A comprehensive analysis of deletions, multiplications, and copy number variations in PARK2.

OBJECTIVES: To perform a comprehensive population genetic study of PARK2. PARK2 mutations are associated with juvenile parkinsonism, Alzheimer disease, cancer, leprosy, and diabetes mellitus, yet ironically, there has been no comprehensive study of PARK2 in control subjects; and to resolve controversial association of PARK2 heterozygous mutations with Parkinson disease (PD) in a well-powered study. METHODS: We studied 1,686 control subjects (mean age 66.1 ± 13.1 years) and 2,091 patients with PD (mean onset age 58.3 ± 12.1 years). We tested for PARK2 deletions/multiplications/copy number variations (CNV) using semiquantitative PCR and multiplex ligation-dependent probe amplification, and validated the mutations by real-time quantitative PCR. Subjects were tested for point mutations previously. Association with PD was tested as PARK2 main effect, and in combination with known PD risk factors: SNCA, MAPT, APOE, smoking, and coffee intake. RESULTS: A total of 0.95% of control subjects and 0.86% of patients carried a heterozygous CNV mutation. CNV mutations found in 16 control subjects were all in exons 1-4, sparing exons that encode functionally critical protein domains. Thirteen patients had 2 CNV mutations, 5 had 1 CNV and 1 point mutation, and 18 had 1 CNV mutation. Mutations found in patients spanned exons 2-9. In whites, having 1 CNV was not associated with increased risk (odds ratio 1.05, p = 0.89) or earlier onset of PD (64.7 ± 8.6 heterozygous vs 58.5 ± 11.8 normal). CONCLUSIONS: This comprehensive population genetic study in control subjects fills the void for a PARK2 reference dataset. There is no compelling evidence for association of heterozygous PARK2 mutations, by themselves or in combination with known risk factors, with PD.

parkin mutation analysis in clinic patients with early-onset Parkinson [corrected] disease.

parkin Mutations are the most common identified cause of Parkinson's disease (PD). It has been suggested that patients with young-onset PD be screened for parkin mutations as a part of their clinical work-up. The aim of this study was to assess parkin mutation frequency in a clinical setting, correlate genotype with phenotype, and evaluate the current justification for clinical parkin testing. Patients were selected from a movement disorder clinic based on diagnosis of PD and onset age

Parkinson's disease, CYP2D6 polymorphism, and age.

OBJECTIVE: PD may be caused by genetic susceptibility to neurotoxins. CYP2D6 is a candidate gene for PD because it regulates drug and toxin metabolism, but association studies have been inconsistent. The aim of this study was to test if the CYP2D6*4 allele (poor metabolizer phenotype) is associated with earlier age at onset. METHODS: Five hundred seventy-six patients with PD and 247 subjects without PD were studied using standard diagnostic, genotyping, and statistical techniques. RESULTS: Surprisingly, mean onset age was significantly later in *4-positive patients. Frequency of *4 was significantly higher in late-onset PD than early-onset PD. When early- and late-onset PD were analyzed separately, *4 had no effect on onset age; hence, the association with delayed onset was likely an artifact of an elevated *4 frequency in late-onset PD. Contrary to a common assumption that CYP2D6 frequencies do not change with age, *4 frequency rose significantly with advancing age, both in patients with PD (from 0.16 at mean age of 56.5 years to 0.21 at mean age of 72) and subjects without PD (from 0.09 at mean age of 45.5 years to 0.21 at mean age of 72). *4 Frequencies in patients with early- and late-onset PD, although different from each other, were in agreement with similarly aged subjects without PD, suggesting the elevated *4 frequency in late-onset PD was likely an age effect, unrelated to PD. CONCLUSION: The CYP2D6*4 allele is not associated with earlier PD onset. *4 May be associated with survival. Inconsistent results from allelic association studies may have been due to an unrecognized age effect.

Efficacy and tolerability of a novel sublingual apomorphine preparation in patients with fluctuating Parkinson's disease.

We tested a novel preparation of sublingual apomorphine hydrochloride (APO) in 10 patients with advanced Parkinson's disease complicated by motor fluctuations and dyskinesias. After dose titration, patients underwent a blinded comparison of APO versus placebo, and an unblinded comparison of APO versus optimally dosed carbidopa/levodopa using timed tapping and walking paradigms. APO was significantly better than placebo in both measures: Tapping speed was 30.8% faster than with placebo (p < .0005), and ambulation speed was 45.2% faster than with placebo (p < .05). Ambulation speed with APO was also 15.9% faster than that with optimal doses of carbidopa/levodopa (p < .05). The latency to onset of clinical improvement with each APO dose was 10 to 40 minutes, and the duration of effect was 60 to 130 minutes. Adverse events included nausea, orthostatic hypotension, and disagreeable taste in the patient's mouth. Aside from the bitter taste, all other side effects resolved with continued use and did not limit dosing in any case. We feel that the good short-term efficacy and tolerability demonstrated in this study warrant further study of this new preparation, as there are several potential advantages of sublingual administration compared with traditional APO preparations.

Digoxin prevents MPTP-induced dopamine depletion in mouse striatum.

Inhibition of Na+/K+ ATPase by cardiac glycosides has been shown to potentiate toxic effects of excitatory amino acids and mitochondrial poisons in neurons in vitro. The present study tested the hypothesis that the systemic administration of the cardiac glycoside, digoxin, potentiates effects of the dopamine neurotoxin 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP) in vivo. Mice were injected with digoxin (1 mg/kg) or vehicle followed by MPTP (20 mg/kg) or saline 1 h later. After 1 or 8 days, mice were euthanized and dopamine levels in the striatum were measured by high-performance liquid chromatography with electrochemical detection. MPTP caused a significant 35-45% reduction in striatal dopamine levels compared to those in control mice. However, pretreatment with digoxin completely prevented the MPTP-induced dopamine depletion. This result was unexpected and suggests that cardiac glycosides may protect against MPTP neurotoxicity.

The effect of nigral implantation on sensitization to dopamine agonists in 6-hydroxydopamine-lesioned rats.

The implantation of fetal nigral tissue into the striatum of patients with Parkinson's disease is a promising approach to treatment which may produce clinical benefit partly by influencing drug responsiveness. The purpose of the present study was to determine the pharmacological mechanisms which drug response changes by measuring to what extent sensitization produced by repeated apomorphine treatment was attenuated by tissue implantation in rats with nigrostriatal lesions. Prior to implantation of nigral cell suspensions, the daily administration of apomorphine to rats with unilateral 6-hydroxydopamine lesions produced a progressive increase in the magnitude and duration of rotational behaviour. After implantation, apomorphine-induced rotational effects were reduced to levels observed upon the initial exposure to drug and did not increase following repeated treatment. Attenuated responses to selective D1 and D2 agonists were also observed after implantation. In vehicle-implanted rats, the initial response to apomorphine was attenuated but then increased following repeated apomorphine administration. No attenuation in responses to selective D1 and D2 agonists was observed in this group. Cell suspensions prepared from fresh and cyropreserved tissue produced similar behavioural effects, even though the volume of transplanted striatum exhibiting tyrosine hydroxylase activity was greater with fresh tissue. The duration of rotational behaviour induced by apomorphine was not affected by cell implantation. These findings suggest that the expression of sensitization in an animal model of parkinsonism may disappear after a period without drug treatment. Implantation of nigral tissue may produce beneficial results in parkinsonism by limiting the development of dopamine agonist-induced sensitization.

Changes in apomorphine pharmacodynamics following repeated treatment in 6-hydroxydopamine-lesioned rats.

Rats with 6-OHDA lesions were repeatedly treated with apomorphine and dose-response relationships compared before and after treatment to better understand the pharmacological mechanisms which result in sensitization. A progressive increase in the magnitude and duration of rotational behavior was observed following repeated treatment and was associated with a nonparallel (upward) shift in apomorphine dose-response curves. These changes are inconsistent with simple shifts in drug potency, and instead are more consistent with changes in the ability of apomorphine to stimulate postsynaptic, striatal efferent pathways which result from repeated drug administration.

Apomorphine tolerance in Parkinson's disease: lack of a dose effect.

The development of tolerance to dopaminergic drugs may be important in the long-term therapy of Parkinson's disease. In this study, we sought to determine whether tolerance developed during infusions of apomorphine and if there was evidence of any dose dependency. Eight patients with Parkinson's disease received 4- to 6-h infusions of apomorphine at low, medium, and high rates on consecutive days. Before and after each infusion, test boluses of apomorphine were administered to measure sensitivity to the drug. The duration of motor effects after the postinfusion boluses were reduced in comparison to those of the preinfusion boluses, indicating that tolerance developed during the infusions. The infusion rate did not affect the responses to the postinfusion test boluses. Our observations indicate that tolerance develops to the antiparkinsonian effect of apomorphine after several hours of its constant infusion, but is not influenced by the dose of drug administered.

Sensitization to apomorphine-induced rotational behavior in 6-OHDA-lesioned rats: effects of NMDA antagonists on drug response.

The development of drug-induced behavioral sensitization is thought to underlie many of the motor complications that accompany chronic L-DOPA treatment of patients with Parkinson's disease. As the development of sensitization to some dopaminergic behaviors has been linked to alterations in NMDA neurotransmission in animal models, we sought to determine whether or not NMDA antagonists can block the development of sensitization to rotational effects of dopamine agonists in rodents with unilateral nigrostriatal lesions. Rats with unilateral 6-hydroxydopamine lesions received either a single dose or eight daily doses of apomorphine, each dose preceded by the NMDA antagonists MK-801 or CPP. Three days after the last apomorphine dose, the circling behavior produced by the D1 agonist SKF 38393 was measured. A single dose of MK-801 (0.1 mg/kg) prevented the subsequent response to SKF 38393 but neither repeated treatment with MK-801 (0.1 or 0.3 mg/kg) nor CPP (0.1 mg/kg) preceding apomorphine prevented the subsequent response to SKF 38393 or attenuated the response in comparison to a control group. Each of the chronic treatment groups exhibited an increase in rotational effects of apomorphine despite MK-801 or CPP pretreatment. These data suggest behavioral sensitization in unilateral nigrostriatally lesioned rats chronically treated with apomorphine is not dependent upon stimulation of NMDA receptors.

Dose- and duration-dependent tolerance to rotational effects of apomorphine in a rat model of Parkinsonism.

We sought to determine what temporal and dose factors influence the development of tolerance to dopaminergic agents in parkinsonism. Apomorphine was administered at varying doses and durations to rats with unilateral 6-hydroxydopamine-induced nigrostriatal lesions and rotational behavior was monitored. Ten rats were studied across seven daily, intermittent treatment sessions, during which four equal boluses of apomorphine were injected at 1- to 2-hr intervals; increasing doses were used on different days. The total number of rotations were reduced by approximately 25% after repeated 0.8- and 3.2-mg/kg doses, but not after doses ranging from 0.1 to 0.4 mg/kg; neither the peak rate nor the duration of responses were altered. Nine other, untreated rats received four, 0.8-mg/kg boluses of apomorphine and did not exhibit any decrement in response. An 8-hr constant treatment (0.2-mg/kg boluses every 10 min) resulted in a 70% reduction in rotational response; plasma levels remained stable in five unlesioned rats who underwent a similar constant treatment. These results suggest that tolerance to dopaminergic stimulation is more apt to develop with constant than with intermittent treatment and that tolerance may require previous drug exposure in order to occur. Optimal treatment of patients with severe parkinsonism may require periods without dopaminergic effect in order to maintain drug response.

Apomorphine infusional therapy in Parkinson's disease: clinical utility and lack of tolerance.

We assessed the clinical utility of apomorphine infusional therapy in patients with parkinsonism and motor fluctuations and sought evidence for alterations in drug response resulting from chronic treatment. Six patients with Parkinson's disease were treated for 3 months with s.c. infusions of apomorphine administered during waking hours. At the beginning and the end of the study, test doses of apomorphine (12.5-100 micrograms/kg) were administered to establish a dose-response curve. Over the study, the patients reported a significant improvement in the number of "on" hours experienced per day and substantially reduced the dose and frequency of levodopa and other antiparkinsonian medications. No average change in apomorphine dose-response relationships or pharmacokinetics was observed during the study. However, two patients lowered the infusion rate during the 3-month observation and exhibited higher drug levels and longer responses following test doses of apomorphine given at the end of the study. Although pragmatic concerns with the use of infusion pumps solutions and adverse effects limited the overall benefit afforded by the treatment, this kind of drug treatment may be useful in selected patients with severe parkinsonism and fluctuations.

Pharmacokinetics of apomorphine in Parkinson's disease.

The pharmacokinetic properties of apomorphine in patients with Parkinson's disease are described. Apomorphine is lipophilic; it has a large volume of distribution and is rapidly cleared from plasma, with an elimination half life of 33 minutes. It is rapidly absorbed following subcutaneous injection, with peak levels achieved within 5-10 minutes in most patients. There is a large variation in absorption between patients but is more constant within patients following repeated dosing. Apomorphine rapidly equilibrates between plasma and brain. Like levodopa, the response tends to be largely "all or none"; larger doses produce a longer duration of effect within a 30-90 minute range. Apomorphine may be administered intranasally and sublingually; of these routes, the former is more quickly and completely absorbed. Other routes of administration, including rectally, are not as well absorbed but may also be used effectively.

Episodic ataxia/myokymia syndrome is associated with point mutations in the human potassium channel gene, KCNA1.

Episodic ataxia (EA) is a rare, familial disorder producing attacks of generalized ataxia, with normal or near-normal neurological function between attacks. One type of EA is characterized by brief episodes of ataxia with myokymia (rippling of muscles) evident between attacks. Linkage studies in four such families suggested localization of an EA/myokymia gene near the voltage gated K+ channel gene, KCNA1 (Kv1.1), on chromosome 12p. Mutation analysis of the KCNA1 coding region in these families identified four different missense point mutations present in the heterozygous state, indicating that EA/myokymia can result from mutations in this gene.

A gene for episodic ataxia/myokymia maps to chromosome 12p13.

Episodic ataxia (EA) is a rare, familial disorder producing attacks of generalized ataxia, with normal or near-normal neurological function between attacks. Families with autosomal dominant EA represent at least two distinct clinical syndromes. One clinical type of EA (MIM 160120) includes individuals who have episodes of ataxia and dysarthria lasting seconds to minutes. In addition, myokymia (rippling of muscles, diagnosable by electromyography) is evident during and between attacks. Since K+ channel genes are candidate genes for EA, we tested markers near known K+ channel genes for linkage. Using a group of Genethon markers from one such region--chromosome 12p--we found evidence of linkage in four EA/myokymia families. A maximum combined lod score of 13.6 was obtained at theta = 0, with the marker D12S99. A human Ca++ channel gene, CACNL1A1, and three human K+ channel genes--KCNA5, KCNA6, and KCNA1--map close to D12S99, but the Ca++ channel gene is unlikely to be the site of the defect, because crossovers have been observed to occur between the disease gene and a CA-repeat marker located close to this gene. Studies of a large EA family with a different clinical phenotype (MIM 108500), which lacks myokymia but is associated with nystagmus, have excluded the gene causing that disease from the chromosome 12p locus.

Controlled release levodopa/carbidopa 25/100 (Sinemet CR 25/100): pharmacokinetics and clinical efficacy in untreated parkinsonian patients.

The pharmacokinetics of the clinically determined optimal dose of controlled release levodopa/carbidopa 25/100 (Sinemet CR 25/100) after 12 weeks of therapy was studied in nine parkinsonian patients without prior exposure to levodopa. The pharmacokinetics of single oral doses of controlled release levodopa/carbidopa 25/100 and 50/200 were also compared. As predicted from the plasma half-life (1.7 +/- 0.3 h) and confirmed by morning trough levels, levodopa did not accumulate when controlled released levodopa/carbidopa 25/100 was administered twice daily. The absorption and bioavailability of CR 25/100 are minimally greater than CR 50/200. Controlled released levodopa/carbidopa 25/100 levodopa plasma levels peak slightly faster than controlled release levodopa/carbidopa 50/200.

Effect of peripheral catechol-O-methyltransferase inhibition on the pharmacokinetics and pharmacodynamics of levodopa in parkinsonian patients.

Catechol-O-methyltransferase (COMT) metabolizes a portion of administered levodopa and thus makes it unavailable for conversion to dopamine in the brain. In an open-label trail, we examined the effects of entacapone, a peripheral inhibitor of COMT, administered acutely or for 8 weeks, on the pharmacokinetics and pharmacodynamics of levodopa in 15 parkinsonian subjects with a fluctuating response to levodopa. Acutely and chronically administered entacapone similarly decreased the plasma elimination of orally and intravenously administered levodopa. Absorption of levodopa was minimally affected. During chronic entacapone treatment, daily levodopa dosages were reduced by 27% yet mean plasma levodopa concentrations were increased by 23%. Plasma 3-O-methyldopa concentrations were decreased by 60%. Entacapone increased the duration of action of single doses of levodopa by a mean of 56%. The percent of the day "on" after 8 weeks of entacapone treatment was 77%; it dropped to 44% upon withdrawal of entacapone. We conclude that inhibition of COMT by entacapone increases the plasma half-life of levodopa and augments the antiparkinsonian effects of single and repeated doses of levodopa.