RNA science and its applications-a look toward the future: Albany, NY USA, November 3-4, 2011.

On November 3-4, 2011, the Symposium RNA Science and its Applications: A look toward the Future was held at the University at Albany-SUNY in the capital of New York State. Unique to this Symposium's format were panel discussions following each of the four platform sessions: RNA Technological Innovation: Analysis, Delivery, Nanotechnologies, IT; Infectious and other diseases: The future of small molecule intervention; RNA Discovery and Innovation: Cell and Molecular Biology; and Cancer and Neurological Disease: The future of small RNAs as therapeutics and tools of investigation. The meeting was organized by Thomas Begley, Marlene Belfort, Daniele Fabris, Melinda Larsen, Pan T.X. Li, Albert Millis, Li Niu, David Shub, and Carla Theimer of The RNA Institute at University at Albany-SUNY, Paul F. Agris, Director, and Jennifer S. Montimurro, Program Manager.

Genome-wide gene-environment study identifies glutamate receptor gene GRIN2A as a Parkinson's disease modifier gene via interaction with coffee.

Our aim was to identify genes that influence the inverse association of coffee with the risk of developing Parkinson's disease (PD). We used genome-wide genotype data and lifetime caffeinated-coffee-consumption data on 1,458 persons with PD and 931 without PD from the NeuroGenetics Research Consortium (NGRC), and we performed a genome-wide association and interaction study (GWAIS), testing each SNP's main-effect plus its interaction with coffee, adjusting for sex, age, and two principal components. We then stratified subjects as heavy or light coffee-drinkers and performed genome-wide association study (GWAS) in each group. We replicated the most significant SNP. Finally, we imputed the NGRC dataset, increasing genomic coverage to examine the region of interest in detail. The primary analyses (GWAIS, GWAS, Replication) were performed using genotyped data. In GWAIS, the most significant signal came from rs4998386 and the neighboring SNPs in GRIN2A. GRIN2A encodes an NMDA-glutamate-receptor subunit and regulates excitatory neurotransmission in the brain. Achieving P(2df) = 10(-6), GRIN2A surpassed all known PD susceptibility genes in significance in the GWAIS. In stratified GWAS, the GRIN2A signal was present in heavy coffee-drinkers (OR = 0.43; P = 6×10(-7)) but not in light coffee-drinkers. The a priori Replication hypothesis that "Among heavy coffee-drinkers, rs4998386_T carriers have lower PD risk than rs4998386_CC carriers" was confirmed: OR(Replication) = 0.59, P(Replication) = 10(-3); OR(Pooled) = 0.51, P(Pooled) = 7×10(-8). Compared to light coffee-drinkers with rs4998386_CC genotype, heavy coffee-drinkers with rs4998386_CC genotype had 18% lower risk (P = 3×10(-3)), whereas heavy coffee-drinkers with rs4998386_TC genotype had 59% lower risk (P = 6×10(-13)). Imputation revealed a block of SNPs that achieved P(2df)<5×10(-8) in GWAIS, and OR = 0.41, P = 3×10(-8) in heavy coffee-drinkers. This study is proof of concept that inclusion of environmental factors can help identify genes that are missed in GWAS. Both adenosine antagonists (caffeine-like) and glutamate antagonists (GRIN2A-related) are being tested in clinical trials for treatment of PD. GRIN2A may be a useful pharmacogenetic marker for subdividing individuals in clinical trials to determine which medications might work best for which patients.

Disease-related and genetic correlates of psychotic symptoms in Parkinson's disease.

Our aim was to examine disease-related and genetic correlates of the development of psychotic symptoms in a large population of patients with Parkinson's disease. We studied 500 patients with Parkinson's disease from the NeuroGenetics Research Consortium using logistic regression models. Predictors were demographic, clinical (motor/nonmotor features), and genetic, measured as continuous or dichotomous variables. Continuous measures were divided into population-based tertiles. Results are given as odds ratios (95% confidence intervals) for dichotomous variables and by ascending tertile for continuous variables. Psychotic symptoms were associated with increasing age: 4.86 (1.62-14.30) and 6.25 (2.09-18.74) (test for trend: P = 0.01); and duration of disease: 3.81 (1.23-11.76) and 5.33 (1.68-16.89) (test for trend: P = 0.03). For nonmotor features, we demonstrated positive trends with depression: 1.31 (0.47-3.61) and 5.01 (2.04-12.33) (test for trend: P < 0.0001); cognitive dysfunction: 0.69 (0.26-1.84) and 2.51 (1.00-6.29) (test for trend: P = 0.03); and an excess for those with sleep disorders: 2.00 (1.03-3.89) (P = 0.04). Psychotic symptoms were not associated with tremor or postural instability scores, but there was an association with freezing of gait: 3.83 (1.67-8.75) (P < 0.002). Psychotic symptoms were not associated with the presence of any examined polymorphisms in the apolipoprotein, alpha-synuclein, or microtubule associated protein tau genes. This is the largest study to examine correlates of psychotic symptoms in Parkinson's disease. We discovered a novel association with freezing of gait. We demonstrated an association with depression and duration of disease, both of which were inconsistently related in previous studies, and confirmed the association with age, cognitive dysfunction, and sleep disorders.

Postural instability/gait disturbance in Parkinson's disease has distinct subtypes: an exploratory analysis.

OBJECTIVE: To test the hypothesis that postural instability with falling (PIF) and freezing of gait (FOG) are distinct subtypes of the postural instability/gait disturbance (PIGD) form of Parkinson's disease (PD). METHODS: 499 PD subjects from the NeuroGenetics Research Consortium were studied using logistic regression to examine, in a cross sectional analysis, predictors of FOG and PIF. Potential predictors were from four spheres; demographic, clinical motor, clinical non-motor and genetic. RESULTS: FOG and PIF were both associated with greater gait subscores and lower tremor subscores on the Unified Parkinson's Disease Rating Scale (p ≤ 0.02). However, they differed with regard to demographic, non-motor and genetic predictors. FOG was associated with greater duration of disease, with ORs of 3.01 (95% CI 1.35 to 6.72) and 4.91 (95% CI 2.29 to 10.54) for third and fourth quartiles of duration, respectively, versus the lowest half of duration. The risk of having psychotic symptoms was also significantly increased (OR 3.02, 95% CI 1.41 to 6.49; p=0.004). FOG was inversely associated with the presence of the CYP2D6*4 allele (OR 0.41, 95% CI 0.21 to 0.80; p=0.009) suggesting a protective effect. PIF was associated with depression (OR 1.08, 95% CI 1.01 to 1.15; p<0.02) and was inversely associated with APOE ε4 (OR 0.21, 95% CI 0.05 to 0.87; p=0.03), again suggesting a protective effect. CONCLUSION: FOG and PIF have different demographic, non-motor and genetic predictors suggesting that they may be pathophysiologically distinct subtypes of PIGD. These findings have implications in the discovery of therapeutic targets for these disabling features as well as for predicting outcomes of PD.

Common genetic variation in the HLA region is associated with late-onset sporadic Parkinson's disease.

Parkinson's disease is a common disorder that leads to motor and cognitive disability. We performed a genome-wide association study of 2,000 individuals with Parkinson's disease (cases) and 1,986 unaffected controls from the NeuroGenetics Research Consortium (NGRC). We confirmed associations with SNCA and MAPT, replicated an association with GAK (using data from the NGRC and a previous study, P = 3.2 x 10(-9)) and detected a new association with the HLA region (using data from the NGRC only, P = 2.9 x 10(-8)), which replicated in two datasets (meta-analysis P = 1.9 x 10(-10)). The HLA association was uniform across all genetic and environmental risk strata and was strong in sporadic (P = 5.5 x 10(-10)) and late-onset (P = 2.4 x 10(-8)) disease. The association peak we found was at rs3129882, a noncoding variant in HLA-DRA. Two studies have previously suggested that rs3129882 influences expression of HLA-DR and HLA-DQ. The brains of individuals with Parkinson's disease show upregulation of DR antigens and the presence of DR-positive reactive microglia, and nonsteroidal anti-inflammatory drugs reduce Parkinson's disease risk. The genetic association with HLA supports the involvement of the immune system in Parkinson's disease and offers new targets for drug development.

Genetic association between alpha-synuclein and idiopathic Parkinson's disease.

Point mutations and copy number variations in SNCA, the gene encoding alpha-synuclein, cause familial Parkinson's disease (PD). A dinucleotide polymorphism (REP1) in the SNCA promoter may be a risk factor for common forms of PD. We studied 1,802 PD patients and 2,129 controls from the NeuroGenetics Research Consortium, using uniform, standardized protocols for diagnosis, subject recruitment, data collection, genotyping, and data analysis. Three common REP1 alleles (257, 259, and 261 bp, with control frequencies of 0.28, 0.65, and 0.06) and several rare alleles (combined frequency <0.01) were detected. We confirmed association of REP1 with PD risk [odds ratio (OR) = 0.86, P = 0.006 for 257-carriers; OR = 1.25, P = 0.022 for 261-carriers]. Using a normalization procedure, we showed that the 257 and 261 alleles are both independently associated with PD risk (for 257, P = 0.002 in overall data, 0.003 in non-familial PD, 0.001 in early-onset PD; for 261, P = 0.056 in overall data, 0.024 in non-familial PD, 0.052 in early-onset PD). The 257-associated risk was consistent with a dominant model [hazard ratio (HR) = 0.99, P = 0.91 for 257/257 vs. 257/X where X denotes all other common alleles; HR = 1.16, P = 0.004 for X/X vs. 257/X]. The 261-associated risk was consistent with a recessive model (HR = 1.89, P = 0.026 for 261/261 vs. 261/X; HR = 0.95, P = 0.42 for X/X vs. 261/X). Genotype-specific mean onset ages (+/-SD) ranged from 54.8 +/- 12.1 for 261/261 to 59.4 +/- 11.5 for 257/257, displaying a trend of decreasing onset age with increasing allele size (P = 0.055). Genetic variation in SNCA and its regulatory regions play an important role in both familial and sporadic PD.

Exploring gene-environment interactions in Parkinson's disease.

The objective of this study was to explore combined effects of four candidate susceptibility genes and two exposures on Parkinson's disease (PD) risk; namely, alpha-synuclein (SNCA) promoter polymorphism REP1, microtubule-associated protein tau (MAPT) H1/H2 haplotypes, apolipoprotein E (APOE) epsilon2/epsilon3/epsilon4 polymorphism, ubiquitin carboxy-terminal esterase L1 (UCHL1) S18Y variant, cigarette smoking and caffeinated coffee consumption. 932 PD patients and 664 control subjects from the NeuroGenetics Research Consortium, with complete data on all six factors, were studied. Uniform protocols were used for diagnosis, recruitment, data collection and genotyping. A logistic regression model which included gene-exposure interactions was applied. Likelihood ratio tests (LRTs) were used for significance testing and Bayesian inference was used to estimate odds ratios (ORs). MAPT (P = 0.007), SNCA REP1 (P = 0.012), smoking (P = 0.001), and coffee (P = 0.011) were associated with PD risk. Two novel interactions were detected: APOE with coffee (P = 0.005), and REP1 with smoking (P = 0.021). While the individual main effects were modest, each yielding OR < 1.6, the effects were cumulative, with some combinations reaching OR = 12.6 (95% CI: 5.9-26.8). This study provides evidence for the long-held notion that PD risk is modulated by cumulative and interactive effects of genes and exposures. Furthermore, the study demonstrates that while interaction studies are useful for exploring risk relationships that might otherwise go undetected, results should be interpreted with caution because of the inherent loss of power due to multiple testing. The novel findings of this study that warrant replication are the evidence for interaction of coffee with APOE, and of smoking with REP1 on PD risk.

Lack of evidence for an association between UCHL1 S18Y and Parkinson's disease.

UCHL1 has been proposed as a candidate gene for Parkinson's disease (PD). A meta-analysis of white and Asian subjects reported an inverse association between the non-synonymous UCHL1 S18Y polymorphism and PD risk. However, this finding was not replicated in a large case-control study and updated meta-analysis restricted to white subjects. We performed a case-control study of 1757 PD patients recruited from movement disorder clinics and 2016 unrelated controls from four regions of the United States. All subjects self-reported as white. We did not observe evidence for an association between S18Y genotypes and PD (overall P-value for association: P = 0.42). After adjustment for age, sex, and recruitment region, the odds ratio for Y/S versus S/S was 0.91 (95% CI: 0.78-1.06) and for Y/Y versus S/S was 0.87 (95% CI: 0.58-1.29). We also did not observe a significant association for recessive or dominant models of inheritance, or after stratification by age at onset, age at blood draw, sex, family history of PD, or recruitment region. Our results suggest that UCHL1 S18Y is not a major susceptibility factor for PD in white populations although we cannot exclude the possibility that the S18Y variant exerts weak effects on risk, particularly in early-onset disease.

Combined effects of smoking, coffee, and NSAIDs on Parkinson's disease risk.

Inverse associations of Parkinson's disease (PD) with cigarette smoking, coffee drinking, and nonsteroidal anti-inflammatory drug (NSAID) use have been reported individually, but their joint effects have not been examined. To quantify associations with PD for the individual, two-way and three-way combinations of these factors, a case-control association study with 1,186 PD patients and 928 controls was conducted. The study setting was the NeuroGenetics Research Consortium. Subjects completed a structured questionnaire regarding smoking, coffee, and NSAID consumption. Odds ratios were calculated using unconditional logistic regression. Smoking, coffee, and over the counter NSAID use as individual factors exhibited significantly reduced risks of 20% to 30%. The two-way and three-way combinations were associated with risk reduction of 37% to 49%, and 62%, respectively. Smoking and coffee exhibited significant inverse risk trends with increasing cumulative exposures, suggesting dose-response relations. With respect to the combination of all three exposures, persons who were at the highest exposure strata for smoking and coffee and used NSAIDs had an estimated 87% reduction in risk (OR = 0.13, 95% CI = 0.06-0.29). Whether this finding reflects true biologic protection needs to be investigated.

Association analysis of MAPT H1 haplotype and subhaplotypes in Parkinson's disease.

OBJECTIVE: An inversion polymorphism of approximately 900 kb on chromosome 17q21, which includes the microtubule-associated protein tau (MAPT) gene defines two haplotype clades, H1 and H2. Several small case-control studies have observed a marginally significant excess of the H1/H1 diplotype among patients with Parkinson's disease (PD), and one reported refining the association to a region spanning exons 1 to 4 of MAPT. We sought to replicate these findings. METHODS: We genotyped 1,762 PD patients and 2,010 control subjects for a single nucleotide polymorphism (SNP) that differentiates the H1 and H2 clades. We also analyzed four SNPs that define subhaplotypes within H1 previously reported to associate with PD or other neurodegenerative disorders. RESULTS: After adjusting for age, sex, and site, we observed a robust association between the H1/H1 diplotype and PD risk (odds ratio for H1/H1 vs H1/H2 and H2/H2, 1.46; 95% confidence interval, 1.25-1.69; p = 8 x 10(-7)). The effect was evident in both familial and sporadic subgroups, men and women, and early- and late-onset disease. Within H1/H1 individuals, there was no significant difference between cases and control subjects in the overall frequency distribution of H1 subhaplotypes. INTERPRETATION: Our data provide strong evidence that the H1 clade, which contains MAPT and several other genes, is a risk factor for PD. However, attributing this finding to variants within a specific region of MAPT is premature. Thorough fine-mapping of the H1 clade in large numbers of individuals is now needed to identify the underlying functional variant(s) that alter susceptibility for PD.

Heterozygous parkin point mutations are as common in control subjects as in Parkinson's patients.

OBJECTIVE: Homozygous or compound heterozygous parkin mutations cause juvenile parkinsonism. Heterozygous parkin mutations are also found in patients with typical Parkinson's disease (PD), but it is unclear whether a single "mutation" in a patient is related to disease or is coincidental, because the mutation frequency in control subjects is unknown. We present a comprehensive sequence analysis of parkin in control subjects. METHODS: A total of 302 patients and 301 control subjects were sequenced, and findings were replicated in 1,260 additional patients and 1,657 control subjects. RESULTS: Thirty-four variants were detected, of which 21 were novel; 12 were polymorphisms and 22 were rare variants. Patients and control subjects did not differ in the frequency, type, or functional location of the variants. Even P437L, a common mutation thought to be pathogenic, was present in unaffected control subjects. INTERPRETATION: parkin point mutations are not exclusive to PD. The mere presence of a single point mutation in a patient, in the absence of a second mutation, should not be taken as a cause of disease unless corroborated by family data and functional studies. This study does not support the notion that heterozygous parkin sequence variants (mutations or polymorphisms) are risk factors for PD. Whether heterozygous dosage anomalies are associated with PD remains to be determined.

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 Parkinson's 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 Parkinson's Research-funded genetics consortia-comprising 14 teams-contributed DNA samples from 5526 patients with Parkinson's 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 Parkinson's disease.

Validity and utility of a LRRK2 G2019S mutation test for the diagnosis of Parkinson's disease.

The G2019S mutation in the LRRK2 gene, the most common known cause of Parkinson's disease (PD), will soon be widely available as a molecular clinical test for PD. The objective of this study was to assess performance characteristics of G2019S as a clinical test for PD in the setting of typical movement disorder clinics in the United States. Subjects included 1,518 sequentially recruited PD patients from seven movement disorder clinics in the United States, and 1,733 unaffected subjects. All 3,251 subjects were genotyped for the G2019S mutation using a TaqMan assay, and mutations were verified by direct sequencing. Test validity estimates were calculated using standard methods. A total of 20/1518 patients and 1/1733 controls carried the G2019S mutation. Specificity was 99.9% (95% CI, 99.6-100%), sensitivity was 1.3% (0.8-2.1%), and the positive likelihood ratio was 22.8. A positive family history of PD increased the positive likelihood ratio to 82.5. Information on gender, age at disease onset, or age at testing did not improve test performance. The gene test was highly accurate in classifying mutation carriers as PD, but it performed poorly in predicting the phenotype of non-mutation carriers. A G2019S molecular test for PD would be highly specific, technically simple, and inexpensive. Test interpretation is straightforward when used for diagnosis of symptomatic individuals, but is more complex for risk assessment and predictive testing in asymptomatic individuals. Test results can have psychological, social, and economical ramifications; thus, proper counseling is essential.

One step closer to fixing association studies: evidence for age- and gender-specific allele frequency variations and deviations from Hardy-Weinberg expectations in controls.

Association studies are the most powerful method available for identifying modest gene effects in complex disorders, but they often produce inconsistent results. With the rapidly growing SNP databases, haplotype maps and high throughput genotyping, the use of association studies is expected to increase; therefore, it is critical and timely that the problems with study design are identified and fixed. We questioned if unrecognized allele and genotype frequency variations in controls could be responsible for some of the inconsistent association findings. We performed a population genetic study of apolipoprotein E (APOE) and cytochrome P450 2D6 (CYP2D6) in 1,748 individuals ranging in age from newborns to centenarians. Although APOE and CYP2D6 are two of the most commonly used candidate genes, this is the first study to examine age- and gender-specific frequency distributions over the entire age spectrum, using a large, ethnically and geographically uniform population. We found significant, previously unrecognized variations in APOE allele frequencies, and deviations from Hardy-Weinberg expectations in CYP2D6 genotype frequencies starting at birth. The allele frequency variations within controls were larger than some reported case-control differences. We demonstrate that unrecognized frequency fluctuations in controls are a serious and potentially common confounder whose impact on association studies has not been appreciated, and one that can be addressed with proper study design. We recommend that population genetic studies be performed on commonly used candidate markers and that rigorous standards be applied for case-control matching.

Parkin mutation dosage and the phenomenon of anticipation: a molecular genetic study of familial parkinsonism.

BACKGROUND: parkin mutations are a common cause of parkinsonism. Possessing two parkin mutations leads to early-onset parkinsonism, while having one mutation may predispose to late-onset disease. This dosage pattern suggests that some parkin families should exhibit intergenerational variation in age at onset resembling anticipation. A subset of familial PD exhibits anticipation, the cause of which is unknown. The aim of this study was to determine if anticipation was due to parkin mutation dosage. METHODS: We studied 19 kindreds that had early-onset parkinsonism in the offspring generation, late-onset parkinsonism in the parent generation, and > or = 20 years of anticipation. We also studied 28 early-onset parkinsonism cases without anticipation. Patients were diagnosed by neurologists at a movement disorder clinic. parkin analysis included sequencing and dosage analysis of all 12 exons. RESULTS: Only one of 19 cases had compound parkin mutations, but contrary to our postulate, the affected relative with late-onset parkinsonism did not have a parkin mutation. In effect, none of the anticipation cases could be attributed to parkin. In contrast, 21% of early-onset parkinsonism patients without anticipation had parkin mutations. CONCLUSION: Anticipation is not linked to parkin, and may signify a distinct disease entity.

Synergistic computational and experimental proteomics approaches for more accurate detection of active serine hydrolases in yeast.

An analysis of the structurally and catalytically diverse serine hydrolase protein family in the Saccharomyces cerevisiae proteome was undertaken using two independent but complementary, large-scale approaches. The first approach is based on computational analysis of serine hydrolase active site structures; the second utilizes the chemical reactivity of the serine hydrolase active site in complex mixtures. These proteomics approaches share the ability to fractionate the complex proteome into functional subsets. Each method identified a significant number of sequences, but 15 proteins were identified by both methods. Eight of these were unannotated in the Saccharomyces Genome Database at the time of this study and are thus novel serine hydrolase identifications. Three of the previously uncharacterized proteins are members of a eukaryotic serine hydrolase family, designated as Fsh (family of serine hydrolase), identified here for the first time. OVCA2, a potential human tumor suppressor, and DYR-SCHPO, a dihydrofolate reductase from Schizosaccharomyces pombe, are members of this family. Comparing the combined results to results of other proteomic methods showed that only four of the 15 proteins were identified in a recent large-scale, "shotgun" proteomic analysis and eight were identified using a related, but similar, approach (neither identifies function). Only 10 of the 15 were annotated using alternate motif-based computational tools. The results demonstrate the precision derived from combining complementary, function-based approaches to extract biological information from complex proteomes. The chemical proteomics technology indicates that a functional protein is being expressed in the cell, while the computational proteomics technology adds details about the specific type of function and residue that is likely being labeled. The combination of synergistic methods facilitates analysis, enriches true positive results, and increases confidence in novel identifications. This work also highlights the risks inherent in annotation transfer and the use of scoring functions for determination of correct annotations.