Marian Beekman

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.

Evidence of genetic enrichment for exceptional survival using a family approach : the Leiden Longevity study

We conducted a sib pair study in very old subjects for the purpose of mapping longevity loci. In the present analysis, we explore whether our recruitment strategy has resulted in a population enriched for a heritable component for exceptional longevity. Our study includes families with at least two long-living siblings (men aged 89 years or above; women aged 91 years or above). Data were collected on date of birth and, if applicable, date of death of parents, brothers and sisters, offspring, and spouses of the long-living participants. Standardised mortality ratios (SMRs) compared with the general Dutch population, were calculated. The SMR for all siblings of the long-living participants was 0.66 (95% CI 0.60–0.73). A similar survival benefit was also observed in the parents (SMR=0.76, 95% CI 0.66–0.87) and in the offspring of the long-living subjects (SMR=0.65, 95% CI 0.51–0.80). The SMR of the spouses of the long-living subjects was 0.95 (95% CI 0.82–1.12). The familial clustering of extended survival is unlikely to be caused by ascertainment bias, because in all analyses the long-living participants were excluded. Moreover, it is also unlikely to be caused by environmental factors, because the spouses of the long-living participants had a mortality risk comparable with the general Dutch population, whereas they share the same environment. We conclude that our sample is genetically enriched for extreme survival.

Reduced insulin/IGF-1 signalling and human longevity

Evidence is accumulating that aging is hormonally regulated by an evolutionarily conserved insulin/IGF‐1 signalling (IIS) pathway. Mutations in IIS components affect lifespan in Caenorhabditis elegans, Drosophila melanogaster and mice. Most long‐lived IIS mutants also show increased resistance to oxidative stress. In D. melanogaster and mice, the long‐lived phenotype of several IIS mutants is restricted to females. Here, we analysed the relationship between IIS signalling, body height and longevity in humans in a prospective follow‐up study. Based on the expected effects (increased or decreased signalling) of the selected variants in IIS pathway components (GHRHR, GH1, IGF1, INS, IRS1), we calculated composite IIS scores to estimate IIS pathway activity. In addition, we analysed the relative impact on lifespan and body size of the separate variants in multivariate models. In women, lower IIS scores are significantly associated with lower body height and improved old age survival. Multivariate analyses showed that these results were most pronounced for the GH1 SNP, IGF1 CA repeat and IRS1 SNP. In females, for variant allele carriers of the GH1 SNP, body height was 2 cm lower (P = 0.007) and mortality 0.80‐fold reduced (P = 0.019) when compared with wild‐type allele carriers. Thus, in females, genetic variation causing reduced IIS activation is beneficial for old age survival. This effect was stronger for the GH1 SNP than for variation in the conserved IIS genes that were found to affect longevity in model organisms.

Variation in the human TP53 gene affects old age survival and cancer mortality

Longevity may depend on a balance between tumor suppression and tissue renewal mechanisms [Campisi, J., 2003. Cancer and ageing: rival demons? Nat. Rev. Cancer 3 (5), 339-349]. Mice with constitutively activated p53 are almost cancer free but their life span is reduced and accompanied by early tissue atrophy [Tyner et al., 2002. p53 mutant mice that display early ageing-associated phenotypes. Nature 415 (6867) 45-53]. Replacement of arginine (Arg) by proline (Pro) at position 72 of human p53 decreases its apoptotic potential [Dumont et al., 2003. The codon 72 polymorphic variants of p53 have markedly different apoptotic potential. Nat. Genet. 33 (3), 357-365] providing a tool to test for a similar trade-off in humans. Using a formal meta-analysis of the published literature we show that carriers of the TP53 codon 72 Pro/Pro genotype have an increased cancer risk compared to Arg/Arg carriers (p<0.05). Next, in a prospective study of 1226 people aged 85 years and over we show that carriers of the Pro/Pro genotype have a 41% increased survival (p = 0.032) despite a 2.54 fold increased (p = 0.007) proportional mortality from cancer. It is suggested that human p53 protect against cancer but at a cost of longevity.

Heritabilities of Apolipoprotein and Lipid Levels in Three Countries

This study investigated the influence of genes and environment on the variation of apolipoprotein and lipid levels, which are important intermediate phenotypes in the pathways toward cardiovascular disease. Heritability estimates are presented, including those for apolipoprotein E and AII levels which have rarely been reported before. We studied twin samples from the Netherlands (two cohorts; n = 160 pairs, aged 13-22 and n = 204 pairs, aged 34-62), Australia (n = 1362 pairs, aged 28-92) and Sweden (n = 302 pairs, aged 42-88). The variation of apolipoprotein and lipid levels depended largely on the influences of additive genetic factors in each twin sample. There was no significant evidence for the influence of common environment. No sex differences in heritability estimates for any phenotype in any of the samples were observed. Heritabilities ranged from 0.48-0.87, with most heritabilities exceeding 0.60. The heritability estimates in the Dutch samples were significantly higher than in the Australian sample. The heritabilities for the Swedish were intermediate to the Dutch and the Australian samples and not significantly different from the heritabilities in these other two samples. Although sample specific effects are present, we have shown that genes play a major role in determining the variance of apolipoprotein and lipid levels in four independent twin samples from three different countries.

Effect of the stromelysin-1 promoter on efficacy of pravastatin in coronary atherosclerosis and restenosis.

It has proved difficult to identify high-risk patients for atherosclerosis and to determine how they might respond to medication. Recently, a common promoter variant of the human stromelysin-1 gene has been reported, which has been shown to affect the transcription. We investigated whether this polymorphism had any impact on the risk of events, especially restenosis and progression of coronary artery disease and whether the effect was modulated by treatment with pravastatin. The stromelysin-1 genotype was determined for 496 men with coronary artery disease and cholesterol levels between 4.0 and 8.0 mmol/L, participating in the Regression Growth Evaluation Statin Study (REGRESS) study, a clinical trial assessing the effect of the lipid-lowering drug pravastatin on the progression of atherosclerosis. Patients in the placebo group with 5A6A or 6A6A genotypes had more clinical events than patients with the 5A5A genotype (26% and 12%, respectively, p = 0.03). In the pravastatin group, the risk of clinical events in patients with 5A6A or 6A6A genotypes was lower, compared with placebo, whereas it was unchanged in those with a 5A5A genotype (p value for interaction: 0.038). Also, the incidence of repeat angioplasty in the placebo group was greater in patients with the 6A6A or 5A6A genotypes, compared with 5A homozygotes (38% and 40%, respectively, vs 11%, p = 0.09). Again, treatment substantially reduced the incidence in heterozygotes and 6A homozygotes (0% and 15%, respectively), whereas it was unchanged in 5A homozygotes (28%, p for interaction: 0.002). These effects were independent of the effects of pravastatin on the lipid levels. Thus, this study suggests that the stomelysin-1 promoter polymorphism confers a genotype-specific response to medication in determining clinical event-free survival and the risk for symptom-driven repeat angioplasty. This variant may therefore act as a predictor, not only of disease progression, but also of response to therapy and risk of restenosis.

Genome-wide association meta-analysis of human longevity identifies a novel locus conferring survival beyond 90 years of age

The genetic contribution to the variation in human lifespan is ∼25%. Despite the large number of identified disease-susceptibility loci, it is not known which loci influence population mortality. We performed a genome-wide association meta-analysis of 7729 long-lived individuals of European descent (≥85 years) and 16 121 younger controls (<65 years) followed by replication in an additional set of 13 060 long-lived individuals and 61 156 controls. In addition, we performed a subset analysis in cases aged ≥90 years. We observed genome-wide significant association with longevity, as reflected by survival to ages beyond 90 years, at a novel locus, rs2149954, on chromosome 5q33.3 (OR = 1.10, P = 1.74 × 10−8). We also confirmed association of rs4420638 on chromosome 19q13.32 (OR = 0.72, P = 3.40 × 10−36), representing the TOMM40/APOE/APOC1 locus. In a prospective meta-analysis (n = 34 103), the minor allele of rs2149954 (T) on chromosome 5q33.3 associates with increased survival (HR = 0.95, P = 0.003). This allele has previously been reported to associate with low blood pressure in middle age. Interestingly, the minor allele (T) associates with decreased cardiovascular mortality risk, independent of blood pressure. We report on the first GWAS-identified longevity locus on chromosome 5q33.3 influencing survival in the general European population. The minor allele of this locus associates with low blood pressure in middle age, although the contribution of this allele to survival may be less dependent on blood pressure. Hence, the pleiotropic mechanisms by which this intragenic variation contributes to lifespan regulation have to be elucidated.

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.

Genome-wide linkage analysis for human longevity: Genetics of Healthy Aging Study

Clear evidence exists for heritability of human longevity, and much interest is focused on identifying genes associated with longer lives. To identify such longevity alleles, we performed the largest genome‐wide linkage scan thus far reported. Linkage analyses included 2118 nonagenarian Caucasian sibling pairs that have been enrolled in 15 study centers of 11 European countries as part of the Genetics of Healthy Aging (GEHA) project. In the joint linkage analyses, we observed four regions that show linkage with longevity; chromosome 14q11.2 (LOD = 3.47), chromosome 17q12‐q22 (LOD = 2.95), chromosome 19p13.3‐p13.11 (LOD = 3.76), and chromosome 19q13.11‐q13.32 (LOD = 3.57). To fine map these regions linked to longevity, we performed association analysis using GWAS data in a subgroup of 1228 unrelated nonagenarian and 1907 geographically matched controls. Using a fixed‐effect meta‐analysis approach, rs4420638 at the TOMM40/APOE/APOC1 gene locus showed significant association with longevity (P‐value = 9.6 × 10−8). By combined modeling of linkage and association, we showed that association of longevity with APOEε4 and APOEε2 alleles explain the linkage at 19q13.11‐q13.32 with P‐value = 0.02 and P‐value = 1.0 × 10−5, respectively. In the largest linkage scan thus far performed for human familial longevity, we confirm that the APOE locus is a longevity gene and that additional longevity loci may be identified at 14q11.2, 17q12‐q22, and 19p13.3‐p13.11. As the latter linkage results are not explained by common variants, we suggest that rare variants play an important role in human familial longevity.

The impact of low-frequency and rare variants on lipid levels

Using a genome-wide screen of 9.6 million genetic variants achieved through 1000 Genomes Project imputation in 62,166 samples, we identify association to lipid traits in 93 loci, including 79 previously identified loci with new lead SNPs and 10 new loci, 15 loci with a low-frequency lead SNP and 10 loci with a missense lead SNP, and 2 loci with an accumulation of rare variants. In six loci, SNPs with established function in lipid genetics (CELSR2, GCKR, LIPC and APOE) or candidate missense mutations with predicted damaging function (CD300LG and TM6SF2) explained the locus associations. The low-frequency variants increased the proportion of variance explained, particularly for low-density lipoprotein cholesterol and total cholesterol. Altogether, our results highlight the impact of low-frequency variants in complex traits and show that imputation offers a cost-effective alternative to resequencing.