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Original Contribution |

Failure to Find α-Synuclein Gene Dosage Changes in 190 Patients With Familial Parkinson Disease FREE

Suzana Gispert, PhD; Claudia Trenkwalder, MD; Luisa Mota-Vieira, PhD; Vladimir Kostic, MD; Georg Auburger, MD
[+] Author Affiliations

Author Affiliations: From the Institute for Experimental Neurobiology (Dr Gispert) and Section of Molecular Neurogenetics, Clinic for Neurology (Dr Auburger), University Hospital, Frankfurt/Main, Germany; Paracelsus Elena Clinic, Kassel, Germany (Dr Trenkwalder); Genetics and Molecular Pathology Unit, Hospital of Divino Espírito Santo, Ponta Delgada, Azores, Portugal (Dr Mota-Vieira); and Institute of Neurology, Clinical Center at Serbia, Belgrade, Yugoslavia (Dr Kostic).


Arch Neurol. 2005;62(1):96-98. doi:10.1001/archneur.62.1.96.
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Published online

Background  Recently, a triplication of the α-synuclein locus was found associated with autosomal dominant Parkinson disease in a large family.

Objective  To determine whether a triplication or some other dosage alteration in the α-synuclein gene is pres-ent in one or more patients with familial PD in a large multinational collective.

Design  Retrospective recruitment of the largest families who were willing to cooperate with the study.

Setting  Centers with specialization in movement disorders genetics.

Patients  One hundred ninety unrelated patients with familial PD from Germany, Portugal, and Yugoslavia.

Main Outcome Measures  α-Synuclein gene dosage values measured with real-time polymerase chain reaction.

Results  None of the samples showed α-synuclein triplication, duplication, or deletion.

Conclusion  Alterations in α-synuclein gene dosage are rare in familial PD.

In recent years, investigations of families in which several members were affected by Parkinson disease (PD) have led to the chromosomal localization or identification of a series of Parkinson genes (Park1 to Park11). Because mutations in Park1 (α-synuclein) can lead to early onset, severe clinical picture, and high penetrance of autosomal dominant PD, α-synuclein was the first gene discovered to underlie a monogenic form of PD. The missense mutations A53T,1 A30P,2 and E46K3 in α-synuclein have each been described in single families with autosomal dominant PD from Italy, Germany, and Spain. Recently, a triplication of the α-synuclein locus4 was reported in a family with autosomal dominant PD from Iowa who had early onset (Park4). All mutations of Park genes have so far proved to be rare,5 and only fragmentary evidence of interactions between the respective Park proteins permits some hypotheses about pathogenetic pathways in this common disease. Since abnormally aggregated α-synuclein in Lewy bodies and Lewy neurites is a diagnostic hallmark in all patients with PD, and since efficient degradation of α-synuclein by ubiquitination or the proteasome appears to be com-promised in PD, the strict control of α-synuclein protein and transcript levels might be a key issue in the pathogenesis of PD in all patients. Thus, it is important to determine the frequency of triplications and other possible gene dosage alterations of α-synuclein in PD collectives. For this purpose, we used a large multinational collective of 190 familial PD cases.

The diagnosis of idiopathic PD was based on the presence of akinetic-rigid symptoms according to the UK Brain Bank Criteria6 with asymmetric onset, resting tremor, and positive response to levodopa. The positive family history was based on information from the index patient regarding other family members with PD or tremor. Patients with diagnoses of atypical or vascular Parkinson syndrome, multisystem atrophy, or subacute arteriosclerotic encephalopathy were excluded from the recruitment.

The 190 unrelated patients with PD characterized in this study included 84 women and 106 men, manifested the disease at ages 23 to 78 years (average, 53 ± 11 years), and originated from Germany (180 cases), Portugal (6 cases), and Yugoslavia (4 cases). Sixty-nine patients had early onset, before 50 years of age. Among 156 patients with vertical transmission in the family history, there were 95 patients with a first-degree relative affected and 40 patients with 2 to 4 affected relatives; among 34 patients with horizontal transmission there were 29 patients with a first-degree relative affected and 8 patients with 2 affected relatives.

After approval of the recruitment protocol by the local ethics committee, written informed consent was obtained from each family member. Venous whole blood samples in EDTA were obtained, stored frozen at –80°C, and extracted for genomic DNA by conventional salt methods. In previous studies, the A53T mutation in α-synuclein and the I93M mutation in the Park5 gene have been demonstrated in our collective,7 and both cases were included in the study as negative controls. Furthermore, the same DNA of one male individual with no signs of PD and a molecular diagnosis of spinocerebellar ataxia type 2 served as standard in all polymerase chain reaction rounds.

For real-time polymerase chain reaction amplification of the test locus α-synuclein, we used the following primers and minor groove binder (MGB) probes previously described by Singleton et al4 :

  • Q-synuclein-3-VIC (5′-AGCCATGGATGTATTC-3′)

  • Q-synuclein-3F (5′-TTCCAGTGTGGTGTAAAGAAATTCAT-3′)

  • Q-synuclein-3R (5′-CCTTGGCCTTTGAAAGTCCTT-3′)

  • Q-synuclein-4-VIC (5′-TGTCTTGAATTTGTTTTTGTAGGC-3′)

  • Q-synuclein-4F (5′-CAGCAATTTAAGGCTAGCTTGGACT-3′)

  • Q-synuclein-4R (5′-CCACTCCCTCCTTGGTTTTG-3′)

For the amplification of a reference locus, we used β-globin primers and a TAMRA probe:

  • β-Globin-FAM (5′-CTCATGGCAAGAAAGTGCTCGGTGC-3′)

  • β-Globin-F (5′-TGGGCAACCCTAAGGTGAAG-3′)

  • β-Globin-R (5′-GTGAGCCAGGCCATCACTAAA-3′)

The polymerase chain reaction was carried out under real-time fluorescent conditions (ABI Prism 5700 sequence detection system with TaqMan master mix, 96-well MicroAmp optical plates, and optical adhesives cover from Applied Biosystems, Foster City, Calif) in a reaction volume of 20 μL with 25 ng of genomic DNA, 900nM primers, and 250nM probes. Each test sample and control sample were amplified in triplicate; the reactions for the test locus and the reference locus were prepared and run in parallel, with the same standard individual DNA included in every run. Polymerase chain reaction conditions were 95°C for 10 minutes, 95°C for 15 seconds, and 60°C for 1 minute (40 cycles). The dosage of each ampliprimer relative to β-globin and normalized to control DNA was determined by means of the 2-ΔΔCt method.8 For all samples with values less than or equal to 0.6 or greater than or equal to 1.3 in a first round of exon 3 amplification, we assessed the reproducibility in a second round of exon 3 amplification, and the validity through exon 4 amplification. In each experiment we took care to use independently diluted DNA aliquots.

Two-fold differences in the α-synuclein gene dosage were resolved in a linear fashion at genomic DNA concentrations of 25, 12.5, 6.25, and 3.125 ng per 20-μL reaction volume, decreasing or increasing the cycle number by 1 cycle at the threshold. The screening of 190 patients with familial PD for dosage changes of α-synuclein exon 3 showed 22 cases with 2−ΔΔCt values outside the normal range (from 0.7 to 1.2). Independent experiments were carried out to reproduce these values for exon 3 and to validate them for exon 4. Consistency supporting triplication, duplication, or deletion events was not observed in any of these 22 cases (Table).

Table Graphic Jump LocationTable. α-Synuclein Gene Dosage in 190 Patients With Familial PD

Not a single case among 190 cases of familial PD was observed in which a triplication, duplication, or deletion event could be substantiated in DNA from whole-blood samples. Like the A53T, A30P, and E46K mutations, the triplication of α-synuclein seems to be limited to individual families. Clearly, this negative evidence on DNA dosage does not rule out that elevated levels of α-synuclein transcript or protein play a key role in the initial pathogenesis in common forms of PD. Indeed, 3 lines of evidence have implicated abnormal α-synuclein levels in both sporadic and familial PD: First, alleles of a complex microsatellite polymorphism within the α-synuclein promoter are associated with the risk of sporadic PD in studies of large populations.9 Second, the transcript level of α-synuclein was shown to be decreased in tissue of patients with PD with the A53T and A30P mutation of α-synuclein, and a haploinsufficiency mechanism was postulated to be central to pathogenesis.10,11 Third, the triplication event (Park4) of the α-synuclein gene locus in the large autosomal dominant family from Iowa has proved sufficient to cause α-synuclein aggregates that appear identical to the Lewy bodies and Lewy neurites found in common sporadic PD cases.4 Recently, an additional family of Swedish-American descent with autosomal dominant Lewy body–positive PD was found to segregate an α-synuclein gene triplication. Patients from both the Iowa and the Swedish-American families developed dementia in the later disease course and had a characteristic loss of neurons in the cornu ammonis region 2/3 hippocampal area.12 Thus, the characteristic Lewy pathological findings of PD and dementia in rare cases may result from α-synuclein overproduction (Park4) or result from ubiquitin-dependent degradation of α-synuclein (Park2). Also, in rare cases it may be the consequence of missense mutations of α-synuclein resulting in enhanced aggregation tendency (Park1). However, in most sporadic cases the cause of PD probably depends on gene-environment interactions, where posttranslational modifications of α-synuclein caused by oxidative stress13 and mitochondrial alterations14 may play a substantial role.

Correspondence: Georg Auburger, MD, Section of Molecular Neurogenetics, Building 26, University Hospital, Theodor Stern Kai 7, 60590 Frankfurt/Main, Germany (auburger@em.uni-frankfurt.de).

Accepted for Publication: April 19, 2004.

Author Contributions:Study concept and design: Gispert, Auburger. Acquisition of data: Gispert, Trenkwalder, Mota-Vieira, Kostic. Analysis and interpretation of data: Gispert, Auburger. Drafting of the manuscript: Gispert, Auburger. Critical revision of the manuscript for important intellectual content: Gispert, Trenkwalder, Mota-Vieira, Kostic, Auburger. Statistical analysis: Gispert, Auburger. Obtained funding: Gispert, Auburger. Administrative, technical, and material support: Gispert, Mota-Vieira, Auburger. Study supervision: Trenkwalder, Kostic, Auburger.

Funding/Support: This study was supported by grants GI342/1-1 and Au96/4-1 from the Deutsche Forschungsgemeinschaft, Bonn, Germany.

Acknowledgment: We are grateful to the patients for their cooperation.

Polymeropoulos  MHLavedan  CLeroy  E  et al.  Mutation in the α-synuclein gene identified in families with Parkinson’s disease. Science 1997;2762045- 2047
PubMed Link to Article
Kruger  RKuhn  WMuller  T  et al.  Ala30Pro mutation in the gene encoding α-synuclein in Parkinson's disease. Nat Genet 1998;18106- 108
PubMed Link to Article
Zarranz  JAlegre  JGómez-Esteban  JC  et al.  The new mutation, E46K, of α-synuclein causes Parkinson and Lewy body dementia. Ann Neurol 2004;55164- 173
PubMed Link to Article
Singleton  ABFarrer  MJohnson  J  et al.  α-Synuclein locus triplication causes Parkinson’s disease. Science 2003;302841
PubMed Link to Article
French Parkinson’s Disease Genetics Study Group, α-Synuclein gene and Parkinson’s disease. Science 1998;2791116- 1117
PubMed
Gibb  WRLees  AJ The significance of the Lewy body in the diagnosis of idiopathic Parkinson’s disease. Neuropathol Appl Neurobiol 1989;1527- 44
PubMed Link to Article
Leroy  EBoyer  RAuburger  G  et al.  A mutation in the ubiquitin-hydrolase L1 gene in a family with Parkinson’s disease suggests a role for the ubiquitin pathway in neurodegenerative disorders. Nature 1998;395451- 452
PubMed Link to Article
Livak  KJSchmittgen  TD Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCt Method. Methods 2001;25402- 408
PubMed Link to Article
Chiba-Falek  ONussbaum  RL Effect of allelic variation at the NACP-Rep1 repeat upstream of the α-synuclein gene (SNCA) on transcription in a cell culture luciferase reporter system. Hum Mol Genet 2001;103101- 3109
PubMed Link to Article
Markopoulou  KWszolek  ZKPfeiffer  RFChase  BA Reduced expression of the G209A α-synuclein allele in familial parkinsonism. Ann Neurol 1999;46374- 381
PubMed Link to Article
Kobayashi  HKruger  RMarkopoulou  K  et al.  Haploinsufficiency at the α-synuclein gene underlies phenotypic severity in familial Parkinson’s disease. Brain 2003;12632- 42
PubMed Link to Article
Farrer  MKachergus  JForno  L  et al.  Comparison of kindreds with parkinsonism and α-synuclein genomic multiplications. Ann Neurol 2004;55174- 179
PubMed Link to Article
Giasson  BIDuda  JEMurray  IV  et al.  Oxidative damage linked to neurodegeneration by selective α-synuclein nitration in synucleinopathy lesions. Science 2000;290985- 989
PubMed Link to Article
van der Walt  JMNicodemus  KKMartin  RR  et al.  Mitochondrial polymorphisms significantly reduce the risk of Parkinson’s disease. Am J Hum Genet 2003;72804- 811
PubMed Link to Article

Figures

Tables

Table Graphic Jump LocationTable. α-Synuclein Gene Dosage in 190 Patients With Familial PD

References

Polymeropoulos  MHLavedan  CLeroy  E  et al.  Mutation in the α-synuclein gene identified in families with Parkinson’s disease. Science 1997;2762045- 2047
PubMed Link to Article
Kruger  RKuhn  WMuller  T  et al.  Ala30Pro mutation in the gene encoding α-synuclein in Parkinson's disease. Nat Genet 1998;18106- 108
PubMed Link to Article
Zarranz  JAlegre  JGómez-Esteban  JC  et al.  The new mutation, E46K, of α-synuclein causes Parkinson and Lewy body dementia. Ann Neurol 2004;55164- 173
PubMed Link to Article
Singleton  ABFarrer  MJohnson  J  et al.  α-Synuclein locus triplication causes Parkinson’s disease. Science 2003;302841
PubMed Link to Article
French Parkinson’s Disease Genetics Study Group, α-Synuclein gene and Parkinson’s disease. Science 1998;2791116- 1117
PubMed
Gibb  WRLees  AJ The significance of the Lewy body in the diagnosis of idiopathic Parkinson’s disease. Neuropathol Appl Neurobiol 1989;1527- 44
PubMed Link to Article
Leroy  EBoyer  RAuburger  G  et al.  A mutation in the ubiquitin-hydrolase L1 gene in a family with Parkinson’s disease suggests a role for the ubiquitin pathway in neurodegenerative disorders. Nature 1998;395451- 452
PubMed Link to Article
Livak  KJSchmittgen  TD Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCt Method. Methods 2001;25402- 408
PubMed Link to Article
Chiba-Falek  ONussbaum  RL Effect of allelic variation at the NACP-Rep1 repeat upstream of the α-synuclein gene (SNCA) on transcription in a cell culture luciferase reporter system. Hum Mol Genet 2001;103101- 3109
PubMed Link to Article
Markopoulou  KWszolek  ZKPfeiffer  RFChase  BA Reduced expression of the G209A α-synuclein allele in familial parkinsonism. Ann Neurol 1999;46374- 381
PubMed Link to Article
Kobayashi  HKruger  RMarkopoulou  K  et al.  Haploinsufficiency at the α-synuclein gene underlies phenotypic severity in familial Parkinson’s disease. Brain 2003;12632- 42
PubMed Link to Article
Farrer  MKachergus  JForno  L  et al.  Comparison of kindreds with parkinsonism and α-synuclein genomic multiplications. Ann Neurol 2004;55174- 179
PubMed Link to Article
Giasson  BIDuda  JEMurray  IV  et al.  Oxidative damage linked to neurodegeneration by selective α-synuclein nitration in synucleinopathy lesions. Science 2000;290985- 989
PubMed Link to Article
van der Walt  JMNicodemus  KKMartin  RR  et al.  Mitochondrial polymorphisms significantly reduce the risk of Parkinson’s disease. Am J Hum Genet 2003;72804- 811
PubMed Link to Article

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