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Recent advances in sequencing technologies and collaborative efforts have led to substantial progress in identifying the genetic causes of amyotrophic lateral sclerosis (ALS). This momentum has, in turn, fostered the development of putative molecular therapies. In this Review, we outline the current genetic knowledge, emphasizing recent discoveries and emerging concepts such as the implication of distinct types of mutation, variability in mutated genes in diverse genetic ancestries and gene–environment interactions. We also propose a high-level model to synthesize the interdependent effects of genetics, environmental and lifestyle factors, and ageing into a unified theory of ALS. Furthermore, we summarize the current status of therapies developed on the basis of genetic knowledge established for ALS over the past 30 years, and we discuss how developing treatments for ALS will advance our understanding of targeting other neurological diseases.

Amyotrophic lateral sclerosis (ALS) is known to represent a collection of overlapping syndromes. Various classification systems based on empirical observations have been proposed, but it is unclear to what extent they reflect ALS population substructures. We aimed to use machine-learning techniques to identify the number and nature of ALS subtypes to obtain a better understanding of this heterogeneity, enhance our understanding of the disease, and improve clinical care.

IMPORTANCE: Juvenile amyotrophic lateral sclerosis (ALS) is a rare form of ALS characterized by age of symptom onset less than 25 years and a variable presentation. OBJECTIVE: To identify the genetic variants associated with juvenile ALS. DESIGN, SETTING, AND PARTICIPANTS: In this multicenter family-based genetic study, trio whole-exome sequencing was performed to identify the disease-associated gene in a case series of unrelated patients diagnosed with juvenile ALS and severe growth retardation. The patients and their family members were enrolled at academic hospitals and a government research facility between March 1, 2016, and March 13, 2020, and were observed until October 1, 2020. Whole-exome sequencing was also performed in a series of patients with juvenile ALS. A total of 66 patients with juvenile ALS and 6258 adult patients with ALS participated in the study. Patients were selected for the study based on their diagnosis, and all eligible participants were enrolled in the study. None of the participants had a family history of neurological disorders, suggesting de novo variants as the underlying genetic mechanism. MAIN OUTCOMES AND MEASURES: De novo variants present only in the index case and not in unaffected family members. RESULTS: Trio whole-exome sequencing was performed in 3 patients diagnosed with juvenile ALS and their parents. An additional 63 patients with juvenile ALS and 6258 adult patients with ALS were subsequently screened for variants in the SPTLC1 gene. De novo variants in SPTLC1 (p.Ala20Ser in 2 patients and p.Ser331Tyr in 1 patient) were identified in 3 unrelated patients diagnosed with juvenile ALS and failure to thrive. A fourth variant (p.Leu39del) was identified in a patient with juvenile ALS where parental DNA was unavailable. Variants in this gene have been previously shown to be associated with autosomal-dominant hereditary sensory autonomic neuropathy, type 1A, by disrupting an essential enzyme complex in the sphingolipid synthesis pathway. CONCLUSIONS AND RELEVANCE: These data broaden the phenotype associated with SPTLC1 and suggest that patients presenting with juvenile ALS should be screened for variants in this gene.

BackgroundA 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.ObjectiveTo evaluate the diagnostic yield and advantages of whole-genome sequencing (WGS) as a standard diagnostic genetic test for ALS.MethodsIn 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.ResultsA 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.ConclusionsOur 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.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with a lifetime risk of one in 350 people and an unmet need for disease-modifying therapies. We conducted a cross-ancestry genome-wide association study (GWAS) including 29,612 patients with ALS and 122,656 controls, which identified 15 risk loci. When combined with 8,953 individuals with whole-genome sequencing (6,538 patients, 2,415 controls) and a large cortex-derived expression quantitative trait locus (eQTL) dataset (MetaBrain), analyses revealed locus-specific genetic architectures in which we prioritized genes either through rare variants, short tandem repeats or regulatory effects. ALS-associated risk loci were shared with multiple traits within the neurodegenerative spectrum but with distinct enrichment patterns across brain regions and cell types. Of the environmental and lifestyle risk factors obtained from the literature, Mendelian randomization analyses indicated a causal role for high cholesterol levels. The combination of all ALS-associated signals reveals a role for perturbations in vesicle-mediated transport and autophagy and provides evidence for cell-autonomous disease initiation in glutamatergic neurons.

The genetic basis of Lewy body dementia (LBD) is not well understood. Here, we performed whole-genome sequencing in large cohorts of LBD cases and neurologically healthy controls to study the genetic architecture of this understudied form of dementia, and to generate a resource for the scientific community. Genome-wide association analysis identified five independent risk loci, whereas genome-wide gene-aggregation tests implicated mutations in the gene GBA. Genetic risk scores demonstrate that LBD shares risk profiles and pathways with Alzheimer’s disease and Parkinson’s disease, providing a deeper molecular understanding of the complex genetic architecture of this age-related neurodegenerative condition.

ObjectiveTo identify the metabolic changes related to the various levels of cognitive deficits in amyotrophic lateral sclerosis (ALS) using 18F-2-fluoro-2-deoxy-D-glucose positron emission tomography (18F-FDG-PET) imaging.Methods274 ALS patients underwent neuropsychological assessment and brain 18F-FDG-PET at diagnosis. According to the criteria published in 2017, cognitive status was classified as ALS with normal cognition (ALS-Cn, n=132), ALS with behavioural impairment (ALS-Bi, n=66), ALS with cognitive impairment (ALS-Ci, n=30), ALS with cognitive and behavioural impairment (ALS-Cbi, n=26), ALS with frontotemporal dementia (ALS–FTD, n=20). We compared each group displaying some degree of cognitive and/or behavioural impairment to ALS-Cn patients, including age at PET, sex and ALS Functional Rating Scale-Revised as covariates.ResultsWe identified frontal lobe relative hypometabolism in cognitively impaired patients that resulted more extensive and significant across the continuum from ALS-Ci, through ALS-Cbi, to ALS–FTD. ALS–FTD patients also showed cerebellar relative hypermetabolism. ALS-Bi patients did not show any difference compared with ALS-Cn.ConclusionsThese data support the concept that patients with cognitive impairment have a more widespread neurodegenerative process compared with patients with a pure motor disease: the more severe the cognitive impairment, the more diffuse the metabolic changes. Otherwise, metabolic changes related to pure behavioural impairment need further characterisation.

IMPORTANCE: During a time with the potential for novel treatment strategies, early detection of disease manifestations at an individual level in presymptomatic carriers of a hexanucleotide repeat expansion in the C9orf72 gene (preSxC9) is becoming increasingly relevant. OBJECTIVES: To evaluate changes in glucose metabolism before symptom onset of amyotrophic lateral sclerosis or frontotemporal dementia in preSxC9 using simultaneous fluorine 18–labeled fluorodeoxyglucose ([18F]FDG positron emission tomographic (PET) and magnetic resonance imaging as well as the mutation’s association with clinical and fluid biomarkers. DESIGN, SETTING, AND PARTICIPANTS: A prospective, case-control study enrolled 46 participants from November 30, 2015, until December 11, 2018. The study was conducted at the neuromuscular reference center of the University Hospitals Leuven, Leuven, Belgium. MAIN OUTCOMES AND MEASURES: Neuroimaging data were spatially normalized and analyzed at the voxel level at a height threshold of P < .001, cluster-level familywise error–corrected threshold of P < .05, and statistical significance was set at P < .05 for the volume-of-interest level analysis, using Benjamini-Hochberg correction for multiple correction. W-score maps were computed using the individuals serving as controls as a reference to quantify the degree of [18F]FDG PET abnormality. The threshold for abnormality on the W-score maps was designated as an absolute W-score greater than or equal to 1.96. Neurofilament levels and performance on cognitive and neurologic examinations were determined. All hypothesis tests were 1-sided. RESULTS: Of the 42 included participants, there were 17 with the preSxC9 mutation (12 women [71%]; mean [SD] age, 51 [9] years) and 25 healthy controls (12 women [48%]; mean [SD] age, 47 [10] years). Compared with control participants, significant clusters of relative hypometabolism were found in frontotemporal regions, basal ganglia, and thalami of preSxC9 participants and relative hypermetabolism in the peri-Rolandic region, superior frontal gyrus, and precuneus cortex. W-score frequency maps revealed reduced glucose metabolism with local maxima in the insular cortices, central opercular cortex, and thalami in up to 82% of preSxC9 participants and increased glucose metabolism in the precentral gyrus and precuneus cortex in up to 71% of preSxC9 participants. Other findings in the preSxC9 group were upper motor neuron involvement in 10 participants (59%), cognitive abnormalities in 5 participants (29%), and elevated neurofilament levels in 3 of 16 individuals (19%) who underwent lumbar puncture. CONCLUSIONS AND RELEVANCE: The results suggest that [18F]FDG PET can identify glucose metabolic changes in preSxC9 at an individual level, preceding significantly elevated neurofilament levels and onset of symptoms.

ObjectiveThe lack of prognostic biomarkers in patients with amyotrophic lateral sclerosis (ALS) induced researchers to develop clinical evaluation tools for stratification and survival prediction. We assessed the correlation between patterns of functional involvement, considered as a cumulative number of body regions involved, and overall survival in a population-based series of patients with ALS (PARALS).MethodsWe derived the functional involvement of four body regions at diagnosis using ALSFRS-R subscores for bulbar, upper limbs, lower limbs and respiratory/thoracic regions. We analysed the effect of number of body regions involved (NBRI) at diagnosis on overall survival, adjusting for age at onset, sex, site of onset, diagnostic delay, forced vital capacity, body mass index, mutational status, cognition and comparing it with King’s staging system.ResultsThe NBRI was strongly related to survival, with a progressive increase of death/tracheostomy risk among groups (two body regions HR=1.24, 95% CI 1.06 to 1.45, p=0007; three body regions HR=1.65, 95% CI 1.38 to 1.98, p<0.001; four body regions HR=2.68, 95% CI 2.11 to 3.39, p<0.001). Using ALSFRS-R score, the consistency between the number of regions involved and King’s clinical stage at diagnosis was very high (81%). The evaluation of respiratory/thoracic region and cognition allowed to subdivide patients into different prognostic categories. Regional spreading of the disease is associated with survival, independently from the initial region involved.ConclusionsThe evaluation of NBRI, with the inclusion of initial respiratory/thoracic involvement and cognition, can be useful in many research fields, improving the stratification of patients. Our findings highlight the importance of the spatial spreading of functional impairment in the prediction of ALS outcome.

Background: Amyotrophic lateral sclerosis (ALS), Alzheimer’s, and Parkinson’s diseases are age-related neurodegenerative diseases. ALS is not a single entity but a syndrome with many different causes. In all 3 diseases, gene mutations account for only 10–15% of cases. Many environmental and lifestyle factors have been implicated as risk factors for ALS, though none have been proven to cause the disease. It is generally believed that ALS results from interactions between environmental risk factors and genetic predisposing factors. The advent of next-generation sequencing and recent advances in research into environmental risk factors offer the opportunity to investigate these interactions. Summary: We propose a hypothesis to explain the syndrome of ALS based on the interaction of many individual environmental risk factors with many individual genetic predisposing factors. We hypothesize that there are many such combinations of individual, specific, genetic, and environmental factors, and that each combination can lead to the development of the syndrome of ALS. We also propose a hypothesis that explains the overlap between the age-related neurodegenerations and their genetic underpinnings. Age and duration of exposure are crucial factors in these age-related neurodegenerative diseases, and we consider how these may relate to gene-environment interactions. Key Messages: To date, genetic studies and environmental studies have investigated the causes of ALS separately. We argue that this univariate approach will not lead to discoveries of important gene-environment interactions. We propose new research approaches to investigating gene-environment interactions based on these hypotheses.

ObjectivesSeveral 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.MethodsEuro-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.Results1557 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.ConclusionWith 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.

ObjectivesTo assess the role of body mass index (BMI) and of the rate of weight loss as prognostic factors in amyotrophic lateral sclerosis (ALS) and to explore the clinical correlates of weight loss in the early phases of the disease.MethodsThe study cohort included all ALS patients in Piemonte/Valle d’Aosta in the 2007–2011 period. Overall survival and the probability of death/tracheostomy at 18 months (logistic regression model) were calculated.ResultsOf the 712 patients, 620 (87.1%) were included in the study. Patients ’ survival was related to the mean monthly percentage of weight loss at diagnosis (p<0.0001), but not to pre-morbid BMI or BMI at diagnosis. Spinal onset patients with dysphagia at diagnosis had a median survival similar to bulbar onset patients. About 20% of spinal onset patients without dysphagia at diagnosis had severe weight loss and initial respiratory impairment, and had a median survival time similar to bulbar onset patients.ConclusionsThe rate of weight loss from onset to diagnosis was found to be a strong and independent prognostic factor in ALS. Weight loss was mainly due to the reduction of nutritional intake related to dysphagia, but a subgroup of spinal onset patients without dysphagia at diagnosis had a severe weight loss and an outcome similar to bulbar patients. According to our findings, we recommend that in clinical trials patients should be stratified according to the presence of dysphagia at the time of enrolment and not by site of onset of symptoms.

ObjectiveTo investigate whether exposure to particulates and combustion products may explain the association between certain occupations and amyotrophic lateral sclerosis (ALS) risk in a large, multicentre, population-based, case–control study, based on full job histories, using job-exposure matrices, with detailed information on possible confounders.MethodsPopulation-based patients with ALS and controls were recruited from five registries in the Netherlands, Ireland and Italy. Demographics and data regarding educational level, smoking, alcohol habits and lifetime occupational history were obtained using a validated questionnaire. Using job-exposure matrices, we assessed occupational exposure to silica, asbestos, organic dust, contact with animals or fresh animal products, endotoxins, polycyclic aromatic hydrocarbons and diesel motor exhaust. Multivariate logistic regression models adjusting for confounding factors were used to determine the association between these exposures and ALS risk.ResultsWe included 1557 patients and 2922 controls. Associations were positive for all seven occupational exposures (ORs ranging from 1.13 to 1.73 for high vs never exposed), and significant on the continuous scale for silica, organic dust and diesel motor exhaust (p values for trend ≤0.03). Additional analyses, adding an exposure (one at a time) to the model in the single exposure analysis, revealed a stable OR for silica. We found similar results when patients with a C9orf72mutation were excluded.ConclusionIn a large, multicentre study, using harmonised methodology to objectively quantify occupational exposure to particulates and combustion products, we found an association between ALS risk and exposure to silica, independent of the other occupational exposures studied.

ObjectiveTo investigate the association between physical activity (PA) and amyotrophic lateral sclerosis (ALS) in population-based case–control studies in three European countries using a validated and harmonised questionnaire.MethodsPatients with incident ALS and controls were recruited from five population-based registers in The Netherlands, Ireland and Italy. Demographic and data regarding educational level, smoking, alcohol habits and lifetime PA levels in both leisure and work time were gathered by questionnaire, and quantified using metabolic equivalent of task scores. Logistic regression models adjusting for PA-related factors were used to determine the association between PA and ALS risk, and forest plots were used to visualise heterogeneity between regions.Results1557 patients and 2922 controls were included. We found a linear association between ALS and PA in leisure time (OR 1.07, P=0.01) and occupational activities (OR 1.06, P<0.001), and all activities combined (OR 1.06, P<0.001), with some heterogeneity between regions: the most evident association was seen in the Irish and Italian cohorts. After adjustment for other occupational exposures or exclusion of patients with a C9orf72mutation, the ORs remained similar.ConclusionWe provide new class I evidence for a positive association between PA and risk of ALS in a large multicentre study using harmonised methodology to objectively quantify PA levels, with some suggestions for population differences.

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.

IMPORTANCE: Various factors have been proposed as possible candidates associated with the prognosis of amyotrophic lateral sclerosis (ALS); however, there is still no consensus on which biomarkers are reliable prognostic factors. C-reactive protein (CRP) is a biomarker of the inflammatory response that shows significant prognostic value for several diseases. OBJECTIVE: To examine the prognostic significance of CRP in ALS. DESIGN, SETTING, AND PARTICIPANTS: Patients’ serum CRP levels were evaluated from January 1, 2009, to June 30, 2015, in a large cohort of patients with ALS observed by an Italian tertiary multidisciplinary center. Results were replicated in an independent cohort obtained from a population-based registry of patients with ALS. A post hoc analysis was performed of the phase 2 trial of NP001 to determine whether stratification by levels of CRP improves differentiation of responders and nonresponders to the drug. MAIN OUTCOMES AND MEASURES: Serum CRP levels from the first examination were recorded to assess their effect on disease progression and survival. RESULTS: A total of 394 patients with ALS (168 women and 226 men; mean [SD] age at diagnosis, 60.18 [13.60] years) were observed in a tertiary multidisciplinary center, and the analysis was replicated in an independent cohort of 116 patients with ALS (50 women and 66 men; mean [SD] age at diagnosis, 67.00 [10.74] years) identified through a regional population-based registry. Serum CRP levels in the 394 patients with ALS correlated with severity of functional impairment, as measured by total score on the ALS Functional Rating Scale–Revised, at first evaluation (r = –0.14818; P = .004), and with patient survival (hazard ratio, 1.129; 95% CI, 1.033-1.234; P = .007). Similar results were found in the independent cohort (hazard ratio, 1.044; 95% CI, 1.016-1.056; P ≤ .001). Moreover, a post hoc analysis of the phase 2 trial of NP001 using the same CRP threshold showed that patients with elevated baseline CRP levels receiving the higher dose of NP001 had significantly less functional impairment after the treatment period compared with patients with normal baseline CRP, regardless of whether patients with normal CRP levels received NP001 or placebo (3.00 [3.62] vs –7.31 [6.23]; P = .04). CONCLUSIONS AND RELEVANCE: These findings suggest that patients with ALS and elevated serum CRP levels progress more rapidly than do those with lower CRP levels and that this elevation may reflect a neuroinflammatory state potentially responsive to the immune regulators such as NP001.

IMPORTANCE: Amyotrophic lateral sclerosis (ALS) is a devastating adult-onset neurodegenerative disorder with a poor prognosis and a median survival of 3 years. However, a significant proportion of patients survive more than 10 years from symptom onset. OBJECTIVE: To identify gene variants influencing survival in ALS. DESIGN, SETTING, AND PARTICIPANTS: This genome-wide association study (GWAS) analyzed survival in data sets from several European countries and the United States that were collected by the Italian Consortium for the Genetics of ALS and the International Consortium on Amyotrophic Lateral Sclerosis Genetics. The study population included 4256 patients with ALS (3125 [73.4%] deceased) with genotype data extended to 7 174 392 variants by imputation analysis. Samples of DNA were collected from January 1, 1993, to December 31, 2009, and analyzed from March 1, 2014, to February 28, 2015. MAIN OUTCOMES AND MEASURES: Cox proportional hazards regression under an additive model with adjustment for age at onset, sex, and the first 4 principal components of ancestry, followed by meta-analysis, were used to analyze data. Survival distributions for the most associated genetic variants were assessed by Kaplan-Meier analysis. RESULTS: Among the 4256 patients included in the analysis (2589 male [60.8%] and 1667 female [39.2%]; mean [SD] age at onset, 59 [12] years), the following 2 novel loci were significantly associated with ALS survival: at 10q23 (rs139550538; P = 1.87 × 10−9) and in the CAMTA1 gene at 1p36 (rs2412208, P = 3.53 × 10−8). At locus 10q23, the adjusted hazard ratio for patients with the rs139550538 AA or AT genotype was 1.61 (95% CI, 1.38-1.89; P = 1.87 × 10−9), corresponding to an 8-month reduction in survival compared with TT carriers. For rs2412208 CAMTA1, the adjusted hazard ratio for patients with the GG or GT genotype was 1.17 (95% CI, 1.11-1.24; P = 3.53 × 10−8), corresponding to a 4-month reduction in survival compared with TT carriers. CONCLUSIONS AND RELEVANCE: This GWAS robustly identified 2 loci at genome-wide levels of significance that influence survival in patients with ALS. Because ALS is a rare disease and prevention is not feasible, treatment that modifies survival is the most realistic strategy. Therefore, identification of modifier genes that might influence ALS survival could improve the understanding of the biology of the disease and suggest biological targets for pharmaceutical intervention. In addition, genetic risk scores for survival could be used as an adjunct to clinical trials to account for the genetic contribution to survival.

IMPORTANCE: Amyotrophic lateral sclerosis (ALS) is a heterogeneous disease with a wide spectrum of involvement of cognitive functions. The mechanisms of this heterogeneity are still largely unknown, but genetic variants may account for this variability. OBJECTIVE: To assess the influence of the apolipoprotein E (APOE) and C9ORF72 genotypes on cognitive impairment in a population-based series of Italian patients with ALS. DESIGN, SETTING, AND PARTICIPANTS: All 504 patients with ALS living in Piemonte, Italy, diagnosed between January 1, 2009, and December 31, 2013, and identified through the Piemonte and Valle d’Aosta register for ALS, were eligible to participate in the study. Controls were 223 age- and sex-matched individuals identified through the patients’ general practitioners. Data analysis was performed from June 1 to December 31, 2014. MAIN OUTCOMES AND MEASURES: The presence of APOE and C9ORF72 genotypes was assessed. Patients were cognitively classified as having ALS with normal cognition, ALS with frontotemporal dementia (FTD), ALS with executive or nonexecutive impairment, and ALS with behavioral impairment. RESULTS: Of the 504 patients with incident ALS, 357 (70.8%) were included in the study; 154 were women, 203 were men, they had a mean (SD) age at onset of 64.8 (10.2) years, and 37 of them carried a C9ORF72 repeat expansion. Cognitive testing revealed that 184 patients (51.5%) had ALS with normal cognition, 51 (14.3%) had ALS with FTD, 103 (28.9%) had ALS with executive or nonexecutive impairment, and 19 (5.3%) had ALS with behavioral impairment. Distribution of APOE haplotypes did not significantly differ between patients and controls or among patients with different levels of cognitive impairment. According to multivariate logistic regression, the presence of C9ORF72 repeat expansions was the strongest determinant of FTD (odds ratio, 13.08; 95% CI, 4.75-36.02; P &lt; .001); however, the presence of an APOE ε2 allele significantly increased the risk of FTD (odds ratio, 2.61; 95% CI, 1.14-6.10; P = .03). Presence of an APOE ε4 allele was ineffectual. CONCLUSIONS AND RELEVANCE: C9ORF72 repeat expansions have a primary role in increasing the risk of cognitive impairment in patients with ALS; the APOE ε2 allele, to a lesser extent, also increases the risk of FTD. These study findings highlight the importance of considering the genetic background of patients with ALS when analyzing the putative effect of genetic modifiers.

Patient-derived induced pluripotent stem cells (iPSCs) provide an opportunity to study human diseases mainly in those cases for which no suitable model systems are available. Here, we have taken advantage of in vitro iPSCs derived from patients affected by amyotrophic lateral sclerosis (ALS) and carrying mutations in the RNA-binding protein FUS to study the cellular behavior of the mutant proteins in the appropriate genetic background. Moreover, the ability to differentiate iPSCs into spinal cord neural cells provides an in vitro model mimicking the physiological conditions. iPSCs were derived from FUSR514S and FUSR521C patient fibroblasts, whereas in the case of the severe FUSP525L mutation, in which fibroblasts were not available, a heterozygous and a homozygous iPSC line were raised by TALEN-directed mutagenesis. We show that aberrant localization and recruitment of FUS into stress granules (SGs) is a prerogative of the FUS mutant proteins and occurs only upon induction of stress in both undifferentiated iPSCs and spinal cord neural cells. Moreover, we show that the incorporation into SGs is proportional to the amount of cytoplasmic FUS, strongly correlating with the cytoplasmic delocalization phenotype of the different mutants. Therefore, the available iPSCs represent a very powerful system for understanding the correlation between FUS mutations, the molecular mechanisms of SG formation and ALS ethiopathogenesis.

IMPORTANCE: Myasthenia gravis is a chronic, autoimmune, neuromuscular disease characterized by fluctuating weakness of voluntary muscle groups. Although genetic factors are known to play a role in this neuroimmunological condition, the genetic etiology underlying myasthenia gravis is not well understood. OBJECTIVE: To identify genetic variants that alter susceptibility to myasthenia gravis, we performed a genome-wide association study. DESIGN, SETTING, AND PARTICIPANTS: DNA was obtained from 1032 white individuals from North America diagnosed as having acetylcholine receptor antibody–positive myasthenia gravis and 1998 race/ethnicity-matched control individuals from January 2010 to January 2011. These samples were genotyped on Illumina OmniExpress single-nucleotide polymorphism arrays. An independent cohort of 423 Italian cases and 467 Italian control individuals were used for replication. MAIN OUTCOMES AND MEASURES: We calculated P values for association between 8 114 394 genotyped and imputed variants across the genome and risk for developing myasthenia gravis using logistic regression modeling. A threshold P value of 5.0 × 10−8 was set for genome-wide significance after Bonferroni correction for multiple testing. RESULTS: In the overall case-control cohort, we identified association signals at CTLA4 (rs231770; P = 3.98 × 10−8; odds ratio, 1.37; 95% CI, 1.25-1.49), HLA-DQA1 (rs9271871; P = 1.08 × 10−8; odds ratio, 2.31; 95% CI, 2.02 - 2.60), and TNFRSF11A (rs4263037; P = 1.60 × 10−9; odds ratio, 1.41; 95% CI, 1.29-1.53). These findings replicated for CTLA4 and HLA-DQA1 in an independent cohort of Italian cases and control individuals. Further analysis revealed distinct, but overlapping, disease-associated loci for early- and late-onset forms of myasthenia gravis. In the late-onset cases, we identified 2 association peaks: one was located in TNFRSF11A (rs4263037; P = 1.32 × 10−12; odds ratio, 1.56; 95% CI, 1.44-1.68) and the other was detected in the major histocompatibility complex on chromosome 6p21 (HLA-DQA1; rs9271871; P = 7.02 × 10−18; odds ratio, 4.27; 95% CI, 3.92-4.62). Association within the major histocompatibility complex region was also observed in early-onset cases (HLA-DQA1; rs601006; P = 2.52 × 10−11; odds ratio, 4.0; 95% CI, 3.57-4.43), although the set of single-nucleotide polymorphisms was different from that implicated among late-onset cases. CONCLUSIONS AND RELEVANCE: Our genetic data provide insights into aberrant cellular mechanisms responsible for this prototypical autoimmune disorder. They also suggest that clinical trials of immunomodulatory drugs related to CTLA4 and that are already Food and Drug Administration approved as therapies for other autoimmune diseases could be considered for patients with refractory disease.

We investigated a large sample of patients with amyotrophic lateral sclerosis (ALS) at rest in order to assess the value of 18F-2-fluoro-2-deoxy-D-glucose (18F-FDG) PET as a biomarker to discriminate patients from controls.

IMPORTANCE: There is an urgent need to identify reliable biomarkers of amyotrophic lateral sclerosis (ALS) progression for clinical practice and pharmacological trials. OBJECTIVES: To correlate several hematological markers evaluated at diagnosis with ALS outcome in a population-based series of patients (discovery cohort) and replicate the findings in an independent validation cohort from an ALS tertiary center. DESIGN, SETTING, AND PARTICIPANTS: The discovery cohort included 712 patients with ALS from the Piemonte and Valle d’Aosta Register for Amyotrophic Lateral Sclerosis from January 1, 2007, to December 31, 2011. The validation cohort comprised 122 patients with ALS at different stages of disease consecutively seen at an ALS tertiary center between January 1, 2007, and January 1, 2009. MAIN OUTCOMES AND MEASURES: The following hematological factors were investigated and correlated with survival: total leukocytes, neutrophils, lymphocytes, monocytes, glucose, creatinine, uric acid, albumin, bilirubin, total cholesterol, triglycerides, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, creatine kinase, thyroid-stimulating hormones, and erythrocyte sedimentation rate; all analyses were performed separately by sex. The patient of the validation cohort also underwent bioelectrical impedance analysis for the calculation of fat-free mass. RESULT: Of the 712 patients in the examined period in Piemonte and Valle d’Aosta, 638 (89.6%) were included in the study. Only serum albumin (men: ≤4.3 vs >4.3 mg/dL, P &lt; .001; women: ≤4.3 vs >4.3 mg/dL, P &lt; .001) and creatinine levels (men: ≤0.82 vs >0.82 mg/dL, P = .004; women: ≤0.65 vs >0.05 mg/dL, P = .004) and lymphocyte count (men: ≤1700 vs >1700/μL, P = .04; women: ≤1700 vs >1700/μL, P = .02) were significantly associated with ALS outcome in both sexes with a dose-response effect (better survival with increasing levels). These findings were confirmed in the validation cohort. Multivariable analysis showed that serum albumin (men: hazard ratio [HR], 1.39; 95% CI, 1.05-1.90; P = .02; women: HR, 1.73; 95 % CI, 1.35-2.39; P = .001) and creatinine (men: HR, 1.47; 95% CI, 1.11-1.95; P = .007; women: HR, 1.49; 95% CI, 1.07-2.05; P = .02) were independent predictors of survival in both sexes; no other hematological factor was retained in the model. In patients with ALS, serum albumin was correlated with markers of inflammatory state while serum creatinine was correlated with fat-free mass, which is a marker of muscle mass. CONCLUSIONS AND RELEVANCE: In ALS, serum albumin and creatinine are independent markers of outcome in both sexes. Creatinine reflects the muscle waste whereas albumin is connected with inflammatory state. Both creatinine and albumin are reliable markers of the severity of clinical status in patients with ALS and can be used in defining prognosis at the time of diagnosis.

IMPORTANCE: Considerable advances have been made in our understanding of the genetics underlying amyotrophic lateral sclerosis (ALS). Nevertheless, for the majority of patients who receive a diagnosis of ALS, the role played by genetics is unclear. Further elucidation of the genetic architecture of this disease will help clarify the role of genetic variation in ALS populations. OBJECTIVE: To estimate the relative importance of genetic factors in a complex disease such as ALS by accurately quantifying heritability using genome-wide data derived from genome-wide association studies. DESIGN, SETTING, AND PARTICIPANTS: We applied the genome-wide complex trait analysis algorithm to 3 genome-wide association study data sets that were generated from ALS case-control cohorts of European ancestry to estimate the heritability of ALS. Cumulatively, these data sets contained genotype data from 1223 cases and 1591 controls that had been previously generated and are publically available on the National Center for Biotechnology Information database of genotypes and phenotypes website (http://www.ncbi.nlm.nih.gov/gap). The cohorts genotyped as part of these genome-wide association study efforts include the InCHIANTI (aging in the Chianti area) Study, the Piemonte and Valle d’Aosta Register for Amyotrophic Lateral Sclerosis, the National Institute of Neurological Disorders and Stroke Repository, and an ALS specialty clinic in Helsinki, Finland. MAIN OUTCOMES AND MEASURES: A linear mixed model was used to account for all known single-nucleotide polymorphisms simultaneously and to quantify the phenotypic variance present in ostensibly outbred individuals. Variance measures were used to estimate heritability. RESULTS: With our meta-analysis, which is based on genome-wide genotyping data, we estimated the overall heritability of ALS to be approximately 21.0% (95% CI, 17.1-24.9) (SE = 2.0%), indicating that additional genetic variation influencing risk of ALS loci remains to be identified. Furthermore, we identified 17 regions of the genome that display significantly high heritability estimates. Eleven of these regions represent novel candidate regions for ALS risk. CONCLUSIONS AND RELEVANCE: We found the heritability of ALS to be significantly higher than previously reported. We also identified multiple, novel genomic regions that we hypothesize may contain causative risk variants that influence susceptibility to ALS.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with an estimated heritability between 40 and 50%. DNA methylation patterns can serve as proxies of (past) exposures and disease progression, as well as providing a potential mechanism that mediates genetic or environmental risk. Here, we present a blood-based epigenome-wide association study meta-analysis in 9706 samples passing stringent quality control (6763 patients, 2943 controls). We identified a total of 45 differentially methylated positions (DMPs) annotated to 42 genes, which are enriched for pathways and traits related to metabolism, cholesterol biosynthesis, and immunity. We then tested 39 DNA methylation–based proxies of putative ALS risk factors and found that high-density lipoprotein cholesterol, body mass index, white blood cell proportions, and alcohol intake were independently associated with ALS. Integration of these results with our latest genome-wide association study showed that cholesterol biosynthesis was potentially causally related to ALS. Last, DNA methylation at several DMPs and blood cell proportion estimates derived from DNA methylation data were associated with survival rate in patients, suggesting that they might represent indicators of underlying disease processes potentially amenable to therapeutic interventions.

To assess the frequency and clinical characteristics of patients with mutations of major amyotrophic lateral sclerosis (ALS) genes in a prospectively ascertained, population-based epidemiologic series of cases.

There is increasing evidence that common genetic risk factors underlie frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). Recently, mutations in the sequestosome 1 (SQSTM1) gene, which encodes p62 protein, have been reported in patients with ALS. P62 is a multifunctional adapter protein mainly involved in selective autophagy, oxidative stress response, and cell signaling pathways. The purpose of our study was to evaluate the frequency of SQSTM1mutations in a dataset of unrelated patients with FTLD or ALS, in comparison with healthy controls and patients with Paget disease of bone (PDB).

SUMMARY Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder of adult life, characterized by the progressive loss of upper motor neurons in the primary motor cortex and of the spinal and bulbar lower motor neurons. The cause of sporadic cases and of the majority of hereditary cases remains elusive. So far a total of 17 genes or genetic loci of familial ALS have been identified, related to DNA/RNA processing, protein aggregation, membrane trafficking and axonal transport and mitochondrial dysfunction. A large range of environmental factors have also been studied, with inconclusive results, with the possible exception of cigarette smoking. ALS is now considered a complex multifactorial neurodegenerative disorder, but studies analyzing genetic and environmental factors together are still underway.

BACKGROUND TAR DNA-binding protein 43, encoded by the TARDBP gene, has been identified as the major pathological protein of frontotemporal lobar dementia (FTLD) with or without amyotrophic lateral sclerosis (ALS) and sporadic ALS. Subsequently, mutations in the TARDBP gene have been detected in 2% to 3% of patients with ALS (both familial and sporadic ALS). However, to our knowledge, there is only 1 description of 2 patients with FTLD and TARDBP gene mutations who later developed motor neuron disease. OBJECTIVE To describe cognitive abnormalities in 3 Italian families with familial ALS and TARDBP gene mutations. DESIGN, SETTING, AND PARTICIPANTS Genetic, neuropsychological, and neuroimaging analyses in 36 patients with familial non–superoxide dismutase 1 gene (SOD1) ALS and 280 healthy controls. MAIN OUTCOME MEASURE We identified 3 index cases of familial ALS carrying the p.Ala382Thr missense mutation of the TARDBP gene and with clinical, neuroimaging, and neuropsychological features of FTLD. RESULTS The p.Ala382Thr missense mutation of the TARDBP gene was absent in the 280 controls. It was present in all affected members of the 3 families for whom DNA was available. All affected members of the 3 families developed FTLD after the onset of ALS, confirmed by neuropsychological testing and hypometabolism in frontal associative areas assessed with fludeoxyglucose F 18 positron emission tomography and computed tomography. CONCLUSIONS Three apparently unrelated families with familial ALS carrying the p.Ala382Thr TARDBP missense mutation developed FTLD. In these families, FTLD cosegregates with ALS. Patients with ALS carrying TARDBP mutations may develop FTLD.Arch Neurol. 2010;67(8):1002-1009--

The cause of amyotrophic lateral sclerosis (ALS) is still unknown. A possible relationship between ALS and sport participation has been supposed, but never definitely demonstrated. We studied a cohort of 7325 male professional football players engaged by a football team from the Italian First or Second Division in the period 1970–2001. ALS cases were identified using different concurrent sources. Standardized morbidity ratios (SMRs) were calculated. During the 137 078 person-years of follow-up, five ALS cases were identified (mean age of onset, 43.4 years). Three cases had a bulbar onset, significantly more than expected (P = 0.003). Since the number of expected cases was 0.77, the overall SMR was 6.5 [95% confidence interval (CI), 2.1–15.1]. The SMR was significantly increased for an ALS onset before 49 years, but not for older subjects. A significant increase of the SMR was found in the periods 1980–1989 and 1990–2001, whereas no ALS case was found in the 1970–1979 period. A dose–response relationship between the duration of professional football activity and the risk of ALS was found (>5 years, 15.2, 95% CI, 3.1–44.4; ≤5 years, 3.5, 95% CI, 0.4–12.7). Our findings seem to indicate that playing professional football is a strong risk factor for ALS.

Sixty-one glioblastomas have been studied, subdivided into the categories of classic glioblastomas (GBM) and glioblastomas with astrocytic (GBA) and oligodendroglial (GBO) differentiated areas. On surgical samples, TP53, Mdm2, CDKN2A/p16–p14 alterations were studied by molecular biology techniques and by immunohistochemistry. It has been found that Mdm2 amplification was more frequent in GBM than in GBA and GBO, that p14ARFwas inactivated in a high percentage of cases in the three tumor categories. Both these and other alterations did not reach a statistical significance, with the exception of CDKN2A/p16 homozygous deletion which showed the highest frequency in GBO. The latter finding could be in line with the observation that CDKN2A/p16 inactivation is a step in the molecular pathway to tumor progression in oligodendrogliomas. TP53 mutations and Mdm2 amplifications were mutually exclusive, whereas TP53 mutations and CDKN2A/p14 inactivation coexisted in 5 cases. The alterations of the p53/Mdm2/p14ARFpathway occurred in 73% of cases and in 80% of cases if CDKN2A homozygous deletions were associated. All glioblastomas with gemistocytic areas showed p14ARFinactivation. Immunohistochemistry showed higher percentages of positivity in comparison with molecular genetics, but with similar variations.

The cell-cycle regulator p16 inhibits the complex cdk4–cyclin D1 and controls G1–S transition. In human tumors, p16 inactivation is often accomplished by homozygous deletion (HD) of its encoding gene, CDKN2A. Methylation of the 5` CpG island promoter has been proposed as an alternative mechanism of inactivation. Expression of p16, CDKN2A HD and 5` CDKN2A CpG island methylation was studied in 25 oligodendrogliomas by immunohistochemistry and PCR amplification. Ten oligodendrogliomas were p16-immunonegative, and CDKN2A HD was determined in 8 of these cases. In the 2 immunonegative cases without HD, no CpG island methylation was found. The absence of CpG island methylation in the p16-immunonegative cases without HD suggests either non-genetic regulation of p16 or different genetic changes. CDKN2A HD did not correlate with histological grading (p = n.s.); however, it showed a correlation with survival (p = 0.03), supporting an important role of CDKN2A in the prognosis of oligodendrogliomas. Int. J. Cancer 88:554–557, 2000. © 2000 Wiley-Liss, Inc.

Abstract: p27/kip1 regulates the G1-S transition of the cell cycle by inhibiting cyclinD-CDK4, cyclinE-CDK2 and cyclinA-CDK2 complexes. Regulation of p27 levels occurs mainly post-translationally by ubiquitin-mediated proteasomal proteolysis. Although genetic changes of p27/kip1 are extremely rare, in many human carcinomas p27 levels are reduced, correlate with histological malignancy, and are associated with poor prognosis. In astrocytic gliomas, p27 decreases with anaplasia and is almost absent in glioblastomas. p27/kip1 was immunohistochemically studied in 37 oligodendrogliomas, categorized according to WHO classification. In this series, the immunohistochemical reaction for p27 was confined to nuclei. p27 score showed a tendency to decrease with malignancy. When the p27 score was considered as high versus low expression (cut-off of p27 labeling index, LI, at 25%), it represented an independent prognostic factor in univariate (P = 0.02) and in multivariate analysis (P = 0.04). The risk ratio suggested that the p27 low expression group had a threefold increased possibility to show a reduced survival. Moreover, p27 levels did not correlate with MIB-1 LI, suggesting that p27 is not merely associated with the control of proliferation.

The recognition of the anaplastic variant of oligodendroglioma is difficult, since it is not easy to identify histological prognostic factors. Among the latter, vascular productive changes have been inconsistently put in relation with survival. In 95 cases of operated oligodendrogliomas, endothelial cell hyperplasia, microvascular proliferations and capillary density were studied by histological and immunohistochemical methods. Capillary density was evaluated on CD31-stained sections by a grid of 100 squares placed in the ocular of the microscope. Statistical analysis was performed in order to compare these parameters with survival. A nodular growth pattern was observed more frequently among tumor grades 3–4 than among tumor grades 1–2. Endothelial cell hyperplasia was more frequent in nodular growth pattern, but it did not correlate with survival. The highest capillary density was found in nodular growth pattern, but it did not correlate with survival as well. Microvascular proliferations correlated with survival only in univariate, but not in multivariate analysis. Age, extent of surgical removal, year of surgery, post-operative Karnofsky score and MIB-1 LI remained associated with survival, as observed in a previous study.

The appearance of medulloblastoma in adult age and the uncertainoverlapping of prognostic factors in pediatric and adult populationsstimulate the question of whether medulloblastoma is different in adults andin children. The pathologic features, proliferation potential andglial/neuronal differentiation have been investigated in 42 adultmedulloblastomas and 42 medulloblastomas of children; the quantitative datahave been compared between the two groups of age. Homer-Wright rosettes,nuclear polymorphism and histologic signs of neuronal differentiation weremore frequent in children cases; GFAP-positive tumor cells and desmoplastictype were more frequent in adult cases. The mean, median and rage of LIs,with PCNA and MIB-1 were significantly (p < 0.05) higher in adults thanin children. All cases, independently from age of the patients wereimmunoreactive with markers of neuronal commitment (class III beta tubulinisotype, MAP-2, neurofilaments). The immunoreactivity pattern suggested amore mature neuronal character in desmoplastic cases of adults than ofchildren and in classic cases of children than of adults. In conclusion,some phenotypic differences between childhood and adult medulloblastomaexist, but do not support a substantially different course of the disease.The higher proliferation potential in adult than in childhood cases isunexpected in a tumor of embryonal origin, and reduces the applicability ofCollin‘s law to medulloblastoma.

Well-differentiated astrocytomas may transform into malignant astrocytomas in time. In surgical specimens, when the histological picture strictly corresponds to that of grade II glioma, the transformation is unpredictable. Clinically, the bad outcome of a quota of astrocytomas is a well known phenomenon. The use of proliferation markers, and recently of MIB-1 LI, for detecting the proliferation potential comes out to be a useful tool for prognosis. A survival analysis of fifty astrocytomas grade II according to the WHO classification was performed with univariate and multivariate analysis of a series of clinical and histological parameters. MIB-1 LI was calculated and compared with all the other parameters. A cut-off of 8% of MIB-1 LI divided the astrocytomas in two groups with significantly different survival (p=0.0066): median survival time of 1062 versus 1686 days. According to multivariate analysis MIB-1 LI resulted to be an independent factor (p=0.002) along with extension of surgical removal (partial versus total), postoperative Karnofsky status (≥ 70 versus < 70) and age (≤ 30 versus > 30). The interpretation of well-differentiated astrocytomas with high MIB-1 LI is that the increasing number of cycling cells precedes phenotypic transformation. MIB-1 LI can be used as a prognostic factor.

Aims and background A clear line cannot be drawn between well-differentiated and anaplastic astrocytomas, and a subset of low-grade tumors, histologically indistinguishable from the others, behaves similarly to anaplastic astrocytomas. The proliferative index could aid in the identification of this subgroup, for which a different therapeutic approach would be indicated.Methods We immunohistochemically evaluated the proliferating ceil nuclear antigen (PCNA) expression in 77 well-differentiated astrocytomas, since PCNA has been considered a good proliferation marker. The prognostic significance of PCNA labeling index (LI) was assessed in univariate and multivariate analysis, taking into consideration some clinical and histologic factors known to affect prognosis.Results PCNA immunostaining identified a subgroup of tumors, characterized by a LI > 5%, with a median survival close to that observed in anaplastic astrocytomas. The survival table of such a group was significantly different from that of the group with a lower LI (p = 0.0009). Multivariate analysis confirmed that PCNA-LI is an independent prognostic factor (p = 0.001).Conclusion These data suggest that PCNA immunostaining can be a useful tool to define the prognosis of low-grade astrocytomas on routine biopsy material.

The prevalence of idiopathic Parkinson's disease (PD) in an area of Northwestern Italy (Socio‐Sanitary District of Cossato, 61, 830 inhabitants) was analyzed and the reliability of “drug tracer methodology” evaluated. The patients were identified by both conventional epidemiological methodology (general practitioners, consultant neurologist, computerized archives) and drug tracer methodology (registered levodopa prescriptions). The diagnosis of PD was clinically verified by examining all patients. Of 146 patients examined, 104 were affected by PD; the remaining by other types of parkinsonism. The crude point prevalence rate (October 20, 1991) was 168 per 100,000 population (95% confidence interval [95% CI] 138–204), with a progressive increase up to the 80–89 age group. Using “tracer” methodology, the estimated crude point prevalence rate was 196 (95% CI 163–235), with an overestimation of the prevalence in older patients and women and an underestimation in younger and less seriously affected cases. The uneven distribution of PD in Italy appears to be the result of, at least in part, methodological factors. “Tracer” methodology may cause biases in the evaluation of the epidemiological characteristics of PD, especially if the clinical diagnosis of cases identified by this method is not carefully verified. Nevertheless, it appears useful as an additional source of cases for descriptive surveys.

The distribution of proliferating cell nuclear antigen (PCNA)-(clone PC 10)- and Ki-67- (clone MIB-1)-positive nuclei was investigated in 60 medulloblastomas of childhood. Although the labeling index of the two markers did not coincide, both showed a wide range of parallel variations. The percentage of positive nuclei was similar in both classic and desmoplastic tumors. A variable proliferation capacity was found in the different tumor structures. Areas with neuronal and glial differentiation showed very few positive nuclei; these were very abundant in the infiltration areas, and along penetrating vessels from subarachnoidal growths. Pale islands were negative or positive only in their peripheral part. Large-cell areas were richer in positive nuclei than classic ones, accounting for their more malignant character. Hyperchromatic round nuclei, not belonging to necrotic foci and called lymphocyte-like nuclei, differently interpreted in the past, were vairiably found in every case. They are known, from previous experience, to stain orange with Acridine Orange fluorochroming, like single-stranded DNA. They were not easily distinguishable from mitoses and were stained by in situ end-labeling of DNA strand breaks, as demonstrated by incorporation of labeled nucleotides. They were regarded as possible apoptotic nuclei, representing either a peculiar type of cell death or the preservation of the cell deletion capacity, typical of the embryonal tissue of origin.

Aims and background. Medulloblastoma in adults is a rare tumor. The small number of cases in the reported series has not permitted a definite assessment of the prognostic role of clinical, pathologic and cell kinetics factors. The largest series of medulloblastoma in adults treated in a single institution is herein reported.Methods. The clinical, therapeutic, pathologic and proliferation features of medulloblastoma in 44 adult patients (> 18 years) were analyzed retrospectively with regard to postoperative survival. The proliferation potential of each tumor was evaluated by the immunohistochemical demonstration of proliferating cell nuclear antigen (PCNA) and Ki-67, clone MIB-1, in paraffin sections.Results. The overall 5- and 10-year survival rates were 40% and 35.6%, respectively. Significant factors in predicting a longer postoperative survival were: age < 37 years, decade of management (1977-1990), radiotherapy (50-55 Gy on the posterior fossa and 30-35 Gy on the spinal cord) and nuclear isomorphism. When corrected for adequacy of radiotreatment, desmoplastic type and differentiation were significantly correlated with a shorter survival. The PCNA-labelling index (LI) ranged from 34.5 to 82.2%, the MIB-1-LI ranged from 9.6 to 64.7%. No association was found between PCNA- or MIB-1-LI values and microscopic features, or between LI values and prognosis.Conclusions. Contrary to a general assumption, desmoplastic medulloblastoma and differentiated medulloblastoma are negative prognostic factors in adequately radiotreated adult patients. This may possibly be referred to lower radiosensitivity of these tumor variants. The LI with PCNA or Ki-67 is of no help in identifying aggressive tumors.

The clinico-pathologic data of 37 primary lymphomas of the brain were retrospectively reviewed. The tumors were classified according to the Kiel classification and the Working Formulation System. They represented 1.02% of all primary intracranial tumors of our series. The radiologic prediction appeared to be difficult: the suspicion was maximal when the absence of pathologic vessels at angiography occurred in a meningioma-like lesion at CT. Median survival was 4.53 months in the 16 cases who underwent surgery only versus 25.7 months in the 8 cases operated and irradiated with 40-60 Gy (p < 0.01). The prognosis of lymphomas of the CNS, even if radioresponsive tumors, remains poor. Most patients relapse after treatment, most often locally in the brain, with a variable frequency of spinal or systemic localization.