Systematic rare variant analyses identify RAB32 as a susceptibility gene for familial Parkinson’s disease

Despite substantial progress, causal variants are identified only for a minority of familial Parkinson’s disease (PD) cases, leaving high-risk pathogenic variants unidentified1,2. To identify such variants, we uniformly processed exome sequencing data of 2,184 index familial PD cases and 69,775 controls. Exome-wide analyses converged on RAB32 as a novel PD gene identifying c.213C > G/p.S71R as a high-risk variant presenting in ~0.7% of familial PD cases while observed in only 0.004% of controls (odds ratio of 65.5). This variant was confirmed in all cases via Sanger sequencing and segregated with PD in three families. RAB32 encodes a small GTPase known to interact with LRRK2 (refs. 3,4). Functional analyses showed that RAB32 S71R increases LRRK2 kinase activity, as indicated by increased autophosphorylation of LRRK2 S1292. Here our results implicate mutant RAB32 in a key pathological mechanism in PD—LRRK2 kinase activity5–7—and thus provide novel insights into the mechanistic connections between RAB family biology, LRRK2 and PD risk

The project MinE databrowser: bringing large-scale whole-genome sequencing in ALS to researchers and the public

Amyotrophic lateral sclerosis (ALS) is a rapidly progressive fatal neurodegenerative disease affecting one in 350 people. The aim of Project MinE is to elucidate the pathophysiology of ALS through whole-genome sequencing at least 15,000 ALS patients and 7500 controls at 30× coverage. Here, we present the Project MinE data browser ( databrowser.projectmine.com ), a unique and intuitive one-stop, open-access server that provides detailed information on genetic variation analyzed in a new and still growing set of 4366 ALS cases and 1832 matched controls. Through its visual components and interactive design, the browser specifically aims to be a resource to those without a biostatistics background and allow clinicians and preclinical researchers to integrate Project MinE data into their own research. The browser allows users to query a transcript and immediately access a unique combination of detailed (meta)data, annotations and association statistics that would otherwise require analytic expertise and visits to scattered resources

ATXN1 repeat expansions confer risk for amyotrophic lateral sclerosis and contribute to TDP-43 mislocalization

Increasingly, repeat expansions are being identified as part of the complex genetic architecture of amyotrophic lateral sclerosis. To date, several repeat expansions have been genetically associated with the disease: intronic repeat expansions in C9orf72, polyglutamine expansions in ATXN2 and polyalanine expansions in NIPA1. Together with previously published data, the identification of an amyotrophic lateral sclerosis patient with a family history of spinocerebellar ataxia type 1, caused by polyglutamine expansions in ATXN1, suggested a similar disease association for the repeat expansion in ATXN1. We, therefore, performed a large-scale international study in 11 700 individuals, in which we showed a significant association between intermediate ATXN1 repeat expansions and amyotrophic lateral sclerosis (P = 3.33 × 10-7). Subsequent functional experiments have shown that ATXN1 reduces the nucleocytoplasmic ratio of TDP-43 and enhances amyotrophic lateral sclerosis phenotypes in Drosophila, further emphasizing the role of polyglutamine repeat expansions in the pathophysiology of amyotrophic lateral sclerosis

Unexpected frequency of the pathogenic AR CAG repeat expansion in the general population

CAG repeat expansions in exon 1 of the AR gene on the X chromosome cause spinal and bulbar muscular atrophy, a male-specific progressive neuromuscular disorder associated with a variety of extra-neurological symptoms. The disease has a reported male prevalence of 1:30,303 or less, but the AR repeat expansion frequency is unknown. We established a pipeline, which combines the use of the ExpansionHunter tool and visual validation, to detect AR CAG expansion on whole-genome sequencing data, benchmarked it to fragment PCR sizing, and applied it to 74,277 unrelated individuals from four large cohorts. Our pipeline showed sensitivity of 100% (95% C.I. 90.8-100%), specificity of 99% (95% C.I. 94.2-99.7%), and positive predictive value of 97.4% (95% C.I. 84.4-99.6%). We found the mutation frequency to be 1:3,182 (95% C.I. 1:2,309-1:4,386, n=117,734) X chromosomes - ten times more frequent than the reported disease prevalence. Modelling using the novel mutation frequency led to estimate disease prevalence of 1:6,887 males, more than four times more frequent than the reported disease prevalence. This discrepancy is possibly due to underdiagnosis of this neuromuscular condition, reduced prevalence, and/or pleomorphic clinical manifestations

Value of systematic genetic screening of patients with amyotrophic lateral sclerosis

Objective The clinical utility of routine genetic sequencing in amyotrophic lateral sclerosis (ALS) is uncertain. Our aim was to determine whether routine targeted sequencing of 44 ALS-relevant genes would have a significant impact on disease subclassification and clinical care. Methods We performed targeted sequencing of a 44-gene panel in a prospective case series of 100 patients with ALS recruited consecutively from the Sheffield Motor Neuron Disorders Clinic, UK. All participants were diagnosed with ALS by a specialist Consultant Neurologist. 7/100 patients had familial ALS, but the majority were apparently sporadic cases. Results 21% of patients with ALS carried a confirmed pathogenic or likely pathogenic mutation, of whom 93% had no family history of ALS. 15% met the inclusion criteria for a current ALS genetic-therapy trial. 5/21 patients with a pathogenic mutation had an additional variant of uncertain significance (VUS). An additional 21% of patients with ALS carried a VUS in an ALS-associated gene. Overall, 13% of patients carried more than one genetic variant (pathogenic or VUS). Patients with ALS carrying two variants developed disease at a significantly earlier age compared with patients with a single variant (median age of onset=56 vs 60 years, p=0.0074). Conclusions Routine screening for ALS-associated pathogenic mutations in a specialised ALS referral clinic will impact clinical care in 21% of cases. An additional 21% of patients have variants in the ALS gene panel currently of unconfirmed significance after removing non-specific or predicted benign variants. Overall, variants within known ALS-linked genes are of potential clinical importance in 42% of patients

Genome-wide identification of the genetic basis of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a complex disease that leads to motor neuron death. Despite heritability estimates of 52%, genome-wide association studies (GWASs) have discovered relatively few loci. We developed a machine learning approach called RefMap, which integrates functional genomics with GWAS summary statistics for gene discovery. With transcriptomic and epigenetic profiling of motor neurons derived from induced pluripotent stem cells (iPSCs), RefMap identified 690 ALS-associated genes that represent a 5-fold increase in recovered heritability. Extensive conservation, transcriptome, network, and rare variant analyses demonstrated the functional significance of candidate genes in healthy and diseased motor neurons and brain tissues. Genetic convergence between common and rare variation highlighted KANK1 as a new ALS gene. Reproducing KANK1 patient mutations in human neurons led to neurotoxicity and demonstrated that TDP-43 mislocalization, a hallmark pathology of ALS, is downstream of axonal dysfunction. RefMap can be readily applied to other complex diseases

Rare variant burden analysis within enhancers identifies CAV1 as an ALS risk gene

Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disease. CAV1 and CAV2 organize membrane lipid rafts (MLRs) important for cell signaling and neuronal survival, and overexpression of CAV1 ameliorates ALS phenotypes in vivo. Genome-wide association studies localize a large proportion of ALS risk variants within the non-coding genome, but further characterization has been limited by lack of appropriate tools. By designing and applying a pipeline to identify pathogenic genetic variation within enhancer elements responsible for regulating gene expression, we identify disease-associated variation within CAV1/CAV2 enhancers, which replicate in an independent cohort. Discovered enhancer mutations reduce CAV1/CAV2 expression and disrupt MLRs in patient-derived cells, and CRISPR-Cas9 perturbation proximate to a patient mutation is sufficient to reduce CAV1/CAV2 expression in neurons. Additional enrichment of ALS-associated mutations within CAV1 exons positions CAV1 as an ALS risk gene. We propose CAV1/CAV2 overexpression as a personalized medicine target for ALS.peer-reviewe

Rare and common genetic determinants of mitochondrial function determine severity but not risk of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease involving selective vulnerability of energy-intensive motor neurons (MNs). It has been unclear whether mitochondrial function is an upstream driver or a downstream modifier of neurotoxicity. We separated upstream genetic determinants of mitochondrial function, including genetic variation within the mitochondrial genome or autosomes; from downstream changeable factors including mitochondrial DNA copy number (mtCN). Across three cohorts including 6,437 ALS patients, we discovered that a set of mitochondrial haplotypes, chosen because they are linked to measurements of mitochondrial function, are a determinant of ALS survival following disease onset, but do not modify ALS risk. One particular haplotype appeared to be neuroprotective and was significantly over-represented in two cohorts of long-surviving ALS patients. Causal inference for mitochondrial function was achievable using mitochondrial haplotypes, but not autosomal SNPs in traditional Mendelian randomization (MR). Furthermore, rare loss-of-function genetic variants within, and reduced MN expression of, ACADM and DNA2 lead to ∼50 % shorter ALS survival; both proteins are implicated in mitochondrial function. Both mtCN and cellular vulnerability are linked to DNA2 function in ALS patient-derived neurons. Finally, MtCN responds dynamically to the onset of ALS independently of mitochondrial haplotype, and is correlated with disease severity. We conclude that, based on the genetic measures we have employed, mitochondrial function is a therapeutic target for amelioration of disease severity but not prevention of ALS

Rare and common genetic determinants of mitochondrial function determine severity but not risk of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease involving selective vulnerability of energy-intensive motor neurons (MNs). It has been unclear whether mitochondrial function is an upstream driver or a downstream modifier of neurotoxicity. We separated upstream genetic determinants of mitochondrial function, including genetic variation within the mitochondrial genome or autosomes; from downstream changeable factors including mitochondrial DNA copy number (mtCN). Across three cohorts including 6,437 ALS patients, we discovered that a set of mitochondrial haplotypes, chosen because they are linked to measurements of mitochondrial function, are a determinant of ALS survival following disease onset, but do not modify ALS risk. One particular haplotype appeared to be neuroprotective and was significantly over-represented in two cohorts of long-surviving ALS patients. Causal inference for mitochondrial function was achievable using mitochondrial haplotypes, but not autosomal SNPs in traditional Mendelian randomization (MR). Furthermore, rare loss-of-function genetic variants within, and reduced MN expression of, ACADM and DNA2 lead to ∼50 % shorter ALS survival; both proteins are implicated in mitochondrial function. Both mtCN and cellular vulnerability are linked to DNA2 function in ALS patient-derived neurons. Finally, MtCN responds dynamically to the onset of ALS independently of mitochondrial haplotype, and is correlated with disease severity. We conclude that, based on the genetic measures we have employed, mitochondrial function is a therapeutic target for amelioration of disease severity but not prevention of ALS

Mutations in the tail and rod domains of the neurofilament heavy-chain gene increase the risk of ALS

OBJECTIVE: Neurofilament heavy-chain gene (NEFH) variants are associated with multiple neurodegenerative diseases, however, their relationship with ALS has not been robustly explored. Still, NEFH is commonly included in genetic screening panels worldwide. We therefore aimed to determine if NEFH variants modify ALS risk. METHODS: Genetic data of 11,130 people with ALS and 7,416 controls from the literature and Project MinE were analysed. We performed meta-analyses of published case-control studies reporting NEFH variants, and variant analysis of NEFH in Project MinE whole-genome sequencing data. RESULTS: Fixed-effects meta-analysis found that rare (MAF <1%) missense variants in the tail domain of NEFH increase ALS risk (OR 4.55, 95% CI 2.13-9.71, p < 0.0001). In Project MinE, ultrarare NEFH variants increased ALS risk (OR 1.37 95% CI 1.14-1.63, p = 0.0007), with rod domain variants (mostly intronic) appearing to drive the association (OR 1.45 95% CI 1.18-1.77, pMadsen-Browning = 0.0007, pSKAT-O = 0.003). While in the tail domain, ultrarare (MAF <0.1%) pathogenic missense variants were also associated with higher risk of ALS (OR 1.94, 95% CI 0.86-4.37, pMadsen-Browning = 0.039), supporting the meta-analysis results. Finally, several tail in-frame deletions were also found to affect disease risk, however, both protective and pathogenic deletions were found in this domain, highlighting an intricate architecture that requires further investigation. INTERPRETATION: We showed that NEFH tail missense and in-frame deletion variants, and intronic rod variants are risk factors for ALS. However, they are not variants of large effect, and their functional impact needs to be clarified in further studies. Therefore, their inclusion in routine genetic screening panels should be reconsidered