Michele De Mari

Comprehensive analysis of the LRRK2 gene in sixty families with Parkinson's disease

Mutations in the gene leucine-rich repeat kinase 2 (LRRK2) have been recently identified in families with Parkinsons disease (PD). However, the prevalence and nature of LRRK2 mutations, the polymorphism content of the gene, and the associated phenotypes remain poorly understood. We performed a comprehensive study of this gene in a large sample of families with Parkinsons disease compatible with autosomal dominant inheritance (ADPD). The full-length open reading frame and splice sites of the LRRK2 gene (51 exons) were studied by genomic sequencing in 60 probands with ADPD (83% Italian). Pathogenic mutations were identified in six probands (10%): the heterozygous p.G2019S mutation in four (6.6%), and the heterozygous p.R1441C mutation in two (3.4%) probands. A further proband carried the heterozygous p.I1371 V mutation, for which a pathogenic role could not be established with certainty. In total, 13 novel disease-unrelated variants and three intronic changes of uncertain significance were also characterized. The phenotype associated with LRRK2 pathogenic mutations is the one of typical PD, but with a broad range of onset ages (mean 55.2, range 38–68 years) and, in some cases, slow disease progression. On the basis of the comprehensive study in a large sample, we conclude that pathogenic LRRK2 mutations are frequent in ADPD, and they cluster in the C-terminal half of the encoded protein. These data have implications both for understanding the molecular mechanisms of PD, and for directing the genetic screening in clinical practice.

Freezing Gait in Parkinson's Disease

Freezing is a well-known problem in Parkinsons disease (PD) and is characterized by an abrupt difficulty in starting or continuing rhythmic and repetitive movements. We utilized a questionnaire in order to assess the occurrence of the freezing gait phenomenon (FG) in a population of 100 consecutive PD patients. Our PD population included 70 males and 30 females, with a mean age of 61.1 +/- 9.1 years. Mean duration of PD was 6.5 +/- 4.0 years. 92/100 patients were under L-Dopa treatment. The FG phenomenon occurred in 60% of patients. It appeared on average 4.8 years after the beginning of PD; in 16% of the cases it was evident before starting L-Dopa treatment. FG was more frequent among female patients. There was no significant correlation between the occurrence of FG and the age of the patients; on the other hand, a significant correlation was found with the duration of the disease (p < 0.001). FG occurred more frequently in the subgroup of patients with the akinetic form (odds ratio: 3.05); whilst an opposite tendency was evident in the subgroup with the tremor predominant form (odds ratio: 0.29).

Effect of resveratrol on mitochondrial function: implications in parkin-associated familiar Parkinson's disease

Mitochondrial dysfunction and oxidative stress occur in Parkinsons disease (PD), but the molecular mechanisms controlling these events are not completely understood. Peroxisome proliferator-activated receptor-gamma coactivator-1α (PGC-1α) is a transcriptional coactivator known as master regulator of mitochondrial functions and oxidative metabolism. Recent studies, including one from our group, have highlighted altered PGC-1α activity and transcriptional deregulation of its target genes in PD pathogenesis suggesting it as a new potential therapeutic target. Resveratrol, a natural polyphenolic compound proved to improve mitochondrial activity through the activation of several metabolic sensors resulting in PGC-1α activation. Here we have tested in vitro the effect of resveratrol treatment on primary fibroblast cultures from two patients with early-onset PD linked to different Park2 mutations. We show that resveratrol regulates energy homeostasis through activation of AMP-activated protein kinase (AMPK) and sirtuin 1 (SIRT1) and raise of mRNA expression of a number of PGC-1αs target genes resulting in enhanced mitochondrial oxidative function, likely related to a decrease of oxidative stress and to an increase of mitochondrial biogenesis. The functional impact of resveratrol treatment encompassed an increase of complex I and citrate synthase activities, basal oxygen consumption, and mitochondrial ATP production and a decrease in lactate content, thus supporting a switch from glycolytic to oxidative metabolism. Moreover, resveratrol treatment caused an enhanced macro-autophagic flux through activation of an LC3-independent pathway. Our results, obtained in early-onset PD fibroblasts, suggest that resveratrol may have potential clinical application in selected cases of PD-affected patients.

Effects of levodopa and COMT inhibitors on plasma homocysteine in Parkinson's disease patients

Homocysteine (Hcy) is a risk factor for vascular diseases, cognitive impairment, and dementia. Elevated plasma concentrations of Hcy have been found recently in Parkinsons disease (PD) patients treated with levodopa, suggesting that levodopa is a cause of hyperhomocysteinemia (HHcy). The mechanism underlying HHcy in PD is the O‐methylation of levodopa catalyzed by catechol‐O‐methyltransferase (COMT) that produces S‐adenosylhomocysteine, which is hydrolyzed rapidly to Hcy. COMT inhibitors (COMT‐I) are used currently in the treatment of PD; however, no study has assessed the effects of COMT‐I administration on Hcy concentrations in PD patients. We compared plasma levels of Hcy, B12, and folate in 26 PD patients treated with levodopa, 20 PD patients treated with levodopa + COMT‐I, and 32 controls. No significant differences were found in vitamin B12 levels, whereas folate concentrations were significantly lower in the levodopa‐treated group. Plasma Hcy was increased significantly in the two groups of PD patients and was significantly lower in the group treated with levodopa + COMT‐I. Statistical analysis showed that the difference in mean Hcy levels observed among PD patients was related to the addition of COMT‐I, rather than to folate concentrations. We conclude that levodopa treatment increases plasma Hcy and the addition of COMT‐I effectively reduces HHcy.

Case–control study of multiple system atrophy

The epidemiology of multiple system atrophy (MSA) is scarcely known, and risk factors have not been definitely identified. We investigated the effect of family history for neurodegenerative diseases and environmental factors on MSA risk in a multicentric case–control study. A total of 73 MSA patients (42 men, 31 women; age, 64.3 ± 8.1 years; disease duration, 4.8 ± 3.9 years), 146 hospital controls (84 men, 62 women; age, 64.9 ± 8.4 years), and 73 population controls (42 men, 31 women; age, 63.7 ± 8.9 years) matched for sex, age (±3 years), and province of residence were enrolled consecutively at seven neurological centers from 1 January 1994 to 31 July 1998. The following variables were investigated: family history of neurodegenerative diseases, education, smoking habits, hobbies, and occupational history. Occupational history of farming was significantly more frequent among MSA cases than controls (OR adj = 2.52; 95% CI, 1.25 to 5.07, MSA vs. hospital controls; OR adj = 4.53; 95% CI, 1.68 to12.2, MSA cases vs. population controls). A dose–response analysis for years of farming corroborated this association. We recently found that smoking is significantly less frequent among MSA cases than controls (Vanacore et al. [2000] Neurology 54:114–119). Here, we report that the effects of farming and smoking on MSA risk do not interact. Our results suggest that occupational history of farming is a risk factor for MSA. Smoking and farming seem to influence MSA risk independently. Further epidemiological studies might provide clues on the etiopathogenesis of MSA.

Antiparkinsonian treatment in pregnancy

We read with interest the article by Shulman and colleagues in a 2000 issue of MovementDisorders. 1 In that article, one case of pregnancy in a patient with Parkinson’s disease (PD) on levodopa therapy was reported. After prospective and quantitative neurological examination of the patient, the authors concluded that pregnancy exacerbates PD and could have a longterm negative impact on the course of the illness. A few cases of pregnancy in parkinsonian women have been described; all of these patients were treated with levodopa but none demonstrated major drug teratogenity. 1–4 Moreover, dopamine agonist treatment during pregnancy of PD patients has been reported by Benito-Leon and associates 3 as a bromocriptine monotherapy; another patient described by Hagell and coworkers discontinued bromocriptine after 2 months of pregnancy. In neither case did bromocriptine treatment result in any teratogenity. Pergolide administration in parkinsonian women during pregnancy has not been reported previously. We describe a woman with PD treated with combined pergolide and levodopa therapy during pregnancy. A 36-year-old woman developed PD with progressive motor slowness in the left limbs at age 32 years in 1996. At the first examination, she presented a slight rigidity and bradykinesia to the left arm and leg and a mild hypomimia. There was no evidence of resting tremor. Hoehn and Yahr rating was 2. Evaluation for other causes of parkinsonism revealed no significant abnormalities. Family history for PD was not reported. Treatment with pergolide up to a dose of 1 mg three times daily resulted in partial improvement, and after 6 months levodopa was added at a dose of 200 mg per day, resulting in optimal control of her symptoms. After 2 years the patient complained of a predictable wearing-off period in the afternoon, demonstrated as slight bradykinesia, mild rigidity in lower limbs, and gait disturbances such as short steps and shuffling, mainly in her left leg. When the patient became pregnant at age 35 years, the dosage of pergolide and levodopa was continued throughout the pregnancy. During the pregnancy, all wearing-off phenomena disappeared and she had optimal control of motor symptoms throughout the day. She gave birth to a normal-term infant in July 2000 with Apgar scores of 9, by cesarean section, because of a podalic presentation. The child shows no evidence of congenital malformation and remains healthy at this time, 13 months of age, with normal development. During the puerperal period the end-of-dose wearing-off symptom reappeared and reached the same level as before pregnancy. To our knowledge, this is the first description of combined pergolide and levodopa treatment during pregnancy in a woman with PD. Except for one case of osteomalacia and another of spontaneous abortion, for which the cause is not well established, no major complications of pregnancy, nor any adverse effects on the fetus that could primarily be related to levodopa plus carbidopa or benserazide have been reported in the literature. 1–7 Studies of carbidopa–levodopa in laboratory animals demonstrated some increase in skeletal malformation but only with high doses, greater than 500 mg/kg/day. 6

Voluptuary habits and clinical subtypes of Parkinson's disease: The FRAGAMP case–control study

We evaluated the possible association between smoking, coffee drinking, and alcohol consumption and Parkinsons disease (PD). The FRAGAMP study is a large Italian multicenter case–control study carried out to evaluate the possible role of environmental and genetic factors in PD. Adjusted ORs were estimated using unconditional logistic regression. Smoking, coffee, and alcohol consumption were also considered as surrogate markers of lifestyle and analysis was carried out considering the presence of at least one, two, or three factors. This latter analysis was separately performed considering Tremor‐Dominant (TD) and Akinetic‐Rigid (AR) patients. Four hundred ninety‐two PD patients (292 men and 200 women) and 459 controls (160 men and 299 women) were enrolled in the study. Multivariate analysis showed a significant negative association between PD and cigarette smoking (OR 0.51; 95%CI 0.36–0.72), coffee drinking (OR 0.61; 95%CI 0.43–0.87) and wine consumption (OR 0.62; 95%CI 0.44–0.86); a significant trend dose‐effect (P < 0.05) has been found for all the factors studied. We have also found a trend dose‐effect for the presence of at least one, two or three factors with a greater risk reduction (83%) for the presence of three factors. However, a different strength of association between TD and AR was found with a greater risk reduction for the AR patients. We found a significant inverse association between PD smoking, coffee, and alcohol consumption. When analysis was carried out considering the association of these factors as possible surrogate markers of a peculiar lifestyle the association was stronger for the AR phenotype.

Reproductive factors and Parkinson's disease: A multicenter case–control study

The objective of this study was to evaluate the possible association between endogenous and exogenous estrogens and Parkinsons disease (PD).

GIGYF2 mutations are not a frequent cause of familial Parkinson's disease

Mutations in the Grb10-interacting GYF protein 2 (GIGYF2) gene, within the PARK11 locus, have been nominated as a cause of Parkinsons disease in Italian and French populations. By sequencing the whole GIGYF2 coding region in forty-six probands (thirty-seven Italians) with familial Parkinsons disease compatible with an autosomal dominant inheritance, we identified no mutations. Our data add to a growing body of evidence suggesting that GIGYF2 mutations are not a frequent cause of PD.

Elevated plasma homocysteine levels in L-dopa-treated Parkinson's disease patients with dyskinesias

Abstract Background: Elevated plasma homocysteine (Hcy) concentrations are associated with increased risk of systemic vascular diseases, Alzheimers disease and vascular dementia. Several cross-sectional reports and two prospective clinical studies have recently reported elevated plasma Hcy levels in L-dopa-treated Parkinsons disease (PD) patients and Hcy has been proposed as a possible mediator for the development of long-term L-dopa motor complications (such as wearing off and on-off phenomena, and dyskinesias). The aim of the study was to elucidate a possible role of L-dopa-related hyperhomocysteinemia in the development of dyskinesias. Methods: In this cross-sectional study we compared Hcy, B12 and folate levels in 53 PD patients treated with L-dopa (29 with dyskinesias, 24 without dyskinesias). Results: Mean plasma Hcy levels were higher in the group of PD patients with dyskinesias (19 vs. 15.4 T: 2.12; p=0.04). After taking into account potential confounding factors, analysis of the data revealed that the occurrence of dyskinesias progressively increased with plasma Hcy levels (relative risk 1.2, 95% CI 1.015–1.4; p=0.03). Conclusions: Our results raise the possibility that Hcy plays a role in the development of dyskinesias, through its toxic effects on both dopaminergic neurons and non-substantia nigra, non-dopaminergic neurons. Clin Chem Lab Med 2006;44:863–6.