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

Genome-wide identification of directed gene networks using large-scale population genomics data

Identification of causal drivers behind regulatory gene networks is crucial in understanding gene function. Here, we develop a method for the large-scale inference of gene–gene interactions in observational population genomics data that are both directed (using local genetic instruments as causal anchors, akin to Mendelian Randomization) and specific (by controlling for linkage disequilibrium and pleiotropy). Analysis of genotype and whole-blood RNA-sequencing data from 3072 individuals identified 49 genes as drivers of downstream transcriptional changes (Wald P < 7 × 10−10), among which transcription factors were overrepresented (Fisher’s P = 3.3 × 10−7). Our analysis suggests new gene functions and targets, including for SENP7 (zinc-finger genes involved in retroviral repression) and BCL2A1 (target genes possibly involved in auditory dysfunction). Our work highlights the utility of population genomics data in deriving directed gene expression networks. A resource of trans-effects for all 6600 genes with a genetic instrument can be explored individually using a web-based browser

Autosomal genetic variation is associated with DNA methylation in regions variably escaping X-chromosome inactivation

X-chromosome inactivation (XCI), i.e., the inactivation of one of the female X chromosomes, restores equal expression of X-chromosomal genes between females and males. However, ~10% of genes show variable degrees of escape from XCI between females, although little is known about the causes of variable XCI. Using a discovery data-set of 1867 females and 1398 males and a replication sample of 3351 females, we show that genetic variation at three autosomal loci is associated with female-specific changes in X-chromosome methylation. Through cis-eQTL expression analysis, we map these loci to the genes SMCHD1/METTL4, TRIM6/HBG2, and ZSCAN9. Low-expression alleles of the loci are predominantly associated with mild hypomethylation of CpG islands near genes known to variably escape XCI, implicating the autosomal genes in variable XCI. Together, these results suggest a genetic basis for variable escape from XCI and highlight the potential of a population genomics approach to identify genes involved in XCI

Skewed X-inactivation is common in the general female population

X-inactivation is a well-established dosage compensation mechanism ensuring that X-chromosomal genes are expressed at comparable levels in males and females. Skewed X-inactivation is often explained by negative selection of one of the alleles. We demonstrate that imbalanced expression of the paternal and maternal X-chromosomes is common in the general population and that the random nature of the X-inactivation mechanism can be sufficient to explain the imbalance. To this end, we analyzed blood-derived RNA and whole-genome sequencing data from 79 female children and their parents from the Genome of the Netherlands project. We calculated the median ratio of the paternal over total counts at all X-chromosomal heterozygous single-nucleotide variants with coverage ≥10. We identified two individuals where the same X-chromosome was inactivated in all cells. Imbalanced expression of the two X-chromosomes (ratios ≤0.35 or ≥0.65) was observed in nearly 50% of the population. The empirically observed skewing is explained by a theoretical model where X-inactivation takes place in an embryonic stage in which eight cells give rise to the hematopoietic compartment. Genes escaping X-inactivation are expressed from both alleles and therefore demonstrate less skewing than inactivated genes. Using this characteristic, we identified three novel escapee genes (SSR4, REPS2, and SEPT6), but did not find support for many previously reported escapee genes in blood. Our collective data suggest that skewed X-inactivation is common in the general population. This may contribute to manifestation of symptoms in carriers of recessive X-linked disorders. We recommend that X-inactivation results should not be used lightly in the interpretation of X-linked variants

Technology in Motion Tremor Dataset: TIM-Tremor

Data was collected within the Technology in Motion project (protocol registered as NL54281.058.15), aimed at developing patient friendly and unobtrusive techniques to characterize motor function in patients with neurological disorders. In this context we used video cameras combined with depth sensors (RGB-D sensors, Microsoft KinectTM v2) and 2 accelerometer sensors (ACL300, Biometrics Ltd, Newport, UK) to objectively quantify the frequency and amplitude of tremor of the upper extremity. This dataset contains 55 patient recorded at performing a set of tasks. For each patient and each task, we record a short seqeuence and provide: (i) Kinect RGB video recording, (ii) Kinect depth map recording encoded as a video, (iii) Kinect depth map aligned with the RGB video, (iv) accelerometer recordings of the 2 sensors. We aditionally, provide an overall labeling file containing for each patient: tremor ratings per arm, and tremor diagnosis More detailed description can be found in documentation

A linear mixed-model approach to study multivariate gene-environment interactions

Different exposures, including diet, physical activity, or external conditions can contribute to genotype-environment interactions (G×E). Although high-dimensional environmental data are increasingly available and multiple exposures have been implicated with G×E at the same loci, multi-environment tests for G×E are not established. Here, we propose the structured linear mixed model (StructLMM), a computationally efficient method to identify and characterize loci that interact with one or more environments. After validating our model using simulations, we applied StructLMM to body mass index in the UK Biobank, where our model yields previously known and novel G×E signals. Finally, in an application to a large blood eQTL dataset, we demonstrate that StructLMM can be used to study interactions with hundreds of environmental variables

Two-year clinical follow-up of the Multicenter Randomized Clinical Trial of Endovascular Treatment for Acute Ischemic Stroke in The Netherlands (MR CLEAN): design and statistical analysis plan of the extended follow-up study

Background: MR CLEAN was the first randomized trial to demonstrate the short-term clinical effectiveness of endovascular treatment in patients with acute ischemic stroke caused by large vessel occlusion in the anterior circulation. Several other trials confirmed that endovascular treatment improves clinical outcome at three months. However, limited data are available on long-term clinical outcome. We aimed to estimate the effect of endovascular treatment on functional outcome at two-year follow-up in patients with acute ischemic stroke. Secondly, we aimed to assess the effect of endovascular treatment on major vascular events and mortality during two years of follow-up. Methods: MR CLEAN is a multicenter clinical trial with randomized treatment allocation, open-label treatment, and blinded endpoint evaluation. Patients included were 18 years or older with acute ischemic stroke caused by a proven anterior proximal artery occlusion who could be treated within six hours after stroke onset. The intervention contrast was endovascular treatment and usual care versus no endovascular treatment and usual care. The current study extended the follow-up duration from three months to two years. The primary outcome is the score on the modified Rankin scale at two years. Secondary outcomes include all-cause mortality and the occurrence of major vascular events within two years of follow-up. Discussion: The results of our study provide information on the long-term clinical effectiveness of endovascular treatment, which may have implications for individual treatment decisions and estimates of cost-effectiveness. Trial registration:NTR1804. Registered on 7 May 2009; ISRCTN10888758. Registered on 24 July 2012 (main MR CLEAN trial); NTR5073. Registered on 26 February 2015 (extended follow-up study)

Extracranial carotid disease and effect of intra-arterial treatment in patients with proximal anterior circulation stroke in MR CLEAN

Background: The presence of extracranial carotid disease (ECD) is associated with less favorable clinical outcomes in patients with acute ischemic stroke caused by intracranial proximal occlusion. Acute intra-arterial treatment (IAT) in the setting of extracranial and intracranial lesions is considered challenging, and whether it yields improved outcomes remains uncertain. Objective: To examine whether the presence of ECD modified the effect of IAT for intracranial proximal anterior circulation occlusion. Design: Prespecified subgroup analysis of a randomized clinical trial of endovascular treatment for acute ischemic stroke in the Netherlands. (Trial registrations: NTR1804 [Netherlands Trial Register] and ISRCTN10888758) Setting: 16 hospitals in the Netherlands. Patients: Acute ischemic stroke caused by proximal intracranial arterial occlusion of the anterior circulation. Extracranial carotid disease was defined as cervical internal carotid artery stenosis (>50%) or occlusion. Intervention: IAT treatment versus no IAT. Measurements: The primary outcome was functional outcome, as measured by the modified Rankin Scale at 90 days and reported as adjusted common odds ratio (acOR) for a shift in direction of a better outcome. Multivariable ordinal logistic regression analysis with an interaction term was used to estimate treatment effect modification by ECD. Results: The overall acOR was 1.67 (95% CI, 1.21 to 2.30) in favor of the intervention. The acOR was 3.1 (CI, 1.7 to 5.8) in the prespecified subgroup of patients with ECD versus 1.3 (CI, 0.9 to 1.9) in patients presenting without ECD. Both acORs are in favor of the intervention (P for interaction = 0.07). Limitation: The study was not powered for subgroup analysis. Conclusion: Intra-arterial treatment may be at least as effective in patients with ECD as in those without ECD, and it should not be withheld in these complex patients with acute ischemic stroke

Association between thrombus composition and stroke etiology in the MR CLEAN Registry biobank

Purpose: The composition of thrombi retrieved during endovascular thrombectomy (EVT) in acute ischemic stroke (AIS) due to large vessel occlusion (LVO) may differ depending on their origin. In this study, we investigated the association between thrombus composition and stroke etiology in a large population of patients from the Dutch MR CLEAN Registry treated with EVT in daily clinical practice. Methods: The thrombi of 332 patients with AIS were histologically analyzed for red blood cells (RBC), fibrin/platelets (F/P), and white blood cells (leukocytes) using a machine learning algorithm. Stroke etiology was assessed using the Trial of Org 10,172 in acute stroke treatment (TOAST) classification. Results: The thrombi of cardioembolic origin contained less RBC and more F/P than those of non-cardioembolic origin (25.8% vs 41.2% RBC [p = 0.003] and 67.1% vs 54.5% F/P [p = 0.004]). The likelihood of a non-cardioembolic source of stroke increased with increasing thrombus RBC content (OR 1.02; [95% CI 1.00–1.06] for each percent increase) and decreased with a higher F/P content (OR 1.02; [95% CI 1.00–1.06]). Thrombus composition in patients with a cardioembolic origin and undetermined origin was similar. Conclusion: Thrombus composition is significantly associated with stroke etiology, with an increase in RBC and a decrease in F/P raising the odds for a non-cardioembolic cause. No difference between composition of cardioembolic thrombi and of undetermined origin was seen. This emphasizes the need for more extensive monitoring for arrhythmias and/or extended cardiac analysis in case of an undetermined origin