Brain, Vol. 126, No. 1, 32-42,
January 2003
© 2003 Guarantors of Brain
doi: 10.1093/brain/awg010
Haploinsufficiency at the 
-synuclein gene underlies phenotypic severity in familial Parkinson’s disease
1 Department of Neurology and 2 Division of Biochemical Analysis, Juntendo University School of Medicine, Tokyo, Japan, Departments of 3 Neurology and 4 Medical Genetics, University of Tübingen, Tübingen, Germany, 5 Department of Neurological Sciences, University of Nebraska Medical Center and 6 Department of Biology, University of Nebraska at Omaha, Omaha, and 7 Department of Neurology, Mayo Clinic Jacksonville, USA
Correspondence to: Nobutaka Hattori, MD, Department of Neurology Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo, Tokyo 113-8421, Japan E-mail: nhattori{at}med.juntendo.ac.jp
Received June 1, 2002. Revised July 25, 2002. Accepted July 29, 2002.
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To date, two point mutations, G209A and G88C, have been reported in the coding region of the
-synuclein gene in autosomal dominant familial Parkinson’s disease. When translated, these lead to the missense mutations Ala53Thr and Ala30Pro, respectively. Reduced mRNA expression of the G209A allele was reported recently in a Greek–American family. Here, we show that -synuclein mRNA is normally expressed in blood cells and report the results of an analysis of -synuclein mRNA and protein expression in lymphoblastoid cell lines established from kindreds with the G209A and G88C mutations. mRNA expression was characterized using a TaqMan real-time quantitative reverse transcriptase–polymerase chain reaction (RT–PCR) assay. We assessed five affected and three unaffected members of a German family with the G88C mutation and two affected members in different, unrelated Greek families with the G209A mutation. The ratio of wild-type to mutant -synuclein allele expression ranged from 2.2 to 9.2 in the affected individuals with a severe clinical phenotype. The ratios of the expression levels of the wild-type to mutant alleles were only slightly decreased in mild cases and were less than 1.0 in two asymptomatic heterozygotes. Sequence analysis of the RT–PCR products showed only the presence of G in position 88 and G in position 209 in severely affected heterozygotes of the German and Greek families, respectively. High performance liquid chromatography/mass spectrometry demonstrated that, relative to wild-type -synuclein, there is a reduction of Ala30Pro -synuclein in lymphoblastoid cell lines originating from severely affected, but not mildly affected G88C/+ heterozygotes. Taken together, these data indicate that there is haploinsufficiency at the -synuclein gene and that the ratio of expression of the wild-type to mutant alleles correlates with the severity of the clinical phenotype. Furthermore, these findings suggest that haploinsufficiency of -synuclein mutations may contribute to disease progression in these forms of familial Parkinson’s disease.
Keywords: -synuclein; haploinsufficiency; familial Parkinson’s disease; G88C and G209A mutations
Abbreviations: ADFPD= autosomal dominant familial Parkinson’s disease; GAPDH = glyceraldehyde-3-phosphate dehydrogenase; HPLC/MS = high performance liquid chromatography/mass spectrometry; RT–PCR = reverse transcriptase–polymerase chain reaction; RFLP = restriction fragment length polymorphism; SDS–PAGE = sodium dodecyl sulphate-polyacrylamide gel electrophoresis
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Introduction |
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Parkinson’s disease is the second most common neurodegenerative disorder in the ageing population. Although most patients with Parkinson’s disease are sporadic cases, it is now clear that genetic factors contribute to the pathogenesis of Parkinson’s disease (Polymeropoulos et al., 1996
, 1997; Gasser et al., 1998; Kitada et al., 1998). The discovery of two point mutations in the coding region of the -synuclein gene in autosomal dominant familial Parkinson’s disease (ADFPD) prompted a series of studies into the role of -synuclein in both familial and sporadic Parkinson’s disease (Polymeropoulos et al., 1997; Spillantini et al., 1997, 1998; Krüger et al., 1998). The first mutation—a G-to-A transition in exon 4 that, if translated, results in an Ala53Pro missense mutation in -synuclein—was identified in a large Italian–American multigenerational family with an ADFPD known as the Contursi kindred (Polymeropoulos et al., 1996, 1997). It has since been identified in multiple, Greek–American and apparently unrelated Greek kindreds (Polymeropoulos et al., 1997; Athanassiadou et al., 1999; Markopoulou et al., 1999; Papadimitriou et al., 1999). The second mutation—a G-to-C transversion in exon 3 that, if translated, results in an Ala30Pro missense mutation—was identified in a German family (Krüger et al., 1998). Several studies suggested that both mutations in the -synuclein gene are rather rare causes of Parkinson’s disease (Polymeropoulos et al., 1996, 1997; Krüger et al., 1998). However, the identification of these mutations in the -synuclein gene provides evidence that -synuclein is involved in the pathogenesis of Parkinson’s disease. These efforts culminated in the identification of - synuclein as a major component of Lewy bodies in sporadic Parkinson’s disease and dementia with Lewy bodies (Spillantini et al., 1997, 1998).
Recently, we reported results from reverse transcriptase–polymerase chain reaction (RT–PCR) restriction fragment length polymorphism (RFLP) experiments that showed low or absent expression of the G209A -synuclein allele in a large Greek–American family (Family H) and suggested that haploinsufficiency may contribute to the pathogenesis in this form of ADFPD (Markopoulou et al., 1999). Haploinsufficiency refers to a phenotype associated with inactivation of a single allele in a diploid organism (Cook et al., 1998; Hu et al., 1998). To determine whether haploinsufficiency underlies the pathogenesis of Parkinson’s disease in other forms of ADFPD associated with different -synuclein mutations, as well as in other families carrying the G209A mutation, we analysed the relationship between the expression levels of both the G88C and the G209A -synuclein mutations and clinical phenotype in members of the German and additional Greek ADFPD families using quantitative mRNA and high performance liquid chromatography/mass spectrometry (HPLC/MS).
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Patients
The German family investigated in this study is, to our knowledge, the only known pedigree carrying the Ala30Pro mutation in the
-synuclein gene, and has been described previously (Krüger et al., 1998). Individual II-I from the HEL-2 kindred has also been described previously (Chase et al., 1999). In all three families, the parkinsonian phenotype is inherited as an autosomal dominant trait. The pedigrees are shown in Fig. 1 and the clinical characteristics of affected family members are summarized in Tables 1 and 2.
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Cell lines
Human lymphoblastoid cell lines (EBV-transformed) were established, with informed consent, from three unrelated families with one or more individuals using a standard protocol (Neitzel, 1986
). These cells were cultured in ISCOVE medium (Gibco, BRL, Rockville, MD, USA) with 10% fetal bovine serum (FBS) (Gibco), 8.5% NaHCO3 L-glutamine (Gibco), 1% glutamine (Gibco) and 1% penicillin-streptamycin (PS) (Gibco). Cells were incubated at 37°C in a 5–6% CO2 atmosphere.
Nucleic acid extraction and preparation of cDNA templates
Genomic DNA was prepared from lymphoblastoid cell lines using a QIAampTM DNA Mini kit (QIAGEN, Hilden, Germany). RNA was isolated from freshly drawn blood using the PAXgene RNA isolation system (PreAnalytix, Valencia, CA, USA). Oligo(dT) primed cDNA was prepared from this RNA as described previously (Markopoulou et al., 1999). RNA was isolated from lymphoblastoid cells using a RNeasyTM Mini Kit and a RNase-Free DNase Set (QIAGEN). Oligo(dT) primed cDNA was prepared from this RNA by reverse transcription using a RT kit (Takara, Shiga, Japan). The cDNA was used directly in TaqMan assays detecting the G88 and G88C alleles. In TaqMan assays detecting the G209 and G209A alleles, the cDNA obtained from reverse transcription alone was insufficient to perform TaqMan PCR. A PCR amplification was performed as described previously (Polymeropoulos et al., 1997; Markopoulou et al., 1999) using primers 1F and 13R, but using only ten cycles of amplification.
Expression of wild-type and mutant recombinant -synuclein protein
For positive controls, we expressed wild-type and mutant -synuclein recombinant protein using the IMPACTTM-CN system (New England BioLabs, Beverly, MA, USA).
RT–PCR–RFLP and PCR–RFLP methods
RT–PCR on RNA isolated from fresh blood was performed as described previously using primers 1F and 13R (Polymeropoulos et al., 1997; Markopoulou et al., 1999). RT–PCR on RNA isolated from cell lines was performed using the TaKaRa RNA PCR kit (Takara). The conditions and primers for RT–PCR and PCR–RFLP were as described previously (Polymeropoulos et al., 1997; Krüger et al., 1998). PCR and RT–PCR products were digested with MvaI or Tsp45I and size-separated on 2%, 3% or 3.5% agarose gels.
Direct sequencing of RT–PCR products
We sequenced each RT–PCR product directly using a BigDye Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA). Samples were then subjected to electrophoresis using an ABI PRISM 310 genetic analyser (Applied Biosystems).
Quantitative PCR using real-time TaqMan–PCR methods
Quantitative PCR was performed using the real-time TaqMan–PCR method, which has recently been established as a rapid and sensitive technique for the quantitation of gene expression. In this method, AmpliTaq DNA polymerase extends the primer and displaces the TaqMan probe through its 5'-to-3' exonuclease activity (Heid et al., 1996). The probes were labelled with a reporter fluorescent dye [6-carboxy-fluorescein dye (FAM)] at the 5' end and a quencher fluorescent dye (TAMRA) at the 3' end.
The expression level of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as an endogenous control. Primers and a TaqMan probe for GAPDH (TaqMan GAPDH Control Reagent Kit) were purchased from Applied Biosystems. The probes for GAPDH were labelled with a reporter fluorescent dye [2,7-dimethoxy-4,5-dichloro-6-carboxy-fluorescein (JOE)] at the 5' end.
Nuclease degradation of the hybridization probe removed the quenching effect of 6-carboxy-tetramethyl-rhodamine from the FAM or JOE fluorescent emission, thus increasing the peak fluorescent emission at 517 and 554 nm, respectively. No signal was emitted when the probe was intact.
Primers and TaqMan probes
Primers for PCR amplification and the TaqMan probe were designed on the basis of the published sequence of the -synuclein mRNA. To selectively amplify the wild-type (G88, G209) and mutant (G88C and G209A) alleles, PCR reactions used different primers capable of amplifying these alleles selectively.
To amplify the wild-type G88 allele, primer set A (forward: 5'-AAGGACTTTCAAAGGCCAAGG-3'; reverse: 5'-CACCCTCTTTTGTCTTTCCTGC-3') was used. To amplify the mutant G88C allele, primer set B (forward primer from primer set A; reverse: 5'-CACCCTCTTTTGTC TTTCCTGG-3') was used. The TaqMan probe to detect these products had the sequence: 5'-AGTTGTGGC TGCTGCTGAGAAAACCAAACA-3'. To amplify the wild-type G209 allele primer set C (forward: 5'-GGT CTTCTCAGCCACTGTTAC -3'; reverse: 5'-TGGCAGAA GCAGCAGGAAA -3') was used. To amplify the mutant G209A allele, primer set D (forward: 5'-TGGTCTTCT CAGCCACTGTTAT-3'; reverse primer from primer set C) was used. The TaqMan probe to detect these products had the sequence: 5'-CCTTGGTTTTGGAGCCTACATAGAGAA CAC-3'.
The reaction mixture contained 25x TaqMan mixture (purchased from Applied Biosystems), the appropriate primer set, the TaqMan probe and the cDNA template in a total volume of 50 µl. The thermal cycling protocol consisted of 2 min at 95°C and 10 min at 95°C, followed by 45 cycles at 95°C for 15 s and 60°C for 1 min. TaqMan–PCR was analysed using a Model 7700 Sequence Detector (Applied Biosystems), which can measure fluorescence in real-time. The starting quantity of the target mRNA was calculated using the Sequence Detector software package version 1.6.3 (Applied Biosystems). The starting quantity of each target mRNA was normalized to that of the GAPDH endogenous control mRNA, allowing for measurement of the relative expression level of each RNA species.
For control TaqMan studies, we prepared constructs for both wild-type and mutant alleles in the plasmid vector pQBI25 (Takara). To obtain the wild-type -synuclein gene, we performed RT–PCR using total RNA from normal brain tissue as a template. The RT–PCR product was subcloned into pQBI25. Site-directed mutagenesis of this clone was used to make constructs with the G88C and G209A mutations. We confirmed the specificity of the oligonucleotide primers for the wild-type and mutant alleles using three templates: the wild-type allele only; a mixture of wild-type and mutant alleles at a 1:1 ratio; and the mutant allele only.
Immunoprecipitation of -synuclein from lymphoblastoid cells
Cells were washed three times with PBS and lysed on ice for 30 min by incubation in 1.0% NP40, 150 mM NaCl, 50 mM Tris–HCl pH 8.0. After centrifugation (20 000g for 30 min), immunoprecipitation was performed using an anti-- synuclein antibody (BD Transduction Laboratories, Lexington, KY, USA) and the SeizeTM X Protein G Immunoprecipitation Kit (PIERCE, Rockford, IL, USA).
One-dimensional sodium dodecyl sulphate–polyacrylmaide gel electrophoresis (SDS–PAGE)
Immunoprecipitated proteins were separated by SDS–PAGE on a 130 x 130 x 1 mm gel system (Nihon Eido Co., Tokyo, Japan) with separating (10% acrylamide) and stacking (3% acrylamide) gels containing 2.6% piperazine diacrylamide. Proteins in the gel were stained using Coomassie brilliant blue and protein profiles were determined using an image analyser, Master Scan from Scanalytics, Billerica, MA, USA.
In-gel digestion with trypsin and extraction of peptides
Bands containing -synuclein protein were identified by comparison with expressed recombinant wild-type and mutant -synuclein proteins [molecular mass (Mr) 19000)]. The bands were excised and the proteins were digested with trypsin in the gel (Castellanos-Serra et al., 1999).
Protein identification by HPLC/MS
Peptide mapping was carried out using an API QSTAR Pulsar hybrid mass spectrometer system consisting of a nano-electrospray and time-of-flight apparatus (Applied Biosystems). The QSTAR hybrid mass spectrometer was combined with a micro-liquid chromatograph (MAGIC 2002, Michrom Bioresources, Inc., Auburn, CA, USA) utilizing a 0.1 mm internal diameter x 50 mm MagicC18 column. The solvent system consisted of (A) 0.1% formic acid, and (B) 0.1% formic acid/90% acetonitrile. The solvent programme was: 3% B for 2 min; a gradient of 1% B/min for 45 min; and 100% B for 5 min. The flow rate was 1 µl/min. Mass spectrometry conditions were: ion spray voltage, 3 kV; voltage for electron multiplier, 2400 V; and curtain gas nitrogen, 10 psi. Proteolytic fragments were identified using PROWL (ProFound, http://prowl.rockefeller.edu) and databases in the public domain (http://www.ncbi.nlm.nih.gov).
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Detection of
-synuclein mRNA expression in fresh bloodTo address whether
-synuclein mRNA expression in lymphoblastoid cell lines is representative of, and relevant to -synuclein mRNA expression in blood cells, we sought to verify that -synuclein mRNA is expressed in lymphocytes. RT–PCR analyses were performed using total RNA templates isolated from freshly drawn blood. RNA was prepared using a method that preserves the integrity of RNA at the time of the blood draw, thus avoiding alterations in gene expression that can occur during storage of blood samples prior to RNA extraction. The results (Fig. 2) indicate that the expected 500 bp RT–PCR product is obtained in control fresh blood samples, the same size as the product obtained in lymphoblastoid cell lines established from control individuals and two affected G209A/+ heterozygotes (Fig. 2, lanes 1–7). No RT–PCR product was obtained in control reactions without RNA template or reverse transcriptase (Fig. 2, lanes 8–9). Direct sequencing of the RT–PCR products obtained using RNA templates from fresh blood confirmed that they are amplification products of -synuclein mRNA (data not shown). These results demonstrate that -synuclein mRNA is expressed in cells found in whole blood. Although these RT–PCR assays were not performed quantitatively, consistently greater RT–PCR product levels were seen in fresh blood RNA samples, suggesting that -synuclein mRNA may be expressed at higher levels in lymphocytes than those in lymphoblastoid cell lines.
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PCR and RT–PCR analysis with MvaI and Tsp45I.
Genomic DNA from individuals III-9, IV-3, IV-5, IV-6 and V-1 from the German family (Fig. 1A) was amplified by PCR using previously described primers (Krüger et al., 1998
). As previously reported, all five individuals possess the G88C mutation (Krüger et al., 1998) (Fig. 3A). RFLP analysis of the RT–PCR products from these individuals indicated that individual IV-3, who is severely affected, did not exhibit the 379 bp RFLP fragment in MvaI digests of the RT–PCR products, indicating that the G88C allele is not transcribed. Individuals III-9, IV-5, IV-6 and V-1 exhibited the 379 bp RFLP RT–PCR fragment indicating that the G88C allele is transcribed (Fig. 3A). However, the exact amount of the G88C transcript could not be evaluated.
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Genomic DNA from individuals IV-2 from the Greek family HEL-1 (Fig. 1B) and II-1 from the Greek family HEL-2 (Fig. 1C) was amplified by PCR using previously described primers (Polymeropoulos et al., 1997
). Both individuals possess the G209A mutation (Chase et al., 1999). RFLP analysis of the RT–PCR products with Tsp45I (Polymeropoulos et al., 1997) reveals that individual IV-2 (HEL-1), who is severely affected and has dementia, did not exhibit the 185 bp RFLP fragment indicating that the G209A allele is not transcribed, whereas individual II-1 (HEL-2) did exhibit the 185 bp fragment, indicating that the G209A allele is transcribed (Fig. 3B).
Direct sequencing of RT–PCR products
To confirm the above results, we performed direct sequencing of the RT–PCR products. Sequence analysis showed two nucleotides (G/C) at nucleotide position 88 of the coding region corresponding to the Ala30Pro substitution in two individuals (III-9 and IV-5). In contrast, the proband from IV-3, who had a severe phenotype, had only G in position 88. Similarly, in the Greek families, sequence analysis of individual II-1 showed two nucleotides (G/A) at nucleotide position 209 corresponding to the Ala53Thr substitution, whereas sequence analysis of IV-2, who also has a severe phenotype, indicated only a G in position 209 (Fig. 3C and data not shown).
Quantitative PCR using real-time TaqMan-PCR method
Control study
We were able to discriminate between the wild-type and mutant alleles (Tables 3 and 4) using the protocol described above.
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Expression of the mutant G88C and G209A allele
Expression analysis of the relative levels of mutant and wild-type alleles was performed using TaqMan real-time-PCR with mRNA as a template. We found different expression levels among affected members of the three families (Fig. 4). A summary of the clinical characteristics of the German family and the two Greek families is shown in Tables 1 and 2, respectively. The larger Greek kindred (HEL-1, Fig. 1B) has not been described previously, whereas the small kindred (HEL-2, Fig. 2C) has (Chase et al., 1999
). The two kindreds are not related genealogically.
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In the German family, the expression of the mutant G88C allele was markedly reduced relative to the wild-type allele in individual IV-3. It is of interest that this individual presented with the most severe symptoms and longest duration of Parkinson’s disease. In individual IV-5, who had mild symptoms with a disease duration of 2 years, the expression level of the mutant allele was about half that of the wild-type allele. In individual III-9, who had slight rigidity and resting tremor, the expression level of the mutant G88C allele was slightly lower than the wild-type allele. Both IV-6 and V-1 showed bradydiadochokinesis only, but did not fulfil the diagnostic criteria of Parkinson’s disease (Calne et al., 1992
). Therefore, these members may be asymptomatic heterozygotes at present and may develop Parkinson’s disease in the future. The expression of the wild-type allele in these two individuals was slightly reduced compared with the mutant allele. The TaqMan analysis could distinguish between the expression levels of the two alleles, even though it was difficult to distinguish individuals with a mild phenotype (III-9, IV-5) from carriers of the mutation (IV-6, V-1) by the RT–PCR RFLP method alone (Table 3, Figs 3 and 4). Individual II-1 from the HEL-2 family developed parkinsonian symptoms that were responsive to L-dopa at age 39 years. Seven years later, at age 46 years, this individual is able to function independently, requires no assistance with activities of daily living and, to date, has no cognitive impairment. The expression level of the mutant G209A allele was slightly higher than that of the wild-type allele. Individual IV-2 from the HEL-1 family developed parkinsonian symptoms at age 42 years, had a more rapid progression with diminishing response to L-dopa and has been unable to function independently, perform any activities of daily living without assistance and has had significant cognitive impairment for approximately the last 5 years. The expression of the mutant G209A allele was markedly reduced relative to that of the wild-type allele.
In summary, the results from all three families indicate that the ratio of expression of the wild-type to mutant -synuclein alleles in lymphoblastoid cell lines correlates with the severity of symptoms of the affected individuals at the time of the cell line’s establishment (Fig. 4, Tables 1, 2, 3 and 4).
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Isolation of
-synuclein from lymphoblastoid cell linesCoomassie brilliant blue stained SDS–PAGE gels identified protein bands of the size of Mr 19000 (Fig. 5), the same size as that of wild-type and mutant
-synuclein recombinant proteins.
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Characterization of
-synuclein protein by HPLC/MSWestern blot analysis using the
-synuclein antibody detected -synuclein in all the cell lines used in this study, including the normal lymphoblastoid cell lines (data not shown). Peptide mapping studies were undertaken to characterize the form(s) of the -synuclein protein present in a subset of these lines. These results also indicate that -synuclein is present in lymphoblastoid cells. Analysis of the HPLC fractions of trypsinized recombinant wild-type and mutant (Ala30Pro) -synuclein protein demonstrated that the peaks (M+2H)2+ at 415.7062 and 428.7469 corresponded to residues 24–32 of wild-type and mutant -synuclein, respectively. HPLC/MS also identified the sequences of these peaks as QGVAEAAGK and QGVAEAPGK, respectively. Fragment ion data were collected on more than seven peptides from the lymphoblastoid cells established from individual IV-3. In total, these fragment ion masses matched 60% of the -synuclein protein, confirming the presence of wild-type -synuclein. In contrast, we could not find any peptides corresponding to mutant (Ala30Pro) -synuclein in cell lines established from IV-3 (Fig. 6C and D). On the other hand, we could detect the peaks for peptides from both wild-type and mutant -synucleins in the lymphoblastoid cells established from individual IV-6 (Fig. 6E and F). Only the peaks for peptides of the wild-type protein were detected in an unaffected, gene-negative member of this kindred (data not shown).
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Discussion |
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We have used quantitative RT–PCR analysis to demonstrate that the mRNA expression of the mutant G88C and G209A alleles of the
-synuclein gene is significantly reduced relative to the wild-type allele in lymphoblastoid cell lines established from affected heterozygotes with a severe clinical phenotype. In contrast, these mutant alleles are expressed at levels similar to the wild-type allele in lymphoblastoid cell lines established from less severely affected heterozygotes or asymptomatic heterozygotes. HPLC/MS indicates that these allele-specific and disease-status specific alterations in mRNA expression are also reflected at the protein level. These results are consistent with the hypothesis that haploinsufficiency at the -synuclein gene underlies the parkinsonian phenotype in kindreds with the G88C as well as the G209A mutation. To our knowledge, this is the first report in which (i) a quantitative analysis that determines the exact level of mRNA expression of both the wild-type and mutant alleles of -synuclein and (ii) an analysis of the proteins encoded by the wild-type and mutant alleles has been used to support a hypothesis of haploinsufficiency in ADFPD.
While differences in the absolute levels of products obtained from individuals with the G88C and G209A mutations may reflect the different sites of the TaqMan probe and primers (which can influence the efficiency of amplification during RT–PCR) in control experiments involving mixtures of wild-type and mutant constructs, both wild-type and mutant templates could be detected (Tables 3 and 4). Thus, it is significant that we find that the ratios of wild-type to mutant transcripts differ between individual members of the families harbouring the G88C and G209A mutations. It is unlikely that these differences reflect clonal variation between populations of lymphoblastoid cells because they were confirmed in multiple experiments using independently generated cell lines. Rather, the data support the explanation that disease duration and severity may influence the expression of both the wild-type and mutant alleles. Disease progression and the presence of dementia appear to be associated with either increased expression of the wild-type allele or reduced expression of the mutant allele. This suggests that the ratio of expression levels of the wild-type to mutant -synuclein alleles may be able to be developed as a clinical marker of this type of ADFPD, particularly since -synuclein is normally expressed in lymphocytes.
We previously studied members of a large Greek–American kindred (Family H) and reported absent or significantly reduced levels of G209A allele expression in lymphoblastoid cell lines established from affected heterozygotes and some asymptomatic heterozygotes (Markopoulou et al., 1999). Here, decreased levels of expression of the mutant allele at the -synuclein gene are associated with the clinical phenotype in severely affected individuals only. One explanation for this involves methodological differences. We previously used RT–PCR RFLP only. There, the RT–PCR product is processed for RFLP analysis at the end-cycle point and the dynamics of PCR amplification make it difficult to interpret the magnitude of differences accurately. Indeed, in this study, it was difficult to distinguish individuals with a mild phenotype (III-9, IV-5) from carriers of the mutation (IV-6, V-1) by this method alone (Fig. 3). This issue is less of a concern as the TaqMan analysis used in this study allows for a real-time quantitative analysis.
What might be the consequences of differential and/or decreased expression of G88C and G209A mutant alleles? One might be that differential expression of the G88C and G209A alleles could result in different levels of the distinct Ala30Pro and Ala53Thr -synuclein products produced by these alleles. Thus, differences between the functions provided by these mutant proteins and the wild-type proteins could underlie some aspects of the disease phenotype. Indeed, our HPLC/MS analysis revealed both wild-type and mutant proteins in a mildly affected G88C/+ individual, but only wild-type protein in a severely affected G88C/+ individual. That the mutant forms of -synuclein are not functionally equivalent to wild-type -synuclein has been suggested by multiple studies (e.g. Crowther et al., 1998; Feany and Bender, 2000; Conway et al., 2000a, b). However, it is difficult to extrapolate from these studies, which are based on in vitro over-expression or ectopic expression of mutant -synuclein proteins, as to how reduced expression of a mutant form of -synuclein would lead to neurodegeneration. An alternative explanation, which is also consistent with findings in other diseases exhibiting haploinsufficiency (Wilkie, 1994), is that the decreased expression of one allele leads to a critical threshold level of -synuclein that makes a cell more susceptible to being affected by the degenerative process. The sequestration of -synuclein in Lewy bodies may contribute to a decrease in the amount of functional -synuclein in the cell. Thus, compared with a cell having two wild-type alleles, a cell harbouring a mutant allele may be more susceptible to the consequences of sequestration of functional -synuclein protein.
Phenotypes of complex diseases such as Parkinson’s disease may be a manifestation of a complex molecular pathophysiology, and result from more than one contributing mechanism. For example, in myotonic dystrophy, two mechanisms—a trinucleotide repeat expansion causing haploinsufficiency of nearby genes and an abnormal protein kinase (myotonic dystrophy protein kinase) affecting RNA homeostasis (Korade-Mirnics et al., 1998)—appear to be involved. The different phenotypic manifestations in familial parkinsonism may also reflect different molecular mechanisms. Thus, a reduction of -synuclein gene expression may underlie the rapid rate of disease progression seen in this form of ADFPD as well as the presence of dementia, while a missense mutation (Ala30Pro and Ala53Thr) may underlie other aspects of the parkinsonian phenotype.
It is not clear whether the differences in the expression pattern of the -synuclein alleles in lymphoblastoid cell lines reflect their expression patterns in the brain, and particularly in the substantia nigra. However, even though -synuclein mRNA and protein isoform expression can be assessed in the brains of deceased individuals, the pattern will reflect the endpoint of the disease process and not the process that led to it. Our results suggest that as the neurodegenerative process unfolds, the expression of the mutant allele decreases and the expression of the wild-type allele increases to support the wild-type -synuclein function in dopaminergic neurons. If there is a variation in the ratio of wild-type to mutant allele transcription among cells within the substantia nigra, cells that survive the neurodegenerative process may be those that are able to retain similar levels of wild-type and mutant allele transcription. In this event, one might not expect to see dramatic differences in the ratios between wild-type and mutant mRNA or protein expression in surviving cells. Nonetheless, further studies using brain tissue from affected patients to study mRNA and protein isoform levels, and to correlate these with disease severity are necessary to confirm our hypothesis of haploinsufficiency.
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Acknowledgements |
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We wish to thank Drs Noriko Shindoh and Tsutomu Fujimura for valuable advice and Mei Wang for her generous gift of
-synuclein recombinant proteins. This study was supported in part by: a grant-in-aid for Scientific Research on Priority Areas from the Ministry of Education, Science, Sports, and Culture, Japan; a grant-in-aid for Health Science Promotion; a grant-in-aid for neurodegenerative disorders from the Ministry of Health and Welfare, Japan; a grant-in-aid from the UCR at the University of Nebraska-Omaha; a Centre of Excellence Grant from National Parkinson Foundation, Miami, Florida, USA; and an M. H. Udall NIH PD Centre of Excellence grant.
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