as described previously (Miller et al., 1988). Samples were either used immediately or stored at 0207C until analysis. All exons (1 to 8) of NDUFV2 were screened for mutations by direct sequencing polymerase chain reaction (PCR) in 20 PD patients randomly selected from among the PD group. PCR was performed with chimera primers that were specific to the sequence for NDUFV2 and also possessed the sequence of the standard sequencing primers (M13 universal and reverse primers) on their 5* ends. The amplified fragments were sequenced by the dideoxy chain termination method using the fluorescence-based automated dye primer sequencing technique with an Applied Biosystems 373A DNA sequencer according to the manufacturer’s instructions (Applied Biosystems Division of Perkin Elmer, Foster City, CA). PCR products for exons 1, 2, 4, 5, 6, and 7 were obtained by nested PCR using two sets of primers. PCR products for exons 3 and 8 were obtained using a single set of primers. The sequences of the oligonucleotide primers are shown in Table 1. Primers for exR and exU are chimera oligonucleotides that possessed the M13 forward universal sequence and the reverse sequence, respectively, at the 5* end. The sequences of these oligonucleotide primers have been deposited with the DNA Data Bank of Japan (DDBJ) (Accession Nos. D64176 and D88542–D88548). PCR conditions are shown in Table 2. One hundred nanograms of each sample was added to a 50-ml reaction mixture containing 50 mM KCl, 10 mM Tris (ph 8.3), 1.5 mM MgCl2 , 0.01% gelatin with 10 pmol of the paired primers, 10 nmol of each dNTP, and 2.5 units of Taq polymerase. LA Taq DNA polymerase (Takara Co., Kyoto, Japan) was used for the first PCRs except for exons 3 and 8. AmpliTaq DNA polymerase (Applied Biosystems Division of Perkin Elmer, Foster City, CA) was used for all the second PCRs including PCRs for exons 3 and 8. The second PCRs were performed with the same PCR mixture containing 1 ml of the first PCR products. The excess primers and dNTPs were removed from the PCR products by Ultrafree-MC centrifugal filter units (Millipore, Tokyo, Japan). The purified PCR products were used as the direct sequencing templates. RFLP-creating PCR screening for Ala29Val mutation in patients with sporadic PD. As will be shown later, we found a novel Ala29-
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Val polymorphic mutation in NDUFV2. We analyzed the frequency of this polymorphism in 126 PD patients and 113 control subjects by RFLP-creating PCR. The Ala29Val mutation (C-T substitution) found in the present study neither created nor abolished any restriction sites. To develop a simple detection method for the C-T substitution, RFLP was artificially induced at the substitution site by introducing a single base mismatch of an oligonucleotide primer. As shown in Table 1, a mismatched primer (ex2AS-mis) and the second primer (ex2S2) were constructed. After amplification, the PCR products were digested with MaeIII (Boehringer Mannheim GmbH, Mannheim, Germany). The products were electrophoresed on a 3.5% agarose gel and visualized with ethidium bromide to detect the 211and (or) 180-bp bands. DNA sequences, amino acid sequences, and protein secondary structure analysis. The DNA sequences and translated amino acid sequences were analyzed using the genetic information processing software Genetyx-Mac Version 8.0 (Software Development Co., Tokyo, Japan). The protein secondary structure was analyzed by theoretical prediction analysis according to the algorithm of Chou and Fasmann (1974). A hydropathy plot of the amino acid sequence was calculated by the method of Kyte and Doolittle (1982) using a window average of seven residues. The computer program used for this analysis was also Genetyx-Mac Version 8.0. Statistical analysis. Statistical analysis was performed with StatView-J Version 4.02 (Abacus Concept Inc., Barkeley, CA), employing the x2 test and Fisher’s exact probability test.
RESULTS
We found a novel structural mutation (a C-to-T transition) in exon 2, which replaces 29 Ala (GCT) with Val (GTT) in the signal peptide. No other mutations were found in the coding region of NDUFV2 among the 20 randomly selected patients. This alanine is conserved among three mammalian species, i.e., bovine (Pilkington and Walker, 1989), human (Pilkington and Walker, 1989; Toda et al., 1989), and rat (Nishikimi et al., 1988) (Fig. 1). The chemical distance (D) between Ala and Val calculated according to the method of Grantham (1974) was relatively large (D Å 64). Computer analysis suggested that replacement of this amino acid caused a change in the hydropathy profile at the first hydrophilic domain (Fig. 2) and that this replacement also caused changes in the secondary structure of the gene product from an a-helix conformation (residues 24–32) to a bsheet conformation (residues 22–32). Thus, the mutant with a valine residue at position 29 shows disruption of the a-helix conformation and a long b-sheet stretch through residues 24–32 (Fig. 3). To investigate the prevalence of this base substitution in a large population, we developed a simple detection method using PCR with a mismatched primer. This procedure creates an artificial RFLP that is affected by the C-T substitution. The polymorphism can be detected as a MaeIII site in exon 2. The MaeIII digestion distinguishes the respective genotypes by their own distinctive restriction patterns (Fig. 4). The mutant allele was found in 32.7% of the 123 PD patients and 38.9% of the 113 control subjects. The frequencies of the mutant allele were not significantly different between the two groups (x2 Å 1.95, P Å 0.16). However, the distribution of the three genotypes was significantly different (x2 Å 7.53, df Å 2, P Å 0.023)
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TABLE 1 Primers Used for Amplification of 24-kDa Subunit Gene (NDUFV2) Sequence (5* r 3*)
Primersa
Region Exon 1
ext EX1S ext EX2AS int EX1S-R int EX1AS-U ext EX2S ext EX4AS int EX2S-R int EX2AS-U EX3S-R EX3AS-U ext EX4S ext EX5AS int EX4S-R int EX4AS-U int EX5S-R int EX5AS-U ext EX6S ext EX7AS int EX6S-R int EX6AS-U int EX7S-R int EX7AS-U EX8S-R EX8AS-U EX2S2 EX2AS-mis
Exon 2
Exon 3 Exons 4 and 5
Exons 6 and 7
Exon 8 PCR for exon2 using mismatched primer
5*-GCG 5*-GGA 5*-CAG 5*-TGT 5*-AGT 5*-AAC 5*-CAG 5*-TGT 5*-CAG 5*-TGT 5*-TAA 5*-GTG 5*-CAG 5*-TGT 5*-CAG 5*-TGT 5*-ATA 5*-CTA 5*-CAG 5*-TGT 5*-CAG 5*-TGT 5*-CAG 5*-TGT 5*-GAT 5*-ATA
CGG TTT GAA AAA TGA TAA GAA AAA GAA AAA CCC TTA GAA AAA GAA AAA ACC TCC GAA AAA GAA AAA GAA AAA GGA AAG
CTG ACC ACA ACG TTC GGC ACA ACG ACA ACG TGA CAA ACA ACG ACA ACG TGG AAA ACA ACG ACA ACG ACA ACG TAG CTC
GGG CCA GCT ACG CAT AAA GCT ACG GCT ACG TAC ATA GCT ACG GCT ACG TCC GCT GCT ACG GCT ACG GCT ACG GGT CTC
AAG ATT ATG GCC TCC AAT ATG GCC ATG GCC TCC TCA ATG GCC ATG GCC TTA CCT ATG GCC ATG GCC ATG GCC AGA CAG
GTG TCC ACC AGT ATT GAA ACC AGT ACC AGT ATT TGT ACC AGT ACC AGT GAG ACC ACC AGT ACC AGT ACC AGT ATA CTC
AAC AGT GT-3* AAG TC-3* GCG CGG CTG GGG AAG CAG TAT GGC AAC CCT GTT GTA-3* TGC CAG-3* AGT TGA TTC CAT TCC GGA TTT ACC CCA ATT CTT GAG AGA ATT TGG CTG CTG CTT TAT GGC TGA TTT-3* ATT CCC T-3* TAA CCC TGA TAC TCC AAC TAA GGC AAA AAT GCT CCA TTA CTA ACA GTG TTA CAA ATA TCA TGT T-3* AA-3* ATA ACC TGG TCC TTA GAA ACC AAC ATC TAA GCA ACA TAG TGA GAC CTA TCC AAA GCT CCT AAC TGG GCT CTT GTG TTA AAG GCC TGC TTG CCA TAT TCC TTA-3* *TA-3* CAT TTT GCG
GTG AAC AGT GT-3* CCC TCG G-3 *
ATT TCC ATA CTT
GTT AAG AGT CTG
GTA-3* TC-3 * CTG-3* GAT-3 *
ATT GAA CTG TGT
TGA TGC TAA ATT
TTT-3* CAG-3 * TTA T-3* CCC T-3*
GAG GGA CTT ACC AGG TAC
TGT T-3* CAC A-3 * GTC-3 * AA-3 * TAA CCA TTA-3* ACC AAA TCC AG-3*
Note. The M13 forward universal sequence and the reverse sequence are underlined. A single base mismatch is indicated by an asterisk. a ext, external reaction; int, internal reaction.
TABLE 2 Fragment Sizes and Thermal Cycling Protocols Fragment size (bp)
Primers pairs Exon 1 EX1S–EX2AS EX1S-R–EX1AS-U Exon 2 EX2S–EX4S EX2S-R–EXAS-U Exon 3 EX3S-R–EXAS-U Exons 4 and 5 EX4S–EX5AS EX4S-R–EX4AS-U EX5S-R–EX5AS-U Exons 6 and 7 EX6S–EX7AS EX6S-R–EXAS-U EX7S-R–EX7AS-U Exon 8 EX8S-R–EX8AS-U PCR for exon 2 using mismatched primer EX2S–EX2AS-mis
PCR conditions
15K 284a
957C/1 min; 987C/20 s, 657C/12 min (30 cycles); 727C/10 min 957C/5 min; 957C/1 min, 637C/1 min, 727C/1 min (35 cycles); 72/7C/7 min
2K 229a
947C/5 min; 947C/1 min, 557C/2 min, 727C/2 min (30 cycles); 727C/7 min 957C/1 min; 957C/30 s, 587C/30 s, 727C/1 min (35 cycles)
242a
957C/1 min; 957C/30 s, 657C/30 s, 727C/1 min (35 cycles)
4K 271a 249a
947C/5 min; 947C/1 min, 557C/1 min, 727C/2 min (30 cycles); 727C/7 min 957C/1 min; 957C/30 s, 587C/30 s, 727C/1 min (35 cycles) 957C/5 min; 957C/30 s, 587C/30 s, 727C/1 min (35 cycles)
2.3K 239a 280a
947C/5 min; 947C/1 min, 557C/1 min, 727C/2 min (30 cycles); 727C/7 min 957C/1 min; 957C/30 s, 607C/30 s, 727C/1 min (35 cycles) 957C/1 min; 957C/30 s, 607C/30 s, 727C/1 min (35 cycles)
283a
957C/1 min; 957C/30 s, 657C/30 s, 727C/1 min (35 cycles)
211
957C/2 min; 957C/1 min, 607C/1 min, 727C/1 min (35 cycles); 727C/7 min
Note. Fragment size is that of the exon plus flanking intronic sequences. EX-R and EX-U are chimera oligonucleotides that possessed the universal and reverse sequences, respectively, at the 5* end. a Fragment size dose not include the sequences of M13 universal and reverse primers.
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FIG. 1. Homology of the amino acid sequences of the signal peptide for the 24-kDa subunit of human, bovine, and rat complex I. Homologous regions are boxed. The arrow indicates the amino acid replaced by the C-to-T transition in exon 2.
(Table 3). Furthermore, the expected frequencies of the three genotypes, under the assumption of Hardy– Weinberg equilibrium, did not differ from the observed frequencies in the control group (x2 Å 1.95, df Å 2, P Å 0.38). On the other hand, there was extreme deviance from Hardy–Weinberg equilibrium in the PD patients (x2 Å 10.18, df Å 2, P Å 0.006 õ 0.01) (Table 3). The frequency of homozygosity for the mutation was significantly higher in the PD patients (23.8%) than in the control subjects (11.5%, Fisher’s exact probability test, P Å 0.0099 õ 0.01). The risk of developing PD associated with homozygosity for this mutation was calculated as 2.40 (95% CI: 1.18–4.88) (Table 2). The frequency of 029Ala homozygotes did not differ significantly between the PD patients and the controls, while the frequency of 029Ala/Val heterozygotes was significantly less common among the PD patients (30.2%) than among the controls (42.5%) (Fisher’s exact probability test, P Å 0.032 õ 0.05). In other words, homozygous mutants occur more frequently and heterozygotes less frequently than expected in the PD patients. DISCUSSION
In our present study, we found a statistically significant difference in the frequency of the novel polymorphism in NDUFV2 in PD patients compared with control subjects. The presence of homozygosity for the Ala29Val mutation increased the risk of developing PD by 2.40-fold. Our results indicate that homozygosity for
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FIG. 3. Secondary structure of the signal peptide for the 24-kDa subunit. The replacement of 029Ala to Val changes the a-helix (at position 24 to 32) to the b-sheet structure (at position 22 to 32).
the Ala29Val mutation in exon 2 of NDUFV2 confers increased susceptibility to PD. The alanine residue located at the polymorphic site was highly conserved among three mammalian species, suggesting the functional importance of this residue. It is predicted that the Ala29Val mutation causes an alteration in the secondary structure of the gene product from an a-helix conformation located in the C-terminal domain of the signal peptide to a b-sheet structure. In the signal pepetide of the 24-kDa subunit, there are two a-helix structures with one b-sheet structure in between. The N-terminal of the signal peptide, which can form an amphiphilic a-helix, seems to be crucial for targeting–recognition events (von Heijne et al., 1989). On the other hand, the a-helix structure of the C-terminal domain of the signal peptide is essential for processing–recognition events by mitochondrial processing peptidase (MPP) (Sjoling et al., 1994). Furthermore, a study based on theoretical analysis of 37 mitochondrial presequences indicates that the a-helical element of the C-terminal domain participates in helix/helix interactions with the peptidase (von Heijne et al., 1989). Thus, the Ala29Val mutation may affect cleavage of the signal peptide, which in turn influences the level of the mature form of 24-kDa subunit of complex I within the mitochondria. Complex I, consisting of at least 41 subunits, is the most complicated of the electron transfer complexes and is composed of subunits encoded by both mitochondrial and nuclear genomes (Walker et al., 1992). Most of the genetic information required for
FIG. 2. Hydropathy plot of the amino acid sequence of the signal peptide for the 24-k Da subunit of human complex I. Positive values indicate hydrophobic regions, whereas negative values indicate hydrophilic regions. The arrow indicates the change in hydrophobicity that is caused by the replacement of 029Ala with Val in the hydrophilic region.
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FIG. 4. MaeIII RFLP of PCR products with a mismatched primer on agarose gel. w/w, m/m, and w/m indicate wildtype homozygote, mutant homozygote, and heterozygote, respectively. M is a 100-bp ladder marker.
mitochondrial biogenesis in complex I is provided by nuclear DNA (nDNA), while only 7 subunits are encoded by mitochondrial DNA (mtDNA). Complex I deficiency is well known to exist in PD, but the cause of this deficiency is unclear. It may be due to exposure to endogenous or exogenous toxins, but it may well be the result of a genetic predisposition. Both
nuclear and mitochondrial DNA mutations can affect the activity of complex I. We have found several point mutations of mtDNA in PD patients that would cause conformational changes in the structure of the gene products (Ikebe et al., 1995), however, we could not find a predominant mtDNA point mutation associated with PD. Shoffner et al. (1993) found a higher incidence of the A-to-G transition at the tRNA Gln gene in PD; however, we could not reproduce their results (unpublished data). As not all the PD patients have this mutation in NDUFV2, complex I deficiency in PD may be due to distinct clusters of mutations in mtDNA and/or nDNA. Although we have not analyzed the relationship between the activity of complex I and the genotypes of NDUFV2, this mutation may well be one of the genetic factors that leads to premature loss of complex I. Such a possibility needs to be studied further. To our knowledge, this is the first report on the association of PD with the genes encoding subunits of complex I. Our findings may thus open up a new pathway for exploring the molecular basis of complex I deficiency in PD and its role in the pathogenesis of this disease.
TABLE 3 NDUFV2 Allele and Genotype Frequencies in PD Patients and Controls
No. subjects No. chromosomes
Control (%)
PD (%)
Total (%)
113 226
126 252
239 478
154 (67.3) 98 (32.7)
306 (64.0) 172 (36.0)
Allele frequency 152 (61.1) 74 (38.9)
029Ala 029Val
x2 Å 1.95, df Å 1, P Å 0.16a Genotype frequency Control (%)
Ala/Ala Ala/Val Val/Val
PD (%)
Observed
Expected
Observed
Expected
Total observed (%)
52 (46.0) 48 (42.5) 13 (11.5)
42 54 17
58 (46.0)b 38 (30.2)c 30 (23.8)d
57 56 13
110 (46.0) 86 (36.0) 43 (18.0)
x2 Å 1.95, df Å 2, P Å 0.38e
x2 Å 10.18, df Å 2, P Å 0.006 f x2 Å 7.53, df Å 2, P Å 0.023 g
a There was no significant difference in allele frequencies between the PD patients and the controls. Expected values were calculated from the allele frequency in the respective groups. b The frequency of Ala/Val heterozygotes was significantly lower in PD than in other homozygotes combined (Fisher’s exact probability test, P Å 0.032 õ 0.05). c The frequency of Val homozygotes is significantly higher in PD than in other genotypes combined (Fisher’s exact probability test, P Å 0.0099 õ 0.01). Odds ratio Å 2.40, 95% CI: 1.18–4.88. d The frequency of Ala homozygotes did not differ significantly between the two groups compared with other two genotypes combined (x2 Å 0.000005, P Å 0.99). e In the controls, the expected frequencies of the genotype did not differ from the observed frequencies. f There was extreme deviance from Hardy–Weinberg equilibrium in the PD patients. g The distribution of the three genotypes was significantly different between the PD patients and the controls.
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ALLELIC ASSOCIATION OF PARKINSON DISEASE AND NDUFV2
sights into the cause of Parkinson’s disease. Neurology 42: 2241– 2250.
ACKNOWLEDGMENTS This work was supported in part by a Grant-in-Aid for Scientific Research on Priority Areas to Y.M. and by a Grant-in-Aid for Scientific Research (08770474 to N.H.) from the Ministry of Education, Science and Culture, Japan, by a Grant-in-Aid for Neurodegenerative Disorders from the Ministry of Health and Welfare, Japan, and by a ‘‘Center of Excellence’’ Grant from the National Parkinson Foundation, Miami, Florida.
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