Smoking and haptoglobin phenotype modulate serum ferritin and haptoglobin levels in Parkinson disease.

The phenotype Hp 2-1 of haptoglobin has been previously associated with increased risk of Parkinson disease (PD) and with serum iron abnormalities in PD patients. Tobacco smoking has been consistently observed in epidemiology studies to be inversely related to PD risk, with mechanisms that remain uncertain. We recently observed that the protective effect of smoking on PD risk is stronger among subjects of haptoglobin Hp 2-2 and Hp 1-1 phenotypes, and weaker among subjects of haptoglobin Hp 2-1 phenotype. In this PD case-control study, we investigated whether tobacco smoking was associated with changes in serum haptoglobin and ferritin concentration that depended on haptoglobin phenotype among 106 PD patients and 238 controls without PD or other neurodegenerative disorders. Serum ferritin concentration, serum haptoglobin concentration, haptoglobin phenotype, and smoking data information of cases and controls were obtained. Differences in haptoglobin and ferritin concentration by smoking status and pack-years of smoking were calculated as well as regression between pack-years and haptoglobin and ferritin concentration, and the effect of haptoglobin phenotype on these parameters. Tobacco smoking was associated with an elevation in serum haptoglobin concentration, especially among healthy controls of haptoglobin Hp 2-2 phenotype, and with an elevation in ferritin concentration especially among PD patients of haptoglobin Hp 2-1 phenotype. These findings suggest that an elevation in haptoglobin concentration, preferentially among subjects of haptoglobin Hp 2-2 phenotype, could be a contributing factor to the protective effect of smoking on PD risk.

The functional polymorphism of the hemoglobin-binding protein haptoglobin influences susceptibility to idiopathic Parkinson's disease.

Oxidative stress and iron have been widely implicated in the etiology of Parkinson's disease (PD). Hemoglobin is the richest source of iron in the body. The human Haptoglobin (Hp) protein is a plasma alpha-2 glycoprotein that removes free Hemoglobin from the circulation and tissues and is important in protection from oxidative stress, in immune system regulation, and angiogenesis. A common genetic polymorphism of Hp exists in the population, where the Hp 1-1, Hp 2-1, and Hp 2-2 forms exhibit profound functional differences. In this study, the Hp genotype corresponding to phenotypes Hp 1-1, 2-1 and 2-2 was determined in 312 idiopathic PD patients and 420 normal control subjects. A significant increase in the number of subjects carrying the Hp 2-1 genotype was present among PD patients. The distribution of Hp genotypes among PD patients (16.0% Hp 1-1, 56.4% Hp 2-1, 27.6% Hp 2-2) was significantly different from the distribution in controls (15.2% Hp 1-1, 48.1% Hp 2-1, 36.7% Hp 2-2) (chi(2) = 6.99, P = 0.030). The odds ratios for PD risk for Hp 2-1 and Hp 1-1 versus Hp 2-2 genotype were 1.51 (1.07-2.12) and 1.36 (0.86-2.15), respectively. Overall, the association of Hp-1 allele with PD resulted stronger among subjects who were never-smokers as compared to ever-smokers. Also, among ever-smokers, Hp genotypes were significantly associated with PD only among women, but not men, indicating the presence of a gene x gender x smoking interaction. To our knowledge, this is the first study that investigates the association of Hp genotypes with the risk of PD.

Brain iron concentrations in regions of interest and relation with serum iron levels in Parkinson disease.

Brain iron has been previously found elevated in the substantia nigra pars compacta (SNpc), but not in other brain regions, of Parkinson's disease (PD) patients. However, iron in circulation has been recently observed to be lower than normal in PD patients. The regional selectivity of iron deposition in brain as well as the relationship between SNpc brain iron and serum iron within PD patients has not been completely elucidated. In this pilot study we measured brain iron in six regions of interest (ROIs) as well as serum iron and serum ferritin, in 24 PD patients and 27 age- gender-matched controls. Brain iron was measured on magnetic resonance imaging (MRI) with a T2 prime (T2') method. Difference in brain iron deposition between PD cases and controls for the six ROIs were calculated. SNpc/white matter brain iron ratios and SNpc/serum iron ratios were calculated for each study participant, and differences between PD patients and controls were tested. PD patients overall had higher brain iron than controls in the SNpc. PD patients had significantly higher SNpc/white matter brain iron ratios than controls, and significantly higher brain SNpc iron/serum iron ratios than controls. These results indicate that PD patients' iron metabolism is disrupted toward a higher partitioning of iron to the brain SNpc at the expenses of iron in the circulation.

Genotype combinations for monoamine oxidase-B intron 13 polymorphism and dopamine D2 receptor TaqIB polymorphism are associated with ever-smoking status among men.

Tobacco smoke inhibits monoamine oxidase-B (MAO-B) activity in vitro and in vivo, suggesting that MAO-B inhibition is a possible contributing factor to tobacco smoke addiction. Thus, MAO-B is a possible candidate gene for predisposition to smoking. The TaqIB polymorphism for the Dopamine D2 Receptor gene (DRD2) has been previously associated with smoking status, although with some contradictory results. We investigated whether genetic variants of MAO-B intron 13 and DRD2 TaqIB polymorphism could be associated with smoking status among control subjects. There was no association of the intron 13 polymorphism itself with smoking status in either men or women. Similarly, no association with smoking status was observed for the TaqIB polymorphism of DRD2 itself. However, among men, there was an interaction between MAO-B intron 13 polymorphism and the DRD2 TaqIB polymorphisms, in which subjects carrying MAO-B allele A and genotype B12 of DRD2 were 2.50 times (95% CI=1.05-5.95) more likely to be ever-smokers than the pool of men carrying all other genotype combinations. These results demonstrate that particular combinations of genotypes for MAO-B and DRD2 genes are associated with significantly higher risk for smoking behavior in men, but not in women.

Characterization of the in vitro transcriptional activity of polymorphic alleles of the human monoamine oxidase-B gene.

The activity of monoamine oxidase-B (MAO-B) enzyme has a high degree of heritability, although no common genetic polymorphisms are present in the MAO-B coding region. The only known polymorphisms in MAO-B gene are a C-1,114T in the 5' region, a variable number of GT repeats in intron 2, and a G/A change in intron 13. The genetically determined differences in MAO-B activity among subjects most likely derive from functional differences conferred by these three MAO-B genetic polymorphisms, or by polymorphisms in trans-acting factors that regulate MAO-B expression. In order to identify differences in transcriptional activity conferred by the MAO-B genetic variants, we cloned alternative alleles at the three polymorphic loci in -1,114, intron 2 and intron 13, and tested them in transfection-luciferase assays in HepG2 hepatoma and 1321N1 astrocytoma cells. The mutant -1,114 allele T had higher activity than allele C in both cell lines, when cloned in pGL3Enhancer. Significant differences in trascriptional activity by intron 2 GT repeat allele were also observed, and varied by cell type and reporter construct. Allele G of intron 13 had significantly higher activity than allele A in both cell lines. These results indicate transcriptional activity differences between MAO-B variant alleles that are naturally occurring in the population.

Haptoglobin phenotype modifies serum iron levels and the effect of smoking on Parkinson disease risk.

INTRODUCTION: Haptoglobin is a hemoglobin-binding protein that exists in three functionally different phenotypes, and haptoglobin phenotype 2-1 has previously been associated with Parkinson disease (PD) risk, with mechanisms not elucidated. Some evidence is emerging that low levels of serum iron may increase PD risk. In this study we investigated whether PD patients have lower serum iron and ferritin than controls, and whether this is dependent on haptoglobin phenotype. We also investigated the effect of Hp phenotype as a modifier of the effect of smoking on PD risk. METHODS: The study population consisted of 128 PD patients and 226 controls. Serum iron, ferritin, and haptoglobin phenotype were determined, and compared between PD cases and controls. Stratified analysis by haptoglobin phenotype was performed to determine effect of haptoglobin phenotype on serum iron parameter differences between PD cases and controls and to investigate its role in the protective effect of smoking on PD risk. RESULTS: PD cases had lower serum iron than controls (83.28 ug/100 ml vs 94.00 ug/100 ml, p 0.006), and in particular among subjects with phenotype 2-1. The protective effect of smoking on PD risk resulted stronger in subjects with phenotype 1-1 and 2-2, and weakest among subjects with phenotype 2-1. Ferritin levels were higher in PD cases than controls among subjects of White ethnicity. CONCLUSIONS: Our results report for the first time that the haptoglobin phenotype may be a contributor of iron levels abnormalities in PD patients. The mechanisms for these haptoglobin-phenotype specific effects will have to be further elucidated.

Gender difference in the interaction of smoking and monoamine oxidase B intron 13 genotype in Parkinson's disease.

We tested for gender-specific interactions between smoking and genetic polymorphisms of monoamine oxidase B (MAO-B) intron 13 (G or A allele), monoamine oxidase A (MAO-A) EcoRV (Yor N allele), and dopamine D2 recepor (DRD2) Taq1B (B1 or B2 allele) in a case-control study of 186 incident idiopathic Parkinson's disease (PD) cases and 296 age- and gender-matched controls. The odds ratios (ORs) for PD risk for ever smokers versus never smokers were 0.27 (95% CI: 0.13-0.58) for men of genotype G, and 1.26 (0.60-2.63) for men of genotype A (interaction chi2 = 8.14, P = 0.004). In contrast, for women, the OR for ever smokers versus never smokers were 0.62 (95% CI: 0.25-1.34) and 0.64 (95% CI: 0.18-2.21) for women of genotype GG/GA and AA, respectively (interaction chi2 = 0.001, P = 0.975). No interactions were detected between smoking and either MAO-A EcoRV or DRD2 Taq1B genotypes. These results suggest that a strong gender difference exists with respect to the modifying effect of MAO-B genotype on the smoking association with PD.

A diet low in animal fat and rich in N-hexacosanol and fisetin is effective in reducing symptoms of Parkinson's disease.

This study describes how foods rich in fisetin and hexacosanol added to a strict diet reversed most symptoms of Parkinson's disease (PD) in one patient. This is a case report involving outpatient care. The subject was a dietitian diagnosed with idiopathic PD in 2000 at the age of 53 years old, with a history of exposure to neurotoxins and no family history of PD. A basic diet started in 2000 consisted of predominantly fruits, vegetables, 100% whole grains, extra virgin olive oil, nuts, seeds, nonfat milk products, tea, coffee, spices, small amounts of dark chocolate, and less than 25 g of animal fat daily. The basic diet alone failed to prevent decline due to PD. In 2009, the basic diet was enhanced with a good dietary source of both fisetin and hexacosanol. Six months after the patient started the enhanced diet rich in fisetin and hexacosanol, a clinically significant improvement in symptoms was noted; the patient's attending neurologist reported that the clinical presentation of cogwheel rigidity, micrographia, bradykinesia, dystonia, constricted arm swing with gait, hypomimia, and retropulsion appeared to be resolved. The only worsening of symptoms occurred when the diet was not followed precisely. Little improvement in tremor or seborrhea was observed. The clinical improvement has persisted to date. To the best of our knowledge, this is the first case where adjunctive diet therapy resulted in a significant reduction of symptoms of PD without changing the type or increasing the amount of medications.

5' and 3' region variability in the dopamine transporter gene (SLC6A3), pesticide exposure and Parkinson's disease risk: a hypothesis-generating study.

The dopamine transporter gene (SLC6A3) is a candidate gene for Parkinson's disease (PD) on the basis of its critical role in dopaminergic neurotransmission. Previously, we identified 22 SNPs in the 5' region of SLC6A3, which segregate as eight haplotypes that differ in transcriptional activity when transfected in rat dopamine-producing cells. In the present work from a case-control study size of 293 cases and 395 controls, we employed a cladistic approach to examine gene-disease association. First, we found strong evidence of balancing selection in this region, as determined by a Tajima's D statistic of 2.97 (P<0.001). Second, we found that the eight haplotypes fit into two main clades and that diplotypes of these clades were marginally associated with PD. Then, after we classified cases and controls by the number of risk alleles, accounting for the well-known 3' region VNTR polymorphism, we found that having two or more risk alleles resulted in a modest but significant increase in PD risk [odds ratio=1.58; 95% confidence interval (CI): 1.03-2.40]. Finally, we detected a significant interaction between occupational pesticide exposure in men and the number of risk alleles. Among pesticide-exposed subjects, the odds ratio for having two or more risk alleles was 5.66 (95% CI: 1.73-18.53). Thus, allelic variants in SLC6A3, which affect gene expression, are associated with PD in this population and may interact with occupational pesticide exposure to increase PD risk.

Dopamine transporter (SLC6A3) 5' region haplotypes significantly affect transcriptional activity in vitro but are not associated with Parkinson's disease.

The dopamine transporter (DAT) plays a critical role in dopaminergic neurotransmission and is also the major site of action for some drugs of abuse. The coding region of the DAT gene, SLC6A3, is well conserved, but non-coding regions are more variable, most notably a variable number of tandem repeats (VNTR) polymorphism in the 3' untranslated region, which has been studied in a number of dopamine-related neurological disorders, including Parkinson's disease (PD). We aimed to characterize variation in the 5' region of SLC6A3 because little is known about the extent of variation in this region and potential consequences of such variation on gene expression. We identified multiple single nucleotide polymorphisms (SNPs) covering approximately 5000 bp 5' of exon 1 through the start of exon 2 (+2106). These SNPs segregated as eight haplotypes, six of which were common. These haplotypes differed significantly in activity in a reporter gene activity assay. However, we did not observe associations between common SNPs or haplotypes and PD in a case-control study of 261 incident cases and 376 age- and gender-matched unrelated controls. By contrast, we did observe a modest association of the 3' VNTR 9-repeat allele with PD (odds ratio=1.45; 95% confidence interval=1.04-2.03). This association was limited to subjects 60 years of age and greater versus those less than 60 years of age. We conclude that although DAT 5' region SNPs haplotypes significantly alter in vitro transcriptional activity, they are not related to PD risk. In addition, our findings provide further evidence supporting an association of PD with the VNTR polymorphism.

DNA sequence analysis of monoamine oxidase B gene coding and promoter regions in Parkinson's disease cases and unrelated controls.

The allele G of the intron 13 G/A polymorphism of the monoamine oxidase B gene (MAO-B) has been associated with Parkinson's disease (PD) in several studies. Apart from a potential direct effect on splicing processes, the association of this intronic polymorphism with PD is due possibly to linkage disequilibrium with other mutations in the coding or promoter regions of the gene. We addressed this latter hypothesis by determining the DNA sequence of the entire MAO-B coding region comprising 15 exons and partial intronic sequences flanking each exon, in 33 cases with idiopathic PD and 38 unrelated controls. The promoter region of MAO-B gene up to base -1,369 from ATG (start point of mRNA translation) was also sequenced to identify variants with potential functional effects on gene transcription. In the promoter region, a new polymorphism consisting of a C to T single base change was detected in position -1,114 from ATG, with an allelic frequency of 3.5%, but it was not associated with PD risk. No commonly occurring (>10%) polymorphisms were found in the exons or the intronic sequences flanking the exons, although several rare variants were detected in the coding and promoter regions.