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International audience; Objective: Metals have been suggested as a risk factor for amyotrophic lateral sclerosis (ALS), but only retrospective studies are available to date. We compared metal levels in prospectively collected blood samples from ALS patients and controls, to explore whether metals are associated with ALS mortality. Methods: A nested ALS case–control study was conducted within the prospective EPIC (European Prospective Investigation into Cancer and Nutrition) cohort. Cases were identified through death certificates. We analyzed metal levels in erythrocyte samples obtained at recruitment, as a biomarker for metal exposure from any source. Arsenic, cadmium, copper, lead, manganese, mercury, selenium, and zinc concentrations were measured by inductively coupled plasma–mass spectrometry. To estimate ALS risk, we applied conditional logistic regression models. Results: The study population comprised 107 cases (65% female) and 319 controls matched for age, sex, and study center. Median time between blood collection and ALS death was 8 years (range = 1–15). Comparing the highest with the lowest tertile, cadmium (odds ratio [OR] = 2.04, 95% confidence interval [CI] = 1.08–3.87) and lead (OR = 1.89, 95% CI = 0.97–3.67) concentrations suggest associations with increased ALS risk. Zinc was associated with a decreased risk (OR = 0.50, 95% CI = 0.27–0.94). Associations for cadmium and lead remained when limiting analyses to noncurrent smokers. Interpretation: This is the first study to compare metal levels before disease onset, minimizing reverse causation. The observed associations suggest that cadmium, lead, and zinc may play a role in ALS etiology. Cadmium and lead possibly act as intermediates on the pathway from smoking to ALS. ANN NEUROL 20209999:n/a–n/a.

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.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that leads to progressive muscle wasting and weakness, eventually resulting in swallowing difficulties and respiratory failure. There is no cure, and only one drug has been proven irrefutably to increase survival by several months. ALS is considered a complex disease, with both genetic and environmental factors contributing to disease onset, and approximately 5-10% of patients describe a positive family history. This thesis contains multiple studies on the genetic architecture of ALS; ultimately aiming to gain more insight into disease processes and provide a basis for new (personalized) treatments. Using different research methods, we identified new DNA variants (C21orf2, NEK1, MOPB and SCFD1) associated with a higher risk of ALS. Interestingly, the proteins C21OF2 and NEK1 were previously identified as interactors, providing novel insights into ALS disease development. Also, we confirm that the previously identified genetic variant in the gene NIPA1 increases ALS risk, and we show that certain genetic risk factors co-occur more often than expected by chance. This thesis further elucidates the genetic background of ALS, and underlines the importance of future large-scale genetic studies on the disease.

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease resulting in progressive weakness and death in three to five years on average. In most patients there are no family members with ALS, and therefore an interplay of genetic and environmental risk factors is thought to be causal for disease development. Unfortunately many risk factors are still unknown and mechanisms of ALS development are not yet unraveled. This thesis presents multiple new risk factors for ALS, both genetic and environmental factors. Also previously postulated risk factors are checked in replication experiments. The finding of mutations in NEK1 as a novel risk factor points toward cilia formation, DNA-damage response, microtubule stability, neuronal morphology and axonal polarity as potential new disease mechanisms that needs to be followed up in future research. Also, a new approach was postulated towards investigating the exposome, the total of environmental factors. In this study many new environmental risk factors were found and for the first time the associations between risk factors could be determined. This exposome-wide study points towards different and independent environmental risk profiles that all lead to the same disease entity, ALS. It revealed that for instance the risk effect of smoking was independent of the effect of alcohol intake or a beneficial vascular risk profile. This thesis not only gives more insight in the risk factors for ALS, but also provides new insight in disease etiopathogenesis, thereby providing a basis for future treatment development.

Amyotrophic lateral sclerosis (ALS) is an adult-onset, fatal neurodegenerative disease mainly caused by the loss of upper and lower motor neurons, resulting in progressive muscle atrophy and paralysis. Although most ALS cases are sporadic (SALS), ~10% of them are familial (FALS), usually with an autosomal-dominant inheritance pattern. Although genetic studies have partially elucidated the genetic background of the disease, a large part of ALS heritability is still missing. The aim of this thesis was the identification of genetic risk factors in ALS, and to understand their role in shaping the clinical phenotype. To achieve this goal, we initially screened a large cohort of FALS and SALS patients of Italian descent for mutations in the known ALS genes FUS, ATXN2 and PFN1. We established a mutational frequency for FUS of ~5% in familial cases, and described a novel phenotype associated with mutations within the nuclear localization signal of the gene. We confirmed the role of ATXN2 intermediate repeats in SALS susceptibility and refined the CAG-repeat range associated with the disease. We observed that PFN1 mutations are extremely rare in sporadic cases and probably do not play a role in FTD pathogenesis. We then aimed to assess whether mutations in known ALS genes are associated to the appearance of extramotor phenotypes within motor neuron diseases, or contribute to the pathogenesis of neurodegenerative disorders other than ALS and FTD. Our results suggest that rare variants in the ANG gene, previously associated with ALS, also confer a susceptibility to idiopathic Parkinson’s disease (PD). We observed an increased presence of oligoclonal bands in the cerebrospinal fluid (CSF) of ALS patients carrying ANG and TARDBP mutations, suggesting a possible link with inflammatory diseases of the central nervous system. We also confirmed an association of c9orf72 (G4C2)n with ALS-plus syndromes, although we did not find evidence of a pathogenic role in other neurodegenerative disorders beyond TDP-43 protheinopathies. Lastly, we aimed to identify novel causative genes in our ALS cohort, using different methodological approaches. We initially relied upon the candidate gene approach to screen a cohort of unrelated FALS index cases for mutations in TAF15 and EWS, selected because of their homology with FUS, and in the PON cluster, encoding for a family of proteins involved in detoxifying exogenous toxics. Although our results initially suggested an association of TAF15 and PON1-3 with ALS, subsequent evidence has casted significant doubt over these and other genes identified through the candidate gene approach. Faced with these pitfalls, we were forced to adopt a different strategy for gene hunting in ALS, and resorted to an exome-wide, case-control, RVB analysis. The validity of such strategy is highlighted by the identification of the two novel ALS genes TUBA4A and NEK1. Genetic studies in ALS have by far given the largest contribution to our understanding of the pathogenesis of the disease, and it is the hope that further advancements in the field will eventually lead to the identification of therapeutic targets toward finding a cure for ALS.

The aim of this thesis was to identify new genetic susceptibility factors, describe the genetic architecture of ALS and explore the utility of gene expression profiles as a biomarker for ALS. Following the technical advances over time, this thesis includes single gene studies, genome-wide associations studies (GWAS), transcriptome analyses and whole genome sequencing studies. Leveraged together, they have led to several conclusions and perspectives.

In order to study the topology of functional networks, functional connectome maps are often thresholded after which only the strongest functional connections remain for analysis. The experiments described in chapter 2 show that graph metrics computed on proportionally thresholded functional connectomes, effectively filtered to keep a fixed number of strongest connections, are heavily influenced by the overall level of functional connectivity of a subject. In patient-control investigations, a difference in overall functional connectivity between groups is shown to interfere with topological effects. In chapter 4 functional connections were included for analysis if the corresponding region pair was also anatomically directly connected in the structural connectome. The integrated structural and functional connectome analyses of chapter 4 revealed overlapping structural and functional connectivity impairment among direct connections of the motor cortex. Connections more distant from the motor cortex showed structural effects but were functionally mostly spared, possibly indicative of functional effects to be secondary to primary structural connectivity disease effects. Chapter 3 touches upon structure-function relationships of brain networks taking a theoretical approach, simulating functional connectivity as synchrony between regions structurally connected according to the white matter connectome. Anatomical hub regions are shown to have a leading role in establishing synchrony between regions of different functional modules. Introducing structural connectivity impairments to these regions resulted in widespread functional effects hindering global integration in the brain network. These simulation findings, although based on a theoretical model, hugely simplifying neural communication, may help us understand how functional effects could arise from (disease-related) structural connectivity impairments. In a unique cohort of a family of asymptomatic C9orf72 mutation carriers and noncarriers, chapter 5 shows that before any clinical signs of ALS, mutation carriers did reveal reduced cortical thickness as compared with the noncarriers. Strikingly, no white matter involvement was detected in the carriers and cortical thinning was observed not in the anticipated motor cortex, but in temporal and parietal regions. These findings may represent developmental effects that predispose carriers to the neurodegenerative processes underlying ALS later in life. Furthermore, these results are in support of the ALS neuroimaging signature to develop close to the onset of disease as suggested in other studies. In chapter 6 it is argued that the healthy connectome governs patterns of spread across the brain of pathogenic protein aggregates in ALS, showing stages of neuropathology can be predicted from connectome topology. A similar relationship between the wiring of the healthy connectome and stages of neuropathology in bvFTD, Alzheimer and Parkinson was observed (chapter 7). With ALS and all three aforementioned neurodegenerative disorders belonging to the so-called ‘proteinopathies’, a class of protein misfolding diseases, the white matter connectome may be a crucial infrastructure in proteinopathies facilitating disease propagation across the brain. Taken together, the findings presented in this thesis relate to a range of ALS imprints on the brain including structural and functional connectivity impairment, cortical thinning in the asymptomatic phase of C9orf72 repeat expansion carriers and spread patterns of misfolded protein aggregation following the network organization of the white matter connectome.

In this thesis, we focus our biomarker discovery on gene expression in blood of an ALS mouse model and ALS patients to find ALS specific gene activity. 1. To explore the potential of blood or muscle transcriptome changes as a diagnostic biomarker. We hypothesized that blood gene expression reflects gene expression in the brain and in motor neurons. We used an SOD1 mouse model overexpressing human mutant SOD1-G93A to compare differentially expressed genes in spinal cord with differential expression in blood and muscle (Chapter 3). Next we analyzed gene expression in blood of sporadic ALS patients and healthy controls and constructed groups of co-differentially, co-expressed genes (Chapter 5). From the finding of altered gene activity in ALS patients, we profiled a larger dataset and constructed a panel that can discriminate with high accuracy between ALS patients and healthy controls (Chapter 6). 2. To find genetic variation associated with ALS and controlling gene expression in blood of ALS patients We performed a two-stage, genome-wide association study (GWAS) to find genetic risk factors associated with ALS (Chapter 7). Combining the gene expression data with genetic variation of sporadic ALS patients made it possible to select genetic variation causing differences in quantitative levels of expression (Chapter 8). This genetical genomics exercise can fine-map previously found linked loci and has the potential of finding new genetic risk factors. 3. To find the underlying pathological pathways in ALS mouse models. The exact pathological mechanism causing motor neuron degeneration is still elusive. We performed a meta-analysis of all published studies using gene expression in ALS mouse models (Chapter 2). Overlap in differential expression might provide important information on common pathological pathways. Next we determined differential gene expression in spinal cord of an SOD1 mouse model as part of our biomarker study (Chapter 3). 4. To find pathways targeted by riluzole. Since riluzole is the only effective drug in ALS, elucidating the mechanism of efficacy of this drug will immediately be relevant to the etiology of this disease. Gene expression profiles of SOD1-G93A mice treated with riluzole were compared to control animals receiving only tap water (Chapter 4). Expression profiles were determined in lumbar spinal cord at pre-symptomatic and early symptomatic ages.

Amyotrophic lateral sclerosis (ALS) is a progressive disease of the motor system involving both upper motor neurons in the brain and lower motor neurons in the spinal cord. Patients suffer from progressive wasting and weakness of limb, bulbar and respiratory muscles. Onset and disease course in ALS is heterogeneous as each patient has its unique combination of upper and lower motor neuron involvement, site of onset and symptom development. In The Netherlands, 400 to 500 persons are diagnosed each year with this destructive disease. ALS can occur at any age during adulthood with a median age of onset of 63 years and a median survival of three years. This thesis consists of three parts 1) disease spread, 2) biomarkers and 3) clinical trials. 1) Up until now, little is known about how ALS progressively affects the motor system. We assessed spread of disease in ALS both clinically as by examining the changes in the brain. Symptom development was found to be not random, but with preferred spread to the opposite limb. Using MRI techniques, mainly DTI, we reconstructed the brain network. Comparing patients with healthy controls, we found a subnetwork of impaired connectivity in ALS. This impaired subnetwork shows large overlap with the motor network in healthy controls. Over time, we found this impaired subnetwork was expanding including more and more brain regions. Our findings in the first part of this thesis are in support of disease spread guided by the brain’s network structure. These findings might provide new targets for disease modifying treatments. 2) Upper motor neuron involvement is currently assessed on clinical examination only, while it is known that patients can have subclinical involvement as well. An objective marker for upper motor neuron involvement would facilitate and improve diagnosis. We used several MRI techniques to assess the integrity of the upper motor neuron in ALS. The main finding from these studies was a significantly reduced cortical thickness in primary motor regions (precentral gyrus) in ALS, suggesting this measure might be an objective marker for upper motor neuron involvement. 3) Only one drug – riluzole – has proved effective in extending the lifespan of patients with ALS, and it does so by only 3–6 months. A better treatment is urgently needed. In 2008 a pilot study showed a favourable effect of lithium in a transgenic mouse model of ALS and in an open-label study in 44 patients. To examine the true efficacy and safety of lithium in ALS, we performed a randomised, placebo controlled, clinical trial with sequential analysis. Unfortunately our results did not lend support for a beneficial effect of lithium on survival, nor disease progression.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease, for which there is no adequate therapy. The majority of ALS patients are sporadic (i.e. they have no relatives with the disease). Sporadic ALS is considered to be a complex disease that is the result of an interplay of genetic and environmental risk factors, which act together to cause disease. Identifying the implicated genes will lead to a better understanding of the pathogenesis and will facilitate the development of novel therapies. This thesis describes several genetic studies that were aimed to identify genetic risk factors that are associated with ALS susceptibility, including genome-wide association studies (GWAS) to identify associated single-nucleotide polymorphisms (SNPs) and candidate gene studies, investigating the role of suspected genes. The focus of this thesis was the role of copy-number variation as a source of increased risk for ALS. We studied this in a genome-wide manner performed association analyses on rare and common CNVs. We identified several novel genetic loci that confer an increased risk for ALS, including a locus on chromosome 9p and UNC13A. Additionally, we found suggestive evidence for an association of NIPA1 deletions with an increased risk for ALS. We then examined this gene in more detail in a follow-up study. This study showed that expansions of a polyalanine repeat in this gene are associated with a higher risk of ALS and, in addition, with a shorter survival and with a lower age at onset of the disease. In another candidate gene study, we examined the role of SMN1, the causative gene for spinal muscular atrophy, a congenital motor neuron disease with some familiarities with ALS. Previous studies have shown that CNVs in this gene are associated with ALS, but results were contradictory. We found firm evidence for an association of SMN1 duplications and ALS susceptibility. We found no association with SMN1 deletions, which has been suggested previously. In conclusion, this thesis describes several novel risk loci for ALS, together with firm evidence for the implication of the previously suspected gene SMN1 in ALS pathogenesis. These findings are a starting point for functional studies investigating the underlying pathogenic pathways and will hopefully lead to better treatment strategies for this devastating disease

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease of motor neurons leading to progressive weakness of the limbs, the bulbar muscles and the respiratory muscles. Fifty percent of patients die within 3 years after onset of symptoms, mainly due to respiratory failure. In a large, prospective, population-based study we calculated the incidence and prevalence of ALS in the Netherlands using the capture-recapture methodology. The annual incidence rate was 2.77 per 100,000 person years. ALS is considered to be a multifactorial disease. Several molecular mechanisms have been described to mediate motor neuron death. We investigated several possible risk factors in ALS, studied in a large prospective, population-based case-control study in the Netherlands between 2006 and 2010. Cigarette smoke could increase the risk of developing ALS through several mechanisms, including inflammation, oxidative stress, and neurotoxicity caused by heavy metals and other chemical compounds present in cigarette smoke. Our study showed that cigarette smoking is, indeed, independently associated with an increased risk of ALS and alcohol consumption is independently associated with a reduced risk of ALS. Current smoking is also associated with a worse prognosis. ALS is more prevalent in men. This suggests a possible neuroprotective effect of female reproductive hormones. We concluded that a longer reproductive time-span, which may be a proxy for longer exposure to female productive hormones, was found to be independently associated with a decreased risk of ALS and with a prolonged survival of ALS patients. An association with increased parental age might suggest there is a role for specific (epi)genetic changes, however in our large study, parental age was not associated with an increased risk of ALS. Familial aggregation of ALS with neurodegenerative diseases such as Parkinson’s disease or dementia, could suggest shared genetic or environmental risk factors.However, in our study no familial aggregation of ALS with other neurodegenerative diseases could be established. On the other hand, the lowered risk of vascular diseases in relatives of ALS patients supports the hypothesis that a beneficial vascular risk profile is associated with increased susceptibility for ALS. Supportive of this hypothesis, a higher level of leisure time physical activity was found in our study . Diagnosing ALS remains difficult because of the lack of a reference test with a high positive predictive value. The aim of the revisions of the different sets of diagnostic criteria was to enhance clinical research, therapeutic trials and molecular genetic studies. Our study showed that the revised El Escorial criteria as well as the Awaji algorithm, still exclude a large percentage of the patients for clinical trials at time of diagnosis. A higher copy number of the SMN2 gene is associated with a more favorable disease course in ALS patients. Increasing the survival motor neuron (SMN) protein expression by enhancing SMN2 transcription by valproic acid could therefore modify the disease course in ALS patients. In our randomized, placebo-controlled, double-blind, clinical trial, valproic acid, at a dose used in epilepsy, as an adjunct to riluzole, did not improve survival in ALS

Each year approximately 400 patients are diagnosed with amyotrophic lateral sclerosis (ALS) in The Netherlands.Thesepatients are ~65 years of age at time of diagnosis.They develop progressive muscleweakness, frequently affecting their arms, legs and trunk, but also muscles involved in speech, swallowing and breathing. ALS patients usually die within three years due to respiratory failure. There is no cure for ALS and only Riluzole prolongs survival with two to three months. The pathogenesis of ALS is complex and involves both genetic and environmentalfactors. In ~5% of the cases more than one family member is affected. These patients are diagnosed with familial ALS (FALS). In the first part of this thesis, we will discuss eleven ALS-associated genes. The next part of this thesis will address other neurodegenerative diseases, including progressive muscular atrophy (PMA) and Parkinson’s disease (PD). Finally, we will describe several functional studies, investigating the effects of genetic abnormalities in ALS-associated genes.

The main focus of this thesis is to identify susceptibility factors in diseases affecting the motor neuron: both motor neuron disease (MND), in which primarily the cell body is affected, and multifocal motor neuropathy (MMN), in which primarily the axon is affected, are covered. Due to its relentless course the most notorious of this spectrum of disorders is amyotrophic lateral sclerosis (ALS), in which both the lower and upper motor neurons are affected causing progressive muscle weakness. The course of the disease is heterogeneous and varies from patient to patient. As ALS cannot be cured, mean disease duration is 3 years when progressive weakness of the respiratory muscles ultimately leads to death of the patient. ALS does occur in families, but the majority concerns a sporadic form. Sporadic ALS is considered a multifactorial disease in which environmental factors in a genetically susceptible host cause motor neuron degeneration. Both the purely lower motor neuron (LMN) and the upper motor neuron (UMN) variants are heterogeneous, although disease progression is generally slower. These variants may represent the same disease entity and the heterogeneity can be used to identify risk factors. MMN is characterized by the presence of progressive, asymmetric, predominantly distal, atrophy and weakness. Its clinical features can mimic MND. Treatment with intravenously applied immunoglobulin improves muscle strength. A multifactorial etiology is implied with a large role for immune-mediated mechanisms which cause demyelinisation of motor nerves. The aim of this thesis was to identify genetic, environmental and lifestyle factors which increase susceptibility to MND and MMN. Proposed disease mechanisms covered in this thesis are oxidative stress, vascularisation, immune-mediated mechanisms and hypermetabolism. Several candidate gene studies were studied. A positive association between ALS and HFE H63D seems to be population-wide, but the association with VEGF could not be consistently detected in different populations. Studies on occupational risk factors and exposure to chemical agents and metals could not show strong evidence in favor of a specific risk factor for ALS and emphasizes the need for future well-designed studies. Based on findings from this thesis as well as newly published studies, smoking seems a consistently associated risk factor for ALS. In contrary to our hypothesis, vascular risk factors did not increase ALS risk. On the other hand, low lipid status seemed to be associated with increased risk for ALS. HLA-DRB1*15 was associated with MMN; monoclonal immunoglobulin was associated with MMN and slowly progressive muscular atrophy, but not the other forms of MND. These findings suggest a possible role for oxidative stress and hypermetabolism in ALS pathogenesis and immune-mediated mechanisms to play a role in MMN, but also some variants of MND. Future studies are needed to elucidate these findings and suggestions are made for study design.

Amyotrophic lateral sclerosis (ALS), a form of motor neuron disease, is a fatal neurodegenerative disease characterised by the selective loss of motor neurons in the cortex, brainstem and spinal cord. Patients suffer from progressive wasting and weakness of limb, bulbar and respiratory muscles, and die on average within 3 years of symptom onset, usually because of respiratory failure. Although ALS can occur anytime during adulthood, the median age of onset is in the mid-fifties. The only therapeutic strategy to slow progression of ALS is currently riluzole, which delays disease development by 3 to 6 months. Global incidence of ALS is about 1 – 2 per 100,000 and lifetime risk of developing ALS is estimated to be 1/600 – 1/2000, which makes it the most common motor neuron disease. About 10% of patients have a family history of ALS (FALS). Over ten different subtypes of FALS are distinguished and mutation in SOD1, ANG, FUS, VAPB and TARDBP have identified. The remaining 90% of ALS cases are sporadic, and are thought to be multifactorial, with both environmental and genetic components contributing to disease susceptibility. On the basis of concordance rates in twin studies, estimates of the heritability of ALS range from 0.38 to 0.85. The aim of this thesis is to confirm and identify novel genetic risk factors for familial and sporadic ALS by performing candidate gene studies and genome-wide association studies. Mutation analysis in Dutch familial ALS patients demonstrated that SOD1 mutations (found in approximately 20% of familial ALS patients) are very rare in The Netherlands, demonstrating genetic heterogeneity of the disease. We further demonstrate segregation of the ANG K17I mutation with disease in a pedigree with FALS. One affected family member was initially diagnosed with Parkinson’s disease, but went on to develop ALS and frontotemporal dementia. This thesis further describes multiple genome-wide association studies in sporadic ALS, in which genetic variation in ITPR2, DPP6, UNC13A and a locus on chromosome 9p are identified as susceptibility factors. We also show that common genetic variation in KIFAP3 is associated with prolonged survival in ALS. Patients who are homozygous for the associated allele have 14.0 month survival advantage. Using a candidate gene approach we show that rare mutation in ANG confer a large risk for both ALS and Parkinson’s disease (odds ratio > 25.0). We also show elevated serum levels in of angiogenin in ALS, but not PD patients.

Primary lateral sclerosis (PLS) is a diagnosis of exclusion in sporadic patients with an adult-onset gradually progressive upper motor neuron (UMN) syndrome. Differentiation between PLS, amyotrophic lateral sclerosis (ALS), and sporadic presentation of hereditary spastic paraparesis (HSP) is important for prognostication and genetic counseling of patients, but may be problematic. We studied a large cohort of Dutch patients with an sporadic adult-onset UMN syndrome in a 3-year prospective study. Some of our findings support the view that PLS may be a variant of ALS: we observed patients who converted from PLS to ALS, patients who had PLS as a phenotypic manifestation of familial ALS, and a patient who had clinical PLS up to the time of death, but who at autopsy showed ALS pathology. On the other hand, many patients retained a phenotype similar to HSP (UMN signs only in the legs, or at most also in the arms). In addition, we could genetically confirm the diagnosis of HSP in a proportion of the included patients. We found that spastin gene (SPG4) and paraplegin gene (SPG7) mutations are frequent cause of patients with UMN symptoms restricted to the legs (both in around 12% of the patients), but mutations were not found in patients with symptoms in the bulbar region (typical PLS phenotype). Differentiating (sporadic) HSP from PLS based on other clinical characteristics was unreliable and therefore depends on genetic testing. Based on our observations and recent studies by other groups, we designed new diagnostic criteria for PLS, incorporating levels of certainty of diagnosis of PLS instead of HSP based on the distribution of UMN symptoms over body region. Our 3-year prospective study validated that classifying patients with a disease duration ?4 years is able to differentiate PLS from HSP in levels of certainty: typical PLS (UMN signs in at least the bulbar region), probable PLS (UMN signs in arms and legs), and possible PLS (UMN signs only in the legs). A disease duration

One in five amyotrophic lateral sclerosis (ALS) patients die due to euthanasia and physician-assisted suicide (EAS) between 1994 and 1998 in the Netherlands. This relative high proportion of EAS in ALS might be the result of inadequate supportive or palliative care, unrecognized depressions, patients feeling they were a burden on others or hopelessness. This thesis examines the rate of EAS in ALS between 2000 and 2008 in the Netherlands and explores the characteristics of end-stage ALS patients. Furthermore, it studies the determinants of requesting for EAS among terminally ill ALS patients, focusing on symptoms of depression, palliative care characteristics, and personal traits. To study these objectives, we translated and validated the 40-item and 5-item Amyotrophic Lateral Sclerosis Assessment Questionnaire (ALSAQ-40, ALSAQ-5) to measure the health-related quality of life of ALS patients. The results in this thesis show that approximately 20% of the ALS patients die due to EAS. This rate was similar in two retrospective studies performed in the UMC Utrecht between 1994-1998 and between 2000-2005 and one prospective study between 2003-2008. The data in this thesis also showed no significant association between symptoms of a depression and EAS according to the ALS patients, the informal caregivers, and the physicians of ALS patients. Thirteen percent of the patients who requested EAS reported that they felt depressed or had diminished interest or pleasure in all, or almost all, activities for at least two weeks for most of the day; the physicians reported that 11% of the patients who received EAS had a depression or symptoms of a depression; and of the informal caregivers 35% reported that the patient who received EAS felt depressed or lost interest for two weeks for most of the day. Even when death approached, a statistical significant increase in symptoms of depression was not observed. EAS was associated with religion not being important to the patient, more years of education, dying at home and feeling hopeless. The data did not show that patients who requested EAS reported significantly more often dissatisfaction with their health care or financial situation than patients who did not requested EAS. Approximately 2 months before the patients’ death, 84% of all patients thought that the health care in general was good or excellent. Albeit most patients were satisfied with the care, there is still room for improvement.