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Restriction fragment length polymorphism assay for the Ala53Thr mutation in exon 4 of the α-synuclein gene. All DNA samples from 207 Chinese patients with PD and 227 unaffected controls yielded a single DNA band, which corresponded to the undigested 216–base pair (bp) polymerase chain reaction product (lanes 2-11 display representative samples). DNA from an affected member of the Contursi kindred (lane 12) yielded 128- and 88-bp fragments together with the 216-bp fragment, indicating the presence of the Ala53Thr mutation. Lane 1 contains the ϕXI74/Hin fI DNA marker.

Restriction fragment length polymorphism assay for the Ala53Thr mutation in exon 4 of the α-synuclein gene. All DNA samples from 207 Chinese patients with PD and 227 unaffected controls yielded a single DNA band, which corresponded to the undigested 216–base pair (bp) polymerase chain reaction product (lanes 2-11 display representative samples). DNA from an affected member of the Contursi kindred (lane 12) yielded 128- and 88-bp fragments together with the 216-bp fragment, indicating the presence of the Ala53Thr mutation. Lane 1 contains the ϕXI74/Hin fI DNA marker.

1.
Polymeropoulos  MHLavedan  CLeroy  E  et al.  Mutation in the alpha-synuclein gene identified in families with Parkinson's disease. Science. 1997;2762045- 2047Article
2.
Kruger  RKuhn  WMuller  T  et al.  Ala30Pro mutation in the gene encoding alpha-synuclein in Parkinson's disease. Nat Genet. 1998;18106- 108Article
3.
Goedert  M The awakening of alpha-synuclein. Nature. 1997;388232- 233Article
4.
Bennett  PNicholl  DJ Absence of the G209A mutation in the alpha-synuclein gene in British families with Parkinson's disease. Neurology. 1998;5033Article
5.
Chan  PTanner  CMJiang  XLangston  JW Failure to find the alpha-synuclein gene missense mutation (G209A) in 100 patients with younger onset Parkinson's disease. Neurology. 1998;50513- 514Article
6.
Munoz  DOliva  RObach  V  et al.  Identification of Spanish familial Parkinson's disease and screening for the Ala53Thr mutation of the alpha-synuclein gene in early onset patients. Neurosci Lett. 1997;23557- 60Article
7.
Hu  CJSung  SMLiu  HC  et al.  No mutation of G209A in the alpha-synuclein gene in sporadic Parkinson's disease among Taiwan Chinese. Eur Neurol. 1999;4185- 87Article
8.
Maranganore  DMHarding  AEMarsden  CD A clinical and genetic study of familial Parkinson's disease. Mov Disord. 1991;6205- 211Article
9.
Chan  DKYWoo  JHo  SC  et al.  Genetic and environmental risk factors for Parkinson's disease in a Chinese population. J Neurol Neurosurg Psychiatry. 1998;65781- 784Article
10.
Miller  SADykes  DDPolesky  HF A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res. 1988;1633
11.
Vaughan  JRFarrer  MJWszolek  ZK  et al.  Sequencing of the alpha-synuclein gene in a large series of cases of familial Parkinson's disease fails to reveal any further mutations: the European Consortium on Genetic Susceptibility in Parkinson's Disease (GSPD). Hum Mol Genet. 1998;7751- 753Article
12.
French Parkinson's Disease Genetics Study Group, Alpha-synuclein gene and Parkinson's disease. Science. 1998;2791116- 1117
13.
Chan  PJiang  XForno  LSDi Monte  DATanner  CMLanston  JW Absence of mutations in the coding region of the alpha-synuclein gene in pathologically proven Parkinson's disease. Neurology. 1998;501136- 1137Article
Original Contribution
April 2000

The α-Synuclein Gene and Parkinson Disease in a Chinese Population

Author Affiliations

From the Departments of Geriatric Medicine (Dr Chan) and Cardiovascular Medicine (Dr Wang), Prince of Wales Hospital, Sydney, Australia; Departments of Medicine and Neurology, Princess Alexandra Hospital, University of Queensland, Brisbane, Australia (Dr Mellick); Department of Medicine and Geriatrics, United Christian Hospital, Hong Kong (Dr Ng); and Departments of Clinical Pathology (Dr Pang) and Medicine (Drs Woo and Kay), Chinese University of Hong Kong, Hong Kong. Dr Cai is a PhD student at the Prince of Wales Hospital.

Arch Neurol. 2000;57(4):501-503. doi:10.1001/archneur.57.4.501
Abstract

Objective  To study the Ala53Thr and Ala30Pro mutations of the α-synuclein gene in a large number of Chinese patients with Parkinson disease (PD) as well as controls.

Methods  We recruited 183 Chinese patients with sporadic PD, 17 with younger-onset PD (onset age <50 years), and 7 with PD and a positive family history as well as 227 unaffected Chinese control subjects from the outpatient departments of 2 major hospitals in Hong Kong. All subjects were assessed for the the diagnosis of PD by a consultant neurologist or geriatrician. Subjects were interviewed with a standard questionnaire that also questioned for family history. Venous blood samples were obtained from the subjects and genomic DNA was extracted and studied for the presence of Ala53Thr mutation in exon 4 and Ala30Pro mutation in exon 3 of the α-synuclein gene using a polymerase chain reaction restriction fragment length polymorphism method.

Results  None of the Chinese PD patients or controls had either the Ala53Thr (exon 4) or Ala30Pro (exon 3) mutation of the α-synuclein gene.

Conclusion  We failed to discover Ala53Thr or Ala30Pro mutations in a large number of Chinese patients with PD and control subjects, adding to the emerging consensus that variations in the α-synuclein gene are associated with PD in few families worldwide.

THE GENE that codes for the presynaptic protein α-synuclein is associated with dominantly inherited parkinsonism in the Italian Contursi kindred and 3 unrelated Greek families.1 In these families, affected members have a missense mutation G209A in exon 4, which results in an exchange of an alanine to a threonine at position 53 of the amino acid sequence (Ala53Thr). A second α-synuclein mutation associated with Parkinson disease (PD) has been recently described by Kruger et al.2 This variant is characterized by a cytosine-to-guanine transversion at nucleotide position 88 of the coding sequence (exon 3), which leads to a change at position 30 of the amino acid sequence (Ala30Pro). α-Synuclein is a precursor to constituents of Alzheimer disease plaques and is found in Lewy bodies, the eosinophilic inclusion bodies that define neuropathological characteristics of PD. The function of α-synuclein is unknown, although it is thought to be involved with neuronal plasticity and may mediate binding between structural proteins.3 Recent studies have failed to find the Ala53Thr mutation in subjects of European Caucasian ancestry with familial,4 younger-onset,5 and sporadic6 PD. As for the Chinese, there has been only one small study7 looking for the Ala53Thr mutation in 65 cases of sporadic PD in Taiwan Chinese patients. We therefore undertook a large-scale study to look for both Ala53Thr and Ala30Pro mutations. We investigated 183 sporadic, 17 younger-onset (onset age of younger than 50 years), and 7 Chinese patients with a positive family history together with 227 unaffected Chinese subjects for the presence of the Ala53Thr and Ala30Pro mutations in the α-synuclein gene.

SUBJECTS AND METHODS

Patients and controls were recruited from the outpatient departments of the United Christian and Prince of Wales hospitals in Hong Kong. All subjects were assessed by a consultant neurologist or geriatrician for the diagnosis of PD. The criteria for diagnosis is in accordance with those described by Maranganore et al.8 In the selection of controls, 10-year age group, sex, and locality of residence were taken into consideration to make the control group as comparable to the PD group as possible. (For more details of the study, refer to Chan et al.9)

The subjects were interviewed with a standard questionnaire and were questioned for family history of PD. If there was a family history, further details regarding which relatives were affected (ie, parents, siblings, uncles, aunts, nephews, or nieces) were obtained. Venous blood samples were taken from the subjects, and genomic DNA was extracted by the salting out method.10

The patients and controls were screened for the presence of the Ala53Thr mutation in exon 4 of the α-synuclein gene (the Contursi mutation) using the polymerase chain reaction (PCR) Ysp 45I restriction fragment length polymorphism (RFLP) method described by Polymeropoulos et al.1 DNA that was obtained from an affected Contursi kindred member and contained the Ala53Thr mutation was kindly donated by Polymeropoulos et al and used as a positive control. The primer sequences used for the DNA amplification were 5′-GCTAATCAGCAATTTAAGGCTAG-3′ (forward) and 5′-GATATGTTCTTAGATGCTCAG-3′ (reverse). The amplification reaction was performed in a 50-µL volume that contained 100 ng of template DNA, 300 ng of each primer, 2-mmol/L magnesium chloride, 0.24-mmol/L deoxynucleotidetriphosphates, and 2 U of DNA polymerase (Red Hot DNA polymerase; Advanced Biotechnologies, Surrey, England). The samples were subjected to an initial denaturation at 94°C for 5 minutes followed by 17 cycles of denaturation at 94°C for 15 seconds, annealing at 50°C for 30 seconds, and extension at 72°C for 30 seconds. The second phase of the amplification reaction involved 18 cycles of denaturation (94°C for 15 seconds), annealing (50°C for 30 seconds), and extension (72°C for 1 minute 30 seconds). The 216–base pair (bp) PCR product was digested with the restriction enzyme Tsp 45I (New England Bio Labs, Beverly, Mass) according to the supplier's protocol, resolved on a 3% agarose gel (agarose 1000; Life Technologies, Melbourne, Australia), and visualized using ethidium bromide staining. Mutant alleles yield restriction fragments of 128 and 88 bp using this technique (Figure 1).

The Ala30Pro mutation in exon 3 of the α-synuclein gene was examined using the Mva I-based PCR-RFLP method previously described by Kruger et al.2 In this case, the forward and reverse primers were 5′-AAGTGTATTTTATGTTTTCC-3′ and 5′-AACTGACATTTGGGGTTTACC-3′, respectively. The PCR reactions (25 µL) contained 100 ng of template DNA, 20 pmol/L of each primer, 2.1-mmol/L magnesium chloride, 0.225-mmol/L deoxynucleotidetriphosphates, and 1 U of DNA polymerase. The samples were subjected to an initial denaturation at 95°C for 5 minutes followed by 35 cycles of denaturation at 95°C for 1 minute, annealing at 54°C for 1 minute, and extension at 72°C for 1 minute. A final extension period of 72°C for 5 minutes completed the reaction. The 192-bp PCR products were digested with Mva I restriction enzyme and resolved using 8% polyacrylamide gel electrophoresis followed by silver staining. Alleles containing the Ala30Pro mutation yield 136- and 56-bp fragments following Mva I digestion.

RESULTS

The patients with PD and a positive family history consisted of 2 female siblings, 2 with an affected mother, 1 with an affected father, brother, and uncle, and 2 with secondary relatives with PD. Two control subjects reported secondary relatives with PD. None of the Chinese patients with PD or controls possessed either the Ala53Thr (exon 4) or the Ala30Pro (exon 3) mutation of the α-synuclein gene.

Figure 1 shows that DNA from an affected Contursi kindred subject who possesses the Ala53Thr mutation yields RFLPs characteristic of the mutant allele. No such fragments were found for any of the Chinese patients and controls.

COMMENT

The failure to discover the Ala53Thr or Ala30Pro mutations in a large number of Chinese patients with PD and control subjects adds to the emerging consensus that variations in the α-synuclein gene are associated with PD in few families worldwide. Moreover, studies have thus far failed to reveal other mutations in the coding region of the α-synuclein gene in large groups of patients with familial11,12 and sporadic13 PD. Studies are yet to determine whether any variations in the regulatory or intronic regions of the α-synuclein gene are associated with PD. Efforts may be better spent in elucidating the mechanism by which the Ala53Thr and Ala30Pro variants of α-synuclein lead to neurodegeneration in individuals possessing these mutations. Such mechanistic information may suggest other candidate genes with causative variants potentially more common in patients with PD worldwide than the Ala53Thr or Ala30Pro variants of α-synuclein.

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Article Information

Accepted for publication November 5, 1999.

Corresponding author: Daniel K. Y. Chan, MBBS, FRACP, Department of Geriatric Medicine, Prince of Wales Hospital, Level 3, High Street Building, High Street, Randwick NSW 2031, Sydney, Australia.

References
1.
Polymeropoulos  MHLavedan  CLeroy  E  et al.  Mutation in the alpha-synuclein gene identified in families with Parkinson's disease. Science. 1997;2762045- 2047Article
2.
Kruger  RKuhn  WMuller  T  et al.  Ala30Pro mutation in the gene encoding alpha-synuclein in Parkinson's disease. Nat Genet. 1998;18106- 108Article
3.
Goedert  M The awakening of alpha-synuclein. Nature. 1997;388232- 233Article
4.
Bennett  PNicholl  DJ Absence of the G209A mutation in the alpha-synuclein gene in British families with Parkinson's disease. Neurology. 1998;5033Article
5.
Chan  PTanner  CMJiang  XLangston  JW Failure to find the alpha-synuclein gene missense mutation (G209A) in 100 patients with younger onset Parkinson's disease. Neurology. 1998;50513- 514Article
6.
Munoz  DOliva  RObach  V  et al.  Identification of Spanish familial Parkinson's disease and screening for the Ala53Thr mutation of the alpha-synuclein gene in early onset patients. Neurosci Lett. 1997;23557- 60Article
7.
Hu  CJSung  SMLiu  HC  et al.  No mutation of G209A in the alpha-synuclein gene in sporadic Parkinson's disease among Taiwan Chinese. Eur Neurol. 1999;4185- 87Article
8.
Maranganore  DMHarding  AEMarsden  CD A clinical and genetic study of familial Parkinson's disease. Mov Disord. 1991;6205- 211Article
9.
Chan  DKYWoo  JHo  SC  et al.  Genetic and environmental risk factors for Parkinson's disease in a Chinese population. J Neurol Neurosurg Psychiatry. 1998;65781- 784Article
10.
Miller  SADykes  DDPolesky  HF A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res. 1988;1633
11.
Vaughan  JRFarrer  MJWszolek  ZK  et al.  Sequencing of the alpha-synuclein gene in a large series of cases of familial Parkinson's disease fails to reveal any further mutations: the European Consortium on Genetic Susceptibility in Parkinson's Disease (GSPD). Hum Mol Genet. 1998;7751- 753Article
12.
French Parkinson's Disease Genetics Study Group, Alpha-synuclein gene and Parkinson's disease. Science. 1998;2791116- 1117
13.
Chan  PJiang  XForno  LSDi Monte  DATanner  CMLanston  JW Absence of mutations in the coding region of the alpha-synuclein gene in pathologically proven Parkinson's disease. Neurology. 1998;501136- 1137Article
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