The protocol for an observational Australian cohort study of CADASIL: The AusCADASIL study.

Introduction: Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL) is a rare genetic condition with a broad phenotypic presentation. This study aims to establish the first Australian cohort of individuals affected by CADASIL (AusCADASIL) and examine its clinical features and longitudinal course, and to investigate neuroimaging and blood biomarkers to assist in early diagnosis and identify disease progression. Methods: Participants will be recruited from six study centres across Australia for an observational study of CADASIL. We aim to recruit 150 participants with diagnosed CADASIL, family history of CADASIL or suspected CADASIL symptoms, and 150 cognitively normal NOTCH3 negative individuals as controls. Participants will complete: 1) online questionnaires on medical and family history, mental health, and wellbeing; 2) neuropsychological evaluation; 3) neurological examination and brain MRI; 4) ocular examination and 5) blood sample donation. Participants will have annual follow-up for 4 years to assess their progression and will be asked to invite a study partner to corroborate their self-reported cognitive and functional abilities.Primary outcomes include cognitive function and neuroimaging abnormalities. Secondary outcomes include investigation of genetics and blood and ocular biomarkers. Data from the cohort will contribute to an international consortium, and cohort participants will be invited to access future treatment/health intervention trials. Discussion: AusCADASIL will be the first study of an Australian cohort of individuals with CADASIL. The study will identify common pathogenic variants in this cohort, and characterise the pattern of clinical presentation and longitudinal progression, including imaging features, blood and ocular biomarkers and cognitive profile.

Associations between fruit and vegetable intakes and incident depression in middle-aged and older adults from 10 diverse international longitudinal cohorts.

BACKGROUND: Emerging observational evidence supports a role for higher fruit and vegetable intake in protecting against the development of depression. However, there is a scarcity of research in older adults or in low- to middle-income countries (LMICs). METHODS: Participants were 7801 community-based adults (mean age 68.6 ± 8.0 years, 55.8 % female) without depression, from 10 diverse cohorts, including four cohorts from LMICs. Fruit and vegetable intake was self-reported via comprehensive food frequency questionnaire, short food questionnaire or diet history. Depressive symptoms were assessed using validated measures, and depression defined applying validated cut-offs. The associations between baseline fruit and vegetable intakes and incident depression over a follow-up period of three to nine years were examined using Cox regression. Analyses were performed by cohort with results meta-analysed. RESULTS: There were 1630 cases of incident depression (21 % of participants) over 40,258 person-years of follow-up. Higher intake of fruit was associated with a lower risk of incident depression (HR 0.87, 95%CI [0.77, 0.99], I2 = 4 %). No association was found between vegetable intake and incident depression (HR 0.93, 95%CI [0.84, 1.04], I2 = 0 %). LIMITATIONS: Diverse measures used across the different cohorts and the modest sample size of our study compared with prior studies may have prevented an association being detected for vegetable intake. CONCLUSIONS: Our study supports a role for fruit, but not vegetable intake in protecting against depression. Research investigating different types of fruits and vegetables using standardised measures in larger cohorts of older adults from low- and middle-income countries is warranted.

Genetics of environmental sensitivity to psychiatric and neurodevelopmental phenotypes: evidence from GWAS of monozygotic twins.

Individual sensitivity to environmental exposures may be genetically influenced. This genotype-by-environment interplay implies differences in phenotypic variance across genotypes. However, environmental sensitivity genetic variants have proven challenging to detect. GWAS of monozygotic twin differences is a family-based variance analysis method, which is more robust to systemic biases that impact population-based methods. We combined data from up to 21,792 monozygotic twins (10,896 pairs) from 11 studies to conduct the largest GWAS meta-analysis of monozygotic phenotypic differences in children and adolescents/adults for seven psychiatric and neurodevelopmental phenotypes: attention deficit hyperactivity disorder (ADHD) symptoms, autistic traits, anxiety and depression symptoms, psychotic-like experiences, neuroticism, and wellbeing. The SNP-heritability of variance in these phenotypes were estimated (h2: 0% to 18%), but were imprecise. We identified a total of 13 genome-wide significant associations (SNP, gene, and gene-set), including genes related to stress-reactivity for depression, growth factor-related genes for autistic traits and catecholamine uptake-related genes for psychotic-like experiences. Monozygotic twins are an important new source of evidence about the genetics of environmental sensitivity.

Heritability of Gene Expression Measured from Peripheral Blood in Older Adults.

The contributions of genetic variation and the environment to gene expression may change across the lifespan. However, few studies have investigated the heritability of blood gene expression in older adults. The current study therefore aimed to investigate this question in a community sample of older adults. A total of 246 adults (71 MZ and 52 DZ twins, 69.91% females; mean age-75.79 ± 5.44) were studied. Peripheral blood gene expression was assessed using Illumina microarrays. A heritability analysis was performed using structural equation modelling. There were 5269 probes (19.9%) from 4603 unique genes (23.9%) (total 26,537 probes from 19,256 genes) that were significantly heritable (mean h2 = 0.40). A pathway analysis of the top 10% of significant genes showed enrichment for the immune response and ageing-associated genes. In a comparison with two other gene expression twin heritability studies using adults from across the lifespan, there were 38 out of 9479 overlapping genes that were significantly heritable. In conclusion, our study found ~24% of the available genes for analysis were heritable in older adults, with only a small number common across studies that used samples from across adulthood, indicating the importance of examining gene expression in older age groups.

Brain structural covariances in the ageing brain in the UK Biobank.

The morphologic properties of brain regions co-vary or correlate with each other. Here we investigated the structural covariances of cortical thickness and subcortical volumes in the ageing brain, along with their associations with age and cognition, using cross-sectional data from the UK Biobank (N = 42,075, aged 45-83 years, 53% female). As the structural covariance should be estimated in a group of participants, all participants were divided into 84 non-overlapping, equal-sized age groups ranging from the youngest to the oldest. We examined 84 cortical thickness covariances and subcortical covariances. Our findings include: (1) there were significant differences in the variability of structural covariance in the ageing process, including an increased variance, and a decreased entropy. (2) significant enrichment in pairwise correlations between brain regions within the occipital lobe was observed in all age groups; (3) structural covariance in older age, especially after the age of around 64, was significantly different from that in the youngest group (median age 48 years); (4) sixty-two of the total 528 pairs of cortical thickness correlations and 10 of the total 21 pairs of subcortical volume correlations showed significant associations with age. These trends varied, with some correlations strengthening, some weakening, and some reversing in direction with advancing age. Additionally, as ageing was associated with cognitive decline, most of the correlations with cognition displayed an opposite trend compared to age associated patterns of correlations.

Enhanced resolution profiling in twins reveals differential methylation signatures of type 2 diabetes with links to its complications.

BACKGROUND: Type 2 diabetes (T2D) susceptibility is influenced by genetic and environmental factors. Previous findings suggest DNA methylation as a potential mechanism in T2D pathogenesis and progression. METHODS: We profiled DNA methylation in 248 blood samples from participants of European ancestry from 7 twin cohorts using a methylation sequencing platform targeting regulatory genomic regions encompassing 2,048,698 CpG sites. FINDINGS: We find and replicate 3 previously unreported T2D differentially methylated CpG positions (T2D-DMPs) at FDR 5% in RGL3, NGB and OTX2, and 20 signals at FDR 25%, of which 14 replicated. Integrating genetic variation and T2D-discordant monozygotic twin analyses, we identify both genetic-based and genetic-independent T2D-DMPs. The signals annotate to genes with established GWAS and EWAS links to T2D and its complications, including blood pressure (RGL3) and eye disease (OTX2). INTERPRETATION: The results help to improve our understanding of T2D disease pathogenesis and progression and may provide biomarkers for its complications. FUNDING: Funding acknowledgements for each cohort can be found in the Supplementary Note.

Multi-omics and pathway analyses of genome-wide associations implicate regulation and immunity in verbal declarative memory performance.

BACKGROUND: Uncovering the functional relevance underlying verbal declarative memory (VDM) genome-wide association study (GWAS) results may facilitate the development of interventions to reduce age-related memory decline and dementia. METHODS: We performed multi-omics and pathway enrichment analyses of paragraph (PAR-dr) and word list (WL-dr) delayed recall GWAS from 29,076 older non-demented individuals of European descent. We assessed the relationship between single-variant associations and expression quantitative trait loci (eQTLs) in 44 tissues and methylation quantitative trait loci (meQTLs) in the hippocampus. We determined the relationship between gene associations and transcript levels in 53 tissues, annotation as immune genes, and regulation by transcription factors (TFs) and microRNAs. To identify significant pathways, gene set enrichment was tested in each cohort and meta-analyzed across cohorts. Analyses of differential expression in brain tissues were conducted for pathway component genes. RESULTS: The single-variant associations of VDM showed significant linkage disequilibrium (LD) with eQTLs across all tissues and meQTLs within the hippocampus. Stronger WL-dr gene associations correlated with reduced expression in four brain tissues, including the hippocampus. More robust PAR-dr and/or WL-dr gene associations were intricately linked with immunity and were influenced by 31 TFs and 2 microRNAs. Six pathways, including type I diabetes, exhibited significant associations with both PAR-dr and WL-dr. These pathways included fifteen MHC genes intricately linked to VDM performance, showing diverse expression patterns based on cognitive status in brain tissues. CONCLUSIONS: VDM genetic associations influence expression regulation via eQTLs and meQTLs. The involvement of TFs, microRNAs, MHC genes, and immune-related pathways contributes to VDM performance in older individuals.

Molecular biomarkers for vascular cognitive impairment and dementia.

As disease-specific interventions for dementia are being developed, the ability to identify the underlying pathology and dementia subtypes is increasingly important. Vascular cognitive impairment and dementia (VCID) is the second most common cause of dementia after Alzheimer disease, but progress in identifying molecular biomarkers for accurate diagnosis of VCID has been relatively limited. In this Review, we examine the roles of large and small vessel disease in VCID, considering the underlying pathophysiological processes that lead to vascular brain injury, including atherosclerosis, arteriolosclerosis, ischaemic injury, haemorrhage, hypoperfusion, endothelial dysfunction, blood-brain barrier breakdown, inflammation, oxidative stress, hypoxia, and neuronal and glial degeneration. We consider the key molecules in these processes, including proteins and peptides, metabolites, lipids and circulating RNA, and consider their potential as molecular biomarkers alone and in combination. We also discuss the challenges in translating the promise of these biomarkers into clinical application.

Apathy as a determinant of health behaviors in older adults: Implications for dementia risk reduction.

INTRODUCTION: Long-term improvements in physical inactivity and other behavioral risk factors are integral to dementia risk reduction; however, sustained behavior change is challenging. Apathy, depression, and fatigue may impact engagement in health behaviors, but their presentation overlaps. This study investigates whether these symptoms are differentially associated with multiple health behaviors. METHODS: In 1037 community-dwelling older adults without dementia (aged 70-90, 55% women), regression analyses examined apathy, depression, and fatigue as predictors of health behaviors (physical activity, diet, alcohol, smoking) and a behavioral risk index. RESULTS: Apathy was associated with reduced physical activity and alcohol use, and one or multiple behavioral risk factors. No or inconsistent relations were found between depression or fatigue and health behaviors. DISCUSSION: Apathy is relevant to multiple health behaviors and should be considered when designing health promotion for older adults, including interventions for dementia risk reduction. Findings highlight the importance of distinguishing apathy from comorbid symptoms. Highlights: Novel theory-based perspective on behavioural risk factors for dementia.Higher apathy predicted less physical activity and alcohol use, and increased odds of lifestyle risk factors.Depressive symptoms were not associated with any health behavior.Apathy may be a determinant of multiple health behaviors in older adults, distinct from depression and fatigue.Considering apathy in precision prevention of dementia appears warranted.

Genetic and environmental influences on fruit and vegetable consumption and depression in older adults.

BACKGROUND: Prior work suggests that higher fruit and vegetable consumption may protect against depression in older adults. Better understanding of the influence of genetic and environmental factors on fruit and vegetable intakes may lead to the design of more effective dietary strategies to increase intakes. In turn this may reduce the occurrence of depression in older adults. OBJECTIVES: The primary aim of this study is to estimate the genetic and environmental influences on the consumption of fruit and vegetables in older adults. The secondary aim is an exploratory analysis into possible shared genetic influences on fruit and vegetable intakes and depression. METHODS: Analysis of observational data from 374 twins (67.1% female; 208 monozygotic (MZ); 166 dizygotic (DZ)) aged ≥ 65 years drawn from the Older Australian Twins Study. Dietary data were obtained using a validated food frequency questionnaire and depressive symptoms were measured using the 15-item short form Geriatric Depression Scale. The contribution of genetic and environmental influences on fruit and vegetable intake were estimated by comparing MZ and DZ twin intakes using structural equation modelling. A tri-variate twin model was used to estimate the genetic and environmental correlation between total fruit and vegetable intakes and depression. RESULTS: In this study, vegetable intake was moderately influenced by genetics (0.39 95%CI 0.22, 0.54). Heritability was highest for brassica vegetables (0.40 95%CI 0.24, 0.54). Overall fruit intake was not significantly heritable. No significant genetic correlations were detected between fruit and vegetable intake and depressive symptoms. CONCLUSIONS: Vegetable consumption, particularly bitter tasting brassica vegetables, was significantly influenced by genetics, although environmental influences were also apparent. Consumption of fruit was only influenced by the environment, with no genetic influence detected, suggesting strategies targeting the food environment may be particularly effective for encouraging fruit consumption.

Sex differences in dementia risk and risk factors: Individual-participant data analysis using 21 cohorts across six continents from the COSMIC consortium.

INTRODUCTION: Sex differences in dementia risk, and risk factor (RF) associations with dementia, remain uncertain across diverse ethno-regional groups. METHODS: A total of 29,850 participants (58% women) from 21 cohorts across six continents were included in an individual participant data meta-analysis. Sex-specific hazard ratios (HRs), and women-to-men ratio of hazard ratios (RHRs) for associations between RFs and all-cause dementia were derived from mixed-effect Cox models. RESULTS: Incident dementia occurred in 2089 (66% women) participants over 4.6 years (median). Women had higher dementia risk (HR, 1.12 [1.02, 1.23]) than men, particularly in low- and lower-middle-income economies. Associations between longer education and former alcohol use with dementia risk (RHR, 1.01 [1.00, 1.03] per year, and 0.55 [0.38, 0.79], respectively) were stronger for men than women; otherwise, there were no discernible sex differences in other RFs. DISCUSSION: Dementia risk was higher in women than men, with possible variations by country-level income settings, but most RFs appear to work similarly in women and men.

Aging Is Associated With Multidirectional Changes in Social Cognition: Findings From an Adult Life-Span Sample Ranging From 18 to 101 Years.

OBJECTIVES: Normal adult aging is associated with changes in social cognition. Although 4 social cognitive domains have been identified (social perception, theory of mind [ToM], affective empathy, and social behavior), no study has tested all 4 domains concurrently in a life-span sample, limiting understanding of the relative magnitude of age-related changes across domains. This study addresses this gap by providing the first assessment of all 4 social cognitive domains in an adult life-span sample. METHODS: Three hundred and seventy-two participants ranging from 18 to 101 years of age took part in this study. Participants completed a testing battery that assessed social perception, ToM, affective empathy, and social behavior, as well as broader cognitive function and well-being. RESULTS: The results showed that adult aging is associated with multidirectional changes in social cognitive abilities, with ToM and social perception showing nonlinear decline across much of the life-span, and affective empathy and social behavior showing improvement. Age remained a significant predictor of all 4 social cognitive domains, even after accounting for broader cognitive function. Weak associations emerged between some of the social cognitive abilities and and indices of broader well-being. DISCUSSION: These findings provide novel and important evidence that normative aging is associated with both gains and losses in social cognition that occur at distinct points of the adult life-span, and that are at least partially independent of general age-related cognitive decline.

Measuring Empathy Across the Adult Lifespan: A Comparison of Three Assessment Types.

Empathy is a core component of social cognition that can be indexed via behavioral, informant-report, or self-report methods of assessment. However, concerns have been raised regarding the lack of convergence between these assessment approaches for cognitive empathy. Here, we provided the first comparison of all three measurement approaches for cognitive and affective empathy in a large adult sample (N = 371) aged 18 to 101 years. We found that poor convergence was more of a problem for cognitive empathy than affective empathy. While none of the cognitive empathy measures correlated with each other, for affective empathy, self-report was significantly associated with both behavioral and informant-report assessments. However, for both cognitive and affective empathy, there was evidence for poor discriminant validity within the measures. Out of the three assessment approaches, only the informant-report measures were consistently associated with indices of social functioning. Importantly, age did not moderate any of the tested relationships, indicating that both the strengths and the limitations of these different types of assessment do not appear to vary as a function of age. These findings highlight the variation that exists among empathy measures and are discussed in relation to their practical implications for the assessment of empathy.

Epigenetic and integrative cross-omics analyses of cerebral white matter hyperintensities on MRI.

Cerebral white matter hyperintensities on MRI are markers of cerebral small vessel disease, a major risk factor for dementia and stroke. Despite the successful identification of multiple genetic variants associated with this highly heritable condition, its genetic architecture remains incompletely understood. More specifically, the role of DNA methylation has received little attention. We investigated the association between white matter hyperintensity burden and DNA methylation in blood at ∼450 000 cytosine-phosphate-guanine (CpG) sites in 9732 middle-aged to older adults from 14 community-based studies. Single CpG and region-based association analyses were carried out. Functional annotation and integrative cross-omics analyses were performed to identify novel genes underlying the relationship between DNA methylation and white matter hyperintensities. We identified 12 single CpG and 46 region-based DNA methylation associations with white matter hyperintensity burden. Our top discovery single CpG, cg24202936 (P = 7.6 × 10-8), was associated with F2 expression in blood (P = 6.4 × 10-5) and co-localized with FOLH1 expression in brain (posterior probability = 0.75). Our top differentially methylated regions were in PRMT1 and in CCDC144NL-AS1, which were also represented in single CpG associations (cg17417856 and cg06809326, respectively). Through Mendelian randomization analyses cg06809326 was putatively associated with white matter hyperintensity burden (P = 0.03) and expression of CCDC144NL-AS1 possibly mediated this association. Differentially methylated region analysis, joint epigenetic association analysis and multi-omics co-localization analysis consistently identified a role of DNA methylation near SH3PXD2A, a locus previously identified in genome-wide association studies of white matter hyperintensities. Gene set enrichment analyses revealed functions of the identified DNA methylation loci in the blood-brain barrier and in the immune response. Integrative cross-omics analysis identified 19 key regulatory genes in two networks related to extracellular matrix organization, and lipid and lipoprotein metabolism. A drug-repositioning analysis indicated antihyperlipidaemic agents, more specifically peroxisome proliferator-activated receptor-alpha, as possible target drugs for white matter hyperintensities. Our epigenome-wide association study and integrative cross-omics analyses implicate novel genes influencing white matter hyperintensity burden, which converged on pathways related to the immune response and to a compromised blood-brain barrier possibly due to disrupted cell-cell and cell-extracellular matrix interactions. The results also suggest that antihyperlipidaemic therapy may contribute to lowering risk for white matter hyperintensities possibly through protection against blood-brain barrier disruption.

High polygenic risk score for exceptional longevity is associated with a healthy metabolic profile.

Healthy metabolic measures in humans are associated with longevity. Dysregulation leads to metabolic syndrome (MetS) and negative health outcomes. Recent exceptional longevity (EL) genome wide association studies have facilitated estimation of an individual's polygenic risk score (PRS) for EL. We tested the hypothesis that individuals with high ELPRS have a low prevalence of MetS. Participants were from five cohorts of middle-aged to older adults. The primary analyses were performed in the UK Biobank (UKBB) (n = 407,800, 40-69 years). Replication analyses were undertaken using three Australian studies: Hunter Community Study (n = 2122, 55-85 years), Older Australian Twins Study (n = 539, 65-90 years) and Sydney Memory and Ageing Study (n = 925, 70-90 years), as well as the Swedish Gothenburg H70 Birth Cohort Studies (n = 2273, 70-93 years). MetS was defined using established criteria. Regressions and meta-analyses were performed with the ELPRS and MetS and its components. Generally, MetS prevalence (22-30%) was higher in the older cohorts. In the UKBB, high EL polygenic risk was associated with lower MetS prevalence (OR = 0.94, p = 1.84 × 10-42) and its components (p < 2.30 × 10-8). Meta-analyses of the replication cohorts showed nominal associations with MetS (p = 0.028) and 3 MetS components (p < 0.05). This work suggests individuals with a high polygenic risk for EL have a healthy metabolic profile promoting longevity.

Genome-wide meta-analyses reveal novel loci for verbal short-term memory and learning.

Understanding the genomic basis of memory processes may help in combating neurodegenerative disorders. Hence, we examined the associations of common genetic variants with verbal short-term memory and verbal learning in adults without dementia or stroke (N = 53,637). We identified novel loci in the intronic region of CDH18, and at 13q21 and 3p21.1, as well as an expected signal in the APOE/APOC1/TOMM40 region. These results replicated in an independent sample. Functional and bioinformatic analyses supported many of these loci and further implicated POC1. We showed that polygenic score for verbal learning associated with brain activation in right parieto-occipital region during working memory task. Finally, we showed genetic correlations of these memory traits with several neurocognitive and health outcomes. Our findings suggest a role of several genomic loci in verbal memory processes.

Polygenic resilience scores capture protective genetic effects for Alzheimer's disease.

Polygenic risk scores (PRSs) can boost risk prediction in late-onset Alzheimer's disease (LOAD) beyond apolipoprotein E (APOE) but have not been leveraged to identify genetic resilience factors. Here, we sought to identify resilience-conferring common genetic variants in (1) unaffected individuals having high PRSs for LOAD, and (2) unaffected APOE-ε4 carriers also having high PRSs for LOAD. We used genome-wide association study (GWAS) to contrast "resilient" unaffected individuals at the highest genetic risk for LOAD with LOAD cases at comparable risk. From GWAS results, we constructed polygenic resilience scores to aggregate the addictive contributions of risk-orthogonal common variants that promote resilience to LOAD. Replication of resilience scores was undertaken in eight independent studies. We successfully replicated two polygenic resilience scores that reduce genetic risk penetrance for LOAD. We also showed that polygenic resilience scores positively correlate with polygenic risk scores in unaffected individuals, perhaps aiding in staving off disease. Our findings align with the hypothesis that a combination of risk-independent common variants mediates resilience to LOAD by moderating genetic disease risk.

Genetic and Environmental Factors in Ageing and Age-Related Disease.

Globally, the population is growing older [...].

Early life affects late-life health through determining DNA methylation across the lifespan: A twin study.

BACKGROUND: Previous findings for the genetic and environmental contributions to DNA methylation variation were for limited age ranges only. We investigated the lifespan contributions and their implications for human health for the first time. METHODS: 1,720 monozygotic twin (MZ) pairs and 1,107 dizygotic twin (DZ) pairs aged 0-92 years were included. Familial correlations (i.e., correlations between twins) for 353,681 methylation sites were estimated and modelled as a function of twin pair cohabitation history. FINDINGS: The methylome average familial correlation was around zero at birth (MZ pair: -0.01; DZ pair: -0.04), increased with the time of twins living together during childhood at rates of 0.16 (95%CI: 0.12-0.20) for MZ pairs and 0.13 (95%CI: 0.07-0.20) for DZ pairs per decade, and decreased with the time of living apart during adulthood at rates of 0.026 (95%CI: 0.019-0.033) for MZ pairs and 0.027 (95%CI: 0.011-0.043) for DZ pairs per decade. Neither the increasing nor decreasing rate differed by zygosity (both P>0.1), consistent with cohabitation environment shared by twins, rather than genetic factors, influencing the methylation familial correlation changes. Familial correlations for 6.6% (23,386/353,681) sites changed with twin pair cohabitation history. These sites were enriched for high heritability, proximal promoters, and epigenetic/genetic associations with various early-life factors and late-life health conditions. INTERPRETATION: Early life strongly influences DNA methylation variation across the lifespan, and the effects are stronger for heritable sites and sites biologically relevant to the regulation of gene expression. Early life could affect late-life health through influencing DNA methylation. FUNDING: Victorian Cancer Agency, Cancer Australia, Cure Cancer Foundation.

Genome-wide study of DNA methylation shows alterations in metabolic, inflammatory, and cholesterol pathways in 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.