Umberto Manera

UNC13A influences survival in Italian amyotrophic lateral sclerosis patients: A population-based study

The common variant rs12608932, located within an intron of UNC13A gene on chromosome 19p13.3, has been suggested to influence susceptibility to amyotrophic lateral sclerosis (ALS), as well as survival, in patients of north European descent. To examine this possibility further, we evaluated the association of rs12608932 with susceptibility and survival in a population-based cohort of 500 Italian ALS patients and 1457 Italian control samples. Although rs12608932 was not associated with ALS susceptibility in our series (p = 0.124), it was significantly associated with survival under the recessive model (median survival for AA/AC genotypes = 3.5 years [interquartile range, 2.2-6.4]; CC = 2.5 years [interquartile range, 1.6-4.2]; p = 0.017). Furthermore, rs12608932 genotype remained an independent prognostic factor in Cox multivariable analysis adjusting for other factors known to influence survival (p = 0.023). Overall, minor allele carrier status of rs12608932 was strongly associated with an approximate 1-year reduction of survival in ALS patients, making it a significant determinant of phenotype variation. The identification of UNC13A as a modifier of prognosis among sporadic ALS patients potentially provides a new therapeutic target aimed at slowing disease progression.

Genetic architecture of ALS in Sardinia

Conserved populations, such as Sardinians, displaying elevated rates of familial or sporadic amyotrophic lateral sclerosis (ALS) provide unique information on the genetics of the disease. Our aim was to describe the genetic profile of a consecutive series of ALS patients of Sardinian ancestry. All ALS patients of Sardinian ancestry, identified between 2008 and 2013 through the Italian ALS Genetic Consortium, were eligible to be included in the study. Patients and controls underwent the analysis of TARDBP, C9ORF72, SOD1, and FUS genes. Genetic mutations were identified in 155 out of 375 Sardinian ALS cases (41.3%), more commonly the p.A382T and p.G295S mutations of TARDBP and the GGGGCC hexanucleotide repeat expansion of C9ORF72. One patient had both p.G295S and p.A382T mutations of TARDBP and 8 carried both the heterozygous p.A382T mutation of TARDBP and a repeat expansion of C9ORF72. Patients carrying the p.A382T and the p.G295S mutations of TARDBP and the C9ORF72 repeat expansion shared distinct haplotypes across these loci. Patients with cooccurrence of C9ORF72 and TARDBP p.A382T missense mutation had a significantly lower age at onset and shorter survival. More than 40% of all cases on the island of Sardinia carry a mutation of an ALS-related gene, representing the highest percentage of ALS cases genetically explained outside of Scandinavia. Clinical phenotypes associated with different genetic mutations show some distinctive characteristics, but the heterogeneity between and among families carrying the same mutations implies that ALS manifestation is influenced by other genetic and nongenetic factors.

Secular Trends of Amyotrophic Lateral Sclerosis: The Piemonte and Valle d’Aosta Register

Importance This study reports the long-term epidemiologic trends of amyotrophic lateral sclerosis (ALS) based on a prospective register. Objective To examine the 20-year epidemiologic trends of ALS in the Piemonte and Valle d’Aosta regions of Italy. Design, Setting, and Participants The Piemonte and Valle d’Aosta Register for ALS (PARALS) is an epidemiologic prospective register that covers 2 Italian regions (population of 4 476 931 inhabitants according to the 2011 census) from January 1, 1995, through December 31, 2014. Case ascertainment is based on multiple sources (neurologic departments, hospital discharge archives, and mortality records). Incidence rates are age and sex standardized for the Italian population of the 2011 census. Age-period-cohort (APC) analysis was performed using a Poisson regression model. Main Outcomes and Measures The primary study outcomes were long-term incidence and prevalence rates of ALS using a prospective design and their determinants. Results During the study period, a total of 2702 patients (mean [SD] age at onset, 65.7 [11.1] years; 1246 [46.1%] female and 1456 [53.9%] male) received a diagnosis of ALS between 1995 and 2014, corresponding to a crude annual incidence rate of 3.03 per 100 000 population (95% CI, 2.85-3.23) and an adjusted incidence rate of 2.78 per 100 000 population (95% CI, 2.57-2.96). The age-adjusted incidence rate increased in the 2 decades of the study (1995-2004: 2.66; 95% CI, 2.50-2.83; 2005-2014: 2.89; 95% CI, 2.71-3.07; P = .04), mostly in women. The adjusted rate ratio of men to women decreased from 1.27:1 (1995-2004) to 1.17:1 (2005-2014). The analysis of deviance for the APC regression models indicated that the drift variable is relevant in explaining the variation of ALS incidence rates over time in the overall population (change in deviance, 4.6553; P = .03) and in women (change in deviance, 3.8821; P = .05) but not in men (change in deviance, 0.77215; P = .38). A total of 479 patients with ALS were alive and had not undergone tracheostomy at the prevalence day (December 31, 2014), corresponding to a crude prevalence rate of 10.54 per 100 000 population (95% CI, 9.64-11.52). Conclusions and Relevance During the 1995 to 2014 period, the crude and adjusted incidences of ALS increased in Piemonte and Valle d’Aosta, mostly in women. The APC model revealed that the increase of ALS incidence is attributable to a birth cohort effect in women, with a peak in the 1930 cohort. The different increase of ALS incidence in men and women points to an effect of exogenous factors with a differential effect on the 2 sexes, acting on a genetic background.

Monocytes of patients with amyotrophic lateral sclerosis linked to gene mutations display altered TDP-43 subcellular distribution

Cytoplasmic accumulation of the nuclear protein transactive response DNA‐binding protein 43 (TDP‐43) is an early determinant of motor neuron degeneration in most amyotrophic lateral sclerosis (ALS) cases. We previously disclosed this accumulation in circulating lymphomonocytes (CLM) of ALS patients with mutant TARDBP, the TDP‐43‐coding gene, as well as of a healthy individual carrying the parental TARDBP mutation. Here, we investigate TDP‐43 subcellular localization in CLM and in the constituent cells, lymphocytes and monocytes, of patients with various ALS‐linked mutant genes.

A novel p.E121G heterozygous missense mutation of SOD1 in an apparently sporadic ALS case with a 14-year course

Abstract The frequency of SOD1 mutations differs among populations: in Italy they account for 13.6% of familial ALS and 0.7% of sporadic cases. We describe an apparently sporadic Italian ALS patient, carrying a novel p.E121G heterozygous missense mutation of SOD1, with a 14-year disease course and a prevalent lower motor neuron phenotype, which are not uncommon among SOD1 mutations carriers. To our knowledge, no other mutation of codon 121 of SOD1 has ever been reported. Three in silico models suggest a deleterious effect of the p.E121G mutation. Nevertheless, further studies are necessary to confirm its pathogenic role and to evaluate eventual genotype-phenotype correlations.

Influence of arterial hypertension, type 2 diabetes and cardiovascular risk factors on ALS outcome: a population-based study

Abstract Objective: To assess the prognostic influence of pre-morbid type 2 diabetes mellitus, arterial hypertension and cardiovascular (CV) risk profile on ALS phenotype and outcome in a population-based cohort of Italian patients. Methods: A total of 650 ALS patients from the Piemonte/Valle d’Aosta Register for ALS, incident in the 2007–2011 period, were recruited. Information about premorbid presence of type 2 diabetes mellitus, arterial hypertension was collected at the time of diagnosis. Patients’ CV risk profile was calculated according to the Joint British Societies’ guidelines on prevention of cardiovascular disease in clinical practice (JBS2). Results: At the univariate analysis, the presence of pre-morbid arterial hypertension was associated with a higher age at onset of ALS and a shorter survival, and patients with a high CV risk profile had a worse prognosis than those with a low CV risk profile. The Cox multivariable analysis did not confirm such findings. Type 2 diabetes mellitus did not modify either the phenotype or the prognosis of ALS patients. Conclusions: This study performed on a large population-based cohort of ALS patients has demonstrated that arterial hypertension, type 2 diabetes and CV risk factors, calculated using the Framingham equation, do not influence ALS phenotype and prognosis.

NADPH oxidases 2 activation in patients with Parkinson's disease

Fig. 1. Mean fluorescence intensity (MFI) of oxidative burst in granulocytes (up) and monocytes (down) of PD patients and related controls after stimulation with bacterial stimulus (E. Coli). Neutrophils provide the first line of defense of the innate immune system by phagocytosing, killing, and digesting bacteria. Killing was previously believed to be accomplished by oxygen free radicals and other reactive oxygen species (ROS) generated by the NADPH oxidase in a process called “respiratory burst”. NADPH oxidase (NOX) enzymes catalyze the formation of ROS, which play a role in the development of different neurological disorders (ND), particularly whose generated by the phagocytic isoform NOX2 that is highly expressed in activated phagocytes including neutrophils and microglia [1], where it is responsible for the respiratory oxidative burst. Increased ROS has been observed in Amyotrophic Lateral Sclerosis (ALS), and a lower NOX2 activity showed a significant increase of survival in ALS patients [2]. There is also evidence that NOX are expressed and activated in patients with Parkinsons Disease (PD) and other ND, though the underlying mechanisms of NOX2mediated oxidative stress in PD pathogenesis are still unknown [3]. To elucidate the links between NOX2 and PD, the aim of this study was to evaluate the enzyme activity measuring oxidative burst of granulocytes and monocytes (ROS production) in fresh whole blood of patients in comparison with matched healthy controls using PhagoburstTM assay by flow cytometry, according to the manufacturers instructions. A total of 40 consecutive patients, 20 men and 20 women (64.7± 8.4 mean age and SD) with diagnosis of idiopathic PD and 40 healthy sexand age-matched control subjects (65.1± 8.6) were enrolled in the study. Patients were recruited at the Parkinsons andMovement Disorders Clinic, ‘Rita Levi Montalcini’ Department of Neurosciences, University of Turin, Italy. 18 subjects were inpatients, 22 outpatients. The study was approved by local Ethic Committee; both patients and controls provided the signed informed consent and personal data were treated according to the current Italian directives (Law No. 196 of 30 June 2003). Diagnosis of PD was made according to the UK Brain Bank criteria by a neurologist expert in movement disorder. The disease duration was 14.4 ± 6.3 years; 95% of patients (38/ 40) were in levodopa þ benserazide treatment. UPDRS of inpatients was 24± 8 (average of the differences of UPDRS OFF score versus UPDRS ON score calculated for each

The multistep hypothesis of ALS revisited: The role of genetic mutations.

Objective Amyotrophic lateral sclerosis (ALS) incidence rates are consistent with the hypothesis that ALS is a multistep process. We tested the hypothesis that carrying a large effect mutation might account for ≥1 steps through the effect of the mutation, thus leaving fewer remaining steps before ALS begins. Methods We generated incidence data from an ALS population register in Italy (2007–2015) for which genetic analysis for C9orf72, SOD1, TARDBP, and FUS genes was performed in 82% of incident cases. As confirmation, we used data from ALS cases diagnosed in the Republic of Ireland (2006–2014). We regressed the log of age-specific incidence against the log of age with least-squares regression for the subpopulation carrying disease-associated variation in each separate gene. Results Of the 1,077 genetically tested cases, 74 (6.9%) carried C9orf72 mutations, 20 (1.9%) had SOD1 mutations, 15 (1.4%) had TARDBP mutations, and 3 (0.3%) carried FUS mutations. In the whole population, there was a linear relationship between log incidence and log age (r2 = 0.98) with a slope estimate of 4.65 (4.37–4.95), consistent with a 6-step process. The analysis for C9orf72-mutated patients confirmed a linear relationship (r2 = 0.94) with a slope estimate of 2.22 (1.74–2.29), suggesting a 3-step process. This estimate was confirmed by data from the Irish ALS register. The slope estimate was consistent with a 2-step process for SOD1 and with a 4-step process for TARDBP. Conclusion The identification of a reduced number of steps in patients with ALS with genetic mutations compared to those without mutations supports the idea of ALS as a multistep process and is an important advance for dissecting the pathogenic process in ALS.

Association between alcohol exposure and the risk of amyotrophic lateral sclerosis in the Euro-MOTOR study

Objectives Several studies focused on the association between alcohol consumption and amyotrophic lateral sclerosis (ALS), although with inconsistent findings. Antioxidants may play a role since lyophilised red wine was found to prolong SOD1 mice lifespan. The aim of this international population-based case–control study performed in Ireland, The Netherlands and Italy was to assess the role of alcohol, and red wine in particular, in developing ALS. Methods Euro-MOTOR is a case–control study where patients with incident ALS and controls matched for gender, age and area of residency were recruited in a population-based design. Logistic regression models adjusted for sex, age, cohort, education, leisure time physical activity, smoking, heart problems, hypertension, stroke, cholesterol and diabetes were performed. Results 1557 patients with ALS and 2922 controls were enrolled in the study. Exposure to alcohol drinking was not significantly associated with ALS risk. A stratified analysis of exposure to alcohol by cohort revealed significant ORs in The Netherlands and in Apulia, with opposite directions (respectively 0.68 and 2.38). With regard to red wine consumption, only in Apulia the double-fold increased risk (OR 2.53) remained significant. A decreased risk was found for current alcohol drinkers (OR 0.83), while a significantly increased risk was detected among former drinkers (OR 1.63). Analysis of cumulative exposure to alcohol revealed no significant associations with ALS risk. Conclusion With few exceptions, no significant association was found between alcohol consumption and ALS. The study of the association between alcohol and ALS requires a thorough exploration, especially considering the role of different type of alcoholic beverages.

Spatial epidemiology of Amyotrophic Lateral Sclerosis in Piedmont and Aosta Valley, Italy: a population-based cluster analysis

The analysis of the spatial distribution of cases could give important cues on putative environmental causes of a disease. Our aim was to perform a spatial analysis of an amyotrophic lateral sclerosis (ALS) cohort from the Piedmont and Aosta Valley ALS register (PARALS) over a 20‐year period.