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Gispert S, Trenkwalder C, Mota-Vieira L, Kostic V, Auburger G. Failure to Find α-Synuclein Gene Dosage Changes in 190 Patients With Familial Parkinson Disease. Arch Neurol. 2005;62(1):96–98. doi:10.1001/archneur.62.1.96
Recently, a triplication of the α-synuclein locus was found associated with autosomal dominant Parkinson disease in a large family.
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.
Retrospective recruitment of the largest families who were willing to cooperate with the study.
Centers with specialization in movement disorders genetics.
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.
None of the samples showed α-synuclein triplication, duplication, or deletion.
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 :
For the amplification of a reference locus, we used β-globin primers and a TAMRA probe:
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).
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 (firstname.lastname@example.org).
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.
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