Suran Nethisinghe

The role of interruptions in polyQ in the pathology of SCA1.

At least nine dominant neurodegenerative diseases are caused by expansion of CAG repeats in coding regions of specific genes that result in abnormal elongation of polyglutamine (polyQ) tracts in the corresponding gene products. When above a threshold that is specific for each disease the expanded polyQ repeats promote protein aggregation, misfolding and neuronal cell death. The length of the polyQ tract inversely correlates with the age at disease onset. It has been observed that interruption of the CAG tract by silent (CAA) or missense (CAT) mutations may strongly modulate the effect of the expansion and delay the onset age. We have carried out an extensive study in which we have complemented DNA sequence determination with cellular and biophysical models. By sequencing cloned normal and expanded SCA1 alleles taken from our cohort of ataxia patients we have determined sequence variations not detected by allele sizing and observed for the first time that repeat instability can occur even in the presence of CAG interruptions. We show that histidine interrupted pathogenic alleles occur with relatively high frequency (11%) and that the age at onset inversely correlates linearly with the longer uninterrupted CAG stretch. This could be reproduced in a cellular model to support the hypothesis of a linear behaviour of polyQ. We clarified by in vitro studies the mechanism by which polyQ interruption slows down aggregation. Our study contributes to the understanding of the role of polyQ interruption in the SCA1 phenotype with regards to age at disease onset, prognosis and transmission.

A reduction in Drp1 mediated fission compromises mitochondrial health in autosomal recessive spastic ataxia of Charlevoix Saguenay

The neurodegenerative disease autosomal recessive spastic ataxia of Charlevoix Saguenay (ARSACS) is caused by loss of function of sacsin, a modular protein that is required for normal mitochondrial network organization. To further understand cellular consequences of loss of sacsin, we performed microarray analyses in sacsin knockdown cells and ARSACS patient fibroblasts. This identified altered transcript levels for oxidative phosphorylation and oxidative stress genes. These changes in mitochondrial gene networks were validated by quantitative reverse transcription PCR. Functional impairment of oxidative phosphorylation was then demonstrated by comparison of mitochondria bioenergetics through extracellular flux analyses. Moreover, staining with the mitochondrial-specific fluorescent probe MitoSox suggested increased levels of superoxide in patient cells with reduced levels of sacsin. Key to maintaining mitochondrial health is mitochondrial fission, which facilitates the dynamic exchange of mitochondrial components and separates damaged parts of the mitochondrial network for selective elimination by mitophagy. Fission is dependent on dynamin-related protein 1 (Drp1), which is recruited to prospective sites of division where it mediates scission. In sacsin knockdown cells and ARSACS fibroblasts, we observed a decreased incidence of mitochondrial associated Drp1 foci. This phenotype persists even when fission is induced by drug treatment. Mitochondrial-associated Drp1 foci are also smaller in sacsin knockdown cells and ARSACS fibroblasts. These data suggest a model for ARSACS where neurons with reduced levels of sacsin are compromised in their ability to recruit or retain Drp1 at the mitochondrial membrane leading to a decline in mitochondrial health, potentially through impaired mitochondrial quality control.

The ARSACS phenotype can include supranuclear gaze palsy and skin lipofuscin deposits

The autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) phenotype was first characterised in patients from Quebec, all of whom had young onset spasticity and gait ataxia. Other features included distal amyotrophy, extensor plantar responses, cerebellar speech, saccadic intrusion in smooth pursuit and hypermyelinated retinal nerve fibres.1 Seven percent had seizures. Spasticity and ataxia were progressive. Electromyography showed denervation. Nerve conduction studies showed reduced conduction velocities with absent sensory action potentials. Nerve biopsy showed a lack of large myelinated axons.2 Subsequent analysis of this cohort has shown two founder mutations in the SACS gene—c.6594delT and c.5254C>T.3 Identification of mutations in SACS as the cause of ARSACS facilitated the detection of further cases worldwide, revealing greater phenotypic variation. Vermeer et al screened 43 patients presenting with ataxia prior to age 25 (suggesting an autosomal recessive cause4), finding 16 patients with SACS mutations.5 One patient had onset aged 12, two showed dystonia. Baets et al 6 screened 85 patients with at least two of cerebellar ataxia, spasticity and peripheral neuropathy, finding 18 different mutations. In five of these patients, disease onset was at over 20 years, one patient had no signs of peripheral neuropathy, several patients presented primarily with peripheral neuropathy and only one had hypermyelinated retinal nerve fibres using standard fundoscopy. Two patients had mild cognitive impairment and one epilepsy. Breckpot et al 7 detected a deletion of SACS combined with a hemizygous SACS mutation causing early-onset ARSACS with hearing impairment. We describe a 37-year-old patient who first walked at 23 months. Aged seven, he had cerebellar ataxia and brisk reflexes in all limbs. Cognitive function has been normal but ataxia and spasticity have progressed. He also developed epilepsy, myoclonus and …

Sequencing analysis of the spinal bulbar muscular atrophy CAG expansion reveals absence of repeat interruptions

Trinucleotide repeat disorders are a heterogeneous group of diseases caused by the expansion, beyond a pathogenic threshold, of unstable DNA tracts in different genes. Sequence interruptions in the repeats have been described in the majority of these disorders and may influence disease phenotype and heritability. Spinal bulbar muscular atrophy (SBMA) is a motor neuron disease caused by a CAG trinucleotide expansion in the androgen receptor (AR) gene. Diagnostic testing and previous research have relied on fragment analysis polymerase chain reaction to determine the AR CAG repeat size, and have therefore not been able to assess the presence of interruptions. We here report a sequencing study of the AR CAG repeat in a cohort of SBMA patients and control subjects in the United Kingdom. We found no repeat interruptions to be present, and we describe differences between sequencing and traditional sizing methods.

Retinal Imaging in Autosomal Recessive Spastic Ataxia of Charlevoix-Saguenay

A case is described of autosomal recessive spastic ataxia of Charlevoix-Saguenay. Genetic analysis has shown that two mutant genes encoding the sacsin protein have been inherited one from each parent. In the proband the thickness of the nerve fibre layer is quantified using optical coherence tomography. An abnormally thick retinal nerve fibre layer has been previously described in this condition, ascribed to hypermyelination; the authors suggest that there is no evidence of abnormal myelination as the thickened nerve fibre layer is not opaque. Lesser degrees of nerve fibre layer thickening are seen in other family members who do not show any of the phenotypic features of the disorder.

Optical coherence tomography in autosomal recessive spastic ataxia of Charlevoix-Saguenay

Autosomal recessive spastic ataxia of Charlevoix-Saguenay is a rare neurodegenerative disorder caused by mutations in the SACS gene. Thickened retinal nerve fibres visible on fundoscopy have previously been described in these patients; however, thickening of the retinal nerve fibre layer as demonstrated by optical coherence tomography appears to be a more sensitive and specific feature. To test this observation, we assessed 292 individuals (191 patients with ataxia and 101 control subjects) by peripapillary time-domain optical coherence tomography. The patients included 146 with a genetic diagnosis of ataxia (17 autosomal spastic ataxia of Charlevoix-Saguenay, 59 Friedreichs ataxia, 53 spinocerebellar ataxias, 17 other genetically confirmed ataxias) and 45 with cerebellar ataxia of unknown cause. The controls included 13 asymptomatic heterozygotes for SACS mutations and 88 unaffected controls. The cases with autosomal recessive spastic ataxia of Charlevoix-Saguenay included 11 previously unpublished SACS mutations, of which seven were nonsense and four missense mutations. Most patients were visually asymptomatic and had no previous history of ophthalmic complaints and normal or near normal visual test results. None had visual symptoms directly attributable to the retinal changes. Twelve of the 17 cases (70.6%) had thickened retinal nerve fibres visible on fundoscopy. All patients with autosomal recessive spastic ataxia of Charlevoix-Saguenay had thickening of the peripapillary retinal nerve fibre layer on optical coherence tomography, whereas all the remaining cases and controls except one showed normal or reduced average peripapillary retinal nerve fibre layer thickness on optical coherence tomography. We propose a cut-off value of 119 µm in average peripapillary retinal nerve fibre layer thickness, which provides a sensitivity of 100% and specificity of 99.4% amongst patients affected with ataxia. This is the largest cohort of patients with this condition to undergo systematic evaluation by optical coherence tomography. This is a useful tool in identifying cases of autosomal recessive spastic ataxia of Charlevoix-Saguenay from other causes of ataxia. Visualization of thickened retinal fibres by direct fundoscopy is less sensitive. We therefore advocate the use of this technique in the assessment of possible cases of this condition.

Altered organization of the intermediate filament cytoskeleton and relocalization of proteostasis modulators in cells lacking the ataxia protein sacsin

Abstract Autosomal Recessive Spastic Ataxia of Charlevoix-Saguenay (ARSACS) is caused by mutations in the gene SACS, encoding the 520 kDa protein sacsin. Although sacsin’s physiological role is largely unknown, its sequence domains suggest a molecular chaperone or protein quality control function. Consequences of its loss include neurofilament network abnormalities, specifically accumulation and bundling of perikaryal and dendritic neurofilaments. To investigate if loss of sacsin affects intermediate filaments more generally, the distribution of vimentin was analysed in ARSACS patient fibroblasts and in cells where sacsin expression was reduced. Abnormal perinuclear accumulation of vimentin filaments, which sometimes had a cage-like appearance, occurred in sacsin-deficient cells. Mitochondria and other organelles were displaced to the periphery of vimentin accumulations. Reorganization of the vimentin network occurs in vitro under stress conditions, including when misfolded proteins accumulate. In ARSACS patient fibroblasts HSP70, ubiquitin and the autophagy-lysosome pathway proteins Lamp2 and p62 relocalized to the area of the vimentin accumulation. There was no overall increase in ubiquitinated proteins, suggesting the ubiquitin–proteasome system was not impaired. There was evidence for alterations in the autophagy–lysosome pathway. Specifically, in ARSACS HDFs cellular levels of Lamp2 were elevated while levels of p62, which is degraded in autophagy, were decreased. Moreover, autophagic flux was increased in ARSACS HDFs under starvation conditions. These data show that loss of sacsin effects the organization of intermediate filaments in multiple cell types, which impacts the cellular distribution of other organelles and influences autophagic activity.

PolyQ Tract Toxicity in SCA1 is Length Dependent in the Absence of CAG Repeat Interruption

Spinocerebellar ataxia type 1 (SCA1) is an autosomal dominant neurodegenerative disorder caused by an expansion of a polyglutamine tract within the ATXN1 gene. Normal alleles have been reported to range from 6 to 35 repeats, intermediate alleles from 36 to 38 repeats and fully penetrant pathogenic alleles have at least 39 repeats. This distribution was based on relatively few samples and the narrow intermediate range makes the accuracy of the repeat sizing crucial for interpreting and reporting diagnostic tests, which can vary between laboratories. Here, we examine the distribution of 6378 SCA1 chromosomes and identify a very late onset SCA1 family with a fully penetrant uninterrupted pathogenic allele containing 38 repeats. This finding supports the theory that polyQ toxicity is related to the increase of the length of the inherited tracts and not as previously hypothesized to the structural transition occurring above a specific threshold. In addition, the threshold of toxicity shifts to a shorter polyQ length with the increase of the lifespan in SCA1. Furthermore, we show that SCA1 intermediate alleles have a different behavior compared to the other polyglutamine disorders as they do not show reduced penetrance when uninterrupted. Therefore, the pathogenic mechanism in SCA1 is distinct from other cytosine-adenine-guanine (CAG) repeat disorders. Accurately sizing repeats is paramount in precision medicine and can be challenging particularly with borderline alleles. We examined plasmids containing cloned CAG repeat tracts alongside a triplet repeat primed polymerase chain reaction (TP PCR) CAG repeat ladder to improve accuracy in repeat sizing by fragment analysis. This method accurately sizes the repeats irrespective of repeat composition or length. We also improved the model for calculating repeat length from fragment analysis sizing by fragment analyzing 100 cloned repeats of known size. Therefore, we recommend these methods for accurately sizing repeat lengths and restriction enzyme digestion to identify interruptions for interpretation of a given allele’s pathogenicity.

Advances in the understanding of hereditary ataxia – implications for future patients

ABSTRACT Introduction: Hereditary ataxias are caused by mutations in a plethora of different genes. Advances in sequencing technologies have led to an exponential increase in novel gene discoveries, highlighted the genetic overlap with other neurological diseases and improved our understanding of genotype-phenotype relationships. Together, these developments allowed the identification of new therapeutic targets that are subsequently making their way into clinical trials. Areas covered: This review focuses on the shared genetic characteristics and the latest insights into the molecular cause of the most prevalent hereditary ataxias. Furthermore, conventional genetic diagnosis and the gradual implementation of next-generation sequencing (NGS) approaches in clinical practice is discussed. Finally, the latest investigated disease-modifying therapeutic agents are reviewed. A literature search was performed in PubMed and the Cochrane Library. Additional information on previous and on-going trials was obtained from the ClinicalTrials.gov website. Expert opinion: The implementation of NGS in clinical practice has led to an increase in detected sequence variants of unknown clinical significance. Determining their pathogenicity is an expensive and time-consuming process. In accordance with the progresses in genetics, there is a need for the simultaneous definition of novel biomarkers and functional assays that can assist in the interpretation of genetic tests.

121 ARSACS: a novel phenotype causing peripheral neuropathy, ataxia and spasticity with supranuclear gaze palsy, myoclonus and epilepsy

The phenotype of Autosomal Recessive Spastic Ataxia of Charlevoix-Saguenay (ARSACS) was initially reported as early-onset limb ataxia, spastic paraparesis and sensorimotor neuropathy with dysarthria, retinal abnormalities, horizontal nystagmus, urinary dysfunction and mitral valve prolapse in patients from North-Eastern Quebec. The identification of recessive mutations in the SACS gene as the cause of ARSACS has allowed genetically-based diagnoses to be made. Subsequent reports from areas outside of Quebec have broadened the ARSACS phenotype to include adult onset as well as patients presenting with only one or two of the triad of spasticity, ataxia and neuropathy. We describe a patient with genetically confirmed ARSACS whose clinical features comprise spasticity, ataxia and neuropathy but also features not previously described in ARSACS including supranuclear gaze palsy, myoclonus and epilepsy as well as unusual skin biopsy findings. Sequencing of the SACS gene revealed that he was compound heterozygous for two novel mutations, further broadening the phenotype and genotype of this increasingly recognised condition.