MOLECULAR AND GENETIC CHARACTERIZATION OF ALS PATIENTS

Amyotrophic lateral sclerosis (ALS) is an adult\u2011onset, rapidly progressive and ultimately fatal neurodegenerative disorder characterised by degeneration of upper and lower motor neurons. This leads to weakness, muscular atrophy and spasticity, which relentlessly progress to complete paralysis with a low survival rate beyond 5 years from symptom onset. In 10% of cases the disease is considered to be genetically transmitted (FALS) while in the remaining cases it occurs sporadically in the population (SALS). To date cases of hereditary ALS have been attributed to mutations in more than 16 different genes, the most common being SOD1, FUS, TARDBP and C9ORF72; mutations in other genes are rare. The above genes explain 60% of the cases of familial ALS and 15% of sporadic cases. The disease can be subdivided into bulbar (25%) and spinal\u2011onset (75\u201180%) forms. Nevertheless, it is currently recognized that pathological changes are not limited to the motor system: patients with ALS may exhibit cognitive abnormalities ranging from impaired frontal executive dysfunction to overt frontotemporal dementia (FTD). In spite of the above evidence, ALS is regarded as a complex disease in which multiple environmental and genetic risk factors contribute to disease susceptibility. Furthermore it is possible that the phenotypic variability, which is frequently detected within families, could be due to multiple genetic factors as devised in the oligogenic disease model. In the present study we analysed 285 SALS and 17 FALS cases. Globally, our molecular analysis explained 10.3% of all ALS cases (31/302). The genes screened were SOD1, TARDBP, FUS and C9ORF72. Genomic DNA was extracted from peripheral blood through a salting out procedure; coding regions and exon\u2011intron boundaries of SOD1 (5 exons), TARDBP (1 exon), and FUS (5 exons) genes were amplified from genomic DNA and sequenced. Otherwise the repeat\u2011primed PCR assay, used for C9ORF72 gene, was performed in order to screen for the presence of the GGGGCC hexanucleotide repeats expansion in ALS patients. The repeat unit of 6 nucleotides expands up to more than several hundred times in the affected individuals. Fewer than 10 repeats are not associated with a pathological phenotype, while more than 30 repeats are associated. However we still do not know the meaning of intermediate repeat sizes (11\u201129). This fact complicates the attribution of the size expansion to the pathological phenotype observed. According to the oligogenic disease model, all patients were screened for the most common ALS\u2011associated genes and all mutated subjects were tested also for ANG. The affected/unaffected family members, when available for the study, were screened for SOD1, TARDBP, FUS and C9ORF72 in order to identify their genetic difference from the proband and to evaluate if this difference could explain an heterogeneous phenotypic expression of the disease. Following these analyses we selected and described in detail 5 FALS and 2 SALS cases and one SETX case, with different intrafamilial phenotypic expression. In one of our SOD1 mutation carriers, the proband manifested the disease after the delivery; together with a specific angiogenin genetic variant this condition seems to have anticipated the age of disease onset and contributed to the aggressive clinical course observed in the proband compared to other family members. We also found one case in which we observed the phenomenon of anticipation, which could be due to hormonal treatments together with the simultaneous presence of 2 mutations (C9ORF72/ TARDBP), as suggested in the oligogenic disease model. Indeed, the neuroprotective effects of estrogens could account for the later age at onset in women as we have tested in another family harbouring the R521C mutation. In this case a male subject developed the disease, whereas his older sister didn\u2019t show any neurological signs. In another family we observed a wide spectrum of clinical symptoms associated with A382T TARDBP mutation. Some family members showed cognitive impairment without signs of ALS, conversely, other relatives showed a typical ALS without any signs of dementia. In addition we have evidence of TARDBP mutation carriers without a neurological phenotype. Surprisingly, two subjects harbouring the mutation in homozygous state display different spectra of clinical symptoms. The screening of SOD1, FUS, C9ORF72 and ANG in the family members, to identify other mutations that could explain this intrafamilial phenotypic heterogeneity, resulted negative; indicating that other genes/conditions could play a role in ALS disease. These data support the hypothesis that phenotypic variability seen in patients carrying the same mutation, could be attributed to additional genetic and/or environmental factors, which could modify the penetrance of the disease. Furthermore we identified a clinical subgroup of patients that develop the disease during pregnancy (child-bearing age), thus indicating an additional modifier conditions linked to hormonal changes. For all these reasons predictive or presymptomatic testing should be undertaken with caution, especially in unaffected family members. Indeed, it is impossible for the genetic test to predict clinical outcomes based on the genotypic results only. A number of uncertainties remain, due to variability in penetrance, expressivity, and modifying factors, such as gene\u2011gene interactions and environmental influences. This adds to the debate for the ethical and psychological dilemmas about genetic testing, since still more has to be discovered related to this devastating disease

Inflammasome in als skeletal muscle: Nlrp3 as a potential biomarker

Since NLRP3 inflammasome plays a pivotal role in several neurodegenerative disorders, we hypothesized that levels of inflammasome components could help in diagnosis or prognosis of amyotrophic lateral sclerosis (ALS). Gene and protein expression was assayed by RT-PCR and Western blot. Spearman’s correlation coefficient was used to determine the linear correlation of transcriptional expression levels with longevity throughout disease progression in mice models. Kaplan-Meier analysis was performed to evaluate MCC950 effects (NLRP3 inhibitor) on lifespan of SOD1G93A mice. The results showed significant alterations in NLRP3 inflammasome gene and protein levels in the skeletal muscle of SOD1G93A mice. Spearman’s correlation coefficient revealed a positive association between Nlrp3 transcriptional levels in skeletal muscle and longevity of SOD1G93A mice (r = 0.506; p = 0.027). Accordingly, NLRP3 inactivation with MCC950 decreased the lifespan of mice. Furthermore, NLRP3 mRNA levels were significantly elevated in the blood of ALS patients compared to healthy controls (p = 0.03). In conclusion, NLRP3 could be involved in skeletal muscle pathogenesis of ALS, either through inflammasome or independently, and may play a dual role during disease progression. NLRP3 gene expression levels could be used as a biomarker to improve diagnosis and prognosis in skeletal muscle from animal models and also to support diagnosis in clinical practice with the blood of ALS patients

Individuals with familial hypercholesterolemia and cardiovascular events have higher circulating Lp(a) levels

BACKGROUND: Cardiovascular disease (CVD) is a major cause of mortality and morbidity. Increased low-density lipoprotein cholesterol (LDL-C) level is its major risk factor. Familial hypercholesterolemia (FH) is a genetic disorder characterized by elevated LDL-C since birth and subsequent premature CVD. There is a heterogeneity in the CVD onset in patients with FH. This is potentially due to the presence of other independent risk factors. Lipoprotein(a) [Lp(a)] is an LDL-like particle and represents a strong risk factor for CVD. OBJECTIVE: Our objective was to understand the contribution of Lp(a) in the susceptibility to CVD in individuals with genetic diagnosis of FH. METHODS: We measured Lp(a) levels in 2 independent and well-characterized genetic-FH cohorts: the FH-Gothenburg cohort (n = 190) and the FH-CEGP Milan cohort (n = 160). The genetic diagnosis was performed by targeted next-generation sequencing (FH-Gothenburg and part of the FH-CEGP Milan cohort), or by Sanger sequencing. RESULTS: We show that among individuals with genetic diagnosis of FH, those with previous CVD had higher Lp(a) levels. In addition, analyzing the response to the lipid-lowering therapies, we have also shown that statins had the same LDL-C-lowering effect irrespective of the type of FH-causative mutation. However, when we examined the lipid-lowering effect of proprotein convertase subtilisin/kexin type 9 inhibition by antibodies, we observed a trend in a better reduction of the LDL-C level in carriers of nonsense mutations. CONCLUSION: In conclusion, our results suggest that Lp(a) contributes to CVD onset in individuals with genetic diagnosis of FH. Our finding supports the importance to identify an efficacious therapy to lower Lp(a) in patients with FH to prevent CVD onset or recurrence

Predicting functional impairment trajectories in amyotrophic lateral sclerosis: a probabilistic, multifactorial model of disease progression.

To employ Artificial Intelligence to model, predict and simulate the amyotrophic lateral sclerosis (ALS) progression over time in terms of variable interactions, functional impairments, and survival. We employed demographic and clinical variables, including functional scores and the utilisation of support interventions, of 3940 ALS patients from four Italian and two Israeli registers to develop a new approach based on Dynamic Bayesian Networks (DBNs) that models the ALS evolution over time, in two distinct scenarios of variable availability. The method allows to simulate patients' disease trajectories and predict the probability of functional impairment and survival at different time points. DBNs explicitly represent the relationships between the variables and the pathways along which they influence the disease progression. Several notable inter-dependencies were identified and validated by comparison with literature. Moreover, the implemented tool allows the assessment of the effect of different markers on the disease course, reproducing the probabilistically expected clinical progressions. The tool shows high concordance in terms of predicted and real prognosis, assessed as time to functional impairments and survival (integral of the AU-ROC in the first 36 months between 0.80-0.93 and 0.84-0.89 for the two scenarios, respectively). Provided only with measurements commonly collected during the first visit, our models can predict time to the loss of independence in walking, breathing, swallowing, communicating, and survival and it can be used to generate in silico patient cohorts with specific characteristics. Our tool provides a comprehensive framework to support physicians in treatment planning and clinical decision-making. [Abstract copyright: © 2022. The Author(s).

Randomized, double-blind, placebo-controlled trial of rapamycin in amyotrophic lateral sclerosis

In preclinical studies rapamycin was found to target neuroinflammation, by expanding regulatory T cells, and affecting autophagy, two pillars of amyotrophic lateral sclerosis (ALS) pathogenesis. Herein we report a multicenter, randomized, double-blind trial, in 63 ALS patients who were randomly assigned in a 1:1:1 ratio to receive rapamycin 2 mg/m2/day,1 mg/m2/day or placebo (EUDRACT 2016-002399-28; NCT03359538). The primary outcome, the number of patients exhibiting an increase >30% in regulatory T cells from baseline to treatment end, was not attained. Secondary outcomes were changes from baseline of T, B, NK cell subpopulations, inflammasome mRNA expression and activation status, S6-ribosomal protein phosphorylation, neurofilaments; clinical outcome measures of disease progression; survival; safety and quality of life. Of the secondary outcomes, rapamycin decreased mRNA relative expression of the pro-inflammatory cytokine IL-18, reduced plasmatic IL-18 protein, and increased the percentage of classical monocytes and memory switched B cells, although no corrections were applied for multiple tests. In conclusion, we show that rapamycin treatment is well tolerated and provides reassuring safety findings in ALS patients, but further trials are necessary to understand the biological and clinical effects of this drug in ALS

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

Collagen XIX Alpha 1 improves prognosis in amyotrophic lateral sclerosis

The identification of more reliable diagnostic or prognostic biomarkers in age-related neurodegenerative diseases, such as Amyotrophic Lateral Sclerosis (ALS), is urgently needed. The objective in this study was to identify more reliable prognostic biomarkers of ALS mirroring neurodegeneration that could be of help in clinical trials. A total of 268 participants from three cohorts were included in this study. The muscle and blood cohorts were analyzed in two cross-sectional studies, while the serial blood cohort was analyzed in a longitudinal study at 6-monthly intervals. Fifteen target genes and fourteen proteins involved in muscle physiology and differentiation, metabolic processes and neuromuscular junction dismantlement were studied in the three cohorts. In the muscle biopsy cohort, the risk for a higher mortality in an ALS patient that showed high Collagen type XIX, alpha 1 (COL19A1) protein levels and a fast progression of the disease was 70.5% (P < 0.05), while in the blood cohort, this risk was 20% (P < 0.01). In the serial blood cohort, the linear mixed model analysis showed a significant association between increasing COL19A1 gene levels along disease progression and a faster progression during the follow-up period of 24 months (P < 0.05). Additionally, higher COL19A1 levels and a faster progression increased 17.9% the mortality risk (P < 0.01). We provide new evidence that COL19A1 can be considered a prognostic biomarker that could help the selection of homogeneous groups of patients for upcoming clinical trial and may be pointed out as a promising therapeutic target in ALS

The relation of serum lipoprotein (a) concentration and apolipoprotein (a) phenotype with preclinical atherosclerosis

Lipoprotein (a) [Lp(a)] is a plasmatic lipoprotein, comprising a low density lipoprotein-like particle with the addition of an apolipoprotein (a) molecule, covalently bonded to apolipoprotein B100. High levels of Lp(a) have been linked to an increased risk of cardiovascular events. Lp(a) levels are mostly attributed to genetic determinants within the apolipoprotein(a) gene (LPA). We examined whether Lp(a) plasma levels, isoforms and polymorphisms are associated with early atherosclerosis by measuring intima-media thickness (IMT) in an asymptomatic population. We assessed data for 60 adult subjects from our Lipid Clinic, in primary prevention and with Lp(a) levels >35 mg/dL. We collect data for Lp(a) concentrations in plasma, LPA kringle IV type 2 (KIV-2) sums of repeats (affecting isoform size), and carrier status for the LPA single-nucleotide polymorphisms (SNP) rs10455872 and rs3798220. Lp(a) was quantified by ELISA test and isoform size by electrophoresis (SDS-PAGE). Isoforms were classified as F, B, S1, S2, S3, S4 based on electrophoretic mobility compared with apoB100. LPA genotype was assessed using a Human CVD BeadChip. Lp(a) mean plasma level was 117 mg/dL (range 36-375 mg/dL). Small isoform apo(a) size (F,B,S1,S2) was present in the 80% of subjects and confirm the inverse relation with plasmatic Lp(a) concentration. Genetic analysis revealed that 26% of subjects carries SNP rs10455872, while 17% carries rs3798220, which are associated with increased Lp(a) levels in carriers versus non-carriers. Finally the carotid IMTs were determined to analyze the association of Lp(a) and with IMT and we found that IMT increased with increase in quartiles of IMT (p<0.05) confirming a relationship between Lp(a) and development of atherosclerosis

Taste changes in amyotrophic lateral sclerosis and effects on quality of life

The primary aim of the study is to evaluate possible taste changes in a cohort of amyotrophic lateral sclerosis patients (pALS) with dysphagia, focusing on eventual psychological and quality of life (QoL) implications. The second aim is to evaluate the changes of QoL following the use of a specific device that provides food flavour. Thirty-two ALS patients were recruited and divided into two groups: subjects feeding only through enteral tube (ET) and subjects still eating by oral way (OW). A specific set of questionnaires was selected and adapted to investigate possible changes of taste and the impact on psychological status and QoL. Moreover, a specific device that provides food flavours in a safety manner was applied to all patients. We found a perceived reduction of taste in ALS patients, in particular in the ET group. All patients showed a strong interest in the preservation of taste, and its loss negatively related to their QoL. The use of the flavour device improved the perceived QoL showing no side effects, even in the ET group. For the first time, our study revealed changes in taste perception in a cohort of ALS patients and the negative consequences that these changes have on psychological status and QoL. Furthermore, the positive effects of the device used to provide flavours suggest a possible rehabilitative effect, which should be better evaluated and confirmed in further studies