Mechanism-free repurposing of drugs for C9orf72-related ALS/FTD using large-scale genomic data.

Repeat expansions in the C9orf72 gene are the most common genetic cause of (ALS) and frontotemporal dementia (FTD). Like other genetic forms of neurodegeneration, pinpointing the precise mechanism(s) by which this mutation leads to neuronal death remains elusive, and this lack of knowledge hampers the development of therapy for C9orf72-related disease. We used an agnostic approach based on genomic data (n = 41,273 ALS and healthy samples, and n = 1,516 C9orf72 carriers) to overcome these bottlenecks. Our drug-repurposing screen, based on gene- and expression-pattern matching and information about the genetic variants influencing onset age among C9orf72 carriers, identified acamprosate, a γ-aminobutyric acid analog, as a potentially repurposable treatment for patients carrying C9orf72 repeat expansions. We validated its neuroprotective effect in cell models and showed comparable efficacy to riluzole, the current standard of care. Our work highlights the potential value of genomics in repurposing drugs in situations where the underlying pathomechanisms are inherently complex. VIDEO ABSTRACT.

Six-minute walk test as outcome measure of fatigability in adults with spinal muscular atrophy treated with nusinersen.

INTRODUCTION/AIMS: Fatigue (subjective perception) and fatigability (objective motor performance worsening) are relevant aspects of disability in individuals with spinal muscular atrophy (SMA). The effect of nusinersen on fatigability in SMA patients has been investigated with conflicting results. We aimed to evaluate this in adult with SMA3. METHODS: We conducted a multicenter retrospective cohort study, including adult ambulant patients with SMA3, data available on 6-minute walk test (6MWT) and Hammersmith Functional Motor Scale-Expanded (HFMSE) at baseline and at least at 6 months of treatment with nusinersen. We investigated fatigability, estimated as 10% or higher decrease in walked distance between the first and sixth minute of the 6MWT, at baseline and over the 14-month follow-up. RESULTS: Forty-eight patients (56% females) were included. The 6MWT improved after 6, 10, and 14 months of treatment (p < 0.05). Of the 27 patients who completed the entire follow-up, 37% improved (6MWT distance increase ≥30 m), 48.2% remained stable, and 14.8% worsened (6MWT distance decline ≥30 m). Fatigability was found at baseline in 26/38 (68%) patients and confirmed at subsequent time points (p < 0.05) without any significant change over the treatment period. There was no correlation between fatigability and SMN2 copy number, sex, age at disease onset, age at baseline, nor with 6MWT total distance and baseline HFMSE score. DISCUSSION: Fatigability was detected at baseline in approximately 2/3 of SMA3 walker patients, without any correlation with clinical features, included motor performance. No effect on fatigability was observed during the 14-month treatment period with nusinersen.

Long-term treatment of SOD1 ALS with tofersen: a multicentre experience in 17 patients.

BACKGROUND: In Amyotrophic Lateral Sclerosis (ALS) patients with SOD1 mutation the intrathecal administration of tofersen slowed down the progression of disease in a controlled clinical study, but results were not statistically significant. METHODS: In this multicentre, observational study, we evaluated a cohort of 27 ALS-SOD1 patients who were treated with tofersen, focussing on 17 patients who were followed for at least 48 weeks (median period of 84 weeks, range 48-108). We compared the clinical slopes, as measured by ALSFRS-R, MRC scale and Forced Vital Capacity, during tofersen treatment with retrospective data at 1 year prior to therapy. Cerebrospinal fluid (CSF) and serum neurofilament light chains (NFL) were measured in all patients. RESULTS: Cumulative evaluation of the ALSFRS-R and MRC progression rates showed a statistically significant change during treatment with respect to the period prior to therapy (p = 0.023 and p = 0.007, respectively). The analysis of individual patients showed that nine of the seventeen patients substantially stabilized or slightly improved. Four patients deteriorated during treatment, while in the remaining patients the very slow course did not allow to identify significant changes. CSF and serum NFL concentration markedly decreased in the near totality of patients. Increased levels of white blood cells and proteins in the CSF were found in 60% of patients. Such alterations were clinically asymptomatic in all but two patients who showed an acute pure motor radiculitis, which responded to steroid therapy. CONCLUSIONS: Clinical findings and NFL analysis strongly suggest that tofersen may have a disease-modifying effect in a subset of SOD1-ALS patients.

Protein biomarkers for the diagnosis and prognosis of Amyotrophic Lateral Sclerosis.

Amyotrophic Lateral Sclerosis (ALS) is the most common motor neuron disease, still incurable. The disease is highly heterogenous both genetically and phenotypically. Therefore, developing efficacious treatments is challenging in many aspects because it is difficult to predict the rate of disease progression and stratify the patients to minimize statistical variability in clinical studies. Moreover, there is a lack of sensitive measures of therapeutic effect to assess whether a pharmacological intervention ameliorates the disease. There is also urgency of markers that reflect a molecular mechanism dysregulated by ALS pathology and can be rescued when a treatment relieves the condition. Here, we summarize and discuss biomarkers tested in multicentered studies and across different laboratories like neurofilaments, the most used marker in ALS clinical studies, neuroinflammatory-related proteins, p75ECD, p-Tau/t-Tau, and UCHL1. We also explore the applicability of muscle proteins and extracellular vesicles as potential biomarkers.

The HFE p.H63D (p.His63Asp) Polymorphism Is a Modifier of ALS Outcome in Italian and French Patients with SOD1 Mutations.

Background: Data from published studies about the effect of HFE polymorphisms on ALS risk, phenotype, and survival are still inconclusive. We aimed at evaluating whether the p.H63D polymorphism is a modifier of phenotype and survival in SOD1-mutated patients. Methods: We included 183 SOD1-mutated ALS patients. Mutations were classified as severe or mild according to the median survival of the study population. Patients were screened for the HFE p.H63D polymorphism. Survival was calculated using the Kaplan-Meier modeling, and differences were measured by the log-rank test. Multivariable analysis was performed with the Cox proportional hazards model (stepwise backward). Results: SOD1 severe mutation carriers show more frequent familial history for ALS and shorter survival compared to mild mutation carriers. Carriers and non-carriers of the p.H63D polymorphism did not differ in terms of sex ratio, frequency of positive familial history, age at onset, and bulbar/spinal ratio. In univariate and in Cox multivariable analysis using sex, age at onset, site of onset, family history, country of origin, and mutation severity as covariates, p.H63D carriers had a longer survival (p = 0.034 and p = 0.004). Conclusions: We found that SOD1-mutated ALS patients carrying the p.H63D HFE polymorphism have a longer survival compared to non-carriers, independently of sex, age and site of onset, family history, nation of origin, and severity of mutations, suggesting a possible role as disease progression modifier for the p.H63D HFE polymorphism in SOD1-ALS.

Adults with spinal muscular atrophy: a large-scale natural history study shows gender effect on disease.

BACKGROUND: Natural history of spinal muscular atrophy (SMA) in adult age has not been fully elucidated yet, including factors predicting disease progression and response to treatments. Aim of this retrospective, cross-sectional study, is to investigate motor function across different ages, disease patterns and gender in adult SMA untreated patients. METHODS: Inclusion criteria were as follows: (1) clinical and molecular diagnosis of SMA2, SMA3 or SMA4 and (2) clinical assessments performed in adult age (>18 years). RESULTS: We included 64 (38.8%) females and 101 (61.2%) males (p=0.0025), among which 21 (12.7%) SMA2, 141 (85.5%) SMA3 and 3 (1.8%) SMA4. Ratio of sitters/walkers within the SMA3 subgroup was significantly (p=0.016) higher in males (46/38) than in females (19/38). Median age at onset was significantly (p=0.0071) earlier in females (3 years; range 0-16) than in males (4 years; range 0.3-28), especially in patients carrying 4 SMN2 copies. Median Hammersmith Functional Rating Scale Expanded scores were significantly (p=0.0040) lower in males (16, range 0-64) than in females (40, range 0-62); median revised upper limb module scores were not significantly (p=0.059) different between males (24, 0-38) and females (33, range 0-38), although a trend towards worse performance in males was observed. In SMA3 patients carrying three or four SMN2 copies, an effect of female sex in prolonging ambulation was statistically significant (p=0.034). CONCLUSIONS: Our data showed a relevant gender effect on SMA motor function with higher disease severity in males especially in the young adult age and in SMA3 patients.

Quality of life assessment in adult spinal muscular atrophy patients treated with nusinersen.

OBJECTIVE: To retrospectively evaluate quality of life (QoL) in a large multicenter cohort of adult patients affected by spinal muscular atrophy (SMA) during nusinersen treatment. METHODS: We included adult (≥ 18 years) patients clinically and genetically defined as SMA2, SMA3 and SMA4, who started nusinersen treatment in adulthood. QoL was rated by the Individualized Neuromuscular Quality of Life (INQoL) questionnaire. Concurrent motor function evaluation included the Hammersmith Functional Motor Scale Expanded (HFMSE), the Revised Upper Limb Module (RULM), the 6 min walking test (6MWT). RESULTS: 189 completed questionnaires were collected during a 14 months' treatment period. 78 patients were included (7 SMA2 and 69 SMA3 and 2 SMA4) with mean disease duration at first nusinersen administration of 33.2 years (± 12.5 years). All the scores for each INQoL domain (weakness, fatigue, activities, independence, social relationship, emotions, body images) and the derived QoL total score, significantly improved during the observation period, except the muscle locking and pain items. Exploratory analyses suggested that emotions and social relationships were more relevant issues for females compared to males. Social relationships were affected also by a longer disease duration (> 30 years). In SMA3 non-walker patients, activities ameliorate better compared to walkers. The HFMSE and RULM significantly improved from baseline; however, no associations with QoL total score and weakness, activities or independence were demonstrated. CONCLUSION: In our cohort, adult SMA patients showed a global improvement at the INQoL assessment over 14 months of nusinersen treatment. QoL assessment is relevant to SMA multidisciplinary evaluation.

Agrypnia excitata as the main feature in anti-leucine-rich glioma-inactivated 1 encephalitis: a detailed clinical and polysomnographic semiological analysis.

BACKGROUND AND PURPOSE: The core manifestations of leucine-rich glioma-inactivated 1 (LGI1) autoantibody-mediated encephalitis are limbic encephalitis and faciobrachial dystonic seizures. Agrypnia excitata (AE) is a rare syndrome characterized by sleep-wake cycle disruption, autonomic hyperactivation and episodes of oneiric stupor. Only a few diseases are known to present with AE. An autoimmune etiology must be considered when accompanied by neuromyotonia. A case of anti-LGI1 encephalitis presenting with AE is reported. METHODS: Detailed clinical, video-polysomnographic, laboratory, radiological and long-term follow-up assessments were performed. RESULTS: A previously healthy 58-year-old man was referred for a rapidly progressive change in mental status, characterized by persistent drowsiness and confusion, accompanied by frequent episodes of unconscious gestures ranging from simple stereotyped movements to more complex actions mimicking various daily activities. Other symptoms included tachycardia, hyperhidrosis, mild hyponatremia, rare faciobrachial dystonic seizures, and a single generalized tonic-clonic seizure, but no neuromyotonia. Prolonged video-polysomnography excluded epileptic activity and showed continuous monomorphic slowing of background activity not consistent with a regular wakefulness or sleep state. A brain magnetic resonance imaging scan was unremarkable. Brain fluorodeoxyglucose positron emission tomography revealed hypermetabolism of the hippocampi, amygdala and basal ganglia. Anti-LGI1 antibodies were detected in the cerebrospinal fluid. The sleep disorder resolved progressively after starting immunotherapy. CONCLUSIONS: Agrypnia excitata can be a dominant, treatable manifestation of anti-LGI1 encephalitis. Oneiric stupor episodes are a useful clinical feature for establishing diagnostic suspicion and could provide a window to understanding the mechanisms behind some movement disorders in autoimmune encephalitis.

Nusinersen safety and effects on motor function in adult spinal muscular atrophy type 2 and 3.

OBJECTIVE: To retrospectively investigate safety and efficacy of nusinersen in a large cohort of adult Italian patients with spinal muscular atrophy (SMA). METHODS: Inclusion criteria were: (1) clinical and molecular diagnosis of SMA2 or SMA3; (2) nusinersen treatment started in adult age (>18 years); (3) clinical data available at least at baseline (T0-beginning of treatment) and 6 months (T6). RESULTS: We included 116 patients (13 SMA2 and 103 SMA3) with median age at first administration of 34 years (range 18-72). The Hammersmith Functional Rating Scale Expanded (HFMSE) in patients with SMA3 increased significantly from baseline to T6 (median change +1 point, p<0.0001), T10 (+2, p<0.0001) and T14 (+3, p<0.0001). HFMSE changes were independently significant in SMA3 sitter and walker subgroups. The Revised Upper Limb Module (RULM) in SMA3 significantly improved between T0 and T14 (median +0.5, p=0.012), with most of the benefit observed in sitters (+2, p=0.018). Conversely, patients with SMA2 had no significant changes of median HFMSE and RULM between T0 and the following time points, although a trend for improvement of RULM was observed in those with some residual baseline function. The rate of patients showing clinically meaningful improvements (as defined during clinical trials) increased from 53% to 69% from T6 to T14. CONCLUSIONS: Our data provide further evidence of nusinersen safety and efficacy in adult SMA2 and SMA3, with the latter appearing to be cumulative over time. In patients with extremely advanced disease, effects on residual motor function are less clear.

Clinical spectrum and IgG subclass analysis of anti-myelin oligodendrocyte glycoprotein antibody-associated syndromes: a multicenter study.

Anti-myelin oligodendrocyte glycoprotein antibodies (MOG-Ab) recently emerged as a potential biomarker in patients with inflammatory demyelinating diseases of the central nervous system. We here compare the clinical and laboratory findings observed in a cohort of MOG-Ab seropositive and seronegative cases and describe IgG subclass analysis results. Consecutive serum samples referred to Verona University Neuropathology Laboratory for aquaporin-4 (AQP4)-Ab and/or MOG-Ab testing were analysed between March 2014 and May 2017. The presence of AQP4-Ab was determined using a cell-based assay. A live cell immunofluorescence assay was used for the detection of MOG-IgG and IgG subclass analysis. Among 454 analysed samples, 29 were excluded due to AQP4-Ab positivity or to the final demonstration of a disorder not compatible with MOG-Ab. We obtained clinical data in 154 out of 425 cases. Of these, 22 subjects resulted MOG-Ab positive. MOG-Ab positive patients were mainly characterised by the involvement of the optic nerve and/or spinal cord. Half of the cases presented relapses and the recovery was usually partial. Brain MRI was heterogeneous while short lesions were the prevalent observation on spinal cord MRI. MOG-Ab titre usually decreased in non-relapsing cases. In all MOG-IgG positive cases, we observed IgG1 antibodies, which were predominant in most subjects. IgG2 (5/22), IgG3 (9/22) and IgG4 (3/22) antibodies were also detectable. We confirm that MOG-Ab-related syndromes have distinct features in the spectrum of demyelinating conditions, and we describe the possible role of the different IgG subclasses in this condition.

ATNX2 is not a regulatory gene in Italian amyotrophic lateral sclerosis patients with C9ORF72 GGGGCC expansion.

There are indications that both familial amyotrophic lateral sclerosis (ALS) and sporadic ALS phenotype and prognosis are partly regulated by genetic and environmental factors, supporting the theory that ALS is a multifactorial disease. The aim of this article was to assess the role of ATXN2 intermediate length repeats in a large series of Italian and Sardinian ALS patients and controls carrying a pathogenetic C9ORF72 GGGGCC hexanucleotide repeat. A total of 1972 ALS cases were identified through the database of the Italian ALS Genetic consortium, a collaborative effort including 18 ALS centers throughout Italy. The study population included: (1) 276 Italian and 57 Sardinian ALS cases who carried the C9ORF72 expansion; (2) 1340 Italian and 299 Sardinian ALS cases not carrying the C9ORF72 expansion. A total of healthy 1043 controls were also assessed. Most Italian and Sardinian cases and controls were homozygous for 22/22 or 23/23 repeats or heterozygous for 22/23 repeats of the ATXN2 gene. ATXN2 intermediate length repeats alleles (≥28) were detected in 3 (0.6%) Italian ALS cases carrying the C9ORF72 expansion, in none of the Sardinian ALS cases carrying the expansion, in 60 (4.3%) Italian cases not carrying the expansion, and in 6 (2.0%) Sardinian ALS cases without C9ORF72 expansion. Intermediate length repeat alleles were found in 12 (1.5%) Italian controls and 1 (0.84%) Sardinian controls. Therefore, ALS patients with C9ORF72 expansion showed a lower frequency of ATXN2 polyQ intermediate length repeats than both controls (Italian cases, p = 0.137; Sardinian cases, p = 0.0001) and ALS patients without C9ORF72 expansion (Italian cases, p = 0.005; Sardinian cases, p = 0.178). In our large study on Italian and Sardinian ALS patients with C9ORF72 GGGGCC hexanucleotide repeat expansion, compared to age-, gender- and ethnic-matched controls, ATXN2 polyQ intermediate length does not represent a modifier of ALS risk, differently from non-C9ORF72 mutated patients.

HFE p.H63D polymorphism does not influence ALS phenotype and survival.

It has been recently reported that the p.His63Asp polymorphism of the HFE gene accelerates disease progression both in the SOD1 transgenic mouse and in amyotrophic lateral sclerosis (ALS) patients. We have evaluated the effect of HFE p.His63Asp polymorphism on the phenotype in 1351 Italian ALS patients (232 of Sardinian ancestry). Patients were genotyped for the HFE p.His63Asp polymorphism (CC, GC, and GG). All patients were also assessed for C9ORF72, TARDBP, SOD1, and FUS mutations. Of the 1351 ALS patients, 363 (29.2%) were heterozygous (GC) for the p.His63Asp polymorphism and 30 (2.2%) were homozygous for the minor allele (GG). Patients with CC, GC, and GG polymorphisms did not significantly differ by age at onset, site of onset of symptoms, and survival; however, in SOD1 patients with CG or GG polymorphism had a significantly longer survival than those with a CC polymorphism. Differently from what observed in the mouse model of ALS, the HFE p.His63Asp polymorphism has no effect on ALS phenotype in this large series of Italian ALS patients.

CHCH10 mutations in an Italian cohort of familial and sporadic amyotrophic lateral sclerosis patients.

Mutations in CHCHD10 have recently been described as a cause of frontotemporal dementia (FTD) comorbid with amyotrophic lateral sclerosis (ALS). The aim of this study was to assess the frequency and clinical characteristics of CHCHD10 mutations in Italian patients diagnosed with familial (n = 64) and apparently sporadic ALS (n = 224). Three apparently sporadic patients were found to carry c.100C>T (p.Pro34Ser) heterozygous variant in the exon 2 of CHCHD10. This mutation had been previously described in 2 unrelated French patients with FTD-ALS. However, our patients had a typical ALS, without evidence of FTD, cerebellar or extrapyramidal signs, or sensorineural deficits. We confirm that CHCHD10 mutations account for ∼ 1% of Italian ALS patients and are a cause of disease in subjects without dementia or other atypical clinical signs.

Mutational screening of NOTCH3 gene reveals two novel mutations: complexity of CADASIL diagnosis.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an adult onset hereditary vascular disease with neurological manifestations. The classical clinical course is relentlessly progressive with early transient ischaemic attacks (TIA) or strokes, dementia and finally death in the mid-1960s. The disorder is inherited in an autosomal dominant fashion, with high penetrance and broad variable clinical course even within family. It is caused by mutations in the NOTCH3 gene; all causative mutations result in gain or loss of a cysteine residue within the extracellular domain, with exons 3 and 4 reported as hot spot mutational sites. Mutation analysis of the NOTCH3 gene was performed through direct sequencing of the 2-23 exons containing all EGF-like domains. Patients underwent genetic counselling pre and post testing. Here, we report two novel mutations located in exons 6 and 15 of the NOTCH3 gene; clinical description for the probands and for available relatives is enclosed. No reliable data on incidence or prevalence rates of this disease are available: it is therefore essential that the diagnosis is obtained in all suspected cases through the extensive analysis of the NOTCH3 gene and that all cases are brought to the attention of the scientific community.