Freerk van Dijk

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.

Cohort Profile: LifeLines, a three-generation cohort study and biobank

The LifeLines Cohort Study is a large population-based cohort study and biobank that was established as a resource for research on complex interactions between environmental, phenotypic and genomic factors in the development of chronic diseases and healthy ageing. Between 2006 and 2013, inhabitants of the northern part of The Netherlands and their families were invited to participate, thereby contributing to a three-generation design. Participants visited one of the LifeLines research sites for a physical examination, including lung function, ECG and cognition tests, and completed extensive questionnaires. Baseline data were collected for 167 729 participants, aged from 6 months to 93 years. Follow-up visits are scheduled every 5 years, and in between participants receive follow-up questionnaires. Linkage is being established with medical registries and environmental data. LifeLines contains information on biochemistry, medical history, psychosocial characteristics, lifestyle and more. Genomic data are available including genome-wide genetic data of 15 638 participants. Fasting blood and 24-h urine samples are processed on the day of collection and stored at -80 °C in a fully automated storage facility. The aim of LifeLines is to be a resource for the national and international scientific community. Requests for data and biomaterials can be submitted to the LifeLines Research Office [LLscience@umcg.nl].

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.

Disease variants alter transcription factor levels and methylation of their binding sites

Most disease-associated genetic variants are noncoding, making it challenging to design experiments to understand their functional consequences. Identification of expression quantitative trait loci (eQTLs) has been a powerful approach to infer the downstream effects of disease-associated variants, but most of these variants remain unexplained. The analysis of DNA methylation, a key component of the epigenome, offers highly complementary data on the regulatory potential of genomic regions. Here we show that disease-associated variants have widespread effects on DNA methylation in trans that likely reflect differential occupancy of trans binding sites by cis-regulated transcription factors. Using multiple omics data sets from 3,841 Dutch individuals, we identified 1,907 established trait-associated SNPs that affect the methylation levels of 10,141 different CpG sites in trans (false discovery rate (FDR) < 0.05). These included SNPs that affect both the expression of a nearby transcription factor (such as NFKB1, CTCF and NKX2-3) and methylation of its respective binding site across the genome. Trans methylation QTLs effectively expose the downstream effects of disease-associated variants.

Mutations in potassium channel kcnd3 cause spinocerebellar ataxia type 19

To identify the causative gene for the neurodegenerative disorder spinocerebellar ataxia type 19 (SCA19) located on chromosomal region 1p21‐q21.

Identification of context-dependent expression quantitative trait loci in whole blood

Genetic risk factors often localize to noncoding regions of the genome with unknown effects on disease etiology. Expression quantitative trait loci (eQTLs) help to explain the regulatory mechanisms underlying these genetic associations. Knowledge of the context that determines the nature and strength of eQTLs may help identify cell types relevant to pathophysiology and the regulatory networks underlying disease. Here we generated peripheral blood RNA–seq data from 2,116 unrelated individuals and systematically identified context-dependent eQTLs using a hypothesis-free strategy that does not require previous knowledge of the identity of the modifiers. Of the 23,060 significant cis-regulated genes (false discovery rate (FDR) ≤ 0.05), 2,743 (12%) showed context-dependent eQTL effects. The majority of these effects were influenced by cell type composition. A set of 145 cis-eQTLs depended on type I interferon signaling. Others were modulated by specific transcription factors binding to the eQTL SNPs.

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.

Kinetics of cell spreading on protein precoated substrata: a study of interfacial aspects

In this paper, interfacial aspects of spreading and adhesion of human skin fibroblasts on solid substrata after protein precoating have been studied. Three solid substrata were used with different surface free energy (gamma s): Tissue Culture Polystyrene (TCPS) with gamma s = 70 erg.cm-2, Polyvinylfluoride (PVF) with gamma s = 56 erg.cm-2 and Fluoroethylenepropylene (FEP) copolymer with gamma s = 18 erg.cm-2. The substrata were precoated with fetal calf serum, bovine fibronectin or bovine serum albumin. Cell spreading was evaluated by means of light microscopy and scanning electron microscopy (SEM). Adhesion sites were studied by transmission electron microscopy (TEM). In general, spreading was lowest on FEP and highest on TCPS. Although protein precoating markedly increased cell spreading, the relative order in which the cells spread on the protein precoated substrata remained identical to that on the bare substrata. Analysis of the kinetics of spreading demonstrated that spreading was fastest on the high-energy substratum and slowest on the low-energy substratum. In the presence of all three types of protein precoating, the average distance between a cell and a substratum after spreading was smaller (20-50 nm) than without a coating (greater than 100 nm).

The effect of buttermilk consumption on biofilm formation on silicone rubber voice prostheses in an artificial throat.

Abstract Biofilm formation on indwelling silicone rubber voice prostheses in laryngectomized patients is still the main reason for dysfunction of the valve, leading to frequent replacements. Within patient support groups in The Netherlands, laryngectomees have suggested that the consumption of buttermilk prolongs the life-time of indwelling silicone rubber voice prostheses. The aim of the present study was to compare biofilm formation on Groningen button voice prostheses in a so-called artificial throat. Ten prostheses were placed in a simulated control group and ten other prostheses in a group with a simulated consumption of 700 ml buttermilk three times a day. Biofilms were allowed to grow on the prostheses by inoculating two artificial throats with the total cultivable microflora (bacteria and yeasts) isolated from an explanted Groningen button voice prosthesis. After 3 days, one artificial throat was perfused three times daily with phosphate buffer (control group) for 8 days, while the other artificial throat was perfused with buttermilk. Prostheses removed from the artificial throat in the control group were covered with a thick biofilm. Scanning electron microscopy showed microcolonies growing into the silicone rubber, similar to the ingrowth observed on explanted Groningen buttons. The simulated consumption of buttermilk in the other artificial throat almost fully prevented the formation of a biofilm on the prostheses during the experimental period. These in vitro experiments in the artificial throat demonstrate that the deterioration of voice prostheses can be lessened by the daily intake of buttermilk through its inhibitory effects on biofilm formation.

Deleterious Alleles in the Human Genome Are on Average Younger Than Neutral Alleles of the Same Frequency

Large-scale population sequencing studies provide a complete picture of human genetic variation within the studied populations. A key challenge is to identify, among the myriad alleles, those variants that have an effect on molecular function, phenotypes, and reproductive fitness. Most non-neutral variation consists of deleterious alleles segregating at low population frequency due to incessant mutation. To date, studies characterizing selection against deleterious alleles have been based on allele frequency (testing for a relative excess of rare alleles) or ratio of polymorphism to divergence (testing for a relative increase in the number of polymorphic alleles). Here, starting from Maruyamas theoretical prediction (Maruyama T (1974), Am J Hum Genet USA 6:669–673) that a (slightly) deleterious allele is, on average, younger than a neutral allele segregating at the same frequency, we devised an approach to characterize selection based on allelic age. Unlike existing methods, it compares sets of neutral and deleterious sequence variants at the same allele frequency. When applied to human sequence data from the Genome of the Netherlands Project, our approach distinguishes low-frequency coding non-synonymous variants from synonymous and non-coding variants at the same allele frequency and discriminates between sets of variants independently predicted to be benign or damaging for protein structure and function. The results confirm the abundance of slightly deleterious coding variation in humans.