- Email: [email protected]
N167 polymorphism in Australian Parkinson's disease patients and controls
Parkinsonism & Related Disorders Parkinsonism and Related Disorders 7 (2001) 89±91
www.elsevier.com/locate/parkreldis
The parkin gene S/N167 polymorphism in Australian Parkinson's disease patients and controls G.D. Mellick a,*, D.D. Buchanan a, N. Hattori b, A.J. Brookes c, Y. Mizuno b, D.G. Le Couteur d, P.A. Silburn e a
Department of Medicine, University of Queensland, Princess Alexandra Hospital, Woolloongabba, Queensland, Australia b Department of Neurology, Juntendo University, Tokyo, Japan c Centre for Genomics Research, Karolinska Institute, Stockholm, Sweden d Canberra Clinical School of the University of Sydney, The Canberra Hospital, Canberra, Australia e Department of Neurology, Princess Alexandra Hospital, Woolloongabba, Queensland, Australia Received 14 February 2000; revised 4 April 2000; accepted 5 April 2000
Abstract This study determined the frequencies of a G-to-A transition (S/N167) polymorphism in exon 4 of the parkin gene in Australian Parkinson's disease patients and control subjects. The genotype of each subject was determined using the polymerase chain reaction and restriction-fragment-length-polymorphism analysis. Overall, the A allele was signi®cantly less common in the Parkinson's disease group (1.7%) compared with the control group (3.8%, OR 0.43, 95% CI 0.19±1.00, P , 0.05), although the frequency in the young onset Parkinson's disease group (6.6%) was not signi®cantly different to controls. The A allele is less common in Australian Caucasian subjects compared to Japanese Parkinson's disease patients and appears to be under-represented in older-onset Parkinson's disease. q 2001 Elsevier Science Ltd. All rights reserved. Keywords: Parkin gene; Polymorphism; A allele
1. Introduction Many cases of autosomal recessive juvenile parkinsonism (AR-JP) are secondary to abnormalities in the parkin gene which maps to the long arm of chromosome 6 (6q25.2-q27) [1±4]. This gene codes for a protein of 465 amino acids with an ubiquitin-like amino terminal domain and a ring-®nger carboxy terminus [1]. The exact function of the parkin protein is yet to be elucidated. However, its amino acid composition and localisation to the Golgi complex and neuronal processes of the melanin-containing neurons suggest a role in vesicular transport [5]. Homozygous deletions [4], compound heterozygous deletions and point mutations [3] in the parkin gene have now been reported. In addition, certain parkin gene linked families contain affected members with clinical features indistinguishable from classical idiopathic Parkinson's disease (PD). The frequency of a G-to-A transition polymorphism in exon 4 of the parkin gene has been recently reported in Japanese Parkinson's patients and controls [6]. It results in a * Corresponding author. Tel.: 161-7-3240-5346; fax: 161-7-3240-5399. E-mail address: [email protected] (G.D. Mellick).
serine to asparagine substitution at amino acid 167 of the protein. We now present the frequency of this polymorphism in Caucasian Australian controls and Parkinson's disease patients of both young (,50 years) and older onset.
2. Methods Patients with PD were collected from hospitals, private neurology clinics and community support groups throughout the state of Queensland, Australia. The control group consisted of patients' spouses and other healthy communitybased, age-matched volunteers residing in the same areas and from the same ethnic background as the PD patients. None of the control subjects had diagnosable neurological disorders. PD was diagnosed according to standard criteria [7] if the subject had a combination of three of the following features: resting tremor, rigidity, bradykinesia, postural instability. The diagnosis was also made when at least two of these features were present with asymmetry in tremor, rigidity or bradykinesia. All subjects were of Caucasian background and were examined by a neurologist (generally PAS).
1353-8020/01/$ - see front matter q 2001 Elsevier Science Ltd. All rights reserved. PII: S 1353-802 0(00)00018-3
90
G.D. Mellick et al. / Parkinsonism and Related Disorders 7 (2001) 89±91
analysis, Fisher's exact test and odds ratios (OR). Data is presented as mean ^ standard deviation and signi®cance was taken at the level p , 0.05. 3. Results The average age of the control subjects (134 males and 100 females) was 68.9 ^ 11.9 years. The average age of the PD patients (140 males and 96 females) was 67.6 ^ 11.8 years. Old onset PD patients (104 males and 71 females) had an average age of 72.8 ^ 8.0 years (onset age 65.3 ^ 9.3 years). Young onset patients (36 males and 25 females) were 53.9 ^ 9.2 years old (onset age 43.1 ^ 5.7 years). Allele and genotype frequencies for the S/N167 polymorphism are presented in Table 1. All genotype frequencies conformed to the Hardy±Weinberg equilibrium. Chi-square analysis revealed that the frequency of the A allele was signi®cantly less common in the PD group compared to control subjects (x 2 4.04, OR 0.43, 95% CI 0.19±1.00, d.f. 1, P 0.04). This result re¯ects the under-representation of the A allele in the older onset group (OR 0.29, 95% CI-0.1±0.86, P 0.02). In contrast, the distribution of the A allele was not signi®cantly different between the young onset group compared to the control group (OR 0.85, 95% CI 0.28±2.55, P 0.77).
Fig. 1. Agarose gel electrophoresis (3%) showing the various genotypes for the parkin gene S/N167 polymorphism following the PCR/RFLP assay. Lane 1 contains a DNA marker. Lane 2Ða subject homozygous for the wild-type (G) allele (fragments of 95 and 167 bp). Lane 3Ða heterozygote subject (AG, fragments of 95, 167 and 262 bp). Lane 4Ða subject homozygous for the variant (A) allele (262 bp fragment).
PD patients were divided into two groups: older onset (OOPD, onset of symptoms at 50 years of age or after) and young onset (YOPD, onset of symptoms before 50 years of age). Genomic DNA was extracted form venous blood samples [8]. Exon 4 of the parkin gene was ampli®ed from genomic DNA using the polymerase chain reaction (PCR). The primer sequences used were 5 0 -ACAAGCTTTTAAAGAGTTTCTTGT-3 0 (forward) and 5 0 -AGGCAATGTGTTAGTACACA-3 0 (reverse). The PCR consisted of an initial denaturation period of 10 min at 948C, 35 cycles of 948C for 30 s (denaturing), 548C for 30 s (annealing) and 728C for 45 s (extension), followed by a ®nal extension time of 10 minutes at 728C. The variant allele, substitutes adenine for a guanine (G-to-A) at the codon corresponding to amino acid position 167, eliminating an AlwNI restriction site. Therefore, PCR product was subjected to digestion with the endonuclease AlwNI (Gibco BRL, USA) according to the manufacturer's speci®cations. The restriction-fragment-length-polymorphism (RFLP) assay distinguished the wild-type (two DNA fragments of 95 and 167 bp) from the variant alleles (a single fragment of 262 bp), following 3% agarose gel electrophoresis (see Fig. 1). Allele frequencies were analysed using univariate Chi-square
4. Discussion This study reports the frequency of the S/N167 polymorphism in a Caucasian population. We found that the A allele was present in 3.8% of non-parkinsonian Caucasians which is signi®cantly less than the frequency of 44% previously reported in a Japanese cohort [6] (x 2 188.7, d.f. 1, P 0.001). This represents another example of racial variation in the frequency of gene polymorphisms reported in PD studies. Other examples include the detoxi®cation genes CYP2D6 [9], CYP2E1 and ALDH2 [10].
Table 1 Allele and genotype frequencies of the S/N167 polymorphism in exon 4 of the parkin gene Control
OOPD
YOPD
Total Parkinson's disease
Number of subjects Number of alleles
234 468
175 350
61 122
236 472
Genotype frequency (%) GG GA AA
217(92.7) 16(6.8) 1(0.5)
171(97.7) 4(2.3) 0(0.0)
57(93.4) 4(6.6) 0(0.0)
228(96.6) 8(3.4) 0(0.0)
Allele frequency (%) Allele G Allele A
450(96.2) 18(3.8)
346(98.9) 4(1.1)**
118(96.7) 4(6.6)
464(98.3) 8(1.7)*
** *
OR 0.29 (95% CI 0.10±0.86), P 0.03 (Fisher's exact test). OR 0.43 (95% CI 0.19±1.00), P 0.04.
G.D. Mellick et al. / Parkinsonism and Related Disorders 7 (2001) 89±91
Such racial differences are important to consider in any examination of genetic risk factors for idiopathic PD. We also found that the A allele was signi®cantly less common in subjects with PD. Interestingly, the older onset patients showed a signi®cant under-representation of the A allele compared to the young onset patients, who did not signi®cantly differ from the controls. This suggests that the A allele does not afford protection to younger onset patients but may be protective against older onset disease. These results are in contrast to the ®ndings of the Japanese study where there was no difference between PD patients and controls for this polymorphism [6]. Because the frequency of the A allele is low in Caucasians, a small absolute difference in allelic frequency between patients (1.7%) and controls (3.8%) yielded a statistically signi®cant OR. However, considerably higher A allelic frequencies were observed in Japanese subjects, requiring much larger differences in absolute allelic frequency to yield a similarly signi®cant result. Conversely, small absolute differences in allelic frequency between Japanese patients and controls would not be detected unless vast numbers of subjects were included in the analysis. Therefore, the apparent racial difference in the protective affect of the A allele possibly re¯ects the difference in allelic frequency between Japanese and Caucasian subjects and must be viewed with a degree of caution. The Japanese study found that another polymorphism in the parkin gene (C-to-T transition in exon 10 (R/W366)) was also less common in their subjects with PD. It must be recognised that all case-control studies are limited, in particular by biases involved with the ascertainment of cases and controls. Even so, the association of parkin polymorphisms with sporadic PD in both Japanese and Australian patients with PD suggests a genuine link between this gene and the pathogenesis of PD. The molecular basis for such a result is unclear. The effect of the S/N167 polymorphism on parkin gene function is yet to be determined. However, the exchange of a serine for an asparagine at position 167 of the protein may have little effect on the secondary structure or hydrophobicity of the protein [6]. Further study must also establish how this polymorphism interacts with other abnormalities in the parkin
91
gene and, indeed, other genetic abnormalities that may increase susceptibility to PD. Acknowledgements This study was supported by the Geriatric Medical Foundation of Queensland, The Princess Alexandra Hospital Research and Development Foundation, Parkinson's Queensland Incorporated and the National Health and Medical Research Council of Australia. References [1] Kitada T, Asakawa S, Hattori N, et al. Mutations in the parkin gene cause autosomal recessive juvenile parkinsonism (see comments). Nature 1998;392:605±8. [2] Hattori N, Kitada T, Matsumine H, et al. Molecular genetic analysis of a novel Parkin gene in Japanese families with autosomal recessive juvenile parkinsonism: evidence for variable homozygous deletions in the Parkin gene in affected individuals. Ann Neurol 1998;44:935±41. [3] Hattori N, Matsumine H, Asakawa S, et al. Point mutations (Thr240Arg and Gln311Stop). Biochem Biophys Res Commun 1998;249:754±8. [4] Lucking CB, Abbas N, Durr A, et al. Homozygous deletions in parkin gene in European and North African families with autosomal recessive juvenile parkinsonism. Lancet 1998;352:1355±6 (The European Consortium on Genetic Susceptibility in Parkinson's Disease and the French Parkinson's Disease Genetics Study Group). [5] Shimura H, Hattori N, Kubo S, et al. Immunohistochemical and subcellular localization of Parkin protein: absence of protein in autosomal recessive juvenile parkinsonism patients. Ann Neurol 1999;45:668±72. [6] Wang M, Hattori N, Matsumine H, et al. Polymorphism in the parkin gene in sporadic Parkinson's disease. Ann Neurol 1999;45:655±8. [7] Calne DB, Snow BJ, Lee C. Criteria for diagnosing Parkinson's disease. Ann Neurol 1992;32:S125±7. [8] Douglas AM, Georgalis AM, Benton LR, Canavan KL, Atchison BA. Puri®cation of human leucocyte DNA: proteinase K is not necessary. Anal Biochem 1992;201:362±5. [9] Wang SL, Huang JD, Lai MD, Liu BH, Lai ML. Molecular basis of genetic variation in debrisoquin hydroxylation in Chinese subjects: polymorphism in RFLP and DNA sequence of CYP2D6. Clin Pharmacol Ther 1993;53:410±8. [10] Iwahashi K, Matsuo Y, Suwaki H, Nakamura K, Ichikawa Y. CYP2E1 and ALDH2 genotypes and alcohol dependence in Japanese. Alcohol Clin Exp Res 1995;19:564±6.
www.elsevier.com/locate/parkreldis
The parkin gene S/N167 polymorphism in Australian Parkinson's disease patients and controls G.D. Mellick a,*, D.D. Buchanan a, N. Hattori b, A.J. Brookes c, Y. Mizuno b, D.G. Le Couteur d, P.A. Silburn e a
Department of Medicine, University of Queensland, Princess Alexandra Hospital, Woolloongabba, Queensland, Australia b Department of Neurology, Juntendo University, Tokyo, Japan c Centre for Genomics Research, Karolinska Institute, Stockholm, Sweden d Canberra Clinical School of the University of Sydney, The Canberra Hospital, Canberra, Australia e Department of Neurology, Princess Alexandra Hospital, Woolloongabba, Queensland, Australia Received 14 February 2000; revised 4 April 2000; accepted 5 April 2000
Abstract This study determined the frequencies of a G-to-A transition (S/N167) polymorphism in exon 4 of the parkin gene in Australian Parkinson's disease patients and control subjects. The genotype of each subject was determined using the polymerase chain reaction and restriction-fragment-length-polymorphism analysis. Overall, the A allele was signi®cantly less common in the Parkinson's disease group (1.7%) compared with the control group (3.8%, OR 0.43, 95% CI 0.19±1.00, P , 0.05), although the frequency in the young onset Parkinson's disease group (6.6%) was not signi®cantly different to controls. The A allele is less common in Australian Caucasian subjects compared to Japanese Parkinson's disease patients and appears to be under-represented in older-onset Parkinson's disease. q 2001 Elsevier Science Ltd. All rights reserved. Keywords: Parkin gene; Polymorphism; A allele
1. Introduction Many cases of autosomal recessive juvenile parkinsonism (AR-JP) are secondary to abnormalities in the parkin gene which maps to the long arm of chromosome 6 (6q25.2-q27) [1±4]. This gene codes for a protein of 465 amino acids with an ubiquitin-like amino terminal domain and a ring-®nger carboxy terminus [1]. The exact function of the parkin protein is yet to be elucidated. However, its amino acid composition and localisation to the Golgi complex and neuronal processes of the melanin-containing neurons suggest a role in vesicular transport [5]. Homozygous deletions [4], compound heterozygous deletions and point mutations [3] in the parkin gene have now been reported. In addition, certain parkin gene linked families contain affected members with clinical features indistinguishable from classical idiopathic Parkinson's disease (PD). The frequency of a G-to-A transition polymorphism in exon 4 of the parkin gene has been recently reported in Japanese Parkinson's patients and controls [6]. It results in a * Corresponding author. Tel.: 161-7-3240-5346; fax: 161-7-3240-5399. E-mail address: [email protected] (G.D. Mellick).
serine to asparagine substitution at amino acid 167 of the protein. We now present the frequency of this polymorphism in Caucasian Australian controls and Parkinson's disease patients of both young (,50 years) and older onset.
2. Methods Patients with PD were collected from hospitals, private neurology clinics and community support groups throughout the state of Queensland, Australia. The control group consisted of patients' spouses and other healthy communitybased, age-matched volunteers residing in the same areas and from the same ethnic background as the PD patients. None of the control subjects had diagnosable neurological disorders. PD was diagnosed according to standard criteria [7] if the subject had a combination of three of the following features: resting tremor, rigidity, bradykinesia, postural instability. The diagnosis was also made when at least two of these features were present with asymmetry in tremor, rigidity or bradykinesia. All subjects were of Caucasian background and were examined by a neurologist (generally PAS).
1353-8020/01/$ - see front matter q 2001 Elsevier Science Ltd. All rights reserved. PII: S 1353-802 0(00)00018-3
90
G.D. Mellick et al. / Parkinsonism and Related Disorders 7 (2001) 89±91
analysis, Fisher's exact test and odds ratios (OR). Data is presented as mean ^ standard deviation and signi®cance was taken at the level p , 0.05. 3. Results The average age of the control subjects (134 males and 100 females) was 68.9 ^ 11.9 years. The average age of the PD patients (140 males and 96 females) was 67.6 ^ 11.8 years. Old onset PD patients (104 males and 71 females) had an average age of 72.8 ^ 8.0 years (onset age 65.3 ^ 9.3 years). Young onset patients (36 males and 25 females) were 53.9 ^ 9.2 years old (onset age 43.1 ^ 5.7 years). Allele and genotype frequencies for the S/N167 polymorphism are presented in Table 1. All genotype frequencies conformed to the Hardy±Weinberg equilibrium. Chi-square analysis revealed that the frequency of the A allele was signi®cantly less common in the PD group compared to control subjects (x 2 4.04, OR 0.43, 95% CI 0.19±1.00, d.f. 1, P 0.04). This result re¯ects the under-representation of the A allele in the older onset group (OR 0.29, 95% CI-0.1±0.86, P 0.02). In contrast, the distribution of the A allele was not signi®cantly different between the young onset group compared to the control group (OR 0.85, 95% CI 0.28±2.55, P 0.77).
Fig. 1. Agarose gel electrophoresis (3%) showing the various genotypes for the parkin gene S/N167 polymorphism following the PCR/RFLP assay. Lane 1 contains a DNA marker. Lane 2Ða subject homozygous for the wild-type (G) allele (fragments of 95 and 167 bp). Lane 3Ða heterozygote subject (AG, fragments of 95, 167 and 262 bp). Lane 4Ða subject homozygous for the variant (A) allele (262 bp fragment).
PD patients were divided into two groups: older onset (OOPD, onset of symptoms at 50 years of age or after) and young onset (YOPD, onset of symptoms before 50 years of age). Genomic DNA was extracted form venous blood samples [8]. Exon 4 of the parkin gene was ampli®ed from genomic DNA using the polymerase chain reaction (PCR). The primer sequences used were 5 0 -ACAAGCTTTTAAAGAGTTTCTTGT-3 0 (forward) and 5 0 -AGGCAATGTGTTAGTACACA-3 0 (reverse). The PCR consisted of an initial denaturation period of 10 min at 948C, 35 cycles of 948C for 30 s (denaturing), 548C for 30 s (annealing) and 728C for 45 s (extension), followed by a ®nal extension time of 10 minutes at 728C. The variant allele, substitutes adenine for a guanine (G-to-A) at the codon corresponding to amino acid position 167, eliminating an AlwNI restriction site. Therefore, PCR product was subjected to digestion with the endonuclease AlwNI (Gibco BRL, USA) according to the manufacturer's speci®cations. The restriction-fragment-length-polymorphism (RFLP) assay distinguished the wild-type (two DNA fragments of 95 and 167 bp) from the variant alleles (a single fragment of 262 bp), following 3% agarose gel electrophoresis (see Fig. 1). Allele frequencies were analysed using univariate Chi-square
4. Discussion This study reports the frequency of the S/N167 polymorphism in a Caucasian population. We found that the A allele was present in 3.8% of non-parkinsonian Caucasians which is signi®cantly less than the frequency of 44% previously reported in a Japanese cohort [6] (x 2 188.7, d.f. 1, P 0.001). This represents another example of racial variation in the frequency of gene polymorphisms reported in PD studies. Other examples include the detoxi®cation genes CYP2D6 [9], CYP2E1 and ALDH2 [10].
Table 1 Allele and genotype frequencies of the S/N167 polymorphism in exon 4 of the parkin gene Control
OOPD
YOPD
Total Parkinson's disease
Number of subjects Number of alleles
234 468
175 350
61 122
236 472
Genotype frequency (%) GG GA AA
217(92.7) 16(6.8) 1(0.5)
171(97.7) 4(2.3) 0(0.0)
57(93.4) 4(6.6) 0(0.0)
228(96.6) 8(3.4) 0(0.0)
Allele frequency (%) Allele G Allele A
450(96.2) 18(3.8)
346(98.9) 4(1.1)**
118(96.7) 4(6.6)
464(98.3) 8(1.7)*
** *
OR 0.29 (95% CI 0.10±0.86), P 0.03 (Fisher's exact test). OR 0.43 (95% CI 0.19±1.00), P 0.04.
G.D. Mellick et al. / Parkinsonism and Related Disorders 7 (2001) 89±91
Such racial differences are important to consider in any examination of genetic risk factors for idiopathic PD. We also found that the A allele was signi®cantly less common in subjects with PD. Interestingly, the older onset patients showed a signi®cant under-representation of the A allele compared to the young onset patients, who did not signi®cantly differ from the controls. This suggests that the A allele does not afford protection to younger onset patients but may be protective against older onset disease. These results are in contrast to the ®ndings of the Japanese study where there was no difference between PD patients and controls for this polymorphism [6]. Because the frequency of the A allele is low in Caucasians, a small absolute difference in allelic frequency between patients (1.7%) and controls (3.8%) yielded a statistically signi®cant OR. However, considerably higher A allelic frequencies were observed in Japanese subjects, requiring much larger differences in absolute allelic frequency to yield a similarly signi®cant result. Conversely, small absolute differences in allelic frequency between Japanese patients and controls would not be detected unless vast numbers of subjects were included in the analysis. Therefore, the apparent racial difference in the protective affect of the A allele possibly re¯ects the difference in allelic frequency between Japanese and Caucasian subjects and must be viewed with a degree of caution. The Japanese study found that another polymorphism in the parkin gene (C-to-T transition in exon 10 (R/W366)) was also less common in their subjects with PD. It must be recognised that all case-control studies are limited, in particular by biases involved with the ascertainment of cases and controls. Even so, the association of parkin polymorphisms with sporadic PD in both Japanese and Australian patients with PD suggests a genuine link between this gene and the pathogenesis of PD. The molecular basis for such a result is unclear. The effect of the S/N167 polymorphism on parkin gene function is yet to be determined. However, the exchange of a serine for an asparagine at position 167 of the protein may have little effect on the secondary structure or hydrophobicity of the protein [6]. Further study must also establish how this polymorphism interacts with other abnormalities in the parkin
91
gene and, indeed, other genetic abnormalities that may increase susceptibility to PD. Acknowledgements This study was supported by the Geriatric Medical Foundation of Queensland, The Princess Alexandra Hospital Research and Development Foundation, Parkinson's Queensland Incorporated and the National Health and Medical Research Council of Australia. References [1] Kitada T, Asakawa S, Hattori N, et al. Mutations in the parkin gene cause autosomal recessive juvenile parkinsonism (see comments). Nature 1998;392:605±8. [2] Hattori N, Kitada T, Matsumine H, et al. Molecular genetic analysis of a novel Parkin gene in Japanese families with autosomal recessive juvenile parkinsonism: evidence for variable homozygous deletions in the Parkin gene in affected individuals. Ann Neurol 1998;44:935±41. [3] Hattori N, Matsumine H, Asakawa S, et al. Point mutations (Thr240Arg and Gln311Stop). Biochem Biophys Res Commun 1998;249:754±8. [4] Lucking CB, Abbas N, Durr A, et al. Homozygous deletions in parkin gene in European and North African families with autosomal recessive juvenile parkinsonism. Lancet 1998;352:1355±6 (The European Consortium on Genetic Susceptibility in Parkinson's Disease and the French Parkinson's Disease Genetics Study Group). [5] Shimura H, Hattori N, Kubo S, et al. Immunohistochemical and subcellular localization of Parkin protein: absence of protein in autosomal recessive juvenile parkinsonism patients. Ann Neurol 1999;45:668±72. [6] Wang M, Hattori N, Matsumine H, et al. Polymorphism in the parkin gene in sporadic Parkinson's disease. Ann Neurol 1999;45:655±8. [7] Calne DB, Snow BJ, Lee C. Criteria for diagnosing Parkinson's disease. Ann Neurol 1992;32:S125±7. [8] Douglas AM, Georgalis AM, Benton LR, Canavan KL, Atchison BA. Puri®cation of human leucocyte DNA: proteinase K is not necessary. Anal Biochem 1992;201:362±5. [9] Wang SL, Huang JD, Lai MD, Liu BH, Lai ML. Molecular basis of genetic variation in debrisoquin hydroxylation in Chinese subjects: polymorphism in RFLP and DNA sequence of CYP2D6. Clin Pharmacol Ther 1993;53:410±8. [10] Iwahashi K, Matsuo Y, Suwaki H, Nakamura K, Ichikawa Y. CYP2E1 and ALDH2 genotypes and alcohol dependence in Japanese. Alcohol Clin Exp Res 1995;19:564±6.