Recent advances in understanding dominant spinocerebellar ataxias from clinical and genetic points of view [version 1; referees: 3 approved]

Abstract Spinocerebellar ataxias (SCAs) are rare types of cerebellar ataxia with a dominant mode of inheritance. To date, 47 SCA subtypes have been identified, and the number of genes implicated in SCAs is continually increasing. Polyglutamine (polyQ) expansion diseases (ATXN1/SCA1, ATXN2/SCA2, ATXN3/SCA3, CACNA1A/SCA6, ATXN7/SCA7, TBP/SCA17, and ATN1/DRPLA) are the most common group of SCAs. No preventive or curative treatments are currently available, but various therapeutic approaches, including RNA-targeting treatments, such as antisense oligonucleotides (ASOs), are being developed. Clinical trials of ASOs in SCA patients are already planned. There is, therefore, a need to identify valid outcome measures for such studies. In this review, we describe recent advances towards identifying appropriate biomarkers, which are essential for monitoring disease progression and treatment efficacy. Neuroimaging biomarkers are the most powerful markers identified to date, making it possible to reduce sample sizes for clinical trials. Changes on brain MRI are already evident at the premanifest stage in SCA1 and SCA2 carriers and are correlated with CAG repeat size. Other potential biomarkers have also been developed, based on neurological examination, oculomotor study, cognitive assessment, and blood and cerebrospinal fluid analysis. Longitudinal studies based on multimodal approaches are required to establish the relationships between parameters and to validate the biomarkers identified

Methylation-dependent PAD2 upregulation in multiple sclerosis peripheral blood

Background: Peptidylarginine deiminase 2 (PAD2) and peptidylarginine deiminase 4 (PAD4) are two members of PAD family which are over-expressed in the multiple sclerosis (MS) brain. Through its enzymatic activity PAD2 converts myelin basic protein (MBP) arginines into citrullines - an event that may favour autoimmunity - while peptidylarginine deiminase 4 (PAD4) is involved in chromatin remodelling. Objectives: Our aim was to verify whether an altered epigenetic control of PAD2, as already shown in the MS brain, can be observed in peripheral blood mononuclear cells (PBMCs) of patients with MS since some of these cells also synthesize MBP. Methods: The expression of most suitable reference genes and of PAD2 and PAD4 was assessed by qPCR. Analysis of DNA methylation was performed by bisulfite method. Results: The comparison of PAD2 expression level in PBMCs from patients with MS vs. healthy donors showed that, as well as in the white matter of MS patients, the enzyme is significantly upregulated in affected subjects. Methylation pattern analysis of a CpG island located in the PAD2 promoter showed that over-expression is associated with promoter demethylation. Conclusion: Defective regulation of PAD2 in the periphery, without the immunological shelter of the blood-brain barrier, may contribute to the development of the autoimmune responses in MS

Novel homozygous GBA2 mutation in a patient with complicated spastic paraplegia

Hereditary spastic paraplegias (HSPs) are a heterogeneous group of neurological disorders characterized primarily by a pyramidal syndrome with lower limb spasticity, which can manifest as pure HSP or associated with a number of neurological or non-neurological signs (i.e., complicated HSPs). The clinical variability of HSPs is associated with a wide genetic heterogeneity, with more than eighty causative genes known. Recently, next generation sequencing (NGS) has allowed increasing genetic definition in such a heterogeneous group of disorders. We report on a 56- year-old man affected by sporadic complicated HSP consisting of a pyramidal syndrome, cerebellar ataxia, congenital cataract, pes cavus, axonal sensory-motor peripheral neuropathy and cognitive decline. Brain MRI showed cerebellar atrophy and thin corpus callosum. By NGS we found a novel homozygous biallelic c.452-1G > C mutation in the b-glucosidase 2 gene (GBA2), known to be causative for autosomal recessive hereditary spastic paraplegia type 46 (SPG46). The rarity of this inherited form besides reporting on a novel mutation, expands the genetic and clinical spectrum of SPG46 related HSP

Evidence for detrimental cross interactions between reactive oxygen and nitrogen species in Leber's hereditary optic neuropathy cells

Here we have collected evidence suggesting that chronic changes in the NO homeostasis and the rise of reactive oxygen species bioavailability can contribute to cell dysfunction in Leber’s hereditary optic neuropathy (LHON) patients.We report that peripheral blood mononuclear cells (PBMCs), derived froma female LHON patient with bilateral reduced vision and carrying the pathogenic mutation 11778/ND4, display increased levels of reactive oxygen species (ROS) and reactive nitrogen species (RNS), as revealed by flow cytometry, fluorometric measurements of nitrite/nitrate, and 3-nitrotyrosine immunodetection. Moreover, viability assays with the tetrazolium dye MTT showed that lymphoblasts from the same patient are more sensitive to prolonged NO exposure, leading to cell death. Taken together these findings suggest that oxidative and nitrosative stress cooperatively play an important role in driving LHON pathology when excess NO remains available over time in the cell environment

Quantitative oculomotor assessment in hereditary ataxia: systematic review and consensus by the ataxia global initiative working group on digital-motor biomarkers

Oculomotor deficits are common in hereditary ataxia, but disproportionally neglected in clinical ataxia scales and as outcome measures for interventional trials. Quantitative assessment of oculomotor function has become increasingly available and thus applicable in multicenter trials and offers the opportunity to capture severity and progression of oculomotor impairment in a sensitive and reliable manner. In this consensus paper of the Ataxia Global Initiative Working Group On Digital Oculomotor Biomarkers, based on a systematic literature review, we propose harmonized methodology and measurement parameters for the quantitative assessment of oculomotor function in natural-history studies and clinical trials in hereditary ataxia. MEDLINE was searched for articles reporting on oculomotor/vestibular properties in ataxia patients and a study-tailored quality-assessment was performed. One-hundred-and-seventeen articles reporting on subjects with genetically confirmed (n=1134) or suspected hereditary ataxia (n=198), and degenerative ataxias with sporadic presentation (n=480) were included and subject to data extraction. Based on robust discrimination from controls, correlation with disease-severity, sensitivity to change, and feasibility in international multicenter settings as prerequisite for clinical trials, we prioritize a core-set of five eye-movement types: (i) pursuit eye movements, (ii) saccadic eye movements, (iii) fixation, (iv) eccentric gaze holding, and (v) rotational vestibulo-ocular reflex. We provide detailed guidelines for their acquisition, and recommendations on the quantitative parameters to extract. Limitations include low study quality, heterogeneity in patient populations, and lack of longitudinal studies. Standardization of quantitative oculomotor assessments will facilitate their implementation, interpretation, and validation in clinical trials, and ultimately advance our understanding of the evolution of oculomotor network dysfunction in hereditary ataxias

Increasing involvement of CAPN1 variants in spastic ataxias and phenotype-genotype correlations

Spastic ataxias are rare neurogenetic disorders involving spinocerebellar and pyramidal tracts. Many genes are involved. Among them, CAPN1, when mutated, is responsible for a complex inherited form of spastic paraplegia (SPG76). We report the largest published series of 21 novel patients with nine new CAPN1 disease-causing variants and their clinical characteristics from two European university hospitals (Paris and Stockholm). After a formal clinical examination, causative variants were identified by next-generation sequencing and confirmed by Sanger sequencing. CAPN1 variants are a rare cause (~ 1.4%) of young-adult-onset spastic ataxia; however, together with all published cases, they allowed us to better describe the clinical and genetic spectra of this form. Truncating variants are the most frequent, and missense variants lead to earlier age at onset in favor of an additional deleterious effect. Cerebellar ataxia with cerebellar atrophy, dysarthria and lower limb weakness are often associated with spasticity. We also suggest that cognitive impairment and depression should be assessed specifically in the follow-up of SPG76 cases.Identification of new causative genes in spinocerebellar degenerations by combination of whole genome scan, next-generation sequencing and biological validation in vitro and in vivoInfrastructure de Recherche Translationnelle pour les Biothérapies en NeurosciencesEuropean Union’s Horizon 2020 research and innovation programm

Solve-RD: systematic pan-European data sharing and collaborative analysis to solve rare diseases.

For the first time in Europe hundreds of rare disease (RD) experts team up to actively share and jointly analyse existing patient's data. Solve-RD is a Horizon 2020-supported EU flagship project bringing together >300 clinicians, scientists, and patient representatives of 51 sites from 15 countries. Solve-RD is built upon a core group of four European Reference Networks (ERNs; ERN-ITHACA, ERN-RND, ERN-Euro NMD, ERN-GENTURIS) which annually see more than 270,000 RD patients with respective pathologies. The main ambition is to solve unsolved rare diseases for which a molecular cause is not yet known. This is achieved through an innovative clinical research environment that introduces novel ways to organise expertise and data. Two major approaches are being pursued (i) massive data re-analysis of >19,000 unsolved rare disease patients and (ii) novel combined -omics approaches. The minimum requirement to be eligible for the analysis activities is an inconclusive exome that can be shared with controlled access. The first preliminary data re-analysis has already diagnosed 255 cases form 8393 exomes/genome datasets. This unprecedented degree of collaboration focused on sharing of data and expertise shall identify many new disease genes and enable diagnosis of many so far undiagnosed patients from all over Europe

Solving unsolved rare neurological diseases-a Solve-RD viewpoint.

Funder: Durch Princess Beatrix Muscle Fund Durch Speeren voor Spieren Muscle FundFunder: University of Tübingen Medical Faculty PATE programFunder: European Reference Network for Rare Neurological Diseases | 739510Funder: European Joint Program on Rare Diseases (EJP-RD COFUND-EJP) | 44140962

Twist exome capture allows for lower average sequence coverage in clinical exome sequencing

Background Exome and genome sequencing are the predominant techniques in the diagnosis and research of genetic disorders. Sufficient, uniform and reproducible/consistent sequence coverage is a main determinant for the sensitivity to detect single-nucleotide (SNVs) and copy number variants (CNVs). Here we compared the ability to obtain comprehensive exome coverage for recent exome capture kits and genome sequencing techniques. Results We compared three different widely used enrichment kits (Agilent SureSelect Human All Exon V5, Agilent SureSelect Human All Exon V7 and Twist Bioscience) as well as short-read and long-read WGS. We show that the Twist exome capture significantly improves complete coverage and coverage uniformity across coding regions compared to other exome capture kits. Twist performance is comparable to that of both short- and long-read whole genome sequencing. Additionally, we show that even at a reduced average coverage of 70× there is only minimal loss in sensitivity for SNV and CNV detection. Conclusion We conclude that exome sequencing with Twist represents a significant improvement and could be performed at lower sequence coverage compared to other exome capture techniques

Solving patients with rare diseases through programmatic reanalysis of genome-phenome data.

Funder: EC | EC Seventh Framework Programm | FP7 Health (FP7-HEALTH - Specific Programme "Cooperation": Health); doi: https://doi.org/10.13039/100011272; Grant(s): 305444, 305444Funder: Ministerio de Economía y Competitividad (Ministry of Economy and Competitiveness); doi: https://doi.org/10.13039/501100003329Funder: Generalitat de Catalunya (Government of Catalonia); doi: https://doi.org/10.13039/501100002809Funder: EC | European Regional Development Fund (Europski Fond za Regionalni Razvoj); doi: https://doi.org/10.13039/501100008530Funder: Instituto Nacional de Bioinformática ELIXIR Implementation Studies Centro de Excelencia Severo OchoaFunder: EC | EC Seventh Framework Programm | FP7 Health (FP7-HEALTH - Specific Programme "Cooperation": Health)Reanalysis of inconclusive exome/genome sequencing data increases the diagnosis yield of patients with rare diseases. However, the cost and efforts required for reanalysis prevent its routine implementation in research and clinical environments. The Solve-RD project aims to reveal the molecular causes underlying undiagnosed rare diseases. One of the goals is to implement innovative approaches to reanalyse the exomes and genomes from thousands of well-studied undiagnosed cases. The raw genomic data is submitted to Solve-RD through the RD-Connect Genome-Phenome Analysis Platform (GPAP) together with standardised phenotypic and pedigree data. We have developed a programmatic workflow to reanalyse genome-phenome data. It uses the RD-Connect GPAP's Application Programming Interface (API) and relies on the big-data technologies upon which the system is built. We have applied the workflow to prioritise rare known pathogenic variants from 4411 undiagnosed cases. The queries returned an average of 1.45 variants per case, which first were evaluated in bulk by a panel of disease experts and afterwards specifically by the submitter of each case. A total of 120 index cases (21.2% of prioritised cases, 2.7% of all exome/genome-negative samples) have already been solved, with others being under investigation. The implementation of solutions as the one described here provide the technical framework to enable periodic case-level data re-evaluation in clinical settings, as recommended by the American College of Medical Genetics