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OBJECTIVE This study was undertaken to identify susceptibility loci for cluster headache and obtain insights into relevant disease pathways. METHODS We carried out a genome-wide association study, where 852 UK and 591 Swedish cluster headache cases were compared with 5,614 and 1,134 controls, respectively. Following quality control and imputation, single variant association testing was conducted using a logistic mixed model for each cohort. The 2 cohorts were subsequently combined in a merged analysis. Downstream analyses, such as gene-set enrichment, functional variant annotation, prediction and pathway analyses, were performed. RESULTS Initial independent analysis identified 2 replicable cluster headache susceptibility loci on chromosome 2. A merged analysis identified an additional locus on chromosome 1 and confirmed a locus significant in the UK analysis on chromosome 6, which overlaps with a previously known migraine locus. The lead single nucleotide polymorphisms were rs113658130 (p = 1.92 × 10-17 , odds ratio [OR] = 1.51, 95% confidence interval [CI] = 1.37-1.66) and rs4519530 (p = 6.98 × 10-17 , OR = 1.47, 95% CI = 1.34-1.61) on chromosome 2, rs12121134 on chromosome 1 (p = 1.66 × 10-8 , OR = 1.36, 95% CI = 1.22-1.52), and rs11153082 (p = 1.85 × 10-8 , OR = 1.30, 95% CI = 1.19-1.42) on chromosome 6. Downstream analyses implicated immunological processes in the pathogenesis of cluster headache. INTERPRETATION We identified and replicated several genome-wide significant associations supporting a genetic predisposition in cluster headache in a genome-wide association study involving 1,443 cases. Replication in larger independent cohorts combined with comprehensive phenotyping, in relation to, for example, treatment response and cluster headache subtypes, could provide unprecedented insights into genotype-phenotype correlations and the pathophysiological pathways underlying cluster headache. ANN NEUROL 2021;90:193-202.

Purpose of review This review aims to summarise the present cerebellar ataxia, neuropathy, vestibular ataxia syndrome (CANVAS) literature, providing both clinical and genetic insights that might facilitate the timely clinical and genetic diagnosis of this disease. Recent findings Recent advancements in the range of the clinical features of CANVAS have aided the development of a broader, more well-defined clinical diagnostic criteria. Additionally, the identification of a biallelic repeat expansion in RFC1 as the cause of CANVAS and a common cause of late-onset ataxia has opened the door to the potential discovery of a pathogenic mechanism, which in turn, may lead to therapeutic advancements and improved patient care. Summary The developments in the clinical and genetic understanding of CANVAS will aid the correct and timely diagnosis of CANVAS, which continues to prove challenging within the clinic. The insights detailed within this review will raise the awareness of the phenotypic spectrum and currently known genetics. We also speculate on the future directions of research into CANVAS.

Cerebellar ataxia, neuropathy and vestibular areflexia syndrome (CANVAS) is a progressive late-onset, neurological disease. Recently, a pentanucleotide expansion in intron 2 of RFC1 was identified as the genetic cause of CANVAS. We screened an Asian-Pacific cohort for CANVAS and identified a novel RFC1 repeat expansion motif, (ACAGG)exp, in three affected individuals. This motif was associated with additional clinical features including fasciculations and elevated serum creatine kinase. These features have not previously been described in individuals with genetically-confirmed CANVAS. Haplotype analysis showed our patients shared the same core haplotype as previously published, supporting the possibility of a single origin of the RFC1 disease allele. We analysed data from >26 000 genetically diverse individuals in gnomAD to show enrichment of (ACAGG) in non-European populations.

Neuronal intranuclear inclusion disease (NIID) is a clinically heterogeneous neurodegenerative condition characterized by pathological intranuclear eosinophilic inclusions. A CGG repeat expansion inNOTCH2NLCwas recently identified to be associated with NIID in patients of Japanese descent. We screened pathologically confirmed European NIID, cases of neurodegenerative disease with intranuclear inclusions and applied in silico-based screening using whole-genome sequencing data from 20 536 participants in the 100 000 Genomes Project. We identified a single European case harbouring the pathogenic repeat expansion with a distinct haplotype structure. Thus, we propose new diagnostic criteria as European NIID represents a distinct disease entity from East Asian cases.

Aminoacyl-tRNA synthetases (ARSs) are ubiquitous, ancient enzymes that charge amino acids to cognate tRNA molecules, the essential first step of protein translation. Here, we describe 32 individuals from 21 families, presenting with microcephaly, neurodevelopmental delay, seizures, peripheral neuropathy, and ataxia, with de novo heterozygous and bi-allelic mutations in asparaginyl-tRNA synthetase (NARS1). We demonstrate a reduction in NARS1 mRNA expression as well as in NARS1 enzyme levels and activity in both individual fibroblasts and induced neural progenitor cells (iNPCs). Molecular modeling of the recessive c.1633C>T (p.Arg545Cys) variant shows weaker spatial positioning and tRNA selectivity. We conclude that de novo and bi-allelic mutations in NARS1 are a significant cause of neurodevelopmental disease, where the mechanism for de novo variants could be toxic gain-of-function and for recessive variants, partial loss-of-function.

SummaryDystonia, a neurological disorder defined by abnormal postures and disorganised movements, is considered to be a neural circuit disorder with dysfunction arising within and between multiple brain regions. Given that spinal circuits constitute the final pathway for motor control, we sought to determine their contribution to the movement disorder. Focusing on the most common inherited dystonia, DYT1-TOR1A, we confined a conditional knockout ofTor1ato the spinal cord and dorsal root ganglia (DRG) and found that these mice recapitulated the phenotype of the human condition, developing early onset generalised torsional dystonia. Physiologically, these mice bore the hallmark features of dystonia: spontaneous contractions at rest, excessive sustained contractions during voluntary movements including co-contractions of motor antagonists, and altered sensory-motor reflexes. Furthermore, spinal locomotor circuits were impaired. Together, these data challenge current understanding of dystonia, and lead to broader insights into spinal cord function and movement disorder pathophysiology.

Multiple systems atrophy (MSA) is a rare neurodegenerative disorder combining varying presentations of cerebellar impairment, parkinsonism and autonomic dysfunction that relies on pathological examination for a definitive diagnosis.1 2 The second consensus diagnostic criteria (SCDC) subdivide patients into two groups based on a predominance of either parkinsonism (MSA-P) or cerebellar impairment (MSA-C).2 MSA is frequently misdiagnosed, especially early in the disease course, with only 60% of possible and probable MSA meeting the pathological criteria.2 3 A recessive intronic expansion in replication factor C subunit 1 ( RFC1 ) has been found to be a cause of late-onset ataxia and cerebellar ataxia, neuropathy and vestibular areflexia syndrome (CANVAS),4 Due to the overlapping clinical features of MSA and CANVAS, we hypothesised that the recessive RFC1 expansion may be implicated in patients that atypically fit the diagnostic criteria for MSA. Here, we present three cases with biallelic RFC1 expansions: two clinically diagnosed with ‘probable’ MSA and one with ‘probable’ based on the SCDC for MSA.2 ### Samples We enrolled 207 patients with ‘possible’ and ‘probable’ MSA, from the National Hospital for Neurology and Neurosurgery (NHNN). MSA diagnosis based on the SCDC for MSA.2 ### Genetic testing Genomic DNA was extracted from blood and investigated under approval of the joint ethics committee of the UCL Institute of Neurology and The NHNN, London, UK (UCLH: 04/N034). Flanking PCR, repeat-primed PCR and Southern blot were performed on all samples as previously described4 (figure 1A). Figure 1 Clinical presentation of affected patients. (A) Workflow of RFC1 screen; clinical screening of cohort leads to sample gDNA undergoing flanking PCR and those with no amplifiable product and potential carriers are taken forward for RPPCR. Samples with a positive RPPCR trace (hallmark sawtooth pattern) are then Southern blotted, confirming size of expansion. (B) CANVAS RPPCR positive fragment analysis traces for patients …

Wastewater-based epidemiology (WBE) has emerged as a critical public health tool in tracking the SARS-CoV-2 epidemic. Monitoring SARS-CoV-2 variants of concern in wastewater has to-date relied on genomic sequencing, which lacks sensitivity necessary to detect low variant abundances in diluted and mixed wastewater samples. Here, we develop and present an open-source method based on allele specific RT-qPCR (AS RT-qPCR) that detects and quantifies the B.1.1.7 variant, targeting spike protein mutations at three independent genomic loci highly predictive of B.1.1.7 (HV69/70del, Y144del, and A570D). Our assays can reliably detect and quantify low levels of B.1.1.7 with low cross-reactivity, and at variant proportions between 0.1% and 1% in a background of mixed SARS-CoV-2. Applying our method to wastewater samples from the United States, we track B.1.1.7 occurrence over time in 19 communities. AS RT-qPCR results align with clinical trends, and summation of B.1.1.7 and wild-type sequences quantified by our assays strongly correlate with SARS-CoV-2 levels indicated by the US CDC N1/N2 assay. This work paves the path for rapid inexpensive surveillance of B.1.1.7 and other SARS-CoV-2 variants in wastewater.

Spinocerebellar ataxia (SCA) is a heterogeneous group of neurodegenerative ataxic disorders with autosomal dominant inheritance. We aim to provide an update on the recent clinical and scientific progresses in SCA where numerous novel genes have been identified with next-generation sequencing techniques. The main disease mechanisms of these SCAs include toxic RNA gain-of-function, mitochondrial dysfunction, channelopathies, autophagy and transcription dysregulation. Recent studies have also demonstrated the importance of DNA repair pathways in modifying SCA with CAG expansions. In addition, we summarise the latest technological advances in detecting known and novel repeat expansion in SCA. Finally, we discuss the roles of antisense oligonucleotides and RNA-based therapy as potential treatments. Electronic supplementary material The online version of this article (10.1007/s00415-018-9076-4) contains supplementary material, which is available to authorized users.

The inherited cerebellar ataxias comprise of a genetic heterogeneous group of disorders. Pathogenic expansions of cytosine-adenine-guanine (CAG) encoding polyglutamine tracts account for the largest proportion of autosomal dominant cerebellar ataxias, while GAA expansion in the first introns of frataxin gene is the commonest cause of autosomal recessive cerebellar ataxias. Currently, there is no available treatment to alter the disease trajectory, with devastating consequences for affected individuals. Inter- and Intrafamily phenotypic variability suggest the existence of genetic modifiers, which may become targets amendable to treatment. Recent studies have demonstrated the importance of DNA repair pathways in modifying spinocerebellar ataxia with CAG repeat expansions. In this review, we discuss the mechanisms in which DNA repair pathways, epigenetics and other genetic factors may act as modifiers in cerebellar ataxias due to trinucleotide repeat expansions.

Cerebellar ataxia with neuropathy and bilateral vestibular areflexia syndrome (CANVAS) is a recently recognized neurodegenerative disease with onset in mid- to late adulthood. The genetic basis for a large proportion of Caucasian patients was recently shown to be the biallelic expansion of a pentanucleotide (AAGGG)n repeat in RFC1. Here, we describe the first instance of CANVAS genetic testing in New Zealand Māori and Cook Island Māori individuals. We show a novel, possibly population-specific CANVAS configuration (AAAGG)10-25(AAGGG)exp, which was the cause of CANVAS in all patients. There were no apparent phenotypic differences compared with European CANVAS patients. Presence of a common disease haplotype among this cohort suggests this novel repeat expansion configuration is a founder effect in this population, which may indicate that CANVAS will be especially prevalent in this group. Haplotype dating estimated the most recent common ancestor at ∼1430 ce. We also show the same core haplotype as previously described, supporting a single origin of the CANVAS mutation.

See Paisán-Ruiz and Jen (doi:10.1093/brain/awaa015) for a scientific commentary on this article. Cortese et al. describe the full disease phenotype, including progression of ataxia, in 100 confirmed carriers of RFC1 repeat expansions. RFC1 repeat expansion should be considered in all cases of sensory ataxic neuropathy, particularly if cerebellar dysfunction, vestibular involvement and cough coexist., Ataxia, causing imbalance, dizziness and falls, is a leading cause of neurological disability. We have recently identified a biallelic intronic AAGGG repeat expansion in replication factor complex subunit 1 (RFC1) as the cause of cerebellar ataxia, neuropathy, vestibular areflexia syndrome (CANVAS) and a major cause of late onset ataxia. Here we describe the full spectrum of the disease phenotype in our first 100 genetically confirmed carriers of biallelic repeat expansions in RFC1 and identify the sensory neuropathy as a common feature in all cases to date. All patients were Caucasian and half were sporadic. Patients typically reported progressive unsteadiness starting in the sixth decade. A dry spasmodic cough was also frequently associated and often preceded by decades the onset of walking difficulty. Sensory symptoms, oscillopsia, dysautonomia and dysarthria were also variably associated. The disease seems to follow a pattern of spatial progression from the early involvement of sensory neurons, to the later appearance of vestibular and cerebellar dysfunction. Half of the patients needed walking aids after 10 years of disease duration and a quarter were wheelchair dependent after 15 years. Overall, two-thirds of cases had full CANVAS. Sensory neuropathy was the only manifestation in 15 patients. Sixteen patients additionally showed cerebellar involvement, and six showed vestibular involvement. The disease is very likely to be underdiagnosed. Repeat expansion in RFC1 should be considered in all cases of sensory ataxic neuropathy, particularly, but not only, if cerebellar dysfunction, vestibular involvement and cough coexist.

Late-onset ataxia is common, often idiopathic, and can result from cerebellar, proprioceptive, or vestibular impairment; when in combination, it is also termed cerebellar ataxia, neuropathy, vestibular areflexia syndrome (CANVAS). We used non-parametric linkage analysis and genome sequencing to identify a biallelic intronic AAGGG repeat expansion in the replication factor C subunit 1 (RFC1) gene as the cause of familial CANVAS and a frequent cause of late-onset ataxia, particularly if sensory neuronopathy and bilateral vestibular areflexia coexist. The expansion, which occurs in the poly(A) tail of an AluSx3 element and differs in both size and nucleotide sequence from the reference (AAAAG)11 allele, does not affect RFC1 expression in patient peripheral and brain tissue, suggesting no overt loss of function. These data, along with an expansion carrier frequency of 0.7% in Europeans, implies that biallelic AAGGG expansion in RFC1 is a frequent cause of late-onset ataxia.

Aim: An outstanding challenge in epigenome studies is the estimation of cell-type proportions in complex epithelial tissues. Materials & methods: Here, we construct and validate a DNA methylation reference and algorithm for complex tissues that contain epithelial, immune and nonimmune stromal cells. Results: Using this reference, we show that easily accessible tissues such as saliva, buccal and cervix exhibit substantial variation in immune cell (IC) contamination. We further validate our reference in the context of oral cancer, where it correctly predicts an increased IC infiltration in cancer but suppressed in patients with highest smoking exposure. Finally, our method can improve the specificity of differentially methylated CpG calls in epithelial cancer. Conclusion: The degree and variation of IC contamination in complex epithelial tissues is substantial. We provide a valuable resource and tool for assessing the epithelial purity and IC contamination of samples and for identifying differential methylation in such complex tissues.