Jessica van Setten

Whole-genome sequence variation, population structure and demographic history of the Dutch population

Whole-genome sequencing enables complete characterization of genetic variation, but geographic clustering of rare alleles demands many diverse populations be studied. Here we describe the Genome of the Netherlands (GoNL) Project, in which we sequenced the whole genomes of 250 Dutch parent-offspring families and constructed a haplotype map of 20.4 million single-nucleotide variants and 1.2 million insertions and deletions. The intermediate coverage (∼13×) and trio design enabled extensive characterization of structural variation, including midsize events (30–500 bp) previously poorly catalogued and de novo mutations. We demonstrate that the quality of the haplotypes boosts imputation accuracy in independent samples, especially for lower frequency alleles. Population genetic analyses demonstrate fine-scale structure across the country and support multiple ancient migrations, consistent with historical changes in sea level and flooding. The GoNL Project illustrates how single-population whole-genome sequencing can provide detailed characterization of genetic variation and may guide the design of future population studies.

Composition of Carotid Atherosclerotic Plaque Is Associated With Cardiovascular Outcome A Prognostic Study

Background— Identification of patients at risk for primary and secondary manifestations of atherosclerotic disease progression is based mainly on established risk factors. The atherosclerotic plaque composition is thought to be an important determinant of acute cardiovascular events, but no prospective studies have been performed. The objective of the present study was to investigate whether atherosclerotic plaque composition is associated with the occurrence of future vascular events. Methods and Results— Atherosclerotic carotid lesions were collected from patients who underwent carotid endarterectomy and were subjected to histological examination. Patients underwent clinical follow-up yearly, up to 3 years after carotid endarterectomy. The primary outcome was defined as the composite of a vascular event (vascular death, nonfatal stroke, nonfatal myocardial infarction) and vascular intervention. The cumulative event rate at 1-, 2-, and 3-year follow-up was expressed by Kaplan–Meier estimates, and Cox proportional hazards regression analyses were performed to assess the independence of histological characteristics from general cardiovascular risk factors. During a mean follow-up of 2.3 years, 196 of 818 patients (24%) reached the primary outcome. Patients whose excised carotid plaque revealed plaque hemorrhage or marked intraplaque vessel formation demonstrated an increased risk of primary outcome (risk difference=30.6% versus 17.2%; hazard ratio [HR] with [95% confidence interval]=1.7 [1.2 to 2.5]; and risk difference=30.0% versus 23.8%; HR=1.4 [1.1 to 1.9], respectively). Macrophage infiltration (HR=1.1 [0.8 to 1.5]), large lipid core (HR=1.1 [0.7 to 1.6]), calcifications (HR=1.1 [0.8 to 1.5]), collagen (HR=0.9 [0.7 to 1.3]), and smooth muscle cell infiltration (HR=1.3 [0.9 to 1.8]) were not associated with clinical outcome. Local plaque hemorrhage and increased intraplaque vessel formation were independently related to clinical outcome and were independent of clinical risk factors and medication use. Conclusions— The local atherosclerotic plaque composition in patients undergoing carotid endarterectomy is an independent predictor of future cardiovascular events.

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.

Improved imputation quality of low-frequency and rare variants in European samples using the 'Genome of The Netherlands'

Although genome-wide association studies (GWAS) have identified many common variants associated with complex traits, low-frequency and rare variants have not been interrogated in a comprehensive manner. Imputation from dense reference panels, such as the 1000 Genomes Project (1000G), enables testing of ungenotyped variants for association. Here we present the results of imputation using a large, new population-specific panel: the Genome of The Netherlands (GoNL). We benchmarked the performance of the 1000G and GoNL reference sets by comparing imputation genotypes with ‘true’ genotypes typed on ImmunoChip in three European populations (Dutch, British, and Italian). GoNL showed significant improvement in the imputation quality for rare variants (MAF 0.05–0.5%) compared with 1000G. In Dutch samples, the mean observed Pearson correlation, r2, increased from 0.61 to 0.71. We also saw improved imputation accuracy for other European populations (in the British samples, r2 improved from 0.58 to 0.65, and in the Italians from 0.43 to 0.47). A combined reference set comprising 1000G and GoNL improved the imputation of rare variants even further. The Italian samples benefitted the most from this combined reference (the mean r2 increased from 0.47 to 0.50). We conclude that the creation of a large population-specific reference is advantageous for imputing rare variants and that a combined reference panel across multiple populations yields the best imputation results.

A genome-wide association study identifies a functional ERAP2 haplotype associated with birdshot chorioretinopathy

Birdshot chorioretinopathy (BSCR) is a rare form of autoimmune uveitis that can lead to severe visual impairment. Intriguingly, >95% of cases carry the HLA-A29 allele, which defines the strongest documented HLA association for a human disease. We have conducted a genome-wide association study in 96 Dutch and 27 Spanish cases, and 398 unrelated Dutch and 380 Spanish controls. Fine-mapping the primary MHC association through high-resolution imputation at classical HLA loci, identified HLA-A*29:02 as the principal MHC association (odds ratio (OR) = 157.5, 95% CI 91.6-272.6, P = 6.6 × 10(-74)). We also identified two novel susceptibility loci at 5q15 near ERAP2 (rs7705093; OR = 2.3, 95% CI 1.7-3.1, for the T allele, P = 8.6 × 10(-8)) and at 14q32.31 in the TECPR2 gene (rs150571175; OR = 6.1, 95% CI 3.2-11.7, for the A allele, P = 3.2 × 10(-8)). The association near ERAP2 was confirmed in an independent British case-control samples (combined meta-analysis P = 1.7 × 10(-9)). Functional analyses revealed that the risk allele of the polymorphism near ERAP2 is strongly associated with high mRNA and protein expression of ERAP2 in B cells. This study further defined an extremely strong MHC risk component in BSCR, and detected evidence for a novel disease mechanism that affects peptide processing in the endoplasmic reticulum.

Cystatin C and Cardiovascular Disease: A Mendelian Randomization Study.

BACKGROUND Epidemiological studies show that high circulating cystatin C is associated with risk of cardiovascular disease (CVD), independent of creatinine-based renal function measurements. It is unclear whether this relationship is causal, arises from residual confounding, and/or is a consequence of reverse causation. OBJECTIVES The aim of this study was to use Mendelian randomization to investigate whether cystatin C is causally related to CVD in the general population. METHODS We incorporated participant data from 16 prospective cohorts (n = 76,481) with 37,126 measures of cystatin C and added genetic data from 43 studies (n = 252,216) with 63,292 CVD events. We used the common variant rs911119 in CST3 as an instrumental variable to investigate the causal role of cystatin C in CVD, including coronary heart disease, ischemic stroke, and heart failure. RESULTS Cystatin C concentrations were associated with CVD risk after adjusting for age, sex, and traditional risk factors (relative risk: 1.82 per doubling of cystatin C; 95% confidence interval [CI]: 1.56 to 2.13; p = 2.12 × 10−14). The minor allele of rs911119 was associated with decreased serum cystatin C (6.13% per allele; 95% CI: 5.75 to 6.50; p = 5.95 × 10−211), explaining 2.8% of the observed variation in cystatin C. Mendelian randomization analysis did not provide evidence for a causal role of cystatin C, with a causal relative risk for CVD of 1.00 per doubling cystatin C (95% CI: 0.82 to 1.22; p = 0.994), which was statistically different from the observational estimate (p = 1.6 × 10−5). A causal effect of cystatin C was not detected for any individual component of CVD. CONCLUSIONS Mendelian randomization analyses did not support a causal role of cystatin C in the etiology of CVD. As such, therapeutics targeted at lowering circulating cystatin C are unlikely to be effective in preventing CVD.

Concept and design of a genome-wide association genotyping array tailored for transplantation-specific studies

BackgroundIn addition to HLA genetic incompatibility, non-HLA difference between donor and recipients of transplantation leading to allograft rejection are now becoming evident. We aimed to create a unique genome-wide platform to facilitate genomic research studies in transplant-related studies. We designed a genome-wide genotyping tool based on the most recent human genomic reference datasets, and included customization for known and potentially relevant metabolic and pharmacological loci relevant to transplantation.MethodsWe describe here the design and implementation of a customized genome-wide genotyping array, the ‘TxArray’, comprising approximately 782,000 markers with tailored content for deeper capture of variants across HLA, KIR, pharmacogenomic, and metabolic loci important in transplantation. To test concordance and genotyping quality, we genotyped 85 HapMap samples on the array, including eight trios.ResultsWe show low Mendelian error rates and high concordance rates for HapMap samples (average parent-parent-child heritability of 0.997, and concordance of 0.996). We performed genotype imputation across autosomal regions, masking directly genotyped SNPs to assess imputation accuracy and report an accuracy of >0.962 for directly genotyped SNPs. We demonstrate much higher capture of the natural killer cell immunoglobulin-like receptor (KIR) region versus comparable platforms. Overall, we show that the genotyping quality and coverage of the TxArray is very high when compared to reference samples and to other genome-wide genotyping platforms.ConclusionsWe have designed a comprehensive genome-wide genotyping tool which enables accurate association testing and imputation of ungenotyped SNPs, facilitating powerful and cost-effective large-scale genotyping of transplant-related studies.

Human Validation of Genes Associated With a Murine Atherosclerotic Phenotype

Objective— The genetically modified mouse is the most commonly used animal model for studying the pathogenesis of atherosclerotic disease. We aimed to assess if mice atherosclerosis-related genes could be validated in human disease through examination of results from genome-wide association studies. Approach and Results— We performed a systematic review to identify atherosclerosis-causing genes in mice and carried out gene-based association tests of their human orthologs for an association with human coronary artery disease and human large artery ischemic stroke. Moreover, we investigated the association of these genes with human atherosclerotic plaque characteristics. In addition, we assessed the presence of tissue-specific cis-acting expression quantitative trait loci for these genes in humans. Finally, using pathway analyses we show that the putative atherosclerosis-causing genes revealed few associations with human coronary artery disease, large artery ischemic stroke, or atherosclerotic plaque characteristics, despite the fact that the majority of these genes have cis-acting expression quantitative trait loci. Conclusions— A role for genes that has been observed in mice for atherosclerotic lesion development could scarcely be confirmed by studying associations of disease development with common human genetic variants. The value of murine atherosclerotic models for selection of therapeutic targets in human disease remains unclear.

Multiethnic Exome-Wide Association Study of Subclinical Atherosclerosis

Background—The burden of subclinical atherosclerosis in asymptomatic individuals is heritable and associated with elevated risk of developing clinical coronary heart disease. We sought to identify genetic variants in protein-coding regions associated with subclinical atherosclerosis and the risk of subsequent coronary heart disease. Methods and Results—We studied a total of 25 109 European ancestry and African ancestry participants with coronary artery calcification (CAC) measured by cardiac computed tomography and 52 869 participants with common carotid intima–media thickness measured by ultrasonography within the CHARGE Consortium (Cohorts for Heart and Aging Research in Genomic Epidemiology). Participants were genotyped for 247 870 DNA sequence variants (231 539 in exons) across the genome. A meta-analysis of exome-wide association studies was performed across cohorts for CAC and carotid intima–media thickness. APOB p.Arg3527Gln was associated with 4-fold excess CAC (P=3×10−10). The APOE &egr;2 allele (p.Arg176Cys) was associated with both 22.3% reduced CAC (P=1×10−12) and 1.4% reduced carotid intima–media thickness (P=4×10−14) in carriers compared with noncarriers. In secondary analyses conditioning on low-density lipoprotein cholesterol concentration, the &egr;2 protective association with CAC, although attenuated, remained strongly significant. Additionally, the presence of &egr;2 was associated with reduced risk for coronary heart disease (odds ratio 0.77; P=1×10−11). Conclusions—Exome-wide association meta-analysis demonstrates that protein-coding variants in APOB and APOE associate with subclinical atherosclerosis. APOE &egr;2 represents the first significant association for multiple subclinical atherosclerosis traits across multiple ethnicities, as well as clinical coronary heart disease.

Serum Lipid Levels, Body Mass Index, and Their Role in Coronary Artery Calcification: A Polygenic Analysis

Background—Coronary artery calcification (CAC) is widely regarded as a cumulative lifetime measure of atherosclerosis, but it remains unclear what is the relationship between calcification and traditional risk factors for coronary artery disease (CAD) and myocardial infarction (MI). This study characterizes the genetic architecture of CAC by evaluating the overall impact of common alleles associated with CAD/MI and its traditional risk factors. Methods and Results—On the basis of summary-association results from the CARDIoGRAMplusC4D study of CAD/MI, we calculated polygenic risk scores in 2599 participants of the Dutch and Belgian Lung Cancer Screening (NELSON) trial, in whom quantitative CAC levels (Agatston scores) were determined from chest computerized tomographic imaging data. The most significant polygenic model explained ≈14% of the observed CAC variance (P=1.6×10–11), which points to a residual effect because of many as yet unknown loci that overlap between CAD/MI and CAC. In addition, we constructed risk scores based on published single-nucleotide polymorphism associations for traditional cardiovascular risk factors and tested these scores for association with CAC. We found nominally significant associations for genetic risk scores of low-density lipoprotein-cholesterol, total cholesterol, and body mass index, which were successfully replicated in 2182 individuals of the Heinz Nixdorf Recall Study. Conclusions—Pervasive polygenic sharing between CAC and CAD/MI suggests that a substantial fraction of the heritable risk for CAD/MI is mediated through arterial calcification. We also provide evidence that genetic variants associated with serum lipid levels and body mass index influence CAC levels.