Genome-Wide and Transcriptome-Wide Association Studies on Northern New England and Ohio Amyotrophic Lateral Sclerosis Cohorts.

Background and Objectives: Amyotrophic lateral sclerosis (ALS) is an age-associated, fatal neurodegenerative disorder causing progressive paralysis and respiratory failure. The genetic architecture of ALS is still largely unknown. Methods: We performed a genome-wide association study (GWAS) and transcriptome-wide association study (TWAS) to understand genetic risk factors for ALS using a population-based case-control study of 435 ALS cases and 279 controls from Northern New England and Ohio. Single nucleotide polymorphism (SNP) genotyping was conducted using the Illumina NeuroChip array. Odds ratios were estimated using covariate-adjusted logistic regression. We also performed a genome-wide SNP-smoking interaction screening. TWAS analyses used PrediXcan to estimate associations between predicted gene expression levels across 15 tissues (13 brain tissues, skeletal muscle, and whole blood) and ALS risk. Results: GWAS analyses identified the p.A382T missense variant (rs367543041, p = 3.95E-6) in the TARDBP gene, which has previously been reported in association with increased ALS risk and was found to share a close affinity with the Sardinian haplotype. Both GWAS and TWAS analyses suggested that ZNF235 is associated with decreased ALS risk. Discussion: Our results support the need for future evaluation to clarify the role of these potential genetic risk factors for ALS and to understand genetic susceptibility to environmental risk factors.

A new method for estimating under-recruitment of a patient registry: a case study with the Ohio Registry of Amyotrophic Lateral Sclerosis.

We developed a disease registry to collect all incident amyotrophic lateral sclerosis (ALS) cases diagnosed during 2016-2018 in Ohio. Due to incomplete case ascertainment and limitations of the traditional capture-recapture method, we proposed a new method to estimate the number of cases not recruited by the Registry and their spatial distribution. Specifically, we employed three statistical methods to identify reference counties with normal case-population relationships to build a Poisson regression model for estimating case counts in target counties that potentially have unrecruited cases. Then, we conducted spatial smoothing to adjust outliers locally. We validated the estimates with ALS mortality data. We estimated that 119 total cases (95% CI [109, 130]) were not recruited, including 36 females (95% CI [31, 41]) and 83 males (95% CI [74, 99]), and were distributed unevenly across the state. For target counties, including estimated unrecruited cases increased the correlation between the case count and mortality count from r = 0.8494 to 0.9585 for the total, from 0.7573 to 0.8270 for females, and from 0.6862 to 0.9292 for males. The advantage of this method in the spatial perspective makes it an alternative to capture-recapture for estimating cases missed by disease registries.

Long-term survival analysis of masitinib in amyotrophic lateral sclerosis.

BACKGROUND: A randomized, placebo-controlled phase III study (AB10015) previously demonstrated that orally administered masitinib (4.5 mg/kg/day) slowed rate of functional decline, with acceptable safety, in amyotrophic lateral sclerosis (ALS) patients having an ALS Functional Rating Scale-revised (ALSFRS-R) progression rate from disease onset to baseline of <1.1 points/month. Here we assess long-term overall survival (OS) data of all participants from study AB10015 and test whether a signal in OS is evident in an enriched patient population similar to that prospectively defined for confirmatory study AB19001. METHODS: Survival status of all patients originally randomized in AB10015 was collected from participating investigational sites. Survival analysis (using the multivariate log-rank test and Cox proportional hazards model, with stratification factors as covariates) was performed on the intention-to-treat population and enriched subgroups, which were defined according to initial randomization, baseline ALSFRS-R progression rate and baseline disease severity. RESULTS: A significant survival benefit of 25 months (p = 0.037) and 47% reduced risk of death (p = 0.025) was observed for patients receiving 4.5 mg/kg/day masitinib (n = 45) versus placebo (n = 62) in an enriched cohort with ⩾2 on each baseline ALSFRS-R individual component score (i.e. prior to any complete loss or severe impairment of functionality) and post-onset ALSFRS-R progression rate <1.1 (i.e. exclusion of very fast progressors) [median OS of 69 versus 44 months, respectively; hazard ratio, 0.53 [95% CI (0.31-0.92)]]. This corresponds to the population enrolled in confirmatory phase III study, AB19001. CONCLUSIONS: Analysis of long-term OS (75 months average follow-up from diagnosis) indicates that oral masitinib (4.5 mg/kg/day) could prolong survival by over 2 years as compared with placebo, provided that treatment starts prior to severe impairment of functionality.This trial was registered at www.ClinicalTrials.gov under identifier NCT02588677 (28 October 2015).

Amyotrophic Lateral Sclerosis Risk, Family Income, and Fish Consumption Estimates of Mercury and Omega-3 PUFAs in the United States.

Most amyotrophic lateral sclerosis (ALS) cases are considered sporadic, without a known genetic basis, and lifestyle factors are suspected to play an etiologic role. We previously observed increased risk of ALS associated with high nail mercury levels as an exposure biomarker and thus hypothesized that mercury exposure via fish consumption patterns increases ALS risk. Lifestyle surveys were obtained from ALS patients (n = 165) and n = 330 age- and sex-matched controls without ALS enrolled in New Hampshire, Vermont, or Ohio, USA. We estimated their annual intake of mercury and omega-3 polyunsaturated fatty acid (PUFA) via self-reported seafood consumption habits, including species and frequency. In our multivariable model, family income showed a significant positive association with ALS risk (p = 0.0003, adjusted for age, sex, family history, education, and race). Neither the estimated annual mercury nor omega-3 PUFA intakes via seafood were associated with ALS risk. ALS incidence is associated with socioeconomic status; however, consistent with a prior international study, this relationship is not linked to mercury intake estimated via fish or seafood consumption patterns.

The Incidence of Amyotrophic Lateral Sclerosis in Ohio 2016-2018: The Ohio Population-Based ALS Registry.

INTRODUCTION: Amyotrophic lateral sclerosis (ALS) is a fatal, neuromuscular disease with no cure. ALS incidence rates have not been assessed specifically in Ohio, yet the state contains both metropolitan and rural areas with a variety of environmental factors that could contribute to disease etiology. We report the incidence of ALS in Ohio residents diagnosed from October 2016 through September 2018. METHODS: We engaged practitioners from 9 Ohio sites to identify newly diagnosed ALS patients and to complete case report forms with demographic and clinical information. ALS was diagnosed according to the Awaji criteria and classified as either definite, probable, or possible. We developed a method to estimate missing cases using a Poisson regression model to impute cases in counties with evidence of undercounting. RESULTS: We identified 333 newly diagnosed ALS patients residing in Ohio during the 2-year index period and found incidence rates varied in the 88 state counties. After incorporating the estimated 27% of missing cases, the corrected crude annual incidence was 1.96/100,000 person-years, and the age- and gender-standardized incidence was 1.71/100,000 person-years (standardized to the 2010 US census). DISCUSSION/CONCLUSION: The estimated Ohio incidence of ALS is overall similar to that reported in other states in the USA. This study reveals a geospatial variation in incidence within the state, and areas with higher rates warrant future investigation.

Risk factors for amyotrophic lateral sclerosis: A regional United States case-control study.

Most amyotrophic lateral sclerosis (ALS) cases are considered sporadic, without a known genetic basis, and environmental exposures are thought to play a causal role. To learn more about sporadic ALS etiology, we recruited n = 188 ALS patients from northern New England and Ohio and matched controls 2:1 from the general population of the same regions. Questionnaires evaluated the association between a variety of lifestyle, behavioral (ie, hobbies and activities), and occupational factors and the risk of ALS, including the duration of time between exposure and ALS onset, and exposure frequency. Head trauma was associated with increased ALS risk (adjusted odds ratio [OR] 1.60 95% confidence interval [CI] 1.04-2.45), with significantly greater effects for injuries occurring 10 or more years prior to symptom onset (P = .037). ALS risk was increased for those reporting severe electrical burns (adjusted OR 2.86, 95% CI 1.37-6.03), with odds ratios highest for burns after age 30 (OR 3.14), and for burns 10 or more years prior to symptom onset (OR 3.09). Hobbies involving lead were the most strongly associated with ALS risk (adjusted OR 2.92, 95% CI 1.45-5.91). Exposures to lead 20 or more years prior to diagnosis had larger effect sizes compared to those occurring more recently. Holding a job in mechanics, painting, or construction was associated with ALS. The identification of these specific environmental factors associated with ALS highlight the need for future prospective and laboratory studies to assess causality, biological mechanisms, and find prevention or treatment opportunities.

l-Serine Reduces Spinal Cord Pathology in a Vervet Model of Preclinical ALS/MND.

The early neuropathological features of amyotrophic lateral sclerosis/motor neuron disease (ALS/MND) are protein aggregates in motor neurons and microglial activation. Similar pathology characterizes Guamanian ALS/Parkinsonism dementia complex, which may be triggered by the cyanotoxin ß-N-methylamino-l-alanine (BMAA). We report here the occurrence of ALS/MND-type pathological changes in vervets (Chlorocebus sabaeus; n = 8) fed oral doses of a dry powder of BMAA HCl salt (210 mg/kg/day) for 140 days. Spinal cords and brains from toxin-exposed vervets were compared to controls fed rice flour (210 mg/kg/day) and to vervets coadministered equal amounts of BMAA and l-serine (210 mg/kg/day). Immunohistochemistry and quantitative image analysis were used to examine markers of ALS/MND and glial activation. UHPLC-MS/MS was used to confirm BMAA exposures in dosed vervets. Motor neuron degeneration was demonstrated in BMAA-dosed vervets by TDP-43+ proteinopathy in anterior horn cells, by reactive astrogliosis, by activated microglia, and by damage to myelinated axons in the lateral corticospinal tracts. Vervets dosed with BMAA + l-serine displayed reduced neuropathological changes. This study demonstrates that chronic dietary exposure to BMAA causes ALS/MND-type pathological changes in the vervet and coadministration of l-serine reduces the amount of reactive gliosis and the number of protein inclusions in motor neurons.

Cyanobacterial neurotoxin BMAA and brain pathology in stranded dolphins.

Dolphin stranding events occur frequently in Florida and Massachusetts. Dolphins are an excellent sentinel species for toxin exposures in the marine environment. In this report we examine whether cyanobacterial neurotoxin, ß-methylamino-L-alanine (BMAA), is present in stranded dolphins. BMAA has been shown to bioaccumulate in the marine food web, including in the muscles and fins of sharks. Dietary exposure to BMAA is associated with the occurrence of neurofibrillary tangles and ß-amyloid plaques in nonhuman primates. The findings of protein-bound BMAA in brain tissues from patients with Alzheimer's disease has advanced the hypothesis that BMAA may be linked to dementia. Since dolphins are apex predators and consume prey containing high amounts of BMAA, we examined necropsy specimens to determine if dietary and environmental exposures may result in the accumulation of BMAA in the brains of dolphins. To test this hypothesis, we measured BMAA in a series of brains collected from dolphins stranded in Florida and Massachusetts using two orthogonal analytical methods: 1) high performance liquid chromatography, and 2) ultra-performance liquid chromatography with tandem mass spectrometry. We detected high levels of BMAA (20-748 µg/g) in the brains of 13 of 14 dolphins. To correlate neuropathological changes with toxin exposure, gross and microscopic examinations were performed on cortical brain regions responsible for acoustico-motor navigation. We observed increased numbers of ß-amyloid+ plaques and dystrophic neurites in the auditory cortex compared to the visual cortex and brainstem. The presence of BMAA and neuropathological changes in the stranded dolphin brain may help to further our understanding of cyanotoxin exposure and its potential impact on human health.

Toenail mercury Levels are associated with amyotrophic lateral sclerosis risk.

INTRODUCTION: Mercury is a neurotoxic metal that is potentially a risk factor for amyotrophic lateral sclerosis (ALS). Consumption of methylmercury contaminated fish is the primary source of US population exposure to mercury. METHODS: We used inductively coupled plasma mass spectrometry to measure levels of mercury in toenail samples from patients with ALS (n = 46) and from controls (n = 66) as a biomarker of mercury exposure. RESULTS: Patients with ALS had higher toenail mercury levels (odds ratio 2.49, 95% confidence interval 1.18-5.80, P = 0.024) compared with controls, adjusted for age and sex. We also estimated the amount of mercury consumed from finfish and shellfish and found toenail mercury levels elevated overall among patients with ALS and controls in the top quartile for consumption (P = 0.018). DISCUSSION: Biomarker data show that ALS is associated with increased with mercury levels, which were related to estimated methylmercury intake via fish. Replication of these associations in additional populations is warranted. Muscle Nerve, 2018.

Gene-Environment-Time Interactions in Neurodegenerative Diseases: Hypotheses and Research Approaches.

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.

Studies of Environmental Risk Factors in Amyotrophic Lateral Sclerosis (ALS) and a Phase I Clinical Trial of L-Serine.

ß-N-Methylamino-L-alanine (BMAA) has been linked to Guam ALS/PDC and shown to produce neurodegeneration in vitro and in vivo (Drosophila, mice, rats, primates). BMAA misincorporation into neuroproteins produces protein misfolding and is inhibited by L-serine. Case-control studies in Northern New England indicate that living near to water-bodies with cyanobacterial blooms increases the risk of developing amyotrophic lateral sclerosis (ALS). The distribution of addresses of ALS cases in New Hampshire, Vermont, and Florida was compared to that of controls. Areas of statistically significantly increased numbers of ALS cases were examined for sources of environmental toxins. A phase I trial of oral L-serine was performed in 20 ALS patients (0.5 to 15 g twice daily). Safety and tolerability were assessed by comparing the rate of deterioration with 430 matched placebo controls. The distribution of residential addresses of ALS cases in New England and Florida revealed many areas where the age- and gender-adjusted frequency of ALS was greater than expected (P < 0.01). GIS studies of these "hot spots" in relation to sources of environmental pollutants, like cyanobacterial blooms, Superfund and Brownfield sites, and landfills, are ongoing. In the phase I trial of L-serine, two patients withdrew from because of gastrointestinal side effects. Three patients died during the study, which was about the expected number. The ALSFRS-R in the L-serine-treated patients showed a dose-related decrease in the rate of progression (34% reduction in slope, P = 0.044). The non-random distribution of addresses of ALS patients suggests that residential exposure to environmental pollutants may play an important role in the etiology of ALS. L-Serine in doses up to 15 g twice daily appears to be safe in patients with ALS. Exploratory studies of efficacy suggested that L-serine might slow disease progression. A phase II trial is planned.

Medical history of chemotherapy or immunosuppressive drug treatment and risk of amyotrophic lateral sclerosis (ALS).

A recent population-based analysis demonstrated lower risk of the lethal degenerative neuromuscular disease, amyotrophic lateral sclerosis (ALS) associated with history of the use of 'antineoplastic agents' and 'immunosuppressants'. To see if this finding was generalizable to other ALS cohorts, we examined associations between use of these agents and ALS risk in an independent case-control study of n = 414 ALS patients and n = 361 controls in an Eastern US population. Controls were sampled from the general population and among non-neurodegenerative disease patients. A history of chemotherapy treatment was significantly associated with a decreased ALS risk (OR 0.46, 95% CI 0.22-0.89, P = 0.026). We did not observe an association between risk of ALS and immunosuppressant therapy use (OR 0.78, 95% CI 0.50-1.02, P = 0.23). Analyses were adjusted for age, gender, and smoking. Our results support the prior report for chemotherapy treatment and lead to further discussion of the underlying mechanism.

Environmental and Occupational Exposures and Amyotrophic Lateral Sclerosis in New England.

BACKGROUND: Recent data provide support for the concept that potentially modifiable exposures are responsible for sporadic amyotrophic lateral sclerosis (ALS). OBJECTIVE: To evaluate environmental and occupational exposures as risk factors for sporadic ALS. METHODS: We performed a case-control study of ALS among residents of New England, USA. The analysis compared questionnaire responses from 295 patients with a confirmed ALS diagnosis to those of 225 controls without neurodegenerative illness. RESULTS: Self-reported job- or hobby-related exposure to one or more chemicals, such as pesticides, solvents, or heavy metals, increased the risk of ALS (adjusted OR 2.51; 95% CI 1.64-3.89). Industries with a higher toxicant exposure potential (construction, manufacturing, mechanical, military, or painting) were associated with an elevated occupational risk (adjusted OR 3.95; 95% CI 2.04-8.30). We also identified increases in the risk of ALS associated with frequent participation in water sports, particularly waterskiing (adjusted OR 3.89; 95% CI 1.97-8.44). Occupation and waterskiing both retained independent statistical significance in a composite model containing age, gender, and smoking status. CONCLUSIONS: Our study contributes to a growing body of literature implicating occupational- and hobby-related toxicant exposures in ALS etiology. These epidemiologic study results also provide motivation for future evaluation of water-body-related risk factors.

Phase I clinical trial of safety of L-serine for ALS patients.

We performed a randomized, double-blind phase I clinical trial for six months on the effects of oral L-serine in patients with ALS. The protocol called for enrollment of patients with a diagnosis of probable or definite ALS, age 18-85 years, disease duration of less than three years and forced vital capacity (FVC) ≥ 60%. Patients were randomly assigned to four different oral twice-daily dose regimens (0.5, 2.5, 7.5, or 15 g/dose). Blood, urine and CSF samples, ALS Functional Rating Scale-Revised (ALSFRS-R) scores and forced vital capacity (FVC) were obtained throughout the trial. Disease progression was compared with matched historical placebo controls from five previous ALS therapeutic trials. Of 20 patients enrolled, one withdrew before receiving study drug and two withdrew with gastro-intestinal problems. Three patients died during the trial. L-serine was generally well tolerated by the patients and L-serine did not appear to accelerate functional decline of patients as measured by slope of their ALSFRS-R scores. Based on this small study, L-serine appears to be generally safe for patients with ALS.

Detection of cyanobacterial neurotoxin ß-N-methylamino-l-alanine within shellfish in the diet of an ALS patient in Florida.

Cyanobacteria produce the neurotoxic amino acid ß-N-methylamino-l-alanine (BMAA), which in contaminated marine waters has been found to accumulate in shellfish. Exposure to BMAA has been associated with an increased risk of neurodegenerative disease. Analysis of blinded samples found BMAA to be present in neuroproteins of individuals who died from ALS and ALS/PDC, but generally not in the brains of patients who died of causes unrelated to neurodegeneration or Huntington's disease, an autosomal dominant neurodegenerative disease. We here report support for a link between a patient with ALS and chronic exposure to the cyanobacterial neurotoxin BMAA via shellfish consumption. The patient had frequently eaten lobsters collected in Florida Bay for approximately 30 years. LC-MS/MS analysis of two lobsters which this ALS patient had placed in his freezer revealed BMAA at concentrations of 27 and 4 µg/g, respectively, as well as the presence of 2,4-diaminobutyric acid (DAB), a BMAA isomer. Two additional lobsters recently collected from Florida Bay also contained the neurotoxins BMAA and DAB. These data suggest that invertebrates collected in water where cyanobacterial blooms are present, if consumed, may result in direct human exposure to these neurotoxic amino acids. The data support the assertion that prolonged exposure to BMAA may have played a role in the etiology of ALS in this patient.

Is exposure to cyanobacteria an environmental risk factor for amyotrophic lateral sclerosis and other neurodegenerative diseases?

There is a broad scientific consensus that amyotrophic lateral sclerosis (ALS) is caused by gene-environment interactions. Mutations in genes underlying familial ALS (fALS) have been discovered in only 5-10% of the total population of ALS patients. Relatively little attention has been paid to environmental and lifestyle factors that may trigger the cascade of motor neuron death leading to the syndrome of ALS, although exposure to chemicals including lead and pesticides, and to agricultural environments, smoking, certain sports, and trauma have all been identified with an increased risk of ALS. There is a need for research to quantify the relative roles of each of the identified risk factors for ALS. Recent evidence has strengthened the theory that chronic environmental exposure to the neurotoxic amino acid ß-N-methylamino-L-alanine (BMAA) produced by cyanobacteria may be an environmental risk factor for ALS. Here we describe methods that may be used to assess exposure to cyanobacteria, and hence potentially to BMAA, namely an epidemiologic questionnaire and direct and indirect methods for estimating the cyanobacterial load in ecosystems. Rigorous epidemiologic studies could determine the risks associated with exposure to cyanobacteria, and if combined with genetic analysis of ALS cases and controls could reveal etiologically important gene-environment interactions in genetically vulnerable individuals.

Pseudobulbar affect: an under-recognized and under-treated neurological disorder.

INTRODUCTION: Pseudobulbar affect (PBA) is a neurologic syndrome of emotional affect disinhibition, characterized by uncontrollable, exaggerated, and often inappropriate emotional outbursts, which may cause severe distress, embarrassment, and social dysfunction. However, the US prevalence of PBA remains unknown. METHODS: An online survey was conducted primarily to estimate the US prevalence of PBA in patients with the six most commonly associated conditions: Alzheimer's disease, amyotrophic lateral sclerosis, multiple sclerosis, Parkinson's disease, stroke, and traumatic brain injury. Invitations to participate were randomly sent online to adults (aged ≥ 18 years) registered in the Harris Poll Online Panel who were patients or belonged to a household with a patient diagnosed with one of the six conditions (identified through previous screening by Harris Interactive). Participants were screened for PBA using the Pathological Laughing and Crying Scale (PLACS) and the Center for Neurologic Study-Lability Scale (CNS-LS). PBA estimates were made using a cut-off score of ≥ 13 on the PLACS and two different cut-off thresholds on the CNS-LS, a lower one of ≥ 13 and a more rigorous one of ≥ 21. Existing US prevalence data for the six underlying conditions were used to estimate US prevalence of PBA. RESULTS: Of 38,000 individuals invited to participate, 8876 responded (23%) and 2318 (26%) completed the questionnaire. Mean prevalence of PBA across all six conditions was 10.1%, 9.4%, and 37.5% with the PLACS ≥ 13, CNS-LS ≥ 21, and CNS-LS ≥ 13 thresholds, respectively. Using disease population estimates from government agencies and professional organizations, the estimated US population with PBA ranged from 1.8 to 7.1 million. Among patients who discussed their laughing and/or crying episodes with a physician, 41% were diagnosed, and about half received a medication for their episodes. CONCLUSIONS: The overall prevalence of PBA was estimated to be about 10% across these commonly associated underlying neurological conditions and appears to be under-recognized.