Review

Genes and parkinsonism

Genes and parkinsonism

John Hardy, Mark R Cookson, and Andrew Singleton

Genetic studies in families with mendelian inheritance of Parkinson’s disease (PD) have reported the cloning of several disease-associated genes. These studies of rare familial forms of the disease have cast doubt on our understanding of the role of genetics in typical PD and have complicated the classification of the disorder. However, this genetic information might help us to construct a hypothesis for the pathogenetic processes that underlie PD. In this review we describe the molecular genetics of PD as currently understood to help explain the pathways that underlie neurodegeneration. Lancet Neurology 2003; 2: 221–28

Parkinson’s disease (PD) had been considered the archetypal non-genetic disorder until 5 years ago. However, nine genetic linkages have since been reported, and three, or arguably four, genes have been identified. These genetic advances were reviewed recently by our group and others,1–3 so we will deal with this progress only briefly here. In addition to detailing the molecular genetic analysis of PD in this review, we have three goals: to discuss the nosology of this disorder; to discuss the possible reasons for the discrepancy between genetic and epidemiological analyses; and to discuss the hypothesis that dysfunction in the ubiquitin proteasome pathway is central to the pathogenesis of PD.

The molecular genetics of PD Nine genetic loci—PARK-1–8 and PARK-10—have been reported for PD (table). Four other genetic disorders are also included in the table because they have phenotypic overlap with PD—frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17),4 X-linked dystonia parkinsonism (XDP), spinocerebellar ataxia type 2,5 and spinocerebellar ataxia type 3 (Machado-Joseph disease).6 These four genetic diseases rarely present as pure parkinsonism and should be thought of separately from the PARK loci. There are few patients with clear mendelian inheritance compared with the number of sporadic cases. However, identification of the genetic determinants of PD in these rare cases and in their families provides us with important clues to the general pathogenetic processes in PD. In addition to these loci, which have been identified by traditional linkage strategies, three genome screens that use affected pedigree member methods in typical late-onset PD have also been reported.7–9 By use of genomic screening methods, researchers have identified distinct regions that are shared by family members with PD at a greater frequency THE LANCET Neurology Vol 2 April 2003

than expected by chance. However, none of these reported linkages reached statistical significance, and so we have not included them in this review. Genes reported to cause PD

The first reported genetic linkage for PD was PARK-1, a locus on chromosome 4q.10 This region contains the ␣-synuclein gene, in which two mutations (A30P and A53T) were shown to cause autosomal dominant PD.11,12 The discovery of mutations in ␣-synuclein, a protein that was subsequently identified as a major component of Lewy bodies,13 was proof of principle for a genetic contribution to PD and shifted the focus of research. The promise of cloning genes involved in rare mendelian forms of PD will provide insights into the pathogenesis and aetiology of the more common forms of the disease. One of the best examples of this is the genetic association of ␣-synuclein with sporadic PD. There is evidence that ␣-synuclein promoter variants contribute to the lifetime risk of typical sporadic PD.14–17 In general, alleles that raise ␣-synuclein expression are associated with increased risk of PD, although the promoter has a complex repeat structure that complicates analysis. PARK-2, the second locus to be identified, is on chromosome 6q. This locus contains the parkin gene, mutations in which cause autosomal recessive juvenile parkinsonism.18 However, the relation between parkin mutations and PD is more complex. The parkin gene promoter, like that of ␣-synuclein, has functional variants and those that have lower transcriptional activity are associated with an increased risk of PD.19 Very recently the PARK-7 locus on chromosome 1q, associated with autosomal recessive early onset PD, has been cloned.20 Mutations in this region lead to loss-of-function variants of the protein DJ-1.20 Mutations in DJ-1 seem to be a rare cause of PD. Although no pathological examinations of patients with mutations at this locus have been reported, PET studies have shown presynaptic dopaminergic-cell loss in these patients. PARK-5 is linked to a single mutation, I93M, of ubiquitin carboxy-terminal hydrolase L1 (UCHL1) in a small family from Germany with PD.21 There are two common JH, MRC, and AS are all at the Laboratory of Neurogenetics, National Institute on Aging, Bethesda, MD, USA. Correspondence: Mark R Cookson, Laboratory of Neurogenetics, National Institute on Aging, NIH, Building 10 Room 6C103, MSC1589, 9000 Rockville Pike, Bethesda MD 20892. Tel +1 301 451 3870; fax +1 301 480 0315; email [email protected]

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Table. Genetic causes of parkinsonism Locus (protein or location)

Inheritance

Population affected (mutation)

Clinical description Disease type

Genetic variants with parkinsonism as the major clinical sign PARK1 Autosomal Italian, PD, plus (␣-synuclein) dominant Greek (A53T), DLB (A53T) German (A30P) Global

Dementia Asymmetry Resting Response to Other tremor levodopa features ++ (+/no)

+++

+++

+++

Onset typically 1 Mbp19) and contains 12 exons that are translated into a 52 kDa protein. Parkin was cloned after the identification of large deletions at this locus in patients with autosomal recessive juvenile parkinsonism.18 Many patients with the disease were either hemizygous or nullizygous for genetic markers within the locus. It is likely, although not certain because of alternative splicing events, that the alleles with large deletions are simply loss-of-function alleles. Many variants with single aminoacid substitutions have been described43,44 and there is less evidence that these cause complete loss of function. In particular, very few of these variants have been found in affected individuals either as homozygotes or as compound heterozygotes with another point mutation. Some of these variants have been found in patients without other parkin mutations, which makes it difficult to exclude the possibility that they are rare, but harmless, coding polymorphisms. Other mutations segregate as apparent dominant mutations without full penetrance, which suggests that they may be dominant negative mutations, and some have been found both with deletion alleles and as single mutations. Few autopsies on patients with parkin mutations have been reported, although it is clear that some patients have typical autosomal recessive juvenile parkinsonism,46 some have typical PD,43 and others have unusual features such as tau-positive glial-cell inclusions.47 The discovery that parkin is a ubiquitin ligase and our knowledge of ubiquitin-ligase biochemistry allows us to speculate on the different modes of inheritance. Deletion alleles are predominantly simple loss-of-function alleles, but some mutations, especially point mutations, result in parkin that may have partial ubiquitin-ligase activity48 and may thus cause an incomplete loss of function. The complete loss of function of parkin might lead to cell death whereas partial loss of function might be associated with PD and Lewy-body formation, as Lewy bodies are heavily ubiquitinated. In support of this idea, many patients with clinically-typical PD49 and a single patient with Lewy-body parkinsonism43 with parkin mutations have point mutations, whereas the majority of patients with ARJP have parkin deletions. Although this is an attractive hypothesis, more pathological studies of patients with mutations in parkin are needed before this can be taken as anything other than speculation. Patients with the same parkin mutations can show some phenotypic variability, which further emphasises the need to study a large series of cases.50

MPTP-induced parkinsonism, which is a clinical phenocopy of PD although Lewy bodies are not present. Recent epidemiological surveys have shown an increased risk of PD in relatives of patients with PD,51 although the magnitude of this increased risk is much lower than that predicted by mendelian models. The role of genetic factors in PD has been challenged by large twin studies.52 Therefore, the epidemiological evidence for a genetic cause of PD is equivocal at best. So how can we reconcile this with the nine reports of genetic linkages? Although there can be no complete answer, perhaps we can offer some partial explanations. First, PD is not an aetiological entity. If it manifests as dementia or autonomic dysfunction, the disease may not be identified. There have been reports of increased incidence of depression53 and dementia54 in the siblings of patients with PD. Furthermore, even within individual families with clear inheritance there is phenotypic variation between PD and diffuse Lewy-body disease.55 Second, the age at onset can be very variable in a sibpair and even within a pair of twins,56 which makes accurate assessment of disease rates in cross-sectional studies subject to error. The use of dopaminergic-function measurement to define a subclinical phenotype increases the concordance rate in monozygotic twins57 and subclinical dopaminergic loss has been noted in otherwise asymptomatic carriers of ␣-synuclein mutations.58 Age at onset varies between 20 and 85 in the Contursi kindred with the A53T ␣-synuclein mutation.59 Third, mild PD often goes unnoticed, even by the patient. Slowness and stiffness of movement, a shuffling gait, a quiet voice, and an expressionless face are so much a part of our preconception of old age that the disease is likely to be substantially under diagnosed, which is undoubtedly related to subclinical disease expressivity. Finally, the contribution of genetics to the lifetime risk of PD outside monogenic families may be small or involve numerous genes with small effects. The increased risk to siblings with oligogenic disorders would be modest, even if the genetic contribution to risk was high. Furthermore, in an outbred population, recessive diseases often present in the absence of a family history. Recent studies support the contention that typical “sporadic” PD has a strong genetic component.57,60 The most likely explanation for the disparity between epidemiology and genetics is a combination of all of these factors, although if sporadic PD is oligogenic then it may be difficult to establish the contribution of inheritance. However, genetic analysis of “typical” PD yields linkages in well-characterised populations,60 which suggests that genetics can resolve the aetiology of the typical disorder.

From genetics to pathway: DNA to disease The conflict between epidemiological and genetic analyses In Charcot’s time, PD was thought to be a genetic disease, but for most of the 20th century, the importance of genetics has been consistently downplayed. This change in thinking may be partly due to the occurrence of post-encephalitic parkinsonism, which can be confused clinically with PD, and

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The genetic defects that have been identified in familial PD allow us to tentatively reconstruct the pathway that leads to cell death. Perhaps the strongest clue to the pathogenesis of PD comes from the identification of parkin as an E3 ubiquitinprotein ligase.61,62 Parkin is one of several proteins63 that ubiquitinate target proteins before they are degraded by the proteasome.64 As already discussed, many parkin

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Genes and parkinsonism

mutants are recessive, produce severely E2 E1 E1-Ub truncated proteins, and probably repreTarget sent loss-of-function mutations. In these +ATP protein E -Ub Ub 2 cases, decreased parkin function leads to a decreased ability of neurons to regulate proteolysis, which results in the loss of UCH E3 nigral neurons in autosomal recessive (parkin) K29 or juvenile parkinsonism. The substrates of (Ub)n K48 parkin are not known but there are +ATP 65 Protein-(Ub) several candidates, some of which are Protein-(Ub) Ub-peptides n K63 26S proteasome known to induce neuronal damage if Protein-(Ub)n overexpressed.66 In more complex situaPeptides •Protein sorting Ub tions, such as allelic variants with partial •Transcription •DNA repair activity, there may be an accumulation of Amino acids •Endocytosis ubiquitinated proteins. 3 As hypothesised by other authors, Figure 1. A simplified schematic diagram of the major components of the ubiquitin–proteasome with specific attention to familial PD. Ubiquitin (Ub) is activated and binds to the E loss of parkin function suggests that pathway enzyme, and is then transferred to a carrier enzyme (E ). Target proteins are recognised by E proteasome mediated turnover of enzymes, such as parkin, which then catalyse the transfer of Ub from the E enzyme to the target damaging proteins within the cell is a protein. There are several lysine acceptor residues on ubiquitin: K29, K48, and K63 are given as central process in the pathogenetic path- examples. Sequential cycles of covalent additions of Ub molecules via K29 or K48 results in the way of PD. UCHL1 recycles ubiquitin addition of a polyubiquitin chain—protein-(Ub) )—which signals the protein for destruction via the from multiubiquitin chains and is thus proteasome. Both this step and the initial activation of Ub are energy dependent (ie, they require ATP). The targeted proteins are degraded to small peptides and constituent amino acids. After part of this pathway. It has been recently degradation, polyubiquitin chains are recycled to monomeric ubiquitin by ubiquitin carboxyl-terminal shown that UCHL1 also has hydrolases (UCH). Other actions of ubiquitin are also important for cellular function. For example, ubiquitin–ubiquitin ligase activity (ie, it K63-linked polyubiquitin chains can trigger DNA repair or endocytosis. Monoubiquitination, adds ubiquitin molecules to pre-existing represented by protein-(Ub) here, is important in protein sorting and transcriptional effects. Finally, chains on proteins before their destruc- small ubiquitin modifiers may undergo similar (and similarly varied) reactions with targeted proteins. tion). The I93M mutation in UCHL1 marginally decreases this ligase activity, while also substantially independent manner,73 as has been shown for other natively lowering the protein’s hydrolase activity. This could unfolded proteins. There is, however, an important report of potentially mean that polyubiquitin chains are not recycled parkin mediated ubiquitination of ␣-synuclein,74 which and cellular pools of monomeric ubiquitin are depleted. The suggests that misregulation of ␣-synuclein is damaging to putative protective variant, Y18, has a normal hydrolase neurons. However, the form of ␣-synuclein that appears to be activity but a dramatically lowered ligase activity and thus may recognised by parkin is glycosylated. The abundance of this be beneficial in maintaining cellular ubiquitin pools.67 There- glycosylated species in the brain is not known and we do not fore, the role of UCHL1 mutations and polymorphic variants know how mutations in ␣-synuclein affect glycosylation and in the pathogenesis of PD supports the hypothesis that subsequent ubiquitination by parkin. Non-glycosylated changes to proteasome function are central to this process. ␣-synuclein is not recognised by parkin.48 That a dimer of Nothing, however, is more dangerous than a plausible UCHL1 may have a ligase activity directed towards hypothesis that lacks definitive data. Furthermore, since ␣-synuclein67 further suggests that regulation of ␣-synuclein ubiquitin also has other roles in the cell, the data on concentrations within the cell via ubiquitination is important. ubiquitin-mediated reactions could be interpreted in However, the type of ubiquitination regulated by UCHL1 different ways. For example, ubiquitin and related modifiers seems to be via K63, which raises the possibility that the can signal for intracellular sorting events and may affect process of ␣-synuclein ubiquitination in this context does not synaptic function.68 Ubiquitin chains can be formed by the promote degradation but instead affects protein localisation. addition of ubiquitin to different lysine groups. For example, There are other ways in which ␣-synuclein might affect a ubiquitin chain linked by lysine 48 signals for the proteasome function independently of the interactions degradation of proteins, whereas chains linked by lysine 63 between the different proteins associated with PD. First, signal for intracellular sorting.69,70 Recent evidence suggests several groups have shown that ␣-synuclein mutations that UCHL1 adds ubiquitin via K63 linkages.67 decrease net proteasomal activity.75–77 The mechanistic details are unclear, but ␣-synuclein can bind to the proteasome ␣-synuclein and the role of fibril formation directly, 78 although indirect mechanisms are also possible. If the loss-of-function parkin mutations (and perhaps the For example, the A30P ␣-synuclein mutant has effects on UCHL1 variants) support the involvement of proteasomes in mitochondria,75 which could compromise ATP production PD pathology, what role, if any, do ␣-synuclein mutations and indirectly inhibit the heavily ATP-dependent processes have in this process? Previous reports of ␣-synuclein of ubiquitination and proteasome activities (figure 1). metabolism via the proteasome71 have been challenged on Reciprocally, mitochondrial inhibitors cause accumulation technical grounds,72 although recent studies suggests that the of ␣-synuclein79–82 and ubiquitin.81,82 Conversely, ␣-synuclein proteasome might degrade ␣-synuclein in a ubiquitin- knockout mice are resistant to MPTP.83 The implication is 64,69,70

1

2

3

2

n

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Mitochondrial damage

mutations ␣–synuclein via ubiquitination?

(protein)

Oligomers, protofibrils, pores

Dopamine

­

ATP

Decreased proteasome function

Accumulating misfolded, toxic, or damaged proteins

Neuronal dysfunction, cell death

?

Vesicle permeabilisation

Intracellular aggregates (Lewy bodies)

parkin UCHL1 Figure 2. Relation between familial PD gene products.

that both mutant forms (A30P and A53T) of ␣-synuclein decrease net proteasome function within the cell, which supports the idea that misregulation of ubiquitination is linked to the neurotoxic effects of the mutant protein. ␣-synuclein may have multiple effects on proteasome function. Whether direct effects or indirect mechanisms are important and whether the proteasome effects are sufficient to explain how the mutations cause disease are unclear. Bearing this in mind, if reduced proteasome function is associated with mutations in ␣-synuclein and loss-of-function mutations in parkin, we could expect decreased proteasome function in PD in general. Recent reports indicate decreased proteasome activity in brain tissue from patients with idiopathic PD84 and a neurotoxic effect of the proteasome inhibitor lactacystin on dopaminergic neurons in vivo.85 There is also evidence that proteasome inhibitors selectively damage catecholaminergic cells in vitro.77 Furthermore, the toxicity of mutant ␣-synuclein can be decreased by a tyrosine hydroxylase inhibitor,86 which suggests that catecholamines contribute to cell death induced by proteasome inhibitors or ␣-synuclein mutations. Catecholamines can inhibit fibrilisation of ␣-synuclein and promote the formation of smaller oligomeric species87 that may be toxic. What is the role of ␣-synuclein fibrillisation in the pathway of neuronal damage in PD? ␣-synuclein is found as fibrillar material in Lewy bodies,88 presumably due to the formation of insoluble complexes of ␤-pleated-sheet aggregates. It has been argued that such fibrillar inclusions must be pathogenetic. However, as mutations in ␣-synuclein cause disease, one would presume that fibrillisation is increased by these mutations; in fact, this is not the case. Several groups have shown that, although the A53T mutation increases the fibrillisation of ␣-synuclein, the A30P mutation is very similar to wild-type and may even retard fibril formation.89,90 Likewise, in transgenic mouse models, fibrillar forms are easier to induce in A53T mutants compared with A30P mutants or wild-type. The A53T ␣-synuclein mutation induces motor deficits that are not seen with A30P.91 However, both mutants share one biophysical property—they form prefibrillar oligomeric species (also known as protofibrils) more rapidly than the wild-type protein.90 This suggests that

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fibrillisation to the extent of forming large insoluble structures is not the common factor in pathogenesis. Small oligomers of ␣-synuclein form annular ring-like structures92 similar to those formed by the amyloid-␤ protein in Alzheimer’s disease.93 These structures can damage cell membranes by a pore-like activity similar to that of some bacterial toxins.93,94 Consistent with this idea, ␣-synuclein protofibrils permeabilise vesicles in a size-restricted fashion95 and lead to a loss of catecholaminergic vesicles in vitro.76 The fact that ␣-synuclein is associated with lipid vesicles may also be important as lipids promote oligomerisation.96 Protofibrils or small oligomers might be the toxic species, which suggests that fibrils are a safely sequestered form of the same protein, although this has not been proved. Relation between PD and gene products

The evidence from genetic studies allows us to say two things with some certainty (figure 2). First, ␣-synuclein mutations promote the accumulation of toxic species with pleiotropic cellular effects, including inhibition of proteasome function,75–77 vesicle permeabilisation,76 and mitochondrial effects.75 It is important to note that there are two pathways here: depletion of cellular ATP stores, which increases proteasome dysfunction, and release of dopamine into the cytosol, which promotes further oligomerisation events. Second, parkin mutations decrease the ability of nigral neurons to withstand cellular stress,77 presumably due to a loss of parkin’s function as a protein–ubiquitin ligase. Whether this is due to effects on the degradation of toxic proteins (figure 2) or another important function of ubiquitin (figure 1) has not been resolved. Furthermore, the precise relation between the control of ␣-synuclein concentrations within the cell and ubiquitination is unclear (figure 2). The potential contribution of UCHL1 variants is another possible thread and although the genetic foundation for this is not solid, UCHL1 variants might have effects on either net proteasome function or on ␣-synuclein protein concentrations. Thus, on balance, the formation of fibrillar ␣-synuclein inclusions is probably not the main pathogenetic event although the formation of fibrillar inclusions occurs throughout the course of the disease. It is therefore not clear

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Genes and parkinsonism

Search strategy and selection criteria Data for this review were identified from the authors’ own files and by searches of PubMed with the search terms “Parkinson” or “parkinsonism” and “genes”. Articles published before February, 2003, were included. Only articles published in Englis’ were reviewed.

whether formation of ubiquitinated products, such as Lewy bodies, is part of this process, although it is often used as an argument to support a central role of ubiquitination in this disease. For these reasons, we have portrayed Lewy-body formation and neuronal dysfunction as parallel events the precise relation of which is unclear. Figure 2 summarises some of these events in terms of their likely temporal relationships. Our contention is that the early events, which are likely to be mediated by protofibrillar ␣-synuclein, are complex and multiple but in summation (indicated by the dotted lines in figure 2) lead to dysfunction of vulnerable sets of neurons and eventually to cell death. The identification of loss-of-function mutations in DJ-1 will allow us to test whether ubiquitination is important and also to further investigate the role that oligomerisation of ␣-synuclein has in PD. At first glance, DJ-1 appears to have no direct role in either proteasome function or protein aggregation pathways; it has been cloned as an RNA binding protein,97 a fertility associated protein,98 and an interactor with the proto-oncogene c-myc.99 DJ-1 responds to oxidative damage,100,101 which could support the hypothesis that the critical pathway in PD is centred on free-radical-mediated damage resulting from mitochondrial dysfunction or from the presence of catecholamines, such as dopamine, in the cytosol. However, as DJ-1 clearly has multiple functions, there are several ways to reconcile DJ-1 mutations with the functions of ␣-synuclein and parkin, as discussed recently.102 The challenge for the future is to use DJ-1 mutations and other mutations, as they are identified, to critically assess the various hypotheses of neurodegeneration in PD. References 1 2 3 4

5 6

7 8

9

Gwinn-Hardy K. Genetics of parkinsonism. Mov Disord 2002; 17: 645–56. Vaughan JR, Davis MB, Wood NW. Genetics of parkinsonism: a review. Ann Hum Genet 2001; 65: 111–26. Kruger R, Eberhardt O, Riess O, Schulz JB, Riess O. Parkinson’s disease: one biochemical pathway to fit all genes? Trends Mol Med 2002; 8: 236–40. Hutton M, Lendon CL, Rizzu P, et al. Association of missense and 5⬘-splice-site mutations in tau with the inherited dementia FTDP-17. Nature 1998; 393: 702–05. Gwinn-Hardy K, Chen JY, Liu HC, et al. Spinocerebellar ataxia type 2 with parkinsonism in ethnic Chinese. Neurology 2000; 55: 800–05. Subramony SH, Hernandez D, Adam A, et al. Ethnic differences in the expression of neurodegenerative disease: Machado-Joseph disease in Africans and Caucasians. Mov Disord 2002; 17: 1068–71. DeStefano AL, Golbe LI, Mark MH, et al. Genomewide scan for Parkinson’s disease: the GenePD study. Neurology 2001; 57: 1124–26. Pankratz N, Nichols WC, Uniacke SK, et al. Genome screen to identify susceptibility genes for Parkinson disease in a sample without parkin mutations. Am J Hum Genet 2002; 71: 124–35. Scott WK, Nance MA, Watts RL, et al. Complete genomic screen in Parkinson disease: evidence for multiple genes. JAMA 2001; 286: 2239–44.

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Conclusions There are two potential outcomes of the genetic analysis of PD. First, improvement of diagnostic accuracy with earlier and perhaps presymptomatic diagnosis. Second, an understanding of disease pathogenesis to facilitate modelling in cell systems and in animals so that treatments can be developed and tested. Due to the success of dopamine replacement therapy, research into PD has been neglected for 20 years, except for the continuing attempt to improve on levodopa as a therapy. Palliative therapies are effective now, but in the future we have to hope that therapies based on aetiology will complement, and then supplant, them. At present, oligomerisation of ␣-synuclein and dysfunction in the ubiquitin–proteasome system have been implicated but it will only be with the identification of other genes and a fuller understanding of the role of novel mutations, such as those found in DJ-1, that we will be able to identify the main pathway involved. With the public availability of the human genome sequence, and six unresolved genetic linkages, research over the next few years will either confirm or disprove the involvement of this pathway. If the ubiquitin–proteasome system is involved, we then have to hope that some part of it is amenable to therapeutic intervention. This research is likely to take at least 10 years, but the fact that researchers working on many other diseases are following a similar path is likely to be a great help in this endeavour. Authors’ contributions

JH wrote the sections on the nosology and epidemiology of PD. MC wrote the sections on the pathways underlying neurodegeneration in PD. AS wrote the genetics section. All authors contributed to the structure of the review and the revision and editing of the article. Conflict of interest

We have no conflicts of interest. Role of the funding source

All authors are employees of the US Federal Government. No funding source was involved in the preparation of this review or the decision to submit it for publication.

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18 Kitada T, Asakawa S, Hattori N, et al. Mutations in the parkin gene cause autosomal recessive juvenile parkinsonism. Nature 1998; 392: 605–08. 19 West AB, Maraganore D, Crook J, et al. Functional association of the parkin gene promoter with idiopathic Parkinson’s disease. Hum Mol Genet 2002; 11: 2787–92. 20 Bonifati V, Rizzu P, van Baren MJ, et al. Mutations in the DJ-1 gene associated with autosomal recessive early-onset parkinsonism. Science 2003; 299: 256–59. 21 Leroy E, Boyer R, Auburger G, et al. The ubiquitin pathway in Parkinson’s disease. Nature 1998; 395: 451–52. 22 Wintermeyer P, Kruger R, Kuhn W, et al. Mutation analysis and association studies of the UCHL1 gene in German Parkinson’s disease patients. Neuroreport 2000; 11: 2079–82. 23 Maraganore DM, Farrer MJ, Hardy JA, Lincoln SJ, McDonnell SK, Rocca WA. Case-control study of the ubiquitin carboxy-terminal hydrolase L1 gene in Parkinson’s disease. Neurology 1999; 53: 1858–60. 24 Satoh J, Kuroda Y. A polymorphic variation of serine to tyrosine at codon 18 in the ubiquitin C-terminal hydrolase-L1 gene is associated with a reduced risk of sporadic Parkinson’s disease in a Japanese population. J Neurol Sci 2001; 189: 113–17. 25 Lincoln S, Vaughan J, Wood N, et al. Low frequency of pathogenic mutations in the ubiquitin carboxyterminal hydrolase gene in familial Parkinson’s disease. Neuroreport 1999; 10: 427–29.

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