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

The Genome of the Netherlands: Design, and project goals

Within the Netherlands a national network of biobanks has been established (Biobanking and Biomolecular Research Infrastructure-Netherlands (BBMRI-NL)) as a national node of the European BBMRI. One of the aims of BBMRI-NL is to enrich biobanks with different types of molecular and phenotype data. Here, we describe the Genome of the Netherlands (GoNL), one of the projects within BBMRI-NL. GoNL is a whole-genome-sequencing project in a representative sample consisting of 250 trio-families from all provinces in the Netherlands, which aims to characterize DNA sequence variation in the Dutch population. The parent-offspring trios include adult individuals ranging in age from 19 to 87 years (mean=53 years; SD=16 years) from birth cohorts 1910-1994. Sequencing was done on blood-derived DNA from uncultured cells and accomplished coverage was 14-15x. The family-based design represents a unique resource to assess the frequency of regional variants, accurately reconstruct haplotypes by family-based phasing, characterize short indels and complex structural variants, and establish the rate of de novo mutational events. GoNL will also serve as a reference panel for imputation in the available genome-wide association studies in Dutch and other cohorts to refine association signals and uncover population-specific variants. GoNL will create a catalog of human genetic variation in this sample that is uniquely characterized with respect to micro-geographic location and a wide range of phenotypes. The resource will be made available to the research and medical community to guide the interpretation of sequencing projects. The present paper summarizes the global characteristics of the project

CHCHD10 variants in amyotrophic lateral sclerosis: where Is the evidence?

Objective: After the initial report of a CHCHD10 mutation in mitochondrial disease with features resembling amyotrophic lateral sclerosis (ALS), CHCHD10 mutations have been considered to be a frequent cause for ALS. However, the exact pathogenicity and clinical significance of these mutations remain unclear. Here, we aimed to determine the role of CHCHD10 mutations in ALS. Methods: We analyzed 4,365 whole genome sequenced ALS patients and 1,832 controls from 7 different countries and examined all nonsynonymous single nucleotide variants in CHCHD10. These were tested for association with ALS, independently and in aggregate using several genetic burden tests (including sequence kernel association test [SKAT], optimal unified test [SKAT-O], and Firth logistic regression). Results: We identified 3 new variants in cases, but only 1 was ALS-specific. lso, 1 control-specific mutation was identified. There was no increased burden of rare coding mutations among ALS patients compared to controls (p=0.86, p=0.86, and p=0.88 for SKAT, SKAT-O, and Firth, respectively). The few carriers with potential pathogenic CHCHD10 mutations exhibited a slowly progressive ALS-like phenotype with atypical features such as myopathy and deafness. Interpretation: CHCHD10 mutations seem to be a far less prevalent cause of pure ALS than previously suggested, and instead appear related to more complex phenotypes. There appears to be insufficient evidence for the pathogenicity of most previously reported variants in pure ALS. This study shows that routine testing for CHCHD10 mutations in pure ALS is not recommended and illustrates the importance of sufficient genetic and functional evidence in establishing pathogenicity of genetic variants

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

Reconsidering the causality of TIA1 mutations in ALS

International audienc

Project MinE: study design and pilot analyses of a large-scale whole-genome sequencing study in amyotrophic lateral sclerosis

The most recent genome-wide association study in amyotrophic lateral sclerosis (ALS) demonstrates a disproportionate contribution from low-frequency variants to genetic susceptibility to disease. We have therefore begun Project MinE, an international collaboration that seeks to analyze whole-genome sequence data of at least 15 000 ALS patients and 7500 controls. Here, we report on the design of Project MinE and pilot analyses of successfully sequenced 1169 ALS patients and 608 controls drawn from the Netherlands. As has become characteristic of sequencing studies, we find an abundance of rare genetic variation (minor allele frequency < 0.1%), the vast majority of which is absent in public datasets. Principal component analysis reveals local geographical clustering of these variants within The Netherlands. We use the whole-genome sequence data to explore the implications of poor geographical matching of cases and controls in a sequence-based disease study and to investigate how ancestry-matched, externally sequenced controls can induce false positive associations. Also, we have publicly released genome-wide minor allele counts in cases and controls, as well as results from genic burden tests

Serum magnesium and calcium levels in relation to ischemic stroke : Mendelian randomization study

ObjectiveTo determine whether serum magnesium and calcium concentrations are causally associated with ischemic stroke or any of its subtypes using the mendelian randomization approach.MethodsAnalyses were conducted using summary statistics data for 13 single-nucleotide polymorphisms robustly associated with serum magnesium (n = 6) or serum calcium (n = 7) concentrations. The corresponding data for ischemic stroke were obtained from the MEGASTROKE consortium (34,217 cases and 404,630 noncases).ResultsIn standard mendelian randomization analysis, the odds ratios for each 0.1 mmol/L (about 1 SD) increase in genetically predicted serum magnesium concentrations were 0.78 (95% confidence interval [CI] 0.69-0.89; p = 1.3 7 10-4) for all ischemic stroke, 0.63 (95% CI 0.50-0.80; p = 1.6 7 10-4) for cardioembolic stroke, and 0.60 (95% CI 0.44-0.82; p = 0.001) for large artery stroke; there was no association with small vessel stroke (odds ratio 0.90, 95% CI 0.67-1.20; p = 0.46). Only the association with cardioembolic stroke was robust in sensitivity analyses. There was no association of genetically predicted serum calcium concentrations with all ischemic stroke (per 0.5 mg/dL [about 1 SD] increase in serum calcium: odds ratio 1.03, 95% CI 0.88-1.21) or with any subtype.ConclusionsThis study found that genetically higher serum magnesium concentrations are associated with a reduced risk of cardioembolic stroke but found no significant association of genetically higher serum calcium concentrations with any ischemic stroke subtype

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

Deciphering the four-letter code : The genetic basis of complex traits and common disease

Deoxyribonucleic acid (DNA) is made up of four bases: adenine (A), cytosine (C), guanine (G), and thymine (T). Assembled in a strategic fashion, these bases code for the unique genomes of all walks of life, from viruses, to rodents, to primates. The human genome, mapped completely for the first time in April of 2003, is 3 billion bases long and contains approximately 20,000 genes. The genome of a randomly selected individual, checked base by base, will be approximately 99.9% identical to the reference map of the human genome. The other 0.1% (roughly 3 million bases total) will contain changes in the DNA that do not match to the canonical reference genome. These changes, or genetic variants, can have biological consequence, as they can impact the shape and function of the proteins for which genes code. Genetic variation accounts for much of the visible (phenotypic) diversity – such as eye color or height – that can be observed in humans today. Such variation also plays a role in susceptibility to disease in humans. In the last ten years, a major breakthrough in identifying the genetic changes that increase risk of disease has been genome-wide association studies (GWAS). In these studies, genetic variants (called single-nucleotide polymorphisms, or SNPs) observed in individuals with a particular trait or disorder (affected individuals) are compared to genetic variants observed in healthy (unaffected) individuals. SNPs that are observed more often in affected individuals than in unaffected individuals are associated to increased disease risk. They mark regions (called “loci”, singular “locus”) of the genome that can contain clues about a disease. Knowing which genes in a locus, when mutated, can increase risk of disease is a crucial first step in understanding why certain people are susceptible to disease while others are not. To date, GWAS have identified more than 7,000 loci that influence risk of disease. The primary goal of the work in this thesis is to identify genetic variants associated to risk of common disease. Finding such mutations can help identify disease-causing genes, potentially improving our ability to prevent, properly diagnose or treat illness in humans

Loci associated with ischaemic stroke and its subtypes (SiGN): A genome-wide association study.

BACKGROUND: The discovery of disease-associated loci through genome-wide association studies (GWAS) is the leading genetic approach to the identification of novel biological pathways underlying diseases in humans. Until recently, GWAS in ischaemic stroke have been limited by small sample sizes and have yielded few loci associated with ischaemic stroke. We did a large-scale GWAS to identify additional susceptibility genes for stroke and its subtypes. METHODS: To identify genetic loci associated with ischaemic stroke, we did a two-stage GWAS. In the first stage, we included 16 851 cases with state-of-the-art phenotyping data and 32 473 stroke-free controls. Cases were aged 16 to 104 years, recruited between 1989 and 2012, and subtypes of ischaemic stroke were recorded by centrally trained and certified investigators who used the web-based protocol, Causative Classification of Stroke (CCS). We constructed case-control strata by identifying samples that were genotyped on nearly identical arrays and were of similar genetic ancestral background. We cleaned and imputed data by use of dense imputation reference panels generated from whole-genome sequence data. We did genome-wide testing to identify stroke-associated loci within each stratum for each available phenotype, and we combined summary-level results using inverse variance-weighted fixed-effects meta-analysis. In the second stage, we did in-silico lookups of 1372 single nucleotide polymorphisms identified from the first stage GWAS in 20 941 cases and 364 736 unique stroke-free controls. The ischaemic stroke subtypes of these cases had previously been established with the Trial of Org 10 172 in Acute Stroke Treatment (TOAST) classification system, in accordance with local standards. Results from the two stages were then jointly analysed in a final meta-analysis. FINDINGS: We identified a novel locus (G allele at rs12122341) at 1p13.2 near TSPAN2 that was associated with large artery atherosclerosis-related stroke (first stage odds ratio [OR] 1&middot;21, 95% CI 1&middot;13-1&middot;30, p=4&middot;50 &times; 10(-8); joint OR 1&middot;19, 1&middot;12-1&middot;26, p=1&middot;30 &times; 10(-9)). Our results also supported robust associations with ischaemic stroke for four other loci that have been reported in previous studies, including PITX2 (first stage OR 1&middot;39, 1&middot;29-1&middot;49, p=3&middot;26 &times; 10(-19); joint OR 1&middot;37, 1&middot;30-1&middot;45, p=2&middot;79 &times; 10(-32)) and ZFHX3 (first stage OR 1&middot;19, 1&middot;11-1&middot;27, p=2&middot;93 &times; 10(-7); joint OR 1&middot;17, 1&middot;11-1&middot;23, p=2&middot;29 &times; 10(-10)) for cardioembolic stroke, and HDAC9 (first stage OR 1&middot;29, 1&middot;18-1&middot;42, p=3&middot;50 &times; 10(-8); joint OR 1&middot;24, 1&middot;15-1&middot;33, p=4&middot;52 &times; 10(-9)) for large artery atherosclerosis stroke. The 12q24 locus near ALDH2, which has previously been associated with all ischaemic stroke but not with any specific subtype, exceeded genome-wide significance in the meta-analysis of small artery stroke (first stage OR 1&middot;20, 1&middot;12-1&middot;28, p=6&middot;82 &times; 10(-8); joint OR 1&middot;17, 1&middot;11-1&middot;23, p=2&middot;92 &times; 10(-9)). Other loci associated with stroke in previous studies, including NINJ2, were not confirmed. INTERPRETATION: Our results suggest that all ischaemic stroke-related loci previously implicated by GWAS are subtype specific. We identified a novel gene associated with large artery atherosclerosis stroke susceptibility. Follow-up studies will be necessary to establish whether the locus near TSPAN2 can be a target for a novel therapeutic approach to stroke prevention. In view of the subtype-specificity of the associations detected, the rich phenotyping data available in the Stroke Genetics Network (SiGN) are likely to be crucial for further genetic discoveries related to ischaemic stroke. FUNDING: US National Institute of Neurological Disorders and Stroke, National Institutes of Health