George D. Mellick

Parkinson's disease, pesticides, and glutathione transferase polymorphisms

BACKGROUND Parkinsons disease is thought to be secondary to the presence of neurotoxins, and pesticides have been implicated as possible causative agents. Glutathione transferases (GST) metabolise xenobiotics, including pesticides. Therefore, we investigated the role of GST polymorphisms in the pathogenesis of idiopathic Parkinsons disease. METHODS We genotyped by PCR polymorphisms in four GST classes (GSTM1, GSTT1, GSTP1, and GSTZ1) in 95 Parkinsons disease patients and 95 controls. We asked all patients for information about pesticide exposure. FINDINGS The distribution of the GSTP1 genotypes differed significantly between patients and controls who had been exposed to pesticides (controls vs patients: AA 14 [54%] of 26 vs seven [18%] of 39; AB 11 [42%] of 26 vs 22 [56%] of 39; BB 1 [4%] of 26 vs six [15%] of 39; AC 0 vs four [10%] of 39, p=0.009). No association was found with any of the other GST polymorphisms. Pesticide exposure and a positive family history were risk factors for Parkinsons disease. INTERPRETATION GSTP1-1, which is expressed in the blood-brain barrier, may influence response to neurotoxins and explain the susceptibility of some people to the parkinsonism-inducing effects of pesticides.

UCHL1 Is a Parkinson's Disease Susceptibility Gene

An Erratum has been published for this article in Ann Neurol 2004;55:899.

Association of LRRK2 exonic variants with susceptibility to Parkinson's disease: A case-control study

BACKGROUND Background The leucine-rich repeat kinase 2 gene (LRRK2) harbours highly penetrant mutations that are linked to familial parkinsonism. However, the extent of its polymorphic variability in relation to risk of Parkinsons disease (PD) has not been assessed systematically. We therefore assessed the frequency of LRRK2 exonic variants in individuals with and without PD, to investigate the role of the variants in PD susceptibility. METHODS LRRK2 was genotyped in patients with PD and controls from three series (white, Asian, and Arab-Berber) from sites participating in the Genetic Epidemiology of Parkinsons Disease Consortium. Genotyping was done for exonic variants of LRRK2 that were identified through searches of literature and the personal communications of consortium members. Associations with PD were assessed by use of logistic regression models. For variants that had a minor allele frequency of 0·5% or greater, single variant associations were assessed, whereas for rarer variants information was collapsed across variants. FINDINGS 121 exonic LRRK2 variants were assessed in 15 540 individuals: 6995 white patients with PD and 5595 controls, 1376 Asian patients and 962 controls, and 240 Arab-Berber patients and 372 controls. After exclusion of carriers of known pathogenic mutations, new independent risk associations were identified for polymorphic variants in white individuals (M1646T, odds ratio 1·43, 95% CI 1·15-1·78; p=0·0012) and Asian individuals (A419V, 2·27, 1·35-3·83; p=0·0011). A protective haplotype (N551K-R1398H-K1423K) was noted at a frequency greater than 5% in the white and Asian series, with a similar finding in the Arab-Berber series (combined odds ratio 0·82, 0·72-0·94; p=0·0043). Of the two previously reported Asian risk variants, G2385R was associated with disease (1·73, 1·20-2·49; p=0·0026), but no association was noted for R1628P (0·62, 0·36-1·07; p=0·087). In the Arab-Berber series, Y2189C showed potential evidence of risk association with PD (4·48, 1·33-15·09; p=0·012). INTERPRETATION The results for LRRK2 show that several rare and common genetic variants in the same gene can have independent effects on disease risk. LRRK2, and the pathway in which it functions, is important in the cause and pathogenesis of PD in a greater proportion of patients with this disease than previously believed. These results will help discriminate those patients who will benefit most from therapies targeted at LRRK2 pathogenic activity. FUNDING Michael J Fox Foundation and National Institutes of Health.

GSK3B polymorphisms alter transcription and splicing in Parkinson's disease

Parkinsons disease (PD) is a neurodegenerative disorder characterized by a combination of motor symptoms. We identified two functional single nucleotide polymorphisms in the glycogen synthase kinase‐3β gene (GSK3B). A promoter single nucleotide polymorphism (rs334558) is associated with transcriptional strength in vitro in which the T allele has greater activity. An intronic single nucleotide polymorphism (rs6438552) regulates selection of splice acceptor sites in vitro. The T allele is associated with altered splicing in lymphocytes and increased levels of GSK3B transcripts that lack exons 9 and 11 (GSKΔexon9+11). Increased levels of GSKΔexon9+11 correlated with enhanced phosphorylation of its substrate, Tau. In a comparison of PD and control brains, there was increased in frequency of T allele (rs6438552) and corresponding increase in GSKΔexon9+11 and Tau phosphorylation in PD brains. Conditional logistic regression indicated gene–gene interaction between T/T genotype of rs334558 and H1/H1 haplotype of microtubule‐associated protein Tau (MAPT) gene (p = 0.009). There was association between a haplotype (T alleles of both GSK3B polymorphisms) and disease risk after stratification by Tau haplotypes ((H1/H2+H2/H2 individuals: odds ratio, 1.64; p = 0.007; (H1/H1 individuals: odds ratio, 0.68; p < 0.001). Ours results suggest GSK3B polymorphisms alter transcription and splicing and interact with Tau haplotypes to modify disease risk in PD. Ann Neurol 2005;58:829–839

Tau haplotypes regulate transcription and are associated with Parkinson's disease

A primary haplotype (H1) of the microtubule‐associated protein Tau (MAPT) gene is associated with Parkinsons disease (PD). However, the mechanism for disease susceptibility remains unknown. We examined the promoter region of MAPT and identified single nucleotide polymorphisms and insertions of 1 to 11 nucleotides. These polymorphisms corresponded to the previously characterized haplotypes, H1 and H2, as well as a novel variant of the H1 haplotype, H1′. As observed in other studies, we demonstrated a significant association with the H1/H1 promoter genotype and PD in a cohort of 206 idiopathic late‐onset cases. This is in contrast with a panel of 13 early‐onset PD patients, for whom we did not detect any mutations in MAPT. By examining single nucleotide polymorphisms in adjacent genes, we showed that linkage disequilibrium does not extend beyond the MAPT haplotype to neighboring genes. To define the mechanism of disease susceptibility, we examined the transcriptional activity of the promoter haplotypes using a luciferase reporter assay. We demonstrated in two human cell lines, SK‐N‐MC and 293, that the H1 haplotype was more efficient at driving gene expression than the H2 haplotype. Our data suggest that an increase in expression of the MAPT gene is a susceptibility factor in idiopathic PD.

Genome-wide association study confirms BST1 and suggests a locus on 12q24 as the risk loci for Parkinson's disease in the European population

We performed a three-stage genome-wide association study (GWAS) to identify common Parkinsons disease (PD) risk variants in the European population. The initial genome-wide scan was conducted in a French sample of 1039 cases and 1984 controls, using almost 500 000 single nucleotide polymorphisms (SNPs). Two SNPs at SNCA were found to be associated with PD at the genome-wide significance level (P < 3 × 10(-8)). An additional set of promising and new association signals was identified and submitted for immediate replication in two independent case-control studies of subjects of European descent. We first carried out an in silico replication study using GWAS data from the WTCCC2 PD study sample (1705 cases, 5200 WTCCC controls). Nominally replicated SNPs were further genotyped in a third sample of 1527 cases and 1864 controls from France and Australia. We found converging evidence of association with PD on 12q24 (rs4964469, combined P = 2.4 × 10(-7)) and confirmed the association on 4p15/BST1 (rs4698412, combined P = 1.8 × 10(-6)), previously reported in Japanese data. The 12q24 locus includes RFX4, an isoform of which, named RFX4_v3, encodes brain-specific transcription factors that regulate many genes involved in brain morphogenesis and intracellular calcium homeostasis.

Disease-specific, neurosphere-derived cells as models for brain disorders

SUMMARY There is a pressing need for patient-derived cell models of brain diseases that are relevant and robust enough to produce the large quantities of cells required for molecular and functional analyses. We describe here a new cell model based on patient-derived cells from the human olfactory mucosa, the organ of smell, which regenerates throughout life from neural stem cells. Olfactory mucosa biopsies were obtained from healthy controls and patients with either schizophrenia, a neurodevelopmental psychiatric disorder, or Parkinson’s disease, a neurodegenerative disease. Biopsies were dissociated and grown as neurospheres in defined medium. Neurosphere-derived cell lines were grown in serum-containing medium as adherent monolayers and stored frozen. By comparing 42 patient and control cell lines we demonstrated significant disease-specific alterations in gene expression, protein expression and cell function, including dysregulated neurodevelopmental pathways in schizophrenia and dysregulated mitochondrial function, oxidative stress and xenobiotic metabolism in Parkinson’s disease. The study has identified new candidate genes and cell pathways for future investigation. Fibroblasts from schizophrenia patients did not show these differences. Olfactory neurosphere-derived cells have many advantages over embryonic stem cells and induced pluripotent stem cells as models for brain diseases. They do not require genetic reprogramming and they can be obtained from adults with complex genetic diseases. They will be useful for understanding disease aetiology, for diagnostics and for drug discovery.

The Vps35 D620N mutation linked to Parkinson's disease disrupts the cargo sorting function of retromer

The retromer is a trimeric cargo‐recognition protein complex composed of Vps26, Vps29 and Vps35 associated with protein trafficking within endosomes. Recently, a pathogenic point mutation within the Vps35 subunit (D620N) was linked to the manifestation of Parkinsons disease (PD). Here, we investigated details underlying the molecular mechanism by which the D620N mutation in Vps35 modulates retromer function, including examination of retromers subcellular localization and its capacity to sort cargo. We show that expression of the PD‐linked Vps35 D620N mutant redistributes retromer‐positive endosomes to a perinuclear subcellular localization and that these endosomes are enlarged in both model cell lines and fibroblasts isolated from a PD patient. Vps35 D620N is correctly folded and binds Vps29 and Vps26A with the same affinity as wild‐type Vps35. While PD‐linked point mutant Vps35 D620N interacts with the cation‐independent mannose‐6‐phosphate receptor (CI‐M6PR), a known retromer cargo, we find that its expression disrupts the trafficking of cathepsin D, a CI‐M6PR ligand and protease responsible for degradation of α‐synuclein, a causative agent of PD. In summary, we find that the expression of Vps35 D620N leads to endosomal alterations and trafficking defects that may partly explain its action in PD.

Large-scale replication and heterogeneity in Parkinson disease genetic loci

Objective: Eleven genetic loci have reached genome-wide significance in a recent meta-analysis of genome-wide association studies in Parkinson disease (PD) based on populations of Caucasian descent. The extent to which these genetic effects are consistent across different populations is unknown. Methods: Investigators from the Genetic Epidemiology of Parkinsons Disease Consortium were invited to participate in the study. A total of 11 SNPs were genotyped in 8,750 cases and 8,955 controls. Fixed as well as random effects models were used to provide the summary risk estimates for these variants. We evaluated between-study heterogeneity and heterogeneity between populations of different ancestry. Results: In the overall analysis, single nucleotide polymorphisms (SNPs) in 9 loci showed significant associations with protective per-allele odds ratios of 0.78–0.87 (LAMP3, BST1, and MAPT) and susceptibility per-allele odds ratios of 1.14–1.43 (STK39, GAK, SNCA, LRRK2, SYT11, and HIP1R). For 5 of the 9 replicated SNPs there was nominally significant between-site heterogeneity in the effect sizes (I2 estimates ranged from 39% to 48%). Subgroup analysis by ethnicity showed significantly stronger effects for the BST1 (rs11724635) in Asian vs Caucasian populations and similar effects for SNCA, LRRK2, LAMP3, HIP1R, and STK39 in Asian and Caucasian populations, while MAPT rs2942168 and SYT11 rs34372695 were monomorphic in the Asian population, highlighting the role of population-specific heterogeneity in PD. Conclusion: Our study allows insight to understand the distribution of newly identified genetic factors contributing to PD and shows that large-scale evaluation in diverse populations is important to understand the role of population-specific heterogeneity. Neurology® 2012;79:659–667

Lack of replication of thirteen single-nucleotide polymorphisms implicated in Parkinson’s disease: a large-scale international study

BACKGROUND A genome-wide association study identified 13 single-nucleotide polymorphisms (SNPs) significantly associated with Parkinsons disease. Small-scale replication studies were largely non-confirmatory, but a meta-analysis that included data from the original study could not exclude all SNP associations, leaving relevance of several markers uncertain. METHODS Investigators from three Michael J Fox Foundation for Parkinsons Research-funded genetics consortia-comprising 14 teams-contributed DNA samples from 5526 patients with Parkinsons disease and 6682 controls, which were genotyped for the 13 SNPs. Most (88%) participants were of white, non-Hispanic descent. We assessed log-additive genetic effects using fixed and random effects models stratified by team and ethnic origin, and tested for heterogeneity across strata. A meta-analysis was undertaken that incorporated data from the original genome-wide study as well as subsequent replication studies. FINDINGS In fixed and random-effects models no associations with any of the 13 SNPs were identified (odds ratios 0.89 to 1.09). Heterogeneity between studies and between ethnic groups was low for all SNPs. Subgroup analyses by age at study entry, ethnic origin, sex, and family history did not show any consistent associations. In our meta-analysis, no SNP showed significant association (summary odds ratios 0.95 to 1.08); there was little heterogeneity except for SNP rs7520966. INTERPRETATION Our results do not lend support to the finding that the 13 SNPs reported in the original genome-wide association study are genetic susceptibility factors for Parkinsons disease.