👤 Yvonne Jansen

Single nucleotide polymorphisms (SNPs) in genes involved in fatty acid metabolism (FADS1 FADS2 gene cluster) are associated with plasma lipid levels. We aimed to investigate whether these associations are already present early in life and compare the relative contribution of FADS SNPs vs traditional (non-genetic) factors as determinants of plasma lipid levels. Information on infants' plasma total cholesterol levels, genotypes of five FADS SNPs (rs174545, rs174546, rs174556, rs174561, and rs3834458), anthropometric data, maternal characteristics, and breastfeeding history was available for 521 2-year-old children from the KOALA Birth Cohort Study. For 295 of these 521 children, plasma HDLc and non-HDLc levels were also known. Multivariable linear regression analysis was used to study the associations of genetic and non-genetic determinants with cholesterol levels. All FADS SNPs were significantly associated with total cholesterol levels. Heterozygous and homozygous for the minor allele children had about 4% and 8% lower total cholesterol levels than major allele homozygotes. In addition, homozygous for the minor allele children had about 7% lower HDLc levels. This difference reached significance for the SNPs rs174546 and rs3834458. The associations went in the same direction for non-HDLc, but statistical significance was not reached. The percentage of total variance of total cholesterol levels explained by FADS SNPs was relatively low (lower than 3%) but of the same order as that explained by gender and the non-genetic determinants together. FADS SNPs are associated with plasma total cholesterol and HDLc levels in preschool children. This brings a new piece of evidence to explain how blood lipid levels may track from childhood to adulthood. Moreover, the finding that these SNPs explain a similar amount of variance in total cholesterol levels as the non-genetic determinants studied reveals the potential importance of investigating the effects of genetic variations in early life. Show less

Dendritic cells (DCs) are professional antigen presenting cells of the immune system that play a crucial role in initiating immune responses and maintaining self tolerance. Better understanding of the molecular basis of DC immunobiology is required to improve DC-based immunotherapies. We previously described the interaction of transcription factor LUMAN (also known as CREB3 or LZIP) with the DC-specific transmembrane protein DC-STAMP in DCs. Target genes of LUMAN and its role in DCs are currently unknown. In this study we set out to identify genes regulated by LUMAN in DCs using microarray analysis. Expression of a constitutively active form of LUMAN in mouse DC cell line D2SC/1 identified Apolipoprotein A4 (ApoA4) as its target gene. Subsequent validation experiments, bioinformatics-based promoter analysis, and silencing studies confirmed that ApoA4 is a true target gene of LUMAN in bone marrow-derived DCs (BMDCs). Show less

Phospho- and sphingolipids are crucial cellular and intracellular compounds. These lipids are required for active transport, a number of enzymatic processes, membrane formation, and cell signalling. Disruption of their metabolism leads to several diseases, with diverse neurological, psychiatric, and metabolic consequences. A large number of phospholipid and sphingolipid species can be detected and measured in human plasma. We conducted a meta-analysis of five European family-based genome-wide association studies (N = 4034) on plasma levels of 24 sphingomyelins (SPM), 9 ceramides (CER), 57 phosphatidylcholines (PC), 20 lysophosphatidylcholines (LPC), 27 phosphatidylethanolamines (PE), and 16 PE-based plasmalogens (PLPE), as well as their proportions in each major class. This effort yielded 25 genome-wide significant loci for phospholipids (smallest P-value = 9.88×10(-204)) and 10 loci for sphingolipids (smallest P-value = 3.10×10(-57)). After a correction for multiple comparisons (P-value<2.2×10(-9)), we observed four novel loci significantly associated with phospholipids (PAQR9, AGPAT1, PKD2L1, PDXDC1) and two with sphingolipids (PLD2 and APOE) explaining up to 3.1% of the variance. Further analysis of the top findings with respect to within class molar proportions uncovered three additional loci for phospholipids (PNLIPRP2, PCDH20, and ABDH3) suggesting their involvement in either fatty acid elongation/saturation processes or fatty acid specific turnover mechanisms. Among those, 14 loci (KCNH7, AGPAT1, PNLIPRP2, SYT9, FADS1-2-3, DLG2, APOA1, ELOVL2, CDK17, LIPC, PDXDC1, PLD2, LASS4, and APOE) mapped into the glycerophospholipid and 12 loci (ILKAP, ITGA9, AGPAT1, FADS1-2-3, APOA1, PCDH20, LIPC, PDXDC1, SGPP1, APOE, LASS4, and PLD2) to the sphingolipid pathways. In large meta-analyses, associations between FADS1-2-3 and carotid intima media thickness, AGPAT1 and type 2 diabetes, and APOA1 and coronary artery disease were observed. In conclusion, our study identified nine novel phospho- and sphingolipid loci, substantially increasing our knowledge of the genetic basis for these traits. Show less

The long-term role of fatty acids (FAs) in the cause of diabetes remains largely unclear. We aimed to investigate erythrocyte membrane FAs, desaturase activity, and dietary FAs in relation to the incidence of type 2 diabetes. We applied a nested case-cohort design (n = 2724, including 673 incident diabetes cases) within the European Prospective Investigation into Cancer and Nutrition (EPIC)-Potsdam Study, which involves 27,548 middle-aged subjects. Thirty erythrocyte membrane FAs (percentage of total FAs) and FA intake (percentage of total fat) were measured at baseline, and physician-confirmed incident diabetes was assessed during a mean follow-up of 7.0 y. We evaluated Δ⁵ desaturase (D5D) and Δ⁶ desaturase (D6D) activity by using FA product-to-precursor ratios (traditional approach) and by investigating variants in FADS1 and FADS2 genes that encode these desaturases (Mendelian randomization approach). As a main finding, erythrocyte 16:1n-7 and 18:3n-6 and FA ratios, which reflect stearoyl coenzyme A desaturase (SCD) and D6D activity, were directly related to diabetes risk in multivariable-adjusted models [relative risks (95% CIs) comparing extreme quintiles: 16:1n-7, 2.11 (1.46, 3.05); 18:3n-6, 2.00 (1.38, 2.88); SCD, 2.61 (1.75, 3.89); and D6D, 2.46 (1.67, 3.63)], whereas the FA ratio that reflects D5D activity was inversely associated with risk [0.46 (0.31, 0.70)]. The Mendelian randomization approach corroborated the direct relation for D6D activity and tended to support the inverse relation for D5D activity. Proportions of dietary FAs showed only modest to low correlations with erythrocyte FAs and were not significantly associated with risk. The FA profile of erythrocyte membrane phospholipids and activity of desaturase enzymes are strongly linked to the incidence of type 2 diabetes. Show less

Association of genetic-variants in the FADS1-FADS2-gene-cluster with fatty-acid-composition in blood of adult-populations is well established. We analyze this genetic-association in two children-cohort-studies. In addition, the association between variants in the FADS-gene-cluster and blood-fatty-acid-composition with eczema was studied. Data of two population-based-birth-cohorts in The Netherlands and Germany (KOALA, LISA) were pooled (n = 879) and analyzed by (logistic) regression regarding the mutual influence of single-nucleotide-polymorphisms (SNPs) in the FADS-gene-cluster (rs174545, rs174546, rs174556, rs174561, rs3834458), on polyunsaturated fatty acids (PUFA) in blood and parent-reported eczema until the age of 2 years. All SNPs were highly significantly associated with all PUFAs except for alpha-linolenic-acid and eicosapentaenoic-acid, also after correction for multiple-testing. All tested SNPs showed associations with eczema in the LISA-study, but not in the KOALA-study. None of the PUFAs was significantly associated with eczema neither in the pooled nor in the analyses stratified by study-cohort. PUFA-composition in young children's blood is under strong control of the FADS-gene-cluster. Inconsistent results were found for a link between these genetic-variants with eczema. PUFA in blood was not associated with eczema. Thus the hypothesis of an inflammatory-link between PUFA and eczema by the metabolic-pathway of LC-PUFAs as precursors for inflammatory prostaglandins and leukotrienes could not be confirmed by these data. Show less

Delta-5 (D5D) and delta-6 (D6D) desaturases are key enzymes in PUFA metabolism. Several factors (e.g. hyperglycaemia, hypertension, blood lipids, statins and fatty acids in diet and biological tissues) may influence desaturase activity. The goals were to evaluate the associations between variation in genes encoding these desaturases (FADS1 and FADS2) and blood concentrations of n-6 PUFA and estimated D5D and D6D activities (evaluated as product/precursor ratio), and to investigate whether other factors influencing the activity of desaturases modify these associations. A random sample of 2066 participants from the European Prospective Investigation into Cancer and Nutrition-Potsdam study (n 27 548) was utilised in the analyses. Crude and adjusted associations between rs174546 genotypes (reflecting genetic variation in the FADS1 FADS2 gene cluster), n-6 PUFA in erythrocytes and estimated desaturase activities were evaluated using multiple linear regression. Potential effect modification was determined by performing stratified analyses and evaluating interaction terms. We found rs174546 genotypes to be related to linoleic (r² 0·060), γ-linolenic (r² 0·041), eicosadienoic (r² 0·034), arachidonic (r² 0·026), docosatetraenoic acids (r² 0·028), estimated D6D activity (r² 0·052) and particularly strongly to dihomo-γ-linolenic acid (DGLA, r² 0·182) and D5D activity (r² 0·231). We did not observe effect modifications with regard to the estimated D5D activity, DGLA and arachidonic acid (AA) for most of the factors evaluated; however, the genetic effect on D5D activity and DGLA may be modified by the dietary n-6:n-3-ratio (P-values for interaction: 0·008 and 0·002), and the genetic effect on DGLA and AA may be modified by lipid-lowering medication (P-values for interaction: 0·0004 and 0·006). In conclusion, genetic variation in the FADS1 FADS2 gene cluster affects n-6 PUFA profiles in erythrocytes reflecting altered D5D activity. Show less

The genes encoding Delta(5)- and Delta(6)-desaturases (FADS1 FADS2 gene cluster) were reported to be associated with n-3 (omega-3) and n-6 (omega-6) fatty acid proportions in human plasma, tissues, and milk. Docosahexaenoic acid (DHA) can be supplied especially by dietary fish or fish oil and synthesized from alpha-linolenic acid through a pathway involving these desaturases. We evaluated whether FADS gene variants modify the effect of maternal fish and fish-oil intake on plasma and milk DHA proportions. FADS1 rs174561, FADS2 rs174575, and intergenic rs3834458 single nucleotide polymorphisms were genotyped in 309 women from the KOALA Birth Cohort Study in The Netherlands. Plasma was collected at 36 wk of pregnancy, and milk was collected at 1 mo postpartum. Fish and fish-oil intake was assessed by using a food-frequency questionnaire at 34 wk of pregnancy and updated for the week of milk collection. Gene-diet interactions were tested by linear regression analysis. DHA proportions were lower in women homozygous for the minor allele than in women who were homozygous for the major allele (DHA proportions in plasma phospholipids: P < 0.01; DHA proportions in milk: P < 0.05). Fish intake ranged from 0 to 2.5 portions of fatty fish/wk, and 12 women took fish-oil supplements during pregnancy. DHA proportions in plasma phospholipids increased with increasing fish and fish-oil intake, irrespective of the genotype. DHA proportions in milk increased only with fish and fish-oil intake in the major-allele carriers. Lower proportions of DHA in milk from women who were homozygous for the minor allele could not be compensated for by increasing fish and fish-oil intake, possibly because of limited incorporation into milk. Show less

The standard model of Wnt signaling specifies that after receipt of a Wnt ligand at the membranous receptor complex, downstream mediators inhibit a cytoplasmic destruction complex, allowing beta-catenin to accumulate in the cytosol and nucleus and co-activate Wnt target genes. Unexpectedly, shortly after Wnt treatment, we detected the dephosphorylated form of beta-catenin at the plasma membrane, where it displayed a discontinuous punctate labeling. This pool of beta-catenin could only be detected in E-cadherin(-/-) cells, because in E-cadherin(+/+) cells Wnt-induced, membranous beta-catenin was concealed by a constitutive junctional pool. Wnt-signaling-dependent dephosphorylated beta-catenin colocalized at the plasma membrane with two members of the destruction complex -- APC and axin -- and the activated Wnt co-receptor LRP6. beta-catenin induced through the Wnt receptor complex was significantly more competent transcriptionally than overexpressed beta-catenin, both in cultured cells and in early Xenopus embryos. Our data reveal a new step in the processing of the Wnt signal and suggest regulation of signaling output beyond the level of protein accumulation. Show less

Variation in the human apolipoprotein (APO) A5 gene (APOA5) is associated with elevated plasma triglycerides. However, data on the exact role of plasma concentrations of APOA5 in human triglyceride homeostasis are lacking. In the present study, we estimated plasma APOA5 levels in patients with type 2 diabetes at baseline and during atorvastatin treatment, a lipid-lowering treatment that results in a reduction in plasma triglycerides and APOC3. Plasma APOA5 concentration was measured by ELISA in 215 subjects with type 2 diabetes, who were taken from the Diabetes Atorvastatin Lipid-lowering Intervention (DALI) study, a 30-week randomised, double-blind, placebo-controlled study, and given atorvastatin 10 mg or 80 mg daily. At baseline, average plasma APOA5 concentration was 25.7+/-15.6 mug/100 ml. Plasma APOA5 (R (s)=0.40), APOC3 (R (s)=0.72) and APOE (R (s)=0.45) were positively correlated with plasma triglyceride levels (all p<0.001). In multiple linear regression analysis, adjusted for age and sex, the variation in plasma triglycerides was explained mostly by APOC3 (52%) and only to a small extent by APOA5 (6%) and APOE (1%). Atorvastatin treatment decreased plasma triglycerides, APOA5, APOC3 and APOE (all p<0.0001). After treatment, APOC3 remained the major determinant of plasma triglyceride levels (59%), while the contributions of APOA5 and APOE were insignificant (2 and 3%). Our findings reveal a positive association between plasma APOA5 and triglycerides in patients with type 2 diabetes. Treatment with atorvastatin decreased plasma APOA5, APOC3, APOE and triglycerides. In contrast to APOC3, APOA5 is not a major determinant of triglyceride metabolism in these patients. Show less

Insulin resistance is polygenic in origin, and can be observed at an early age. We have shown that variations in APOC3-482T>C and hepatic lipase (LIPC)-514C>T), individually (APOC3 alone) and interactively, modulate insulin and glucose levels after an OGTT in young healthy men participating in the European Atherosclerosis Research Study II (EARSII). Variation in the insulin gene (INS) variable number tandem repeat (VNTR) has been found to predispose to type 1 and type 2 diabetes. We have evaluated the HphI site 23 bp upstream of the INS gene (a surrogate marker for the VNTR) in EARSII (n=822), to determine if variation in INS contributes to insulin resistance. Carriers of the INS VNTR class III (HphI-) allele (frequency=0.29 (95%CI 0.27, 0.31)) had significantly higher 60-min insulin concentrations after the OGTT (P=0.014) and a marginally higher AUC insulin (P=0.07), compared to class I (HphI+) homozygotes. However, this effect on AUC insulin was modified by the level of physical activity, displaying significant gene:environment interaction (P=0.03). We tested for gene:gene interaction between the INS VNTR and both the LIPC-514C>T and APOC3-482T>C. While there was a significant interaction between INS VNTR and LIPC-514C>T on AUC glucose (P=0.013) and on AUC insulin (P=0.015), there was no interaction with APOC3-482T>C. Thus, despite a modest effect of the INS VNTR alone, the influence of this variant on insulin sensitivity was modified by gene:environment and gene:gene interactions, illustrating the biological complexity of insulin resistance. Show less

Multiple gene defects cause Batten disease. Accelerated apoptosis accounts for neurodegeneration in the late infantile and juvenile forms that are due to defects in the CLN3 and CLN2 genes. Extensive neuronal death is seen in CLN2- and CLN3-deficient human brain as well as in CLN6-deficient sheep brain and retina. When neurons in late infantile and juvenile brain survive, they manage to do so by upregulating the neuroprotective molecule Bcl-2. The CLN3 gene has antiapoptotic properties at the molecular level. We show that the CLN2 gene is neuroprotective: it enhances growth of NT2 cells and maintains survival of human postmitotic hNT neurons. Conversely, blocking CLN3 or CLN2 expression in hNT neurons with adenoviral antisense-CLN3 or antisense-CLN2-AAV2 constructs causes apoptosis. The drug flupirtine is a triaminopyridine derivative that acts as a nonopioid analgesic. Flupirtine upregulates Bcl-2, increases glutathione levels, activates an inwardly rectifying potassium channel, and delays loss of intermitochondrial membrane calcium retention capacity. We show that flupirtine aborts etoposide-induced apoptosis in CLN1-, CLN2-, CLN3-, and CLN6-deficient as well as normal lymphoblasts. Flupirtine also prevents the death of CLN3- and CLN2-deficient postmitotic hNT neurons at the mitochondrial level. We show that a mechanism of neuroprotection exerted by flupirtine involves complete functional antagonism of N-methyl-D-aspartate or N-methyl-D-aspartate-induced neuronal apoptosis. Flupirtine may be useful as a drug capable of halting the progression of neurodegenerative diseases caused by dysregulated apoptosis. Show less

Juvenile Batten disease is a neurodegenerative disease caused by accelerated apoptotic death of photoreceptors and neurons attributable to defects in the CLN3 gene. CLN3 is antiapoptotic when overexpressed in NT2 neuronal precursor cells. CLN3 negatively modulates endogenous ceramide levels in NT2 cells and acts upstream of ceramide generation. Because defects in regulation of apoptosis are involved in the development of cancer, we evaluated the expression of CLN3 on both mRNA and protein levels in a variety of cancer cell lines and solid colon cancer tissue. We also observed the effect of the blocking of CLN3 protein expression on cancer cell growth, survival, ceramide production, and apoptosis by using an adenovirus-bearing antisense CLN3 construct. We show that CLN3 mRNA and protein are overexpressed in glioblastoma (U-373G and T98g), neuroblastoma (IMR-32 and SK-N-MC), prostate (Du145, PC-3, and LNCaP), ovarian (SK-OV-3, SW626, and PA-1), breast (BT-20, BT-549, and BT-474), and colon (SW1116, SW480, and HCT 116) cancer cell lines but not in pancreatic (CAPAN and As-PC-1) or lung (A-549 and NCI-H520) cancer cell lines. CLN3 is also up-regulated in mouse melanoma and breast carcinoma cancer cell lines. We found CLN3 expression is 22-330% higher than in corresponding normal colon control tissue in 8 of 10 solid colon tumors. An adenovirus-expressing antisense CLN3 (Ad-AS-CLN3) blocks CLN3 protein expression in DU-145, BT-20, SW1116, and T98g cancer cell lines as seen by Western blot. Blocking of CLN3 expression using Ad-AS-CLN3 inhibits growth and viability of cancer cells. It also causes elevation in endogenous ceramide production through de novo ceramide synthesis and results in increased apoptosis as shown by propidium iodide and JC-1 staining. This suggests that Ad-AS-CLN3 may be an option for therapy in some cancers. More importantly these results suggest that CLN3 is a novel molecular target for cancer drug discovery. Show less

Both hepatic lipase (HL) and apolipoprotein C-III (apoC-III) influence lipid metabolism. Common variation in promoters of both genes, LIPC -514C > T and APOC3 -482C > T, respectively, have been shown to affect plasma lipids and lipoproteins and glucose tolerance. We studied the interaction between both variants on parameters of glucose tolerance and lipid metabolism in 714 healthy young males participating in the second European Atherosclerosis Research Study (EARS-II). Approximately 18% of the subjects were carriers of at least one rare LIPC and APOC3 allele. These subjects exhibited, after fasting and oral fat loading, the highest values of triglyceride-rich lipoproteins, but there was no significant interactive effect on any lipid variable. However, interaction occurred on basal diastolic blood pressure (p =0.036) and, during oral glucose tolerance testing, on peak (p = 0.0065) and area under the curve for glucose (p =0.049), and insulin (p = 0.035). This resulted in the highest diastolic blood pressure and lowest glucose tolerance in carriers of at least one rare allele of both genes. Thus gene:gene interaction between LIPC and APOC3, even in these healthy young males, leads to changes in parameters that are typically characteristic of Syndrome-X. Show less

Neuronal ceroid lipofuscinoses (NCLs) are the most common hereditary neurodegenerative disorders of childhood. The first symptom of this heterogeneous group of devastating lysosomal storage diseases is progressive visual failure. The different forms of NCL can be distinguished by age of onset, clinical features and the characteristics of the accumulated materials. The juvenile form, Batten-Spielmeyer-Vogt disease which is caused by mutations in the CLN3 gene, is the most frequent form of the disease in which loss of vision becomes apparent around the age of 5-8 years. The gene was found to encode a novel integral membrane protein localizing to the lysosomes, confirming that the primary defect in NCL is in lysosomal function. The CLN3 protein function is still unknown, and is examined in several model organisms. We are studying the nematode Caenorhabditis elegans, and have identified three CLN3 homologues. In order to investigate the role of the CLN3 protein in C. elegans, Cecln-3 deletion mutants are being isolated from an ethyl methanesulphonate (EMS)-induced deletion mutant library. Examination of these mutants may provide us with information that will help in dissecting the processes in which the CLN3 protein is involved. In this library two mutated C. elegans Cln-3 loci have been identified, of which one mutant, NL748, was isolated. This mutant contains a deletion of the whole gene. The deletion mutant was characterized with regard to life expectancy, and showed no significant differences when compared with wild-type. Show less

The S2 allele of the SstI polymorphism of the apolipoprotein (apo) C-III gene has been associated with elevated triacylglycerol concentrations, high blood pressure, and increased risk of coronary artery disease, all of which are characteristic of an insulin-resistant state. To study the effect of this mutation on carbohydrate metabolism in healthy persons, we gave 41 male subjects 3 consecutive diets. The first was rich in saturated fat [15% protein, 47% carbohydrate, 38% fat (20% saturated)], the second was a National Cholesterol Education Program Step 1 diet [15% protein, 57% carbohydrate, 28% fat (< 10% saturated)], and the last was rich in monounsaturated fat [15% protein, 47% carbohydrate, 38% fat (22% monounsaturated, < 10% saturated)]. At the end of each dietary period, subjects received an oral-glucose-tolerance test (OGTT). Apo C-III genotype significantly affected basal glucose concentrations (P < 0.045) and insulin concentrations after the OGTT (P < 0.012). APOC3*S1/APOC3*S2 subjects (n = 13) had higher insulin concentrations after the OGTT than APOC3*S1/APOC3*S1 subjects (n = 28) in the 3 periods (diet 1: P < 0.0004; diet 2: P < 0.01; diet 3: P < 0.008). Multiple regression analysis showed that this polymorphism predicted the insulin response to the OGTT (P < 0.031) and the difference between basal insulin concentrations and insulin concentrations after the OGTT (P < 0.002) with the saturated fat diet. In summary, our results suggest that the mutation in the apo C-III gene affects insulin response to an OGTT, which could result in reduced sensitivity to insulin, especially when persons consume diets rich in saturated fat. Show less

The plasma lipid response to changes in dietary fat and cholesterol can vary between individuals. The SstI polymorphism, arising from a cytosine to guanosine substitution in the 3' untranslated region of the APOC3 gene distinguishes between two alleles--S1 and S2. The S2 allele has been associated with elevated plasma triacylglycerol, cholesterol, and apolipoprotein (apo) C-III concentrations. In 90 young men we examined the effect of the same mutation on the response of low-density-lipoprotein (LDL) cholesterol to dietary monounsaturated fat. The frequency for the S2 allele was 0.14. Subjects were fed a low-fat diet for 25 d, followed by a diet rich in monounsaturated fatty acid (22% MUFA, 38% total fat) for 28 d; lipoproteins were measured at the end of each diet. There were no significant differences in initial total cholesterol between subjects with the APOC3*S1/APOC3*S1 (S1/S1) and APOC3*S1/APOC3*S2 (S1/S2) genotypes. After consumption of the diet high in MUFA, significant increases in LDL cholesterol (0.13 mmol/L, P < 0.027) were noted in the S1/S1 subjects whereas a significant decrease was observed in the S1/S2 subjects (-0.18 mmol/L, P < 0.046). Significant genotypic effects were seen for diet-induced changes in LDL cholesterol (P < 0.00034), total cholesterol (P < 0.009), and apo B (P < 0.0014). A study of the effect of the interaction between this mutation with that present in position -76 of the APOA1 gene promoter region (G/A) revealed that both mutations had an additive effect on changes in total cholesterol, LDL cholesterol, and apo B induced by diets. Plasma LDL-cholesterol responsiveness to the diet may be explained, at least in part, by variation at the APOC3 gene locus. Show less