Comparing methods to combine functional loss and mortality in clinical trials for amyotrophic lateral sclerosis

Objective: Amyotrophic lateral sclerosis (ALS) clinical trials based on single end points only partially capture the full treatment effect when both function and mortality are affected, and may falsely dismiss efficacious drugs as futile. We aimed to investigate the statistical properties of several strategies for the simultaneous analysis of function and mortality in ALS clinical trials. Methods: Based on the Pooled Resource Open-Access ALS Clinical Trials (PRO-ACT) database, we simulated longitudinal patterns of functional decline, defined by the revised amyotrophic lateral sclerosis functional rating scale (ALSFRS-R) and conditional survival time. Different treatment scenarios with varying effect sizes were simulated with follow-up ranging from 12 to 18 months. We considered the following analytical strategies: 1) Cox model; 2) linear mixed effects (LME) model; 3) omnibus test based on Cox and LME models; 4) composite time-to-6-point decrease or death; 5) combined assessment of function and survival (CAFS); and 6) test based on joint modeling framework. For each analytical strategy, we calculated the empirical power and sample size. Results: Both Cox and LME models have increased false-negative rates when treatment exclusively affects either function or survival. The joint model has superior power compared to other strategies. The composite end point increases false-negative rates among all treatment scenarios. To detect a 15% reduction in ALSFRS-R decline and 34% decline in hazard with 80% power after 18 months, the Cox model requires 524 patients, the LME model 794 patients, the omnibus test 526 patients, the composite end poi

Comparative interactomics analysis of different ALS-associated proteins identifies converging molecular pathways

Amyotrophic lateral sclerosis (ALS) is a devastating neurological disease with no effective treatment available. An increasing number of genetic causes of ALS are being identified, but how these genetic defects lead to motor neuron degeneration and to which extent they affect common cellular pathways remains incompletely understood. To address these questions, we performed an interactomic analysis to identify binding partners of wild-type (WT) and ALS-associated mutant versions of ATXN2, C9orf72, FUS, OPTN, TDP-43 and UBQLN2 in neuronal cells. This analysis identified several known but also many novel binding partners of these proteins. Interactomes of WT and mutant ALS proteins were very similar except for OPTN and UBQLN2, in which mutations caused loss or gain of protein interactions. Several of the identified interactomes showed a high degree of overlap: shared binding partners of ATXN2, FUS and TDP-43 had roles in RNA metabolism; OPTN- and UBQLN2-interacting proteins were related to protein degradation and protein transport, and C9orf72 interactors function in mitochondria. To conf

The frontotemporal syndrome of ALS is associated with poor survival

Thirty percent of ALS patients have a frontotemporal syndrome (FS), defined as behavioral changes or cognitive impairment. Despite previous studies, there are no firm conclusions on the effect of the FS on survival and the use of non-invasive ventilation (NIV) in ALS. We examined the effect of the FS on survival and the start and duration of NIV in ALS. Behavioral changes were defined as >22 points on the ALS-Frontotemporal-Dementia-Questionnaire or ≥3 points on ≥2 items of the Neuropsychiatric Inventory. Cognitive impairment was defined as below the fifth percentile on ≥2 tests of executive function, memory or language. Classic ALS was defined as ALS without the frontotemporal syndrome. We performed survival analyses from symptom ons

A replication study of genetic risk loci for ischemic stroke in a Dutch population: A case-control study

We aimed to replicate reported associations of 10 SNPs at eight distinct loci with overall ischemic stroke (IS) and its subtypes in an independent cohort of Dutch IS patients. We included 1,375 IS patients enrolled in a prospective multicenter hospital-based cohort in the Netherlands, and 1,533 population-level controls of Dutch descent. We tested these SNPs for association with overall IS and its subtypes (large artery atherosclerosis, small vessel disease and cardioembolic stroke (CE), as classified by TOAST) using an additive multivariable logistic regression model, adjusting for age and sex. We obtained odds ratios (OR) with 95% confidence intervals (95% CI) for the risk allele of each SNP analyzed and exact p-values by permutation. We confirmed the association at 4q25 (PITX2) (OR 1.43; 95% CI, 1.13-1.81, p = 0.029) and 16q22 (ZFHX3) (OR 1.62; 95% CI, 1.26-2.07, p = 0.001) as risk loci for CE. Locus 16q22 was also associated with overall IS (OR 1.24; 95% CI, 1.08-1.42, p = 0.016). Other loci previously associated with IS and/or its subtypes were not confirmed. In conclusion, we validated two loci (4q25, 16q22) associated with CE. In addition, our study may suggest that the association of locus 16q22 may not be limited to CE, but also includes overall IS

Age-related accrual of methylomic variability is linked to fundamental ageing mechanisms

Background: Epigenetic change is a hallmark of ageing but its link to ageing mechanisms in humans remains poorly understood. While DNA methylation at many CpG sites closely tracks chronological age, DNA methylation changes relevant to biological age are expected to gradually dissociate from chronological age, mirroring the increased heterogeneity in health status at older ages. Results: Here, we report on the large-scale identification of 6366 age-related variably methylated positions (aVMPs) identified in 3295 whole blood DNA methylation profiles, 2044 of which have a matching RNA-seq gene expression profile. aVMPs are enriched at polycomb repressed regions and, accordingly, methylation at those positions is associated with the expression of genes encoding components of polycomb repressive complex 2 (PRC2) in trans. Further analysis revealed trans-associations for 1816 aVMPs with an additional 854 genes. These trans-associated aVMPs are characterized by either an age-related

Blood lipids influence DNA methylation in circulating cells

Background: Cells can be primed by external stimuli to obtain a long-term epigenetic memory. We hypothesize that long-term exposure to elevated blood lipids can prime circulating immune cells through changes in DNA methylation, a process that may contribute to the development of atherosclerosis. To interrogate the causal relationship between triglyceride, low-density lipoprotein (LDL) cholesterol, and high-density lipoprotein (HDL) cholesterol levels and genome-wide DNA methylation while excluding confounding and pleiotropy, we perform a stepwise Mendelian randomization analysis in whole blood of 3296 individuals. Results: This analysis shows that differential methylation is the consequence of inter-individual variation in blood lipid levels and not vice versa. Specifically, we observe an effect of triglycerides on DNA methylation at three CpGs, of LDL cholesterol at one CpG, and of HDL cholesterol at two CpGs using multivariable Mendelian randomization. Using RNA-seq data available for a large subset of individuals (N = 2044), DNA methylation of these six CpGs is associated with the expression of CPT1A and SREBF1 (for triglycerides), DHCR24 (for LDL cholesterol) and

Controlling bias and inflation in epigenome- and transcriptome-wide association studies using the empirical null distribution

We show that epigenome- and transcriptome-wide association studies (EWAS and TWAS) are prone to significant inflation and bias of test statistics, an unrecognized phenomenon introducing spurious findings if left unaddressed. Neither GWAS-based methodology nor state-of-the-art confounder adjustment methods completely remove bias and inflation. We propose a Bayesian method to control bias and inflation in EWAS and TWAS based on estimation of the empirical null distribution. Using simulations and real data, we demonstrate that our method maximizes power while properly controlling the false positive rate. We illustrate the utility of our method in large-scale EWAS and TWAS meta-analyses of age and smoking

Validated inference of smoking habits from blood with a finite DNA methylation marker set

Inferring a person’s smoking habit and history from blood is relevant for complementing or replacing self-reports in epidemiological and public health research, and for forensic applications. However, a finite DNA methylation marker set and a validated statistical model based on a large dataset are not yet available. Employing 14 epigenome-wide association studies for marker discovery, and using data from six population-based cohorts (N = 3764) for model building, we identified 13 CpGs most suitable for inferring smoking versus non-smoking status from blood with a cumulative Area Under the Curve (AUC) of 0.901. Internal fivefold cross-validation yielded an average AUC of 0.897 ± 0.137, while external model validation in an independent population-based cohort (

Joint sequencing of human and pathogen genomes reveals the genetics of pneumococcal meningitis

Streptococcus pneumoniae is a common nasopharyngeal colonizer, but can also cause lifethreatening invasive diseases such as empyema, bacteremia and meningitis. Genetic variation of host and pathogen is known to play a role in invasive pneumococcal disease, though to what extent is unknown. In a genome-wide association study of human and pathogen we show that human variation explains almost half of variation in susceptibility to pneumococcal meningitis and one-third of variation in severity, identifying variants in CCDC33 associated with susceptibility. Pneumococcal genetic variation explains a large amount of invasive potential (70%), but has no effect on severity. Serotype alone is insufficient to explain invasiveness, suggesting other pneumococcal factors are involved in progression to invasive disease. We identify pneumococcal genes involved

WGS-based telomere length analysis in Dutch family trios implicates stronger maternal inheritance and a role for RRM1 gene

Telomere length (TL) regulation is an important factor in ageing, reproduction and cancer development. Genetic, hereditary and environmental factors regulating TL are currently widely investigated, however, their relative contribution to TL variability is still understudied. We have used whole genome sequencing data of 250 family trios from the Genome of the Netherlands project to perform computational measurement of TL and a series of regression and genome-wide association analyses to reveal TL inheritance patterns and associated genetic factors. Our results confirm that TL is a largely heritable trait, primarily with mother’s, and, to a lesser extent, with father’s TL having the strongest influence on the offspring. In this cohort, mother’s, but not father’s age at conception was positively linked to offspring TL. Age-related TL attrition of 40 bp/year had relatively small influence on TL variability. Finally, we have identified TL-associated variations in ribonuclease reductase catalytic subunit M1 (RRM1 gene), which is known to regulate telomere maintenance in yeast. We also highlight the importance of multivariate approach and the limitations of existing tools for the analysis of TL as a polygenic heritable quantitative trait