Human Molecular Genetics, 2001, Vol. 10, No. 16 1649-1656
© 2001 Oxford University Press
The importance of gene dosage studies: mutational analysis of the parkin gene in early-onset parkinsonism
1Department of Neurology and 2Department of Human Genetics, Medical University of Lübeck, Ratzeburger Allee 160, 23538 Lübeck, Germany, 3Department of Hematology, Regional General Hospital, 39100 Bolzano, Italy, 4TIB MOLBIOL, 10829 Berlin, Germany, 5Division of Neurology, Department of Medicine, University of Toronto, and Toronto Western Hospital, Toronto M5T 2S8, Canada, 6Molecular Neurogenetics Unit, Massachusetts General Hospital, and Departments of Neurology and Neuroscience Program, Harvard Medical School, Boston, MA 02129, USA, 7Molecular Genetics, Albert Einstein College of Medicine, Bronx, NY 10461, USA and 8Department of Neurology, Regional General Hospital, 39100 Bolzano, Italy
Received March 28, 2001; Revised and Accepted June 6, 2001.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Early-onset parkinsonism (EOP) may be associated with different mutations in the parkin gene, including exon deletions and duplications. To test for gene dosage alterations, we developed a new method of quantitative duplex PCR using the fluorescence resonance energy transfer technique on the LightCycler (Roche Diagnostics). In 21 patients with EOP, three mutations (a single base pair substitution in exon 3 and small deletions in exon 9) were detected by conventional mutational screening (single-strand conformation polymorphism and sequence analysis), while alterations of gene dosage were found in seven patients. We identified heterozygous and compound heterozygous deletions of exons 2, 3, 5 and 7. The latter was also found in the homozygous state. In addition, two heterozygous duplications of exon 4 were observed. Remarkably, two patients carried more than two parkin mutations. This is the first study systematically screening all 12 exons of parkin by real-time, kinetic quantification and clearly shows that mutational analysis of the parkin gene should include gene dosage studies. Furthermore, our method of quantitative PCR is easily applicable to any other gene to be screened for deletions or duplications of whole exons.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Early-onset parkinsonism (EOP) is a neurodegenerative disorder with features of Parkinson’s disease (PD) and additional clinical findings in some patients, such as diurnal fluctuation of symptoms, sleep benefit, dystonia, early levodopa-induced dyskinesias and hyper-reflexia. EOP is usually considered to start below the age of 40 years (1), and it has recently been shown that genetic factors appear to be important in the etiology of PD with an early age of onset [at or before 50 years of age (2)]. Indeed, EOP is frequently inherited in an autosomal recessive fashion (3,4), and a wide range of mutations in the parkin gene may be associated with the disease (3–7). However, we recently described a large kindred (Family LA) with late-onset parkinsonism, clinically indistinguishable from idiopathic PD, with compound heterozygous parkin mutations (8). Interestingly, in asymptomatic carriers of a heterozygous parkin mutation with one apparently normal allele, positron emission tomography studies revealed a mild, but statistically significant decrease of mean FDOPA uptake versus controls in all striatal regions (9), suggesting that even heterozygous parkin mutations may be associated with (subclinical) changes in the dopaminergic system. Supporting this idea, heterozygous mutations were also reported in a considerable percentage of patients (28%) in the largest published study, which included semiquantitative analysis, on the association between EOP and mutations in the parkin gene among 54 index patients with familial or isolated PD (4). In contrast, most studies screening for parkin mutations reported in the literature did not include gene dosage studies and reported homozygous mutations only (3,5,10–12). One additional study showed heterozygous mutations in three out of 35 families, while 23 families were described as mutation-negative (6). Therefore, it remained to be investigated whether patients with only one or no detected mutation carried a heterozygous deletion or duplication of one or more exons on the other allele (or on both alleles), which are not detectable by conventional PCR amplification. To test for such gene dosage alterations, we developed a new, highly accurate screening method using the LightCycler (Roche Diagnostics). Here we present the results of a complete mutational screen on 21 patients with onset of parkinsonism at <50 years of age.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Patients
Twenty-one patients (age at time of sampling: 51.3 ± 10.1 years, 67% male, 33% female; age of onset: 39.4 ± 7.9 years; range: 18–49 years) were included. In addition, family members were available from three of the index cases, including the previously described Family LA (8). Family history of parkinsonism or tremor was positive in seven cases (33%) whereas three family members of patient B-300 showed restless legs syndrome (RLS). Clinical features in 17 of the 21 patients (81%) were consistent with idiopathic Parkinson’s disease, while four patients (19%) had additional neurologic signs and symptoms, such as sleep benefit, early levodopa-induced dyskinesias and hyperreflexia (Table 1).
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Homozygous or compound heterozygous mutations were identified in all patients (4/4) with additional signs of EOP, while only three of the remaining 17 patients (18%) without additional clinical findings carried at least one mutation (Table 1). Furthermore, patients with at least one mutation (n = 7) had a significantly lower age of onset (32.4 ± 9.5 years) compared to those without mutations (n = 14; 41.6 ± 6.5 years; P < 0.05).
Conventional mutational screening
Homozygous mutations were identified in three patients (14%) including a novel missense mutation in exon 3 (346C
A, resulting in the predicted amino acid change Ala82Glu; B-219), two deletions of exon 7 (B-219, B-300), and a 1 bp deletion in exon 9 (1072del T; B-125). B-15 (a member of Family LA) was found to carry the latter mutation in the heterozygous state. Furthermore, one patient (T-1) carried a heterozygous 2 bp deletion in exon 9 (1147–8delAA) (Table 1). The missense mutation was not detected in any of the 350 controls.
Gene dosage analysis
The majority of patients showed a ratio of parkin to beta globin of approximately 1.0 (Fig. 1A), while alterations of gene dosage were found in seven patients.
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2.2 kb, B-217 only shows a very faint band at 1.5 kb (arrow), indicative of the deletion.Patient B-151 carried a compound heterozygous deletion of exons 2 and 5 (parkin/beta globin ratio of 0.51 and 0.48, respectively; Fig. 1B). T-1 was identified to have a heterozygous exon 3 deletion (ratio 0.51). In two other patients (B-61, B-125), we detected a parkin exon 4/beta globin ratio of 1.48 and 1.52, respectively (Fig. 1C), suggesting a duplication of this exon.
In addition, we confirmed the presence of the two homozygous exon 7 deletions in B-219 and B-300 that were first identified by the absence of a PCR product on gel electrophoresis (data not shown). In both cases, one parent was available and showed the expected heterozygous exon 7 deletion which was undetectable by conventional PCR (Fig. 1D and E). In B-217 (parent of B-219), the deletion was also shown by Southern blot analysis (Fig. 1F). B-219 carried a total of four (two different homozygous) mutations and B-125 a homozygous and an additional heterozygous mutation.
Finally, the exonic deletion in Family LA, reported previously to comprise exons 1–7, was redefined to be a deletion of exon 7 only.
All detected mutations are listed in Table 1 and shown in Figure 2.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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This is the first study systematically screening all 12 exons of parkin, combining conventional mutational analysis and gene dosage studies. In total, mutations were found in seven of 21 patients (33%), six of whom (86%) carried homozygous or compound heterozygous mutations, while one patient carried a mutation in the heterozygous state only. It is noteworthy that we found two patients with more than two mutations in the parkin gene. Even in this small patient sample, presence of mutations appears to be associated with a lower age of onset and atypical clinical signs and symptoms.
In this patient sample, five mutations (one 1 bp substitutions, two small deletion and two homozygous exon deletions) were detected by conventional mutational screening, while six alterations of gene dosage were found by quantitative PCR analysis (Fig. 2). Notably, B-151 with a compound heterozygous deletion of exons 2 and 5 was initially considered mutation-negative by conventional screening. Similarly, a heterozygous deletion of exon 3 was identified in T-1, who was previously thought to carry only a heterozygous 2 bp deletion. Unfortunately, neither RNA nor relatives were available from these patients. Thus, the localization of the two mutations on different alleles could not be demonstrated, but they are likely to explain the phenotype. In two other patients, we observed an increase of 50% in gene dosage of exon 4 of parkin, suggesting heterozygous duplications of this exon. In addition, the larger deletion in Family LA was redefined to a deletion of exon 7 rather than a deletion of exons 1–7 (8). A mismatch in the primer annealing site or a reduced turnover time of the mRNA probably prevented the amplification of both alleles in the previously performed RT–PCR analysis (8). Furthermore, quantitative PCR served to confirm two homozygous deletions of exon 7 by demonstrating the segregation of the deletion in available family members.
Homozygous or compound heterozygous mutations in the parkin gene probably result in the absence of functional gene product. Remarkably, patient B-219 carried two homozygous mutations. It remains to be investigated which one of these mutations, or whether the combination of both, is responsible for the phenotype. Furthermore, patient B-125 had a homozygous 1 bp deletion (1072delT) and a heterozygous exon 4 duplication. In this context, their functional role (if expressed) remains to be investigated. Finally, the significance of heterozygous parkin mutations in patients with isolated tremor or RLS (relatives of B-219 and B-300) remains to be elucidated.
The mutations found in our patients are spread over the gene and affect exons 2, 3, 4, 5, 7 and 9. Except for the exon 5 deletion, all exon deletions cause frame shifts and result in truncation of the parkin protein, missing the RING-IBR-RING motif. This motif at the C-terminus is likely to be responsible for the probable function of parkin as an E3 ubiquitin protein ligase (13–15). The same result is expected for the small deletions within exon 9, leading to loss of IBR and RING2. In addition, a deletion of exon 5 will probably result in variations of splicing and consequently in a reduction of functional gene product. The potential consequences of the amino acid change Ala82Glu remain to be investigated because in this case, it occurs in combination with a homozygous exon deletion and, in addition, does not affect any conserved protein motif. However, if present alone it may, for example, alter the conformation of the protein.
To date, only two studies address the question of gene dosage of parkin, using two different approaches (4,7). Lücking et al. (4) performed the first screen for alterations of gene dosage of parkin. However, the method employed, a semi-quantitative multiplex PCR assay, did not include screening of exon 1. In addition, the authors measured the amount of PCR products in the putative log-linear phase of their multiplex PCR, without showing constant amplification efficiency for all primer pairs. The amount of PCR product was only estimated by measuring peak heights on an automated sequencer and they used intronic primers only, thus not ruling out the possibility of intron polymorphisms affecting the primer annealing site. Another group performed a PCR assay with TaqMan probes for exons 1–5 only (7).
Both the TaqMan and the LightCycler systems allow utilization of quantitative real-time PCR assays by employing labeled oligonucleotide probes which generate sequence-specific signals for the quantitative evaluation. However, TaqMan probes are more sensitive to variations within the binding site which may result in loss of signal even in the case of 1 bp mutations. In contrast, in the case of a mismatch, LightCycler hybridization probes would still generate a signal. This mismatch would result in a lower melting temperature of the probe which is detectable by melting curve analysis. Furthermore, unlike hybridization probes in LightCycler assays, TaqMan probes have to be hydrolyzed during PCR by the 5'3' exonuclease activity of Taq DNA polymerase to generate a signal. It has recently been shown that TaqMan probes may be degraded during PCR by the Taq polymerase resulting in unspecific signal (16).
To detect gene dosage changes for the diagnosis of DNA duplications and deletions in the PMP22 gene, leading to Charcot–Marie–Tooth disease type 1A or tomaculous neuropathy, Ruiz-Ponte et al. (17) employed the melting curve modus of the LightCycler. In short, a polymorphic fragment of the PMP22 gene was amplified and the area under the melting curve compared between each allele and a competitor molecule, respectively (17). For this, a polymorphism is needed within the target sequence, and two PCR experiments have to be performed in case of homozygosity. Neither of these prerequisites is required by our method, rendering it the quicker and more versatile approach.
We performed the quantification with hybridization probes using the LightCycler (Roche Diagnostics), enabling real-time measurements during the log-linear phase of a PCR, which only comprises two to five cycles. To exclude false positive detection of gene dosage alterations due to possible sequence changes in intronic primer annealing sites, we used exonic primers, if possible. Sequence changes in the exons were previously excluded by single-strand conformation polymorphism (SSCP)/sequence analysis.
Beta globin proved to be a well suited reference gene since, as a single copy gene, it does not cause any instabilities in gene dosage due to amplification of pseudogenes. In case of a beta globin mutation, most individuals show symptoms of thalassemia which was excluded in our patients. Furthermore, if there was an alteration in gene dosage of beta globin, ratios for all parkin exons would be altered in our assay, which was not the case in any of our samples.
To confirm our ability to detect deletions, we used three different types of positive controls (samples with known heterozygous deletions): (i) members of Family LA with the large deletion described previously, indicated by incompatibilities in haplotype analysis at marker D6S305; (ii) parents of patients with known homozygous exon deletions; and (iii) a sample with a 40 bp deletion in exon 3 which overlapped with one of the primer annealing sites.
In addition, we performed a Southern blot analysis to demonstrate the presence of a heterozygous deletion of exon 7 in one patient. The obvious decrease in band intensity at the 1.5 kb EcoRI fragment is consistent with this deletion. Interestingly, this band was one of four shown to be absent in a similarly designed Southern blot of a patient with a homozygous deletion of exons 3–7 (3). It is conceivable that the 1.5 kb fragment corresponds to exon 7. Furthermore, one expects the generation of a new fragment by the deletion. However, due to the very large size of the parkin introns, this fragment probably consists entirely of intronic sequence and is thus not detectable with the exonic probe used. This idea is supported by similar experiments showing homozygous exon deletions (3,5). Since intronic sequences of the parkin gene are currently only partially available, thus preventing the design of suitable intronic probes for the detection of newly generated fragments, Southern blot analysis does not appear to be the method of choice to identify heterozygous alterations of gene dosage.
The relatively high frequency of alterations in gene dosage even in this small patient sample underlines the necessity of quantitative mutational screening in patients with parkinsonism, as previously indicated elsewhere (4).
Taken together, our new method of quantitative duplex PCR of all exons of parkin with beta globin as internal standard on the LightCycler allows for quick and accurate testing for deletions and duplications of whole exons of parkin using genomic DNA. Systematic screening for parkin mutations, including gene dosage, of large PD and EOP patient cohorts will help to further elucidate the phenotypic spectrum and frequency of parkin mutations, will shed light on the frequency of patients with ‘true’ heterozygous mutations, and may reveal putative phenotype–genotype correlations.
The method of quantitative PCR described here is also applicable to other genes and disorders by designing suitable probes. It is conceivable that patients with dominantly inherited disorders, reported as ‘mutation-negative’, may carry heterozygous deletions of one or more exons. This hypothesis is supported by the detection of exonic deletions with two different laborious methods. (i) An exonic deletion in the GTP cyclohydrolase I gene was recently identified by Southern blotting in a patient with dominantly inherited dopa-responsive dystonia (18). (ii) In patients with holoprosencephaly, large deletions were found by fluorescence in situ hybridization, leading to hemizygosity of the SIX3 gene (19). Our method provides the opportunity to study heterozygous exon deletions with genomic DNA as sample material in any disease-associated gene in a short period of time.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Patients and controls
After giving informed consent, all patients, including one patient from the Family LA reported previously (8), underwent a detailed neurological examination by a specialist in movement disorders (P.P.Pramstaller and A.E.Lang). Patients with an age of onset of PD <50 years were included in the study. The diagnosis of PD was established according to the UK Parkinson’s Disease Society Brain Bank criteria (20,21). Patients were considered as clinically definite if they showed at least two out of three cardinal signs (resting tremor, rigidity, bradykinesia) and a consistent response to levodopa. Secondary parkinsonism and other Parkinson plus syndromes were excluded by history, absence of additional neurological signs not compatible with PD or EOP, normal laboratory findings (full blood count, urine analysis, serum electrolytes, liver, renal and thyroid function tests, vitamin B12 and folate levels, VDRL, copper, ceruloplasmin and acanthocytes), and by neuroimaging (CT or MRI brain scan). A total of 350 healthy, ethnically matched blood donors were included as controls.
Mutational analysis
To detect point mutations and small deletions/insertions, we performed SSCP analysis after amplification of all 12 exons of parkin, using published intronic primers (3). All altered fragments were then sequenced on an automated sequencing machine (LI-COR).
Gene dosage analysis was performed in a quantitative duplex PCR assay of all 12 exons of parkin on the LightCycler (Roche Diagnostics) using the fluorescence resonance energy transfer technique. The beta globin gene was co-amplified with each individual parkin exon and served as internal standard (Table 2). The probes were labeled oligonucleotides emitting light at different wavelengths (Table 3). The following reagents were used for amplification in a 10 µl reaction: 1 µl Hybridization FastStart Mix (Roche Diagnostics), 2–4.5 mM MgCl2, 0.2 µM of each of the hybridization probes (one pair of 3'-fluorescein and 5'-LightCycler Red 705 for the reference gene beta globin, and one exon-specific pair of 3'-fluorescein and 5'-LightCycler Red 640 for parkin), 0.5–1.0 µM of each primer and 1–15 ng of DNA. PCR conditions were as follows: 95°C for 10 min, 95°C for 10 s, 55°C for 10 s, 72°C for 10 s (40 cycles); measurement of fluorescence in each cycle was at 55°C.
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During the log-linear phase, amplification can be described as: N = N0(1 + Econst)n where N = number of amplified molecules; N0 = initial number of molecules; E = amplification efficiency; n = number of cycles. Since amplification efficiency during the log-linear phase is constant, the initial concentration of the sample was calculated based on the above formula, using a standard curve. This standard curve was generated using human genomic DNA (Roche Diagnostics) in concentrations of 5, 1.25 and 0.3125 ng/µl, respectively. All standards were amplified in duplicate and a regression curve was calculated. Sample concentrations were inferred based on this regression curve. Concentrations not within the range of the standard templates were disregarded and adjusted. All samples were also measured in duplicate and results were accepted only within a range of <10% of the standard deviation of the two inferred sample concentrations. Using an internal control (co-amplification of beta globin) provided a relative ratio of concentration parkin/concentration beta globin. Thus, this ratio corresponded to the gene dosage of both DNA fragments. A ratio between 0.8 and 1.2 was considered as normal (Fig. 1A), a heterozygous deletion was expected at a ratio between 0.4 and 0.6, a heterozygous duplication between 1.3 and 1.7, and a homozygous duplication or a triplication at a ratio between 1.8 and 2.2. All detected gene dosage variations were confirmed at least twice. After each PCR, a melting curve was performed between 40 and 80°C to analyze the product for the presence of mutations covered by the pair of hybridization probes.
Southern blotting
Eight micrograms of genomic DNA of one patient and two controls were digested with EcoRI, followed by agarose gel electrophoresis and blotting onto a nylon membrane by standard procedures. A mix of amplicons of all 12 exons of parkin generated with published intronic primers (3) was labeled with [32P]CTP by the random priming method and used as probe. Hybridization was performed as described elsewhere (22).
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ACKNOWLEDGEMENTS |
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We would like to thank the patients and family members who participated in this study, and Dr Anja Haack for excellent technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft (Kl-1134/2-1 to K.H., P.V. and C.K.), the Parkinson’s Disease Foundation (C.K.) and the Neuroepidemology Project South Tyrol (P.P.P.).
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +49 451 500 2993; Fax: +49 451 500 4861; Email: klein_ch@neuro.mu-luebeck.de The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors
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REFERENCES |
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