Sandra Imholz

Dietary n−3 and n−6 polyunsaturated fatty acid intake interacts with FADS1 genetic variation to affect total and HDL-cholesterol concentrations in the Doetinchem Cohort Study

BACKGROUND The delta-5 and delta-6 desaturases, encoded by the FADS1 and FADS2 genes, are rate-limiting enzymes in polyunsaturated fatty acid (PUFA) biosynthesis. Single nucleotide polymorphisms in the FADS gene cluster region have been associated with both PUFA concentrations in plasma or erythrocyte membrane phospholipids and cholesterol concentrations in recent genome-wide association studies. OBJECTIVE We examined whether genetic variations in the FADS gene cluster region interact with dietary intakes of n-3 (omega-3) and n-6 (omega-6) PUFAs to affect plasma total, HDL-, and non-HDL-cholesterol concentrations. DESIGN Dietary intakes of n-3 and n-6 PUFAs, plasma concentrations of total and HDL cholesterol, and rs174546, rs482548, and rs174570 in the FADS gene cluster region were measured in 3575 subjects in the second survey of the Doetinchem Cohort Study. RESULTS Significant associations between rs174546 genotypes and total and non-HDL-cholesterol concentrations were observed in the group with a high intake of n-3 PUFAs (> or =0.51% of total energy; P = 0.006 and 0.047, respectively) but not in the low-intake group (P for interaction = 0.32 and 0.51, respectively). The C allele was associated with high total and non-HDL-cholesterol concentrations. Furthermore, the C allele was significantly associated with high HDL-cholesterol concentrations in the group with a high intake of n-6 PUFAs (> or =5.26% of total energy, P = 0.004) but not in the group with a low intake (P for interaction = 0.02). CONCLUSION Genetic variation in the FADS1 gene potentially interacts with dietary PUFA intakes to affect plasma cholesterol concentrations, which should be investigated further in other studies.

Literature-based genetic risk scores for coronary heart disease: the Cardiovascular Registry Maastricht (CAREMA) prospective cohort study.

Background— Genome-wide association studies (GWAS) have identified many single-nucleotide polymorphisms (SNPs) associated with coronary heart disease (CHD) or CHD risk factors (RF). Using a case-cohort study within the prospective Cardiovascular Registry Maastricht (CAREMA) cohort, we tested if genetic risk scores (GRS) based on GWAS-identified SNPs are associated with and predictive for future CHD. Methods and Results— Incident cases (n=742), that is, participants who developed CHD during a median follow-up of 12.1 years (range, 0.0–16.9 years), were compared with a randomly selected subcohort of 2221 participants selected from the total cohort (n=21 148). We genotyped 179 SNPs previously associated with CHD or CHD RF in GWAS as published up to May 2, 2011. The allele-count GRS, composed of all SNPs, the 153 RF SNPs, or the 29 CHD SNPs were not associated with CHD independent of CHD RF. The weighted 29 CHD SNP GRS, with weights obtained from GWAS for every SNP, were associated with CHD independent of CHD RF (hazard ratio, 1.12 per weighted risk allele; 95% confidence interval, 1.04–1.21) and improved risk reclassification with 2.8% (P=0.031). As an exploratory approach to achieve weighting, we performed least absolute shrinkage and selection operator (LASSO) regression analysis on all SNPs and the CHD SNPs. The CHD LASSO GRS performed equal to the weighted CHD GRS, whereas the Overall LASSO GRS performed slightly better than the weighted CHD GRS. Conclusions— A GRS composed of CHD SNPs improves risk prediction when adjusted for the effect sizes of the SNPs. Alternatively LASSO regression analysis may be used to achieve weighting; however, validation in independent populations is required.Background— Genome-wide association studies (GWAS) have identified many single-nucleotide polymorphisms (SNPs) associated with coronary heart disease (CHD) or CHD risk factors (RF). Using a case-cohort study within the prospective Cardiovascular Registry Maastricht (CAREMA) cohort, we tested if genetic risk scores (GRS) based on GWAS-identified SNPs are associated with and predictive for future CHD. Methods and Results— Incident cases (n=742), that is, participants who developed CHD during a median follow-up of 12.1 years (range, 0.0–16.9 years), were compared with a randomly selected subcohort of 2221 participants selected from the total cohort (n=21 148). We genotyped 179 SNPs previously associated with CHD or CHD RF in GWAS as published up to May 2, 2011. The allele-count GRS, composed of all SNPs, the 153 RF SNPs, or the 29 CHD SNPs were not associated with CHD independent of CHD RF. The weighted 29 CHD SNP GRS, with weights obtained from GWAS for every SNP, were associated with CHD independent of CHD RF (hazard ratio, 1.12 per weighted risk allele; 95% confidence interval, 1.04–1.21) and improved risk reclassification with 2.8% ( P =0.031). As an exploratory approach to achieve weighting, we performed least absolute shrinkage and selection operator (LASSO) regression analysis on all SNPs and the CHD SNPs. The CHD LASSO GRS performed equal to the weighted CHD GRS, whereas the Overall LASSO GRS performed slightly better than the weighted CHD GRS. Conclusions— A GRS composed of CHD SNPs improves risk prediction when adjusted for the effect sizes of the SNPs. Alternatively LASSO regression analysis may be used to achieve weighting; however, validation in independent populations is required.

Restricted diet delays accelerated ageing and genomic stress in DNA-repair-deficient mice

Mice deficient in the DNA excision-repair gene Ercc1 (Ercc1∆/−) show numerous accelerated ageing features that limit their lifespan to 4-6 months. They also exhibit a ‘survival response’, which suppresses growth and enhances cellular maintenance. Such a response resembles the anti-ageing response induced by dietary restriction (also known as caloric restriction). Here we report that a dietary restriction of 30% tripled the median and maximal remaining lifespans of these progeroid mice, strongly retarding numerous aspects of accelerated ageing. Mice undergoing dietary restriction retained 50% more neurons and maintained full motor function far beyond the lifespan of mice fed ad libitum. Other DNA-repair-deficient, progeroid Xpg−/− (also known as Ercc5−/−) mice, a model of Cockayne syndrome, responded similarly. The dietary restriction response in Ercc1∆/− mice closely resembled the effects of dietary restriction in wild-type animals. Notably, liver tissue from Ercc1∆/− mice fed ad libitum showed preferential extinction of the expression of long genes, a phenomenon we also observed in several tissues ageing normally. This is consistent with the accumulation of stochastic, transcription-blocking lesions that affect long genes more than short ones. Dietary restriction largely prevented this declining transcriptional output and reduced the number of γH2AX DNA damage foci, indicating that dietary restriction preserves genome function by alleviating DNA damage. Our findings establish the Ercc1∆/− mouse as a powerful model organism for health-sustaining interventions, reveal potential for reducing endogenous DNA damage, facilitate a better understanding of the molecular mechanism of dietary restriction and suggest a role for counterintuitive dietary-restriction-like therapy for human progeroid genome instability syndromes and possibly neurodegeneration in general.

Genetic variations in regulatory pathways of fatty acid and glucose metabolism are associated with obesity-phenotypes: a population based cohort study

Background:As nuclear receptors and transcription factors have an important regulatory function in adipocyte differentiation and fat storage, genetic variation in these key regulators and downstream pathways may be involved in the onset of obesity.Objective:To explore associations between single nucleotide polymorphisms (SNPs) in candidate genes from regulatory pathways that control fatty acid and glucose metabolism, and repeated measurements of body mass index (BMI) and waist circumference in a large Dutch study population.Methods:Data of 327 SNPs across 239 genes were analyzed for 3575 participants of the Doetinchem cohort, who were examined three times during 11 years, using the Illumina Golden Gate assay. Adjusted random coefficient models were used to analyze the relationship between SNPS and obesity phenotypes. False discovery rate q-values were calculated to account for multiple testing. Significance of the associations was defined as a q-value ⩽0.20.Results:Two SNPs (in NR1H4 and SMARCA2 in women only) were significantly associated with both BMI and waist circumference. In addition, two SNPs (in SIRT1 and SCAP in women only) were associated with BMI alone. A functional SNP, in IL6, was strongly associated with waist.Conclusion:In this explorative study among participants of a large population-based cohort, five SNPs, mainly located in transcription mediator genes, were strongly associated with obesity phenotypes. The results from whole genome and candidate gene studies support the potential role of NR1H4, SIRT1, SMARCA2 and IL6 in obesity. Although replication of our findings and further research on the functionality of these SNPs and underlying mechanism is necessary, our data indirectly suggest a role of GATA transcription factors in weight control.

Markers of Endogenous Desaturase Activity and Risk of Coronary Heart Disease in the CAREMA Cohort Study

Background Intakes of n-3 polyunsaturated fatty acids (PUFAs), especially EPA (C20∶5n-3) and DHA (C22∶6n-3), are known to prevent fatal coronary heart disease (CHD). The effects of n-6 PUFAs including arachidonic acid (C20∶4n-6), however, remain unclear. δ-5 and δ-6 desaturases are rate-limiting enzymes for synthesizing long-chain n-3 and n-6 PUFAs. C20∶4n-6 to C20∶3n-6 and C18∶3n-6 to C18∶2n-6 ratios are markers of endogenous δ-5 and δ-6 desaturase activities, but have never been studied in relation to incident CHD. Therefore, the aim of this study was to investigate the relation between these ratios as well as genotypes of FADS1 rs174547 and CHD incidence. Methods We applied a case-cohort design within the CAREMA cohort, a large prospective study among the general Dutch population followed up for a median of 12.1 years. Fatty acid profile in plasma cholesteryl esters and FADS1 genotype at baseline were measured in a random subcohort (n = 1323) and incident CHD cases (n = 537). Main outcome measures were hazard ratios (HRs) of incident CHD adjusted for major CHD risk factors. Results The AA genotype of rs174547 was associated with increased plasma levels of C204n-6, C20∶5n-3 and C22∶6n-3 and increased δ-5 and δ-6 desaturase activities, but not with CHD risk. In multivariable adjusted models, high baseline δ-5 desaturase activity was associated with reduced CHD risk (P for trend = 0.02), especially among those carrying the high desaturase activity genotype (AA): HR (95% CI) = 0.35 (0.15–0.81) for comparing the extreme quintiles. High plasma DHA levels were also associated with reduced CHD risk. Conclusion In this prospective cohort study, we observed a reduced CHD risk with an increased C20∶4n-6 to C20∶3n-6 ratio, suggesting that δ-5 desaturase activity plays a role in CHD etiology. This should be investigated further in other independent studies.

Exploring genetic determinants of plasma total cholesterol levels and their predictive value in a longitudinal study

BACKGROUND Plasma total cholesterol (TC) levels are highly genetically determined. Although ample evidence of genetic determination of separate lipoprotein cholesterol levels has been reported, using TC level directly as a phenotype in a relatively large broad-gene based association study has not been reported to date. METHODS AND RESULTS We genotyped 361 single nucleotide polymorphisms (SNPs) across 243 genes based on pathways potentially relevant to cholesterol metabolism in 3575 subjects that were examined thrice over 11 years. Twenty-three SNPs were associated with TC levels after adjustment for multiple testing. We used 12 of them (rs7412 and rs429358 in APOE, rs646776 in CELSR2, rs1367117 in APOB, rs6756629 in ABCG5, rs662799 in APOA5, rs688 in LDLR, rs10889353 in DOCK7, rs2304130 in NCAN, rs3846662 in HMGCR, rs2275543 in ABCA1, rs7275 in SMARCA4) that were confirmed in previous candidate association or genome-wide-association studies to define a gene risk score (GRS). Average TC levels increased from 5.23 ± 0.82 mmol/L for those with 11 or less cholesterol raising alleles to 6.03 ± 1.11 mmol/L for those with 18 or more (P for trend<0.0001). The association with TC levels was slightly stronger when the weighted GRS that weighted the magnitude of allelic effects was used. CONCLUSION A panel of common genetic variants in the genes pivotal in cholesterol metabolism could possibly help identify those people who are at risk of high cholesterol levels.

Identification of interleukin-1 beta, but no other inflammatory proteins, as an early onset pre-eclampsia biomarker in first trimester serum by bead-based multiplexed immunoassays.

This study aimed to determine the predictive value of growth factors, cardiovascular, and immunological markers for first trimester identification of early onset pre‐eclampsia (PE).

Bead‐based multiplexed immunoassays to identify new biomarkers in maternal serum to improve first trimester Down syndrome screening

To identify new discriminative biomarkers for Down syndrome (DS) pregnancies using a bead‐based multiplexed immunoassay, and to use the newly identified biomarkers to construct a prediction model for non‐invasive DS screening.

Glucose levels and genetic variants across transcriptional pathways: interaction effects with BMI.

Objective:Much of the genetic variation in glucose levels remains to be discovered. Especially, research on gene–environment interactions is scarce. Overweight is one of the main risk factors for hyperglycemia. As transcriptional regulation is important for both weight maintenance and glucose control, we analyzed 353 single nucleotide polymorphisms (SNPs), occurring in transcriptional pathways of glucose and lipid metabolism in interaction with body mass index (BMI) on glucose levels.Research design and methods:SNPs were measured in 3244 participants of the Doetichem cohort. Non-fasting glucose levels and BMI were measured twice in 6 years. SNP × BMI interactions were analyzed by mixed models and adjusted for age, sex, time since last meal, and follow-up time. False discovery rate (FDR) <0.2 was used to adjust for multiple testing.Results:Two SNPs in the PPARGC1A gene (rs8192678, FDR=0.07; rs3755863, FDR=0.17) showed a significant interaction with BMI. The rare allele of both SNPs was associated with significantly lower glucose levels in subjects with a BMI⩽25 kg m–2 (rs8192678, P=0.02; rs3755863, P=0.03). An inverse association was suggested in subjects with a BMI>28 kg m–2. A small intervention study (n=120) showed similar, though non-significant, results.Conclusions:Using a pathway-based approach, we found that BMI significantly modified the association between two SNPs in the PPARGC1A gene and glucose levels. The association between glucose and PPARGC1A was only present in lean subjects. This suggests that the effect of the PPARGC1A gene, which is involved both in fatty acid oxidation and glucose metabolism, is modified by BMI.

Deletion of individual Ku subunits in mice causes an NHEJ-independent phenotype potentially by altering apurinic/apyrimidinic site repair.

Ku70 and Ku80 form a heterodimer called Ku that forms a holoenzyme with DNA dependent-protein kinase catalytic subunit (DNA-PKCS) to repair DNA double strand breaks (DSBs) through the nonhomologous end joining (NHEJ) pathway. As expected mutating these genes in mice caused a similar DSB repair-defective phenotype. However, ku70-/- cells and ku80-/- cells also appeared to have a defect in base excision repair (BER). BER corrects base lesions, apurinic/apyrimidinic (AP) sites and single stand breaks (SSBs) utilizing a variety of proteins including glycosylases, AP endonuclease 1 (APE1) and DNA Polymerase β (Pol β). In addition, deleting Ku70 was not equivalent to deleting Ku80 in cells and mice. Therefore, we hypothesized that free Ku70 (not bound to Ku80) and/or free Ku80 (not bound to Ku70) possessed activity that influenced BER. To further test this hypothesis we performed two general sets of experiments. The first set showed that deleting either Ku70 or Ku80 caused an NHEJ-independent defect. We found ku80-/- mice had a shorter life span than dna-pkcs-/- mice demonstrating a phenotype that was greater than deleting the holoenzyme. We also found Ku70-deletion induced a p53 response that reduced the level of small mutations in the brain suggesting defective BER. We further confirmed that Ku80-deletion impaired BER via a mechanism that was not epistatic to Pol β. The second set of experiments showed that free Ku70 and free Ku80 could influence BER. We observed that deletion of either Ku70 or Ku80, but not both, increased sensitivity of cells to CRT0044876 (CRT), an agent that interferes with APE1. In addition, free Ku70 and free Ku80 bound to AP sites and in the case of Ku70 inhibited APE1 activity. These observations support a novel role for free Ku70 and free Ku80 in altering BER.