Genomic Analysis Identifies Risk Factors in Restless Legs Syndrome.

OBJECTIVE: Restless legs syndrome (RLS) is a neurological condition that causes uncomfortable sensations in the legs and an irresistible urge to move them, typically during periods of rest. The genetic basis and pathophysiology of RLS are incompletely understood. We sought to identify additional novel genetic risk factors associated with RLS susceptibility. METHODS: We performed a whole-genome sequencing and genome-wide association meta-analysis of RLS cases (n = 9,851) and controls (n = 38,957) in 3 population-based biobanks (All of Us, Canadian Longitudinal Study on Aging, and CARTaGENE). RESULTS: Genome-wide association analysis identified 9 independent risk loci, of which 8 had been previously reported, and 1 was a novel risk locus (LMX1B, rs35196838, OR 1.14, 95% CI 1.09-1.19, p value = 2.2 × 10-9). Furthermore, a transcriptome-wide association study also identified GLO1 and a previously unreported gene, ELFN1. A genetic correlation analysis revealed significant common variant overlaps between RLS and neuroticism (rg = 0.40, se = 0.08, p value = 5.4 × 10-7), depression (rg = 0.35, se = 0.06, p value = 2.17 × 10-8), and intelligence (rg = -0.20, se = 0.06, p value = 4.0 × 10-4). INTERPRETATION: Our study expands the understanding of the genetic architecture of RLS, and highlights the contributions of common variants to this prevalent neurological disorder. ANN NEUROL 2024;96:994-1005.

Rare-variant and polygenic analyses of amyotrophic lateral sclerosis in the French-Canadian genome.

PURPOSE: The genetic etiology of amyotrophic lateral sclerosis (ALS) includes few rare, large-effect variants and potentially many common, small-effect variants per case. The genetic risk liability for ALS might require a threshold comprised of a certain amount of variants. Here, we tested the degree to which risk for ALS was affected by rare variants in ALS genes, polygenic risk score, or both. METHODS: 335 ALS cases and 356 controls from Québec, Canada were concurrently tested by microarray genotyping and targeted sequencing of ALS genes known at the time of study inception. ALS genome-wide association studies summary statistics were used to estimate an ALS polygenic risk score (PRS). Cases and controls were subdivided into rare-variant heterozygotes and non-heterozygotes. RESULTS: Risk for ALS was significantly associated with PRS and rare variants independently in a logistic regression model. Although ALS PRS predicted a small amount of ALS risk overall, the effect was most pronounced between ALS cases and controls that were not heterozygous for a rare variant in the ALS genes surveyed. CONCLUSION: Both PRS and rare variants in ALS genes impact risk for ALS. PRS for ALS is most informative when rare variants are not observed in ALS genes.

Expanded CAG Repeats in ATXN1, ATXN2, ATXN3, and HTT in the 1000 Genomes Project.

BACKGROUND: Spinocerebellar ataxia types 1, 2, 3 and Huntington disease are neurodegenerative disorders caused by expanded CAG repeats. METHODS: We performed an in-silico analysis of CAG repeats in ATXN1, ATXN2, ATXN3, and HTT using 30× whole-=genome sequencing data of 2504 samples from the 1000 Genomes Project. RESULTS: Seven HTT-positive, 3 ATXN2-positive, 1 ATXN3-positive, and 6 possibly ATXN1-positive samples were identified. No correlation was found between the repeat sizes of the different genes. The distribution of CAG alleles varied by ethnicity. CONCLUSION: Our results suggest that there may be asymptomatic small expanded repeats in almost 0.5% of these populations. © 2020 International Parkinson and Movement Disorder Society.

Chronic lithium treatment alters the excitatory/ inhibitory balance of synaptic networks and reduces mGluR5-PKC signalling in mouse cortical neurons.

Background: Bipolar disorder is characterized by cyclical alternation between mania and depression, often comorbid with psychosis and suicide. Compared with other medications, the mood stabilizer lithium is the most effective treatment for the prevention of manic and depressive episodes. However, the pathophysiology of bipolar disorder and lithium's mode of action are yet to be fully understood. Evidence suggests a change in the balance of excitatory and inhibitory activity, favouring excitation in bipolar disorder. In the present study, we sought to establish a holistic understanding of the neuronal consequences of lithium exposure in mouse cortical neurons, and to identify underlying mechanisms of action. Methods: We used a range of technical approaches to determine the effects of acute and chronic lithium treatment on mature mouse cortical neurons. We combined RNA screening and biochemical and electrophysiological approaches with confocal immunofluorescence and live-cell calcium imaging. Results: We found that only chronic lithium treatment significantly reduced intracellular calcium flux, specifically by activating metabotropic glutamatergic receptor 5. This was associated with altered phosphorylation of protein kinase C and glycogen synthase kinase 3, reduced neuronal excitability and several alterations to synapse function. Consequently, lithium treatment shifts the excitatory­inhibitory balance toward inhibition. Limitations: The mechanisms we identified should be validated in future by similar experiments in whole animals and human neurons. Conclusion: Together, the results revealed how lithium dampens neuronal excitability and the activity of the glutamatergic network, both of which are predicted to be overactive in the manic phase of bipolar disorder. Our working model of lithium action enables the development of targeted strategies to restore the balance of overactive networks, mimicking the therapeutic benefits of lithium but with reduced toxicity.

Multiomics Analyses Identify Genes and Pathways Relevant to Essential Tremor.

INTRODUCTION: The genetic factors and molecular mechanisms predisposing to essential tremor (ET) remains largely unknown. OBJECTIVE: The objective of this study was to identify pathways and genes relevant to ET by integrating multiomics approaches. METHODS: Case-control RNA sequencing of 2 cerebellar regions was done for 64 samples. A phenome-wide association study (pheWAS) of the differentially expressed genes was conducted, and a genome-wide gene association study (GWGAS) was done to identify pathways overlapping with the transcriptomic data. Finally, a transcriptome-wide association study (TWAS) was done to identify novel risk genes for ET. RESULTS: We identified several novel dysregulated genes, including CACNA1A and SHF. Pathways including axon guidance, olfactory loss, and calcium channel activity were significantly enriched. The ET GWGAS data found calcium ion-regulated exocytosis of neurotransmitters to be significantly enriched. The TWAS also found calcium and olfactory pathways enriched. The pheWAS identified that the underexpressed differentially expressed gene, SHF, is associated with a blood pressure medication (P = 9.3E-08), which is used to reduce tremor in ET patients. Treatment of cerebellar DAOY cells with the ET drug propranolol identified increases in SHF when treated, suggesting it may rescue the underexpression. CONCLUSION: We found that calcium-related pathways were enriched across the GWGAS, TWAS, and transcriptome. SHF was shown to have significantly decreased expression, and the pheWAS showed it was associated with blood pressure medication. The treatment of cells with propranolol showed that the drug restored levels of SHF. Overall, our findings highlight the power of integrating multiple different approaches to prioritize ET pathways and genes. © 2020 International Parkinson and Movement Disorder Society.

Targeted sequencing reveals expanded genetic diversity of human transfer RNAs.

Transfer RNAs are required to translate genetic information into proteins as well as regulate other cellular processes. Nucleotide changes in tRNAs can result in loss or gain of function that impact the composition and fidelity of the proteome. Despite links between tRNA variation and disease, the importance of cytoplasmic tRNA variation has been overlooked. Using a custom capture panel, we sequenced 605 human tRNA-encoding genes from 84 individuals. We developed a bioinformatic pipeline that allows more accurate tRNA read mapping and identifies multiple polymorphisms occurring within the same variant. Our analysis identified 522 unique tRNA-encoding sequences that differed from the reference genome from 84 individuals. Each individual had ~66 tRNA variants including nine variants found in less than 5% of our sample group. Variants were identified throughout the tRNA structure with 17% predicted to enhance function. Eighteen anticodon mutants were identified including potentially mistranslating tRNAs; e.g., a tRNASer that decodes Phe codons. Similar engineered tRNA variants were previously shown to inhibit cell growth, increase apoptosis and induce the unfolded protein response in mammalian cell cultures and chick embryos. Our analysis shows that human tRNA variation has been underestimated. We conclude that the large number of tRNA genes provides a buffer enabling the emergence of variants, some of which could contribute to disease.

The landscape of regional missense mutational intolerance quantified from 125,748 exomes.

Missense variants can have a range of functional impacts depending on factors such as the specific amino acid substitution and location within the gene. To interpret their deleteriousness, studies have sought to identify regions within genes that are specifically intolerant of missense variation 1-12 . Here, we leverage the patterns of rare missense variation in 125,748 individuals in the Genome Aggregation Database (gnomAD) 13 against a null mutational model to identify transcripts that display regional differences in missense constraint. Missense-depleted regions are enriched for ClinVar 14 pathogenic variants, de novo missense variants from individuals with neurodevelopmental disorders (NDDs) 15,16 , and complex trait heritability. Following ClinGen calibration recommendations for the ACMG/AMP guidelines, we establish that regions with less than 20% of their expected missense variation achieve moderate support for pathogenicity. We create a missense deleteriousness metric (MPC) that incorporates regional constraint and outperforms other deleteriousness scores at stratifying case and control de novo missense variation, with a strong enrichment in NDDs. These results provide additional tools to aid in missense variant interpretation.

A blended genome and exome sequencing method captures genetic variation in an unbiased, high-quality, and cost-effective manner.

We deployed the Blended Genome Exome (BGE), a DNA library blending approach that generates low pass whole genome (1-4× mean depth) and deep whole exome (30-40× mean depth) data in a single sequencing run. This technology is cost-effective, empowers most genomic discoveries possible with deep whole genome sequencing, and provides an unbiased method to capture the diversity of common SNP variation across the globe. To evaluate this new technology at scale, we applied BGE to sequence >53,000 samples from the Populations Underrepresented in Mental Illness Associations Studies (PUMAS) Project, which included participants across African, African American, and Latin American populations. We evaluated the accuracy of BGE imputed genotypes against raw genotype calls from the Illumina Global Screening Array. All PUMAS cohorts had R 2 concordance ≥95% among SNPs with MAF≥1%, and never fell below ≥90% R 2 for SNPs with MAF<1%. Furthermore, concordance rates among local ancestries within two recently admixed cohorts were consistent among SNPs with MAF≥1%, with only minor deviations in SNPs with MAF<1%. We also benchmarked the discovery capacity of BGE to access protein-coding copy number variants (CNVs) against deep whole genome data, finding that deletions and duplications spanning at least 3 exons had a positive predicted value of ~90%. Our results demonstrate BGE scalability and efficacy in capturing SNPs, indels, and CNVs in the human genome at 28% of the cost of deep whole-genome sequencing. BGE is poised to enhance access to genomic testing and empower genomic discoveries, particularly in underrepresented populations.

Genomic analysis identifies risk factors in restless legs syndrome.

Restless legs syndrome (RLS) is a neurological condition that causes uncomfortable sensations in the legs and an irresistible urge to move them, typically during periods of rest. The genetic basis and pathophysiology of RLS are incompletely understood. Here, we present a whole-genome sequencing and genome-wide association meta-analysis of RLS cases (n = 9,851) and controls (n = 38,957) in three population-based biobanks (All of Us, Canadian Longitudinal Study on Aging, and CARTaGENE). Genome-wide association analysis identified nine independent risk loci, of which eight had been previously reported, and one was a novel risk locus (LMX1B, rs35196838, OR = 1.14, 95% CI = 1.09-1.19, p-value = 2.2 × 10-9). A genome-wide, gene-based common variant analysis identified GLO1 as an additional risk gene (p-value = 8.45 × 10-7). Furthermore, a transcriptome-wide association study also identified GLO1 and a previously unreported gene, ELFN1. A genetic correlation analysis revealed significant common variant overlaps between RLS and neuroticism (rg = 0.40, se = 0.08, p-value = 5.4 × 10-7), depression (rg = 0.35, se = 0.06, p-value = 2.17 × 10-8), and intelligence (rg = -0.20, se = 0.06, p-value = 4.0 × 10-4). Our study expands the understanding of the genetic architecture of RLS and highlights the contributions of common variants to this prevalent neurological disorder.

Convergent coexpression of autism-associated genes suggests some novel risk genes may not be detectable in large-scale genetic studies.

Autism spectrum disorder (ASD) is a heritable neurodevelopmental disorder characterized by deficits in social interactions and communication. Protein-altering variants in many genes have been shown to contribute to ASD; however, understanding the convergence across many genes remains a challenge. We demonstrate that coexpression patterns from 993 human postmortem brains are significantly correlated with the transcriptional consequences of CRISPR perturbations in human neurons. Across 71 ASD risk genes, there was significant tissue-specific convergence implicating synaptic pathways. Tissue-specific convergence was further demonstrated across schizophrenia and atrial fibrillation risk genes. The degree of ASD convergence was significantly correlated with ASD association from rare variation and differential expression in ASD brains. Positively convergent genes showed intolerance to functional mutations and had shorter coding lengths than known risk genes even after removing association with ASD. These results indicate that convergent coexpression can identify potentially novel genes that are unlikely to be discovered by sequencing studies.

Transcriptomic effects of propranolol and primidone converge on molecular pathways relevant to essential tremor.

Essential tremor (ET) is one of the most common movement disorders, affecting nearly 5% of individuals over 65 years old. Despite this, few genetic risk loci for ET have been identified. Recent advances in pharmacogenomics have previously been useful to identify disease related molecular targets. Notably, gene expression has proven to be quite successful for the inference of drug response in cell models. We sought to leverage this approach in the context of ET where many patients are responsive to two drugs: propranolol and primidone. In this study, cerebellar DAOY and neural progenitor cells were treated for 5 days with clinical concentrations of propranolol and primidone, after which RNA-sequencing was used to identify convergent differentially expressed genes across treatments. Propranolol was found to affect the expression of genes previously associated with ET and other movement disorders such as TRAPPC11. Pathway enrichment analysis of these convergent drug-targeted genes identified multiple terms related to calcium signaling, endosomal sorting, axon guidance, and neuronal morphology. Furthermore, genes targeted by ET drugs were enriched within cell types having high expression of ET-related genes in both cortical and cerebellar tissues. Altogether, our results highlight potential cellular and molecular mechanisms associated with tremor reduction and identify relevant genetic biomarkers for drug-responsiveness in ET.

Questioning the Association of the STMN2 Dinucleotide Repeat With Amyotrophic Lateral Sclerosis.

Objectives: Recently, the number of dinucleotide CA repeats in an intron of the STMN2 gene was reported to be associated with an increased risk for amyotrophic lateral sclerosis (ALS). Therefore, we sought to replicate this observation in an independent group of ALS patients and a much larger control group. Methods: Here, we used whole-genome sequencing and tested the STMN2 CA repeat in a case-control cohort of the European genetic background and in genomes from various populations in the gnomAD cohort to attempt to replicate this proposed association. Results: We find that repeats well above the previously reported pathogenic threshold of 19 are commonly observed in unaffected individuals across different populations. Furthermore, we did not observe an association between longer STMN2 CA repeats and ALS phenotype. Discussion: In summary, our results do not support a role of STMN2 CA repeats toward ALS risk. As TDP-43 aggregation is central to ALS pathogenesis, lowered expression of STMN2 could be used as a biomarker for ALS. Therefore, a variant associated both with the risk for ALS and the level of STMN2 expression would be clinically useful. However, for a variant to be actionable, it must be strongly replicated in independent cohorts and exceed the rigorous statistical thresholds applied.

Transcriptome-wide association study reveals increased neuronal FLT3 expression is associated with Tourette's syndrome.

Tourette's Syndrome (TS) is a neurodevelopmental disorder that is characterized by motor and phonic tics. A recent TS genome-wide association study (GWAS) identified a genome-wide significant locus. However, determining the biological mechanism of GWAS signals remains difficult. To characterize effects of expression quantitative trait loci (eQTLs) in TS and understand biological underpinnings of the disease. Here, we conduct a TS transcriptome-wide association study (TWAS) consisting of 4819 cases and 9488 controls. We demonstrate that increased expression of FLT3 in the dorsolateral prefrontal cortex (DLPFC) is associated with TS. We further show that there is global dysregulation of FLT3 across several brain regions and probabilistic causal fine-mapping of the TWAS signal prioritizes FLT3 with a posterior inclusion probability of 0.849. After, we proxy the expression with 100 lymphoblastoid cell lines, and demonstrate that TS cells has a 1.72 increased fold change compared to controls. A phenome-wide association study also points toward FLT3 having links with immune-related pathways such as monocyte count. We further identify several splicing events in MPHOSPH9, CSGALNACT2 and FIP1L1 associated with TS, which are also implicated in immune function. This analysis of expression and splicing begins to explore the biology of TS GWAS signals.

Association of Essential Tremor With Novel Risk Loci: A Genome-Wide Association Study and Meta-analysis.

Importance: Essential tremor (ET) is one of the most common movement disorders, affecting 5% of the general population older than 65 years. Common variants are thought to contribute toward susceptibility to ET, but no variants have been robustly identified. Objective: To identify common genetic factors associated with risk of ET. Design, Setting, and Participants: Case-control genome-wide association study. Inverse-variance meta-analysis was used to combine cohorts. Multicenter samples collected from European populations were collected from January 2010 to September 2019 as part of an ongoing study. Included patients were clinically diagnosed with or reported having ET. Control individuals were not diagnosed with or reported to have ET. Of 485 250 individuals, data for 483 054 passed data quality control and were used. Main Outcomes and Measures: Genotypes of common variants associated with risk of ET. Results: Of the 483 054 individuals included, there were 7177 with ET (3693 [51.46%] female; mean [SD] age, 62.66 [15.12] years), and 475 877 control individuals (253 785 [53.33%] female; mean [SD] age, 56.40 [17.6] years). Five independent genome-wide significant loci and were identified and were associated with approximately 18% of ET heritability. Functional analyses found significant enrichment in the cerebellar hemisphere, cerebellum, and axonogenesis pathways. Genetic correlation (r), which measures the degree of genetic overlap, revealed significant common variant overlap with Parkinson disease (r, 0.28; P = 2.38 × 10-8) and depression (r, 0.12; P = 9.78 × 10-4). A separate fine-mapping of transcriptome-wide association hits identified genes such as BACE2, LRRN2, DHRS13, and LINC00323 in disease-relevant brain regions, such as the cerebellum. Conclusions and Relevance: The results of this genome-wide association study suggest that a portion of ET heritability can be explained by common genetic variation and can help identify new common genetic risk factors for ET.

Influence of polygenic risk scores for schizophrenia and resilience on the cognition of individuals at-risk for psychosis.

Cognitive impairment is a core feature of schizophrenia which precedes the onset of full psychotic symptoms, even in the ultra-high-risk stage (UHR). Polygenic risk scores (PRS) can be computed for many psychiatric disorders and phenotyping traits, including scores for resilience. We explored the correlations between several PRS and neurocognition in UHR individuals. We included 107 UHR individuals; 29.9% of them converted to psychosis (UHR-C) while 57.0% did not (UHR-NC) during the 1-year follow-up. Cognitive performances were assessed with the Wechsler Adult Intelligence Scale estimating the Intelligence Quotient (IQ), the Trail Making Test, the verbal fluency, the Stroop test, and the Wisconsin card sorting test. Linear regression models were used to test their association with the PRS for schizophrenia, bipolar disorder, major depression, ADHD, cross-disorders, cognitive performance, intelligence, education attainment, and resilience to schizophrenia. UHR-C had a lower IQ than UHR-NC. The PRS for schizophrenia negatively correlated with IQ, while the PRS for cognitive performance and for resilience positively correlated with IQ. PRS for schizophrenia showed a significant correlation with working memory and processing speed indices. PRS for schizophrenia showed a higher effect on IQ in UHR-NC, and UHR-NC with high PRS for schizophrenia had a similar IQ as UHR-C. Conversely, UHR-C with a high PRS for resilience performed as well as UHR-NC. Our findings suggest that cognitive deficits may predate the onset of psychosis. The genetic architecture of schizophrenia seems to impacts the cognition in UHR-NC. Cognition is also mediated by PRS for resilience.

Genome-Wide Association Study Meta-Analysis for Parkinson Disease Motor Subtypes.

OBJECTIVE: To discover genetic determinants of Parkinson disease (PD) motor subtypes, including tremor dominant (TD) and postural instability/gait difficulty (PIGD) forms. METHODS: In 3,212 PD cases of European ancestry, we performed a genome-wide association study (GWAS) examining 2 complementary outcome traits derived from the Unified Parkinson's Disease Rating Scale, including dichotomous motor subtype (TD vs PIGD) or a continuous tremor/PIGD score ratio. Logistic or linear regression models were adjusted for sex, age at onset, disease duration, and 5 ancestry principal components, followed by meta-analysis. RESULTS: Among 71 established PD risk variants, we detected multiple suggestive associations with PD motor subtype, including GPNMB (rs199351, p subtype = 0.01, p ratio = 0.03), SH3GL2 (rs10756907, p subtype = 0.02, p ratio = 0.01), HIP1R (rs10847864, p subtype = 0.02), RIT2 (rs12456492, p subtype = 0.02), and FBRSL1 (rs11610045, p subtype = 0.02). A PD genetic risk score integrating all 71 PD risk variants was also associated with subtype ratio (p = 0.026, ß = -0.04, 95% confidence interval = -0.07-0). Based on top results of our GWAS, we identify a novel suggestive association at the STK32B locus (rs2301857, p ratio = 6.6 × 10-7), which harbors an independent risk allele for essential tremor. CONCLUSIONS: Multiple PD risk alleles may also modify clinical manifestations to influence PD motor subtype. The discovery of a novel variant at STK32B suggests a possible overlap between genetic risk for essential tremor and tremor-dominant PD.

Transcriptome-wide association study for restless legs syndrome identifies new susceptibility genes.

Restless legs syndrome (RLS) is a common neurological condition, with a prevalence of 5-15% in Central Europe and North America. Although genome-wide association studies (GWAS) have identified some common risk regions for RLS, the causal genes have yet to be fully elucidated. We conducted a transcriptome-wide association study involving 15,126 RLS cases and 95,725 controls, from the most recent meta-analysis of GWAS, and gene expression weights of GTEx v7 and the CMC dorsolateral prefrontal cortex tissue panels. We identified 13 associations (in 8 independent loci) at the transcriptome-wide significant level, of which 6 were not implicated in the previous GWAS: SKAP1, SLC36A1, CCDC57, FN3KRP, NCOA6/TRPC4AP. A fine-mapping approach prioritized CMTR1, RP1-153P14.5, PRPF6, and PPP3R1 - to our knowledge, the latter of which is the first RLS-associated gene directly implicated in dopaminergic pathways. Overall, our findings highlight the power of integrating gene expression data with GWAS to prioritize putative causal genes for functional follow-up studies.

Genetic and epidemiological characterization of restless legs syndrome in Québec.

Currently, a total of 19 genetic loci are associated with the risk for developing RLS. This study aimed to assess these RLS predisposing genetic variants, as well as investigate the epidemiological profile and diagnostic features of individuals with RLS in the Québec population, using an interviewer-administered questionnaire. A total of 18 RLS-associated variants were genotyped in the Québec population-based CARTaGENE cohort. A case-control series consisting of 1,362 RLS cases and 1,379 age-matched unaffected controls was used to conduct a genetic and epidemiological association study that integrated the first four RLS diagnostic features of affected individuals, as well as additional RLS-related questions (e.g. frequency of the symptoms and number of total pregnancies in female). Five RLS-predisposing variants were significantly associated after Bonferroni correction and an additional five variants were nominally associated with RLS (p < 0.05). BTBD9 was the strongest genetic risk factor in our cohort (rs9296249, OR = 1.71, p = 9.57 × 10-10). The patient group that met all four essential diagnostic criteria of RLS provided the most significant genetic findings. These results suggest that employing the questionnaire which included standard diagnostic criteria of RLS could improve the accuracy of the survey-based studies.

Transcriptomic Changes Resulting From STK32B Overexpression Identify Pathways Potentially Relevant to Essential Tremor.

Objective: Essential tremor (ET) is a common movement disorder that has a high heritability. A number of genetic studies have associated different genes and loci with ET, but few have investigated the biology of any of these genes. STK32B was significantly associated with ET in a large genome-wide association study (GWAS) and was found to be overexpressed in ET cerebellar tissue. The objective of this study is to determine the effects of overexpressed STK32B in cerebellar DAOY cells. Methods: Here, we overexpressed STK32B RNA in human cerebellar DAOY cells and used an RNA-Seq approach to identify differentially expressed genes (DEGs) by comparing the transcriptome profile of these cells to one of the control DAOY cells. Results: Pathway and gene ontology enrichment identified axon guidance, olfactory signaling, and calcium-voltage channels as significant. Additionally, we show that overexpressing STK32B affects transcript levels of previously implicated ET genes such as FUS. Conclusion: Our results investigate the effects of overexpressed STK32B and suggest that it may be involved in relevant ET pathways and genes.