Targeting autophagy impairment improves the phenotype of a novel CLN8 zebrafish model.

CLN8 is an endoplasmic reticulum cargo receptor and a regulator of lysosome biogenesis whose loss of function leads to neuronal ceroid lipofuscinosis. CLN8 has been linked to autophagy and lipid metabolism, but much remains to be learned, and there are no therapies acting on the molecular signatures in this disorder. The present study aims to characterize the molecular pathways involved in CLN8 disease and, by pinpointing altered ones, to identify potential therapies. To bridge the gap between cell and mammalian models, we generated a new zebrafish model of CLN8 deficiency, which recapitulates the pathological features of the disease. We observed, for the first time, that CLN8 dysfunction impairs autophagy. Using autophagy modulators, we showed that trehalose and SG2 are able to attenuate the pathological phenotype in mutant larvae, confirming autophagy impairment as a secondary event in disease progression. Overall, our successful modeling of CLN8 defects in zebrafish highlights this novel in vivo model's strong potential as an instrument for exploring the role of CLN8 dysfunction in cellular pathways, with a view to identifying small molecules to treat this rare disease.

Ataxia and Hypogonadism: a Review of the Associated Genes and Syndromes.

The association of hypogonadism and cerebellar ataxia was first recognized in 1908 by Gordon Holmes. Since the seminal description, several heterogeneous phenotypes have been reported, differing for age at onset, associated features, and gonadotropins levels. In the last decade, the genetic bases of these disorders are being progressively uncovered. Here, we review the diseases associating ataxia and hypogonadism and the corresponding causative genes. In the first part of this study, we focus on clinical syndromes and genes (RNF216, STUB1, PNPLA6, AARS2, SIL1, SETX) predominantly associated with ataxia and hypogonadism as cardinal features. In the second part, we mention clinical syndromes and genes (POLR3A, CLPP, ERAL1, HARS, HSD17B4, LARS2, TWNK, POLG, ATM, WFS1, PMM2, FMR1) linked to complex phenotypes that include, among other features, ataxia and hypogonadism. We propose a diagnostic algorithm for patients with ataxia and hypogonadism, and we discuss the possible common etiopathogenetic mechanisms.

A new genetic cause of spastic ataxia: the p.Glu415Lys variant in TUBA4A.

Tubulinopathies encompass neurodevelopmental disorders caused by mutations in genes encoding for different isotypes of α- and ß-tubulins, the structural components of microtubules. Less frequently, mutations in tubulins may underlie neurodegenerative disorders. In the present study, we report two families, one with 11 affected individuals and the other with a single patient, carrying a novel, likely pathogenic, variant (p. Glu415Lys) in the TUBA4A gene (NM_006000). The phenotype, not previously described, is that of spastic ataxia. Our findings widen the phenotypic and genetic manifestations of TUBA4A variants and add a new type of spastic ataxia to be taken into consideration in the differential diagnosis.

Standards of Fluid Biomarker Collection and Pre-analytical Processes in Humans and Mice: Recommendations by the Ataxia Global Initiative Working Group on Biomarkers.

The Ataxia Global Initiative (AGI) aims to serve as a platform to facilitate clinical trial readiness for the hereditary ataxias. Clinical trials for these diseases have been hampered by the lack of objective measures to study disease onset, progression, and treatment efficacy. While these issues are not unique to the genetic ataxias, the relative rarity of these diseases makes the need for such measures even more pressing to achieve statistical power in clinical trials. In this report, we have described the efforts of the AGI fluid biomarker working group (WG) in developing uniform protocols for biomarker sampling and storage, both for human and preclinical studies in mice. By reducing collection variability, we anticipate reduced noise in downstream biomarker analysis that will improve statistical power and minimize the necessary sample size. The emphasis has been on defining and standardizing the sampling and pre-analytical work-up of minimal set of biological samples, specifically blood plasma and serum, keeping in mind the need for harmonization of collection and storage that can be achieved with relatively limited cost and resources. An optional package is detailed for those centers that have the resources and commitment for additional biofluids/sample processing and storage. Finally, we have delineated similar standardized protocols for mice that will be important for preclinical studies in the field.

Power of NGS-based tests in HSP diagnosis: analysis of massively parallel sequencing in clinical practice.

Hereditary spastic paraplegia (HSP) refers to a group of heterogeneous neurological disorders mainly characterized by corticospinal degeneration (pure forms), but sometimes associated with additional neurological and extrapyramidal features (complex HSP). The advent of next-generation sequencing (NGS) has led to huge improvements in knowledge of HSP genetics and made it possible to clarify the genetic etiology of hundreds of "cold cases," accelerating the process of reaching a molecular diagnosis. The different NGS-based strategies currently employed as first-tier approaches most commonly involve the use of targeted resequencing panels and exome sequencing, whereas genome sequencing remains a second-tier approach because of its high costs. The question of which approach is the best is still widely debated, and many factors affect the choice. Here, we aim to analyze the diagnostic power of different NGS techniques applied in HSP, by reviewing 38 selected studies in which different strategies were applied in different-sized cohorts of patients with genetically uncharacterized HSP.

Using Cluster Analysis to Overcome the Limits of Traditional Phenotype-Genotype Correlations: The Example of RYR1-Related Myopathies.

Thanks to advances in gene sequencing, RYR1-related myopathy (RYR1-RM) is now known to manifest itself in vastly heterogeneous forms, whose clinical interpretation is, therefore, highly challenging. We set out to develop a novel unsupervised cluster analysis method in a large patient population. The objective was to analyze the main RYR1-related characteristics to identify distinctive features of RYR1-RM and, thus, offer more precise genotype-phenotype correlations in a group of potentially life-threatening disorders. We studied 600 patients presenting with a suspicion of inherited myopathy, who were investigated using next-generation sequencing. Among them, 73 index cases harbored variants in RYR1. In an attempt to group genetic variants and fully exploit information derived from genetic, morphological, and clinical datasets, we performed unsupervised cluster analysis in 64 probands carrying monoallelic variants. Most of the 73 patients with positive molecular diagnoses were clinically asymptomatic or pauci-symptomatic. Multimodal integration of clinical and histological data, performed using a non-metric multi-dimensional scaling analysis with k-means clustering, grouped the 64 patients into 4 clusters with distinctive patterns of clinical and morphological findings. In addressing the need for more specific genotype-phenotype correlations, we found clustering to overcome the limits of the "single-dimension" paradigm traditionally used to describe genotype-phenotype relationships.

A Schematic Approach to Defining the Prevalence of COL VI Variants in Five Years of Next-Generation Sequencing.

OBJECTIVE: To define the prevalence of variants in collagen VI genes through a next-generation sequencing (NGS) approach in undiagnosed patients with suspected neuromuscular disease and to propose a diagnostic flowchart to assess the real pathogenicity of those variants. METHODS: In the past five years, we have collected clinical and molecular information on 512 patients with neuromuscular symptoms referred to our center. To pinpoint variants in COLVI genes and corroborate their real pathogenicity, we sketched a multistep flowchart, taking into consideration the bioinformatic weight of the gene variants, their correlation with clinical manifestations and possible effects on protein stability and expression. RESULTS: In Step I, we identified variants in COLVI-related genes in 48 patients, of which three were homozygous variants (Group 1). Then, we sorted variants according to their CADD score, clinical data and complementary studies (such as muscle and skin biopsy, study of expression of COLVI on fibroblast or muscle and muscle magnetic resonance). We finally assessed how potentially pathogenic variants (two biallelic and 12 monoallelic) destabilize COL6A1-A2-A3 subunits. Overall, 15 out of 512 patients were prioritized according to this pipeline. In seven of them, we confirmed reduced or absent immunocytochemical expression of collagen VI in cultured skin fibroblasts or in muscle tissue. CONCLUSIONS: In a real-world diagnostic scenario applied to heterogeneous neuromuscular conditions, a multistep integration of clinical and molecular data allowed the identification of about 3% of those patients harboring pathogenetic collagen VI variants.

Integrative Organelle-Based Functional Proteomics: In Silico Prediction of Impaired Functional Annotations in SACS KO Cell Model.

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is an inherited neurodegenerative disease characterized by early-onset spasticity in the lower limbs, axonal-demyelinating sensorimotor peripheral neuropathy, and cerebellar ataxia. Our understanding of ARSACS (genetic basis, protein function, and disease mechanisms) remains partial. The integrative use of organelle-based quantitative proteomics and whole-genome analysis proposed in the present study allowed identifying the affected disease-specific pathways, upstream regulators, and biological functions related to ARSACS, which exemplify a rationale for the development of improved early diagnostic strategies and alternative treatment options in this rare condition that currently lacks a cure. Our integrated results strengthen the evidence for disease-specific defects related to bioenergetics and protein quality control systems and reinforce the role of dysregulated cytoskeletal organization in the pathogenesis of ARSACS.

Episodic ataxia and severe infantile phenotype in spinocerebellar ataxia type 14: expansion of the phenotype and novel mutations.

INTRODUCTION: Spinocerebellar ataxia type 14 (SCA14) is a dominantly inherited neurological disorder characterized by slowly progressive cerebellar ataxia. SCA14 is caused by mutations in PRKCG, a gene encoding protein kinase C gamma (PKCγ), a master regulator of Purkinje cells development. METHODS: We performed next-generation sequencing targeted resequencing panel encompassing 273 ataxia genes in 358 patients with genetically undiagnosed ataxia. RESULTS: We identified fourteen patients in ten families harboring nine pathogenic heterozygous variants in PRKCG, seven of which were novel. We encountered four patients with not previously described phenotypes: one with episodic ataxia, one with a spastic paraparesis dominating her clinical manifestations, and two children with an unusually severe phenotype. CONCLUSIONS: Our study broadens the genetic and clinical spectrum of SCA14.

Implication of folate deficiency in CYP2U1 loss of function.

Hereditary spastic paraplegias are heterogeneous neurodegenerative disorders. Understanding of their pathogenic mechanisms remains sparse, and therapeutic options are lacking. We characterized a mouse model lacking the Cyp2u1 gene, loss of which is known to be involved in a complex form of these diseases in humans. We showed that this model partially recapitulated the clinical and biochemical phenotypes of patients. Using electron microscopy, lipidomic, and proteomic studies, we identified vitamin B2 as a substrate of the CYP2U1 enzyme, as well as coenzyme Q, neopterin, and IFN-α levels as putative biomarkers in mice and fluids obtained from the largest series of CYP2U1-mutated patients reported so far. We also confirmed brain calcifications as a potential biomarker in patients. Our results suggest that CYP2U1 deficiency disrupts mitochondrial function and impacts proper neurodevelopment, which could be prevented by folate supplementation in our mouse model, followed by a neurodegenerative process altering multiple neuronal and extraneuronal tissues.

Efficient Neuroprotective Rescue of Sacsin-Related Disease Phenotypes in Zebrafish.

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is a multisystem hereditary ataxia associated with mutations in SACS, which encodes sacsin, a protein of still only partially understood function. Although mouse models of ARSACS mimic largely the disease progression seen in humans, their use in the validation of effective therapies has not yet been proposed. Recently, the teleost Danio rerio has attracted increasing attention as a vertebrate model that allows rapid and economical screening, of candidate molecules, and thus combines the advantages of whole-organism phenotypic assays and in vitro high-throughput screening assays. Through CRISPR/Cas9-based mutagenesis, we generated and characterized a zebrafish sacs-null mutant line that replicates the main features of ARSACS. The sacs-null fish showed motor impairment, hindbrain atrophy, mitochondrial dysfunction, and reactive oxygen species accumulation. As proof of principle for using these mutant fish in high-throughput screening studies, we showed that both acetyl-DL-leucine and tauroursodeoxycholic acid improved locomotor and biochemical phenotypes in sacs-/- larvae treated with these neuroprotective agents, by mediating significant rescue of the molecular functions altered by sacsin loss. Taken together, the evidence here reported shows the zebrafish to be a valuable model organism for the identification of novel molecular mechanisms and for efficient and rapid in vivo optimization and screening of potential therapeutic compounds. These findings may pave the way for new interventions targeting the earliest phases of Purkinje cell degeneration in ARSACS.

NGS in Hereditary Ataxia: When Rare Becomes Frequent.

The term hereditary ataxia (HA) refers to a heterogeneous group of neurological disorders with multiple genetic etiologies and a wide spectrum of ataxia-dominated phenotypes. Massive gene analysis in next-generation sequencing has entered the HA scenario, broadening our genetic and clinical knowledge of these conditions. In this study, we employed a targeted resequencing panel (TRP) in a large and highly heterogeneous cohort of 377 patients with a clinical diagnosis of HA, but no molecular diagnosis on routine genetic tests. We obtained a positive result (genetic diagnosis) in 33.2% of the patients, a rate significantly higher than those reported in similar studies employing TRP (average 19.4%), and in line with those performed using exome sequencing (ES, average 34.6%). Moreover, 15.6% of the patients had an uncertain molecular diagnosis. STUB1, PRKCG, and SPG7 were the most common causative genes. A comparison with published literature data showed that our panel would have identified 97% of the positive cases reported in previous TRP-based studies and 92% of those diagnosed by ES. Proper use of multigene panels, when combined with detailed phenotypic data, seems to be even more efficient than ES in clinical practice.

A next generation sequencing-based analysis of a large cohort of ataxic patients refines the clinical spectrum associated with spinocerebellar ataxia 21.

BACKGROUND AND PURPOSE: Spinocerebellar ataxia 21 (SCA21) is a rare autosomal dominant neurodegenerative disorder caused by TMEM240 gene mutations. To date, SCA21 has been reported only in a limited number of families worldwide. Here, we describe clinical and molecular findings in five additional SCA21 patients from four unrelated families, diagnosed through a multicentre next generation sequencing-based molecular screening project on a large cohort of patients with degenerative and congenital ataxias. METHODS: A cohort of 393 patients with ataxia of unknown aetiology was selected. Following the identification of heterozygous pathogenic TMEM240 variants using a target resequencing panel, we carried out an in-depth phenotyping of the novel SCA21 patients. RESULTS: Five patients from four unrelated families, three of Italian and one of Libyan origin, were identified. These patients were carriers of previously reported TMEM240 mutations. Clinically, our SCA21 cohort includes both adult onset, slowly progressive cerebellar ataxias associated with cognitive impairment resembling cerebellar cognitive affective syndrome and early onset forms associated with cognitive delay, neuropsychiatric features, or evidence of hypomyelination on brain magnetic resonance imaging. None of our patients exhibited signs of extrapyramidal involvement. The so-called "recurrent" c.509C>T (p.Pro170Leu) mutation was detected in two of four families, corroborating its role as a hot spot. CONCLUSIONS: Our results confirm that SCA21 is present also in Italy, suggesting that it might not be as rare as previously thought. The phenotype of these novel SCA21 patients indicates that slowly progressive cerebellar ataxia, and cognitive and psychiatric symptoms are the most typical clinical features associated with mutations in the TMEM240 gene.

Neuroacanthocytosis Syndromes in an Italian Cohort: Clinical Spectrum, High Genetic Variability and Muscle Involvement.

Neuroacanthocytosis (NA) syndromes are a group of genetically defined diseases characterized by the association of red blood cell acanthocytosis, progressive degeneration of the basal ganglia and neuromuscular features with characteristic persistent hyperCKemia. The main NA syndromes include autosomal recessive chorea-acanthocytosis (ChAc) and X-linked McLeod syndrome (MLS). A series of Italian patients selected through a multicenter study for these specific neurological phenotypes underwent DNA sequencing of the VPS13A and XK genes to search for causative mutations. Where it has been possible, muscle biopsies were obtained and thoroughly investigated with histochemical assays. A total of nine patients from five different families were diagnosed with ChAC and had mostly biallelic changes in the VPS13A gene (three nonsense, two frameshift, three splicing), while three patients from a single X-linked family were diagnosed with McLeod syndrome and had a deletion in the XK gene. Despite a very low incidence (only one thousand cases of ChAc and a few hundred MLS cases reported worldwide), none of the 8 VPS13A variants identified in our patients is shared by two families, suggesting the high genetic variability of ChAc in the Italian population. In our series, in line with epidemiological data, McLeod syndrome occurs less frequently than ChAc, although it can be easily suspected because of its X-linked mode of inheritance. Finally, histochemical studies strongly suggest that muscle pathology is not simply secondary to the axonal neuropathy, frequently seen in these patients, but primary myopathic alterations can be detected in both NA syndromes.

New pathogenic variants in COQ4 cause ataxia and neurodevelopmental disorder without detectable CoQ10 deficiency in muscle or skin fibroblasts.

COQ4 is a component of an enzyme complex involved in the biosynthesis of coenzyme Q10 (CoQ10), a molecule with primary importance in cell metabolism. Mutations in the COQ4 gene are responsible for mitochondrial diseases showing heterogeneous age at onset, clinical presentations and association with CoQ10 deficiency. We herein expand the phenotypic and genetic spectrum of COQ4-related diseases, by reporting two patients harboring bi-allelic variants but not showing CoQ10 deficiency. One patient was found to harbor compound heterozygous mutations (specifically, c.577C>T/p.Pro193Ser and the previously reported c.718C>T/p.Arg240Cys) associated with progressive spasticity, while the other harbored two novel missense (c.284G>A/p.Gly95Asp and c.305G>A/p.Arg102His) associated with a neurodevelopmental disorder. Both patients presented motor impairment and ataxia. To further understand the role of COQ4, we performed functional studies in patient-derived fibroblasts, yeast and "crispant" zebrafish larvae. Micro-oxygraphy showed impaired oxygen consumption rates in one patient, while yeast complementation assays showed that all the mutations were presumably disease related. Moreover, characterization of the coq4 F0 CRISPR zebrafish line showed motor defects and cell reduction in a specific area of the hindbrain, a region reminiscent of the human cerebellum. Our expanded phenotype associated with COQ4 mutations allowed us to investigate, for the first time, the role of COQ4 in brain development in vivo.

The "crab sign": an imaging feature of spinocerebellar ataxia type 48.

PURPOSE: A new form of autosomal dominant hereditary spinocerebellar ataxia (SCA) has been recently described (SCA48), and here we investigate its conventional MRI findings to identify the presence of a possible imaging feature of this condition. METHODS: In this retrospective observational study, we evaluated conventional MRI scans from 10 SCA48 patients (M/F = 5/5; 44.7 ± 7.8 years). For all subjects, atrophy of both supratentorial and infratentorial compartments were recorded, as well as the presence of possible T2-weighted imaging (T2WI) signal alterations. RESULTS: In SCA48 patients, no meaningful supratentorial changes were found, both in terms of volume loss or MRI signal changes. Atrophy of the cerebellum was present in all cases, involving both the vermis and the hemispheres, but particularly affecting the postero-lateral portions of the cerebellar hemispheres. In all patients, with the exception of only one subject (90.0% of the cases), a T2WI hyperintensity of both dentate nuclei was found. The association of such signal alteration with the pattern of cerebellar atrophy resembled the appearance of a crab ("crab sign"). CONCLUSION: Our findings suggest that SCA48 patients are characterized by cerebellar atrophy, mainly involving the postero-lateral hemisphere areas, along with a T2WI hyperintensity of dentate nuclei. We propose that the association of such signal change, along with the atrophy of the lateral portion of the cerebellar hemispheres, resembled the appearance of a crab, and therefore, we propose the "crab sign" as a neuroradiological sign present in SCA48 patients.

Bi-allelic mutations in HARS1 severely impair histidyl-tRNA synthetase expression and enzymatic activity causing a novel multisystem ataxic syndrome.

Mutations in histidyl-tRNA synthetase (HARS1), an enzyme that charges transfer RNA with the amino acid histidine in the cytoplasm, have only been associated to date with autosomal recessive Usher syndrome type III and autosomal dominant Charcot-Marie-Tooth disease type 2W. Using massive parallel sequencing, we identified bi-allelic HARS1 variants in a child (c.616G>T, p.Asp206Tyr and c.730delG, p.Val244Cysfs*6) and in two sisters (c.1393A>C, p.Ile465Leu and c.910_912dupTTG, p.Leu305dup), all characterized by a multisystem ataxic syndrome. All mutations are rare, segregate with the disease, and are predicted to have a significant effect on protein function. Functional studies helped to substantiate their disease-related roles. Indeed, yeast complementation assays showing that one out of two mutations in each patient is loss-of-function, and the reduction of messenger RNA and protein levels and enzymatic activity in patient's skin-derived fibroblasts, together support the pathogenicity of the identified HARS1 variants in the patient phenotypes. Thus, our efforts expand the allelic and clinical spectrum of HARS1-related disease.