Variability in Clinical Phenotype in TARDBP Mutations: Amyotrophic Lateral Sclerosis Case Description and Literature Review.

Mutations in the 43 kDa transactive-response (TAR)-DNA-binding protein (TARDBP) are associated with 2-5% of familial Amyotrophic Lateral Sclerosis (ALS) cases. TAR DNA-Binding Protein 43 (TDP-43) is an RNA/DNA-binding protein involved in several cellular mechanisms (e.g., transcription, pre-mRNA processing, and splicing). Many ALS-linked TARDBP mutations have been described in the literature, but few phenotypic data on monogenic TARDBP-mutated ALS are available. In this paper, (1) we describe the clinical features of ALS patients carrying mutations in the TARDBP gene evaluated at the Tertiary ALS Center at Maggiore della Carità University Hospital, Novara, Italy, from 2010 to 2020 and (2) present the results of our review of the literature on this topic, analyzing data obtained for 267 patients and highlighting their main clinical and demographic features.

Parkinsonism in SCA19/22: Dopamine Transporter Imaging in an Italian Family Harboring a Novel Mutation.

Spinocerebellar ataxia (SCA)19/22 is a channelopathy caused by mutations in the KCND3 gene encoding for the voltage-gated potassium channel Kv4.3. In the present work, we report an Italian family harboring a novel KCND3 missense mutation characterized by ataxia and mild parkinsonism. Patients underwent dopamine transporter single-photon emission computed tomography to assess dopaminergic degeneration. Normal findings were observed, and treatment with levodopa did not yield any benefit, thus suggesting the involvement of other mechanisms to explain parkinsonian symptoms in SCA19/22. Our cases expand the genetic and imaging spectrum of this rare disease and emphasize a cautious approach in managing parkinsonism in these patients.

Neuroinflammatory Pathways in the ALS-FTD Continuum: A Focus on Genetic Variants.

Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal dementia (FDT) are progressive neurodegenerative disorders that, in several cases, overlap in clinical presentation, and genetic and pathological disease mechanisms. About 10-15% of ALS cases and up to 40% of FTD are familial, usually with dominant traits. ALS and FTD, in several cases, share common gene mutations, such as in C9ORF72, TARDBP, SQSTM-1, FUS, VCP, CHCHD10, and TBK-1. Also, several mechanisms are involved in ALS and FTD pathogenesis, such as protein misfolding, oxidative stress, and impaired axonal transport. In addition, neuroinflammation and neuroinflammatory cells, such as astrocytes, oligodendrocytes, microglia, and lymphocytes and, overall, the cellular microenvironment, have been proposed as pivotal players in the pathogenesis the ALS-FTD spectrum disorders. This review overviews the current evidence regarding neuroinflammatory markers in the ALS/FTD continuum, focusing on the neuroinflammatory pathways involved in the genetic cases, moving from post-mortem reports to in vivo biofluid and neuroimaging data. We further discuss the potential link between genetic and autoimmune disorders and potential therapeutic implications.

Association of Copresence of Pathogenic Variants Related to Amyotrophic Lateral Sclerosis and Prognosis.

BACKGROUND AND OBJECTIVES: Despite recent advances, it is not clear whether the various genes/genetic variants related to amyotrophic lateral sclerosis (ALS) interact in modifying patients' phenotype. The aim of this study was to determine whether the copresence of genetic variants related to ALS has interactive effects on the course of the disease. METHODS: The study population includes 1,245 patients with ALS identified through the Piemonte Register for ALS between 2007 and 2016 and not carrying superoxide dismutase type 1, TAR DNA binding protein, and fused in sarcoma pathogenic variants. Controls were 766 Italian participants age-matched, sex-matched, and geographically matched to cases. We considered Unc-13 homolog A (UNC13A) (rs12608932), calmodulin binding transcription activator 1 (CAMTA1) (rs2412208), solute carrier family 11 member 2 (SLC11A2) (rs407135), and zinc finger protein 512B (ZNF512B) (rs2275294) variants, as well as ataxin-2 (ATXN2) polyQ intermediate repeats (≥31) and chromosome 9 open reading frame 72 (C9orf72) GGGGCC intronic expansions (≥30). RESULTS: The median survival time of the whole cohort was 2.67 years (interquartile range [IQR] 1.67-5.25). In univariate analysis, only C9orf72 (2.51 years, IQR 1.74-3.82; p = 0.016), ATXN2 (1.82 years, IQR 1.08-2.33; p < 0.001), and UNC13A C/C (2.3 years, IQR 1.3-3.9; p < 0.001) significantly reduced survival. In Cox multivariable analysis, CAMTA1 also emerged to be independently related to survival (hazard ratio 1.13, 95% CI 1.001-1.30, p = 0.048). The copresence of 2 detrimental alleles/expansions was correlated with shorter survival. In particular, the median survival of patients with CAMTA1 G/G+G/T and UNC13A C/C alleles was 1.67 years (1.16-3.08) compared with 2.75 years (1.67-5.26) of the patients not carrying these variants (p < 0.001); the survival of patients with CAMTA1 G/G+G/T alleles and ATXN2 ≥31 intermediate polyQ repeats was 1.75 years (0.84-2.18) (p < 0.001); the survival of patients with ATXN2 ≥31 polyQ repeats and UNC13A C/C allele was 1.33 years (0.84-1.75) (p < 0.001); the survival of patients with C9ORF72 ≥30 and UNC13A C/C allele was 1.66 years (1.41-2.16). Each pair of detrimental alleles/expansions was associated to specific clinical phenotypes. DISCUSSION: We showed that gene variants acting as modifiers of ALS survival or phenotype can act on their own or in unison. Overall, 54% of patients carried at least 1 detrimental common variant or repeat expansion, emphasizing the clinical impact of our findings. In addition, the identification of the interactive effects of modifier genes represents a crucial clue for explaining ALS clinical heterogeneity and should be considered when designing and interpreting clinical trials results.

Clinical and Metabolic Signature of UNC13A rs12608932 Variant in Amyotrophic Lateral Sclerosis.

Background and Objectives: To characterize the clinical and cognitive behavioral phenotype and brain 18F-2-fluoro-2-deoxy-d-glucose-PET (18F-FDG-PET) metabolism of patients with amyotrophic lateral sclerosis (ALS) carrying the rs12608932 variant of the UNC13A gene. Methods: The study population included 1,409 patients with ALS without C9orf72, SOD1, TARDBP, and FUS mutations identified through a prospective epidemiologic ALS register. Control participants included 1,012 geographically matched, age-matched, and sex-matched participants. Clinical and cognitive differences between patients carrying the C/C rs12608932 genotype and those carrying the A/A + A/C genotype were assessed. A subset of patients underwent 18F-FDG-PET. Results: The C/C genotype was associated with an increased risk of ALS (odds ratio: 1.54, 95% confidence interval 1.18-2.01, p = 0.001). Patients with the C/C genotype were older, had more frequent bulbar onset, and manifested a higher rate of weight loss. In addition, they showed significantly reduced performance in the letter fluency test, fluency domain of Edinburgh Cognitive and Behavioural ALS Screen (ECAS) and story-based empathy task (reflecting social cognition). Patients with the C/C genotype had a shorter survival (median survival time, C/C 2.25 years, interquartile range [IQR] 1.33-3.92; A/A + C/C: 2.90 years, IQR 1.74-5.41; p = 0.0001). In Cox multivariable analysis, C/C genotype resulted to be an independent prognostic factor. Finally, patients with a C/C genotype had a specific pattern of hypometabolism on brain 18F-FDG-PET extending to frontal and precentral areas of the right hemisphere. Discussion: C/C rs12608932 genotype of UNC13A is associated with a specific motor and cognitive/behavioral phenotype, which reflects on 18F-FDG-PET findings. Our observations highlight the importance of adding the rs12608932 variant in UNC13A to the ALS genetic panel to refine the individual prognostic prediction and reduce heterogeneity in clinical trials.

Clinical relevance of single-subject brain metabolism patterns in amyotrophic lateral sclerosis mutation carriers.

BACKGROUND AND OBJECTIVES: The ALS diagnosis requires an integrative approach, combining the clinical examination and supporting tests. Nevertheless, in several cases, the diagnosis proves to be suboptimal, and for this reason, new diagnostic methods and novel biomarkers are catching on. The 18F-fluorodeoxyglucose (18F-FDG)-PET could be a helpful method, but it still requires additional research for sensitivity and specificity. We performed an 18F-FDG-PET single-subject analysis in a sample of familial ALS patients carrying different gene mutations, investigating the genotype-phenotype correlations and exploring metabolism correlations with clinical and neuropsychological data. METHODS: We included ten ALS patients with pathogenic gene mutation who underwent a complete clinical and neuropsychological evaluation and an 18F-FDG-PET scan at baseline. Patients were recruited between 2018 and 2022 at the ALS Tertiary Centre in Novara, Italy. Patients were selected based on the presence of ALS gene mutation (C9orf72, SOD1, TBK1, and KIF5A). Following a validated voxel-based Statistical Parametric Mapping (SPM) procedure, we obtained hypometabolism maps at single-subject level. We extracted regional hypometabolism from the SPM maps, grouping significant hypometabolism regions into three meta-ROIs (motor, prefrontal association and limbic). Then, the corresponding 18F-FDG-PET regional hypometabolism was correlated with clinical and neuropsychological features. RESULTS: Classifying the patients with C9orf72-ALS based on the rate of disease progression from symptoms onset to the time of scan, we observed two different patterns of brain hypometabolism: an extensive motor and prefrontal hypometabolism in patients classified as fast progressors, and a more limited brain hypometabolism in patients grouped as slow progressors. Patients with SOD1-ALS showed a hypometabolic pattern involving the motor cortex and prefrontal association regions, with a minor involvement of the limbic regions. The patient with TBK1-ALS showed an extended hypometabolism, in limbic systems, along with typical motor involvement, while the hypometabolism in the patient with KIF5A-ALS involved almost exclusively the motor regions, supporting the predominantly motor impairment linked to this gene mutation. Additionally, we observed strong correlations between the hypometabolism in the motor, prefrontal association and limbic meta-ROI and the specific neuropsychological performances. CONCLUSIONS: To our knowledge, this is the first study investigating brain hypometabolism at the single-subject level in genetic ALS patients carrying different mutations. Our results show high heterogeneity in the hypometabolism maps and some commonalities in groups sharing the same mutation. Specifically, in patients with C9orf72-ALS the brain hypometabolism was larger in patients classified as fast progressors than slow progressors. In addition, in the whole group, the brain metabolism showed specific correlations with clinical and neuropsychological impairment, confirming the ability of 18F-FDG-PET in revealing pattern of neuronal dysfunction, aiding the diagnostic workup in genetic ALS patients.

Exploring the phenotype of Italian patients with ALS with intermediate ATXN2 polyQ repeats.

OBJECTIVE: To detect the clinical characteristics of patients with amyotrophic lateral sclerosis (ALS) carrying an intermediate ATXN2 polyQ number of repeats in a large population-based series of Italian patients with ALS. METHODS: The study population includes 1330 patients with ALS identified through the Piemonte and Valle d'Aosta Register for ALS, diagnosed between 2007 and 2019 and not carrying C9orf72, SOD1, TARDBP and FUS mutations. Controls were 1274 age, sex and geographically matched Italian subjects, identified through patients' general practitioners. RESULTS: We found 42 cases and 4 controls with≥31 polyQ repeats, corresponding to an estimated OR of 10.4 (95% CI 3.3 to 29.0). Patients with≥31 polyQ repeats (ATXN2+) compared with those without repeat expansion (ATXN2-) had more frequently a spinal onset (p=0.05), a shorter diagnostic delay (p=0.004), a faster rate of ALSFRS-R progression (p=0.004) and King's progression (p=0.004), and comorbid frontotemporal dementia (7 (28.0%) vs 121 (13.4%), p=0.037). ATXN2+ patients had a 1-year shorter survival (ATXN2+ patients 1.82 years, 95% CI 1.08 to 2.51; ATXN2- 2.84 years, 95% CI 1.67 to 5.58, p=0.0001). ATXN2 polyQ intermediate repeats was independently related to a worse outcome in Cox multivariable analysis (p=0.006). CONCLUSIONS: In our population-based cohort, ATXN2+ patients with ALS have a distinctive phenotype, characterised by a more rapid disease course and a shorter survival. In addition, ATXN2+ patients have a more severe impairment of cognitive functions. These findings have relevant implications on clinical practice, including the possibility of refining the individual prognostic prediction and improving the design of ALS clinical trials, in particular as regards as those targeted explicitly to ATXN2.

Systematic evaluation of genetic mutations in ALS: a population-based study.

BACKGROUND: A genetic diagnosis in Amyotrophic Lateral Sclerosis (ALS) can inform genetic counselling, prognosis and, in the light of incoming gene-targeted therapy, management. However, conventional genetic testing strategies are often costly and time-consuming. OBJECTIVE: To evaluate the diagnostic yield and advantages of whole-genome sequencing (WGS) as a standard diagnostic genetic test for ALS. METHODS: In this population-based cohort study, 1043 ALS patients from the Piemonte and Valle d'Aosta Register for ALS and 755 healthy individuals were screened by WGS for variants in 42 ALS-related genes and for repeated-expansions in C9orf72 and ATXN2. RESULTS: A total of 279 ALS cases (26.9%) received a genetic diagnosis, namely 75.2% of patients with a family history of ALS and 21.5% of sporadic cases. The mutation rate among early-onset ALS patients was 43.9%, compared with 19.7% of late-onset patients. An additional 14.6% of the cohort carried a genetic factor that worsen prognosis. CONCLUSIONS: Our results suggest that, because of its high diagnostic yield and increasingly competitive costs, along with the possibility of retrospectively reassessing newly described genes, WGS should be considered as standard genetic testing for all ALS patients. Additionally, our results provide a detailed picture of the genetic basis of ALS in the general population.

Characterization of the p.L145F and p.S135N Mutations in SOD1: Impact on the Metabolism of Fibroblasts Derived from Amyotrophic Lateral Sclerosis Patients.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the loss of the upper and lower motor neurons (MNs). About 10% of patients have a family history (familial, fALS); however, most patients seem to develop the sporadic form of the disease (sALS). SOD1 (Cu/Zn superoxide dismutase-1) is the first studied gene among the ones related to ALS. Mutant SOD1 can adopt multiple misfolded conformation, lose the correct coordination of metal binding, decrease structural stability, and form aggregates. For all these reasons, it is complicated to characterize the conformational alterations of the ALS-associated mutant SOD1, and how they relate to toxicity. In this work, we performed a multilayered study on fibroblasts derived from two ALS patients, namely SOD1L145F and SOD1S135N, carrying the p.L145F and the p.S135N missense variants, respectively. The patients showed diverse symptoms and disease progression in accordance with our bioinformatic analysis, which predicted the different effects of the two mutations in terms of protein structure. Interestingly, both mutations had an effect on the fibroblast energy metabolisms. However, while the SOD1L145F fibroblasts still relied more on oxidative phosphorylation, the SOD1S135N fibroblasts showed a metabolic shift toward glycolysis. Our study suggests that SOD1 mutations might lead to alterations in the energy metabolism.

C9ORF72 Repeat Expansion Affects the Proteome of Primary Skin Fibroblasts in ALS.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive degeneration of the corticospinal motor neurons, which ultimately leads to death. The repeat expansion in chromosome 9 open reading frame 72 (C9ORF72) represents the most common genetic cause of ALS and it is also involved in the pathogenesis of other neurodegenerative disorders. To offer insights into C9ORF72-mediated pathogenesis, we quantitatively analyzed the proteome of patient-derived primary skin fibroblasts from ALS patients carrying the C9ORF72 mutation compared with ALS patients who tested negative for it. Differentially expressed proteins were identified, used to generate a protein-protein interaction network and subjected to a functional enrichment analysis to unveil altered molecular pathways. ALS patients were also compared with patients affected by frontotemporal dementia carrying the C9ORF72 repeat expansion. As a result, we demonstrated that the molecular pathways mainly altered in fibroblasts (e.g., protein homeostasis) mirror the alterations observed in C9ORF72-mutated neurons. Moreover, we highlighted novel molecular pathways (nuclear and mitochondrial transports, vesicle trafficking, mitochondrial bioenergetics, glucose metabolism, ER-phagosome crosstalk and Slit/Robo signaling pathway) which might be further investigated as C9ORF72-specific pathogenetic mechanisms. Data are available via ProteomeXchange with the identifier PXD023866.

An Investigation of the Role of Common and Rare Variants in a Large Italian Multiplex Family of Multiple Sclerosis Patients.

Known multiple sclerosis (MS) susceptibility variants can only explain half of the disease's estimated heritability, whereas low-frequency and rare variants may partly account for the missing heritability. Thus, here we sought to determine the occurrence of rare functional variants in a large Italian MS multiplex family with five affected members. For this purpose, we combined linkage analysis and next-generation sequencing (NGS)-based whole exome and whole genome sequencing (WES and WGS, respectively). The genetic burden attributable to known common MS variants was also assessed by weighted genetic risk score (wGRS). We found a significantly higher burden of common variants in the affected family members compared to that observed among sporadic MS patients and healthy controls (HCs). We also identified 34 genes containing at least one low-frequency functional variant shared among all affected family members, showing a significant enrichment in genes involved in specific biological processes-particularly mRNA transport-or neurodegenerative diseases. Altogether, our findings point to a possible pathogenic role of different low-frequency functional MS variants belonging to shared pathways. We propose that these rare variants, together with other known common MS variants, may account for the high number of affected family members within this MS multiplex family.

Expanding the genetic spectrum of primary familial brain calcification due to SLC2OA2 mutations: a case series.

Primary familial brain calcification (PFBC) is a neurological condition characterized by the presence of intracranial calcifications, mainly involving basal ganglia, thalamus, and dentate nuclei. So far, six genes have been linked to this condition: SLC20A2, PDGFRB, PDGFB, and XPR1 inherited as autosomal-dominant trait, while MYORG and JAM2 present a recessive pattern of inheritance. Patients mainly present with movement disorders, psychiatric disturbances, and cognitive decline or are completely asymptomatic and calcifications may represent an occasional finding. Here we present three variants in SLC20A2, two exonic and one intronic, which we found in patients with PFBC associated to three different clinical phenotypes. One variant is novel and two were already described as variants of uncertain significance. We confirm the pathogenicity of these three variants and suggest a broadening of the phenotypic spectrum associated with mutations in SLC20A2.

Metal(loid)s role in the pathogenesis of amyotrophic lateral sclerosis: Environmental, epidemiological, and genetic data.

Amyotrophic Lateral Sclerosis (ALS) is a progressive neurodegenerative disorder of the motor system. The etiology is still unknown and the pathogenesis remains unclear. ALS is familial in the 10% of cases with a Mendelian pattern of inheritance. In the remaining sporadic cases, a multifactorial origin is supposed in which several predisposing genes interact with environmental factors. The etiological role of environmental factors, such as pesticides, exposure to electromagnetic fields, and metals has been frequently investigated, with controversial findings. Studies in the past two decades have highlighted possible roles of metals, and ionic homeostasis dysregulation has been proposed as the main trigger to motor-neuron degeneration. This study aims at evaluating the possible role of environmental factors in etiopathogenesis of ALS, with a particular attention on metal contamination, focusing on the industrial Briga area in the province of Novara (Piedmont region, North Italy), characterized by: i) a higher incidence of sporadic ALS (sALS) in comparison with the entire province, and ii) the reported environmental pollution. Environmental data from surface, ground and discharge waters, and from soils were collected and specifically analyzed for metal content. Considering the significance of genetic mechanisms in ALS, a characterization for the main ALS genes has been performed to evaluate the genetic contribution for the sALS patients living in the area of study. The main findings of this study are the demonstration that in the Briga area the most common metal contaminants are Cu, Zn, Cr, Ni (widely used in tip-plating processes), that are above law limits in surface waters, discharge waters, and soil. In addition, other metals and metalloids, such as Cd, Pb, Mn, and As show a severe contamination in the same area. Results of genetic analyses show that sALS patients in the Briga area do not carry recurrent mutations or an excess of mutations in the four main ALS causative genes (SOD1, TARDBP, FUS, C9ORF72) and for ATXN2 CAG repeat locus. This study supports the hypothesis that the higher incidence of sALS in Briga area may be related to environmental metal(loid)s contamination, along with other environmental factors. Further studies, implementing analysis of genetic polymorphisms, as well as investigation with long term follow-up, may yield to key aspects into the etiology of ALS. The interplay between different approaches (environmental, chemical, epidemiological, genetic) of our work provides new insights and methodology to the comprehension of the disease etiology.

Variants in the 5'UTR reduce SHOX expression and contribute to SHOX haploinsufficiency.

SHOX haploinsufficiency causes 70-90% of Léri-Weill dyschondrosteosis (LWD) and 2-10% of idiopathic short stature (ISS). Deletions removing the entire gene or enhancers and point mutations in the coding region represent a well-established cause of haploinsufficiency. During diagnostic genetic testing on ISS/LWD patients, in addition to classic SHOX defects, five 5'UTR variants (c.-58G > T, c.-55C > T, c.-51G > A, c.-19G > A, and c.-9del), were detected whose pathogenetic role was unclear and were thus classified as VUS (Variants of Uncertain Significance). The purpose of the present study was to investigate the role of these noncoding variations in SHOX haploinsufficiency. The variants were tested for their ability to interfere with correct gene expression of a regulated reporter gene (luciferase assay). The negative effect on the mRNA splicing predicted in silico for c.-19G > A was assayed in vitro through a minigene splicing assay. The luciferase assay showed that c.-51G > A, c.-19G > A, and c.-9del significantly reduce luciferase activity by 60, 35, and 40% at the homozygous state. Quantification of the luciferase mRNA showed that c.-51G > A and c.-9del might interfere with the correct SHOX expression mainly at the post-transcriptional level. The exon trapping assay demonstrated that c.-19G > A determines the creation of a new branch site causing an aberrant mRNA splicing. In conclusion, this study allowed us to reclassify two of the 5'UTR variants identified during SHOX diagnostic screening as likely pathogenic, one remains as a VUS, and two as likely benign variants. This analysis for the first time expands the spectrum of the genetic causes of SHOX haploinsufficiency to noncoding variations in the 5'UTR.

Mutational Analysis of Known ALS Genes in an Italian Population-Based Cohort.

OBJECTIVE: To assess the burden of rare genetic variants and to estimate the contribution of known amyotrophic lateral sclerosis (ALS) genes in an Italian population-based cohort, we performed whole genome sequencing in 959 patients with ALS and 677 matched healthy controls. METHODS: We performed genome sequencing in a population-based cohort (Piemonte and Valle d'Aosta Registry for ALS [PARALS]). A panel of 40 ALS genes was analyzed to identify potential disease-causing genetic variants and to evaluate the gene-wide burden of rare variants among our population. RESULTS: A total of 959 patients with ALS were compared with 677 healthy controls from the same geographical area. Gene-wide association tests demonstrated a strong association with SOD1, whose rare variants are the second most common cause of disease after C9orf72 expansion. A lower signal was observed for TARDBP, proving that its effect on our cohort is driven by a few known causal variants. We detected rare variants in other known ALS genes that did not surpass statistical significance in gene-wise tests, thus highlighting that their contribution to disease risk in our cohort is limited. CONCLUSIONS: We identified potential disease-causing variants in 11.9% of our patients. We identified the genes most frequently involved in our cohort and confirmed the contribution of rare variants in disease risk. Our results provide further insight into the pathologic mechanism of the disease and demonstrate the importance of genome-wide sequencing as a diagnostic tool.

Author Correction: Next Generation Sequencing of Pooled Samples: Guideline for Variants' Filtering.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.