Role of the repeat expansion size in predicting age of onset and severity in RFC1 disease.

RFC1 disease, caused by biallelic repeat expansion in RFC1, is clinically heterogeneous in terms of age of onset, disease progression and phenotype. We investigated the role of the repeat size in influencing clinical variables in RFC1 disease. We also assessed the presence and role of meiotic and somatic instability of the repeat. In this study, we identified 553 patients carrying biallelic RFC1 expansions and measured the repeat expansion size in 392 cases. Pearson's coefficient was calculated to assess the correlation between the repeat size and age at disease onset. A Cox model with robust cluster standard errors was adopted to describe the effect of repeat size on age at disease onset, on age at onset of each individual symptoms, and on disease progression. A quasi-Poisson regression model was used to analyse the relationship between phenotype and repeat size. We performed multivariate linear regression to assess the association of the repeat size with the degree of cerebellar atrophy. Meiotic stability was assessed by Southern blotting on first-degree relatives of 27 probands. Finally, somatic instability was investigated by optical genome mapping on cerebellar and frontal cortex and unaffected peripheral tissue from four post-mortem cases. A larger repeat size of both smaller and larger allele was associated with an earlier age at neurological onset [smaller allele hazard ratio (HR) = 2.06, P < 0.001; larger allele HR = 1.53, P < 0.001] and with a higher hazard of developing disabling symptoms, such as dysarthria or dysphagia (smaller allele HR = 3.40, P < 0.001; larger allele HR = 1.71, P = 0.002) or loss of independent walking (smaller allele HR = 2.78, P < 0.001; larger allele HR = 1.60; P < 0.001) earlier in disease course. Patients with more complex phenotypes carried larger expansions [smaller allele: complex neuropathy rate ratio (RR) = 1.30, P = 0.003; cerebellar ataxia, neuropathy and vestibular areflexia syndrome (CANVAS) RR = 1.34, P < 0.001; larger allele: complex neuropathy RR = 1.33, P = 0.008; CANVAS RR = 1.31, P = 0.009]. Furthermore, larger repeat expansions in the smaller allele were associated with more pronounced cerebellar vermis atrophy (lobules I-V ß = -1.06, P < 0.001; lobules VI-VII ß = -0.34, P = 0.005). The repeat did not show significant instability during vertical transmission and across different tissues and brain regions. RFC1 repeat size, particularly of the smaller allele, is one of the determinants of variability in RFC1 disease and represents a key prognostic factor to predict disease onset, phenotype and severity. Assessing the repeat size is warranted as part of the diagnostic test for RFC1 expansion.

De novo KCNA6 variants with attenuated KV 1.6 channel deactivation in patients with epilepsy.

OBJECTIVE: Mutations in the genes encoding neuronal ion channels are a common cause of Mendelian neurological diseases. We sought to identify novel de novo sequence variants in cases with early infantile epileptic phenotypes and neurodevelopmental anomalies. METHODS: Following clinical diagnosis, we performed whole exome sequencing of the index cases and their parents. Identified channel variants were expressed in Xenopus oocytes and their functional properties assessed using two-electrode voltage clamp. RESULTS: We identified novel de novo variants in KCNA6 in four unrelated individuals variably affected with neurodevelopmental disorders and seizures with onset in the first year of life. Three of the four identified mutations affect the pore-lining S6 α-helix of KV 1.6. A prominent finding of functional characterization in Xenopus oocytes was that the channel variants showed only minor effects on channel activation but slowed channel closure and shifted the voltage dependence of deactivation in a hyperpolarizing direction. Channels with a mutation affecting the S6 helix display dominant effects on channel deactivation when co-expressed with wild-type KV 1.6 or KV 1.1 subunits. SIGNIFICANCE: This is the first report of de novo nonsynonymous variants in KCNA6 associated with neurological or any clinical features. Channel variants showed a consistent effect on channel deactivation, slowing the rate of channel closure following normal activation. This specific gain-of-function feature is likely to underlie the neurological phenotype in our patients. Our data highlight KCNA6 as a novel channelopathy gene associated with early infantile epileptic phenotypes and neurodevelopmental anomalies.

RFC1 expansions are a common cause of idiopathic sensory neuropathy.

After extensive evaluation, one-third of patients affected by polyneuropathy remain undiagnosed and are labelled as having chronic idiopathic axonal polyneuropathy, which refers to a sensory or sensory-motor, axonal, slowly progressive neuropathy of unknown origin. Since a sensory neuropathy/neuronopathy is identified in all patients with genetically confirmed RFC1 cerebellar ataxia, neuropathy, vestibular areflexia syndrome, we speculated that RFC1 expansions could underlie a fraction of idiopathic sensory neuropathies also diagnosed as chronic idiopathic axonal polyneuropathy. We retrospectively identified 225 patients diagnosed with chronic idiopathic axonal polyneuropathy (125 sensory neuropathy, 100 sensory-motor neuropathy) from our general neuropathy clinics in Italy and the UK. All patients underwent full neurological evaluation and a blood sample was collected for RFC1 testing. Biallelic RFC1 expansions were identified in 43 patients (34%) with sensory neuropathy and in none with sensory-motor neuropathy. Forty-two per cent of RFC1-positive patients had isolated sensory neuropathy or sensory neuropathy with chronic cough, while vestibular and/or cerebellar involvement, often subclinical, were identified at examination in 58%. Although the sensory ganglia are the primary pathological target of the disease, the sensory impairment was typically worse distally and symmetric, while gait and limb ataxia were absent in two-thirds of the cases. Sensory amplitudes were either globally absent (26%) or reduced in a length-dependent (30%) or non-length dependent pattern (44%). A quarter of RFC1-positive patients had previously received an alternative diagnosis, including Sjögren's syndrome, sensory chronic inflammatory demyelinating polyneuropathy and paraneoplastic neuropathy, while three cases had been treated with immune therapies.

[CANVAS: case report on a novel repeat expansion disorder with late-onset ataxia]. / CANVAS: Fallbericht einer neuen Repeat-Erkrankung mit spät beginnender Ataxie.

This article presents the case of a 74-year-old female patient who first developed a progressive disease with sensory neuropathy, cerebellar ataxia and bilateral vestibulopathy at the age of 60 years. The family history was unremarkable. Magnetic resonance imaging (MRI) showed atrophy of the cerebellum predominantly in the vermis and atrophy of the spinal cord. The patient was given the syndromic diagnosis of cerebellar ataxia, neuropathy, vestibular areflexia syndrome (CANVAS). In 2019 the underlying genetic cause of CANVAS was discovered to be an intronic repeat expansion in the RFC1 gene with autosomal recessive inheritance. The patient exhibited the full clinical picture of CANVAS and was tested positive for this repeat expansion on both alleles. The CANVAS is a relatively frequent cause of late-onset hereditary ataxia (estimated prevalence 5­13/100,000). In contrast to the present patient, the full clinical picture is not always present. Therefore, testing for the RFC1 gene expansion is recommended in the work-up of patients with otherwise unexplained late-onset sporadic ataxia. As intronic repeat expansions cannot be identified by next generation sequencing methods, specific testing is necessary.

Increased frequency of repeat expansion mutations across different populations.

Repeat expansion disorders (REDs) are a devastating group of predominantly neurological diseases. Together they are common, affecting 1 in 3,000 people worldwide with population-specific differences. However, prevalence estimates of REDs are hampered by heterogeneous clinical presentation, variable geographic distributions and technological limitations leading to underascertainment. Here, leveraging whole-genome sequencing data from 82,176 individuals from different populations, we found an overall disease allele frequency of REDs of 1 in 283 individuals. Modeling disease prevalence using genetic data, age at onset and survival, we show that the expected number of people with REDs would be two to three times higher than currently reported figures, indicating underdiagnosis and/or incomplete penetrance. While some REDs are population specific, for example, Huntington disease-like 2 in Africans, most REDs are represented in all broad genetic ancestries (that is, Europeans, Africans, Americans, East Asians and South Asians), challenging the notion that some REDs are found only in specific populations. These results have worldwide implications for local and global health communities in the diagnosis and counseling of REDs.

Optical Genome Mapping Enables Detection and Accurate Sizing of RFC1 Repeat Expansions.

A recessive Short Tandem Repeat expansion in RFC1 has been found to be associated with cerebellar ataxia, neuropathy and vestibular areflexia syndrome (CANVAS), and to be a frequent cause of late onset ataxia and sensory neuropathy. The usual procedure for sizing these expansions is based on Southern Blotting (SB), a time-consuming and a relatively imprecise technique. In this paper, we compare SB with Optical Genome Mapping (OGM), a method for detecting Structural Variants (SVs) based on the measurement of distances between fluorescently labelled probes, for the diagnosis of RFC1 CANVAS and disease spectrum. The two methods are applied to 17 CANVAS patients' blood samples and resulting sizes compared, showing a good agreement. Further, long-read sequencing is used for two patients to investigate the agreement of sizes with either SB or OGM. Our study concludes that OGM represents a viable alternative to SB, allowing for a simpler technique, a more precise sizing of the expansion and ability to expand analysis of SV in the entire genome as opposed to SB which is a locus specific method.

CANVAS: a late onset ataxia due to biallelic intronic AAGGG expansions.

The ataxias are a group of disorders that manifest with balance, movement, speech and visual problems. They can arise due to dysfunction of the cerebellum, the vestibular system and/or the sensory neurons. Genetic defects are a common cause of chronic ataxia, particularly common are repeat expansions in this group of conditions. Co-occurrence of cerebellar ataxia with neuropathy and vestibular areflexia syndrome has been termed CANVAS. Although CANVAS is a rare syndrome, on discovery of biallelic expansions in the second intron of replication factor C subunit 1 (RFC1) gene, we and others have found the phenotype is broad and RFC1 expansions are a common cause of late-onset progressive ataxia.We aim to provide a review and update on recent developments in CANVAS and populations, where the disorder has been reported. We have also optimised a protocol for RFC1 expansion screening which is described herein and expanded phenotype after analysing late-onset ataxia patients from around the world.