👤 Rudolf A De Boer

Cascade screening can identify individuals with elevated lipoprotein(a) [Lp(a)], a causal risk factor for atherosclerotic cardiovascular disease (ASCVD). The aim of this study is to explore the effectiveness of cascade screening with asymptomatic children as index cases to identify family members with elevated Lp(a). In this retrospective study we used our database consisting of all children referred for a tentative diagnosis of hereditary dyslipidemia to the Amsterdam University Medical Centers pediatric lipid clinic (1989-2023). Elevated Lp(a) was defined as ≥30 mg/dL or ≥75 nmol/L. We evaluated two cascade screening approaches (opportunistic and systematic), calculated the number needed to screen (NNS) and repeated the analysis exclusively in children with FH as subgroup with particularly high cardiovascular risk. A total of 1,931 children were included (732 indexes, mean age (SD) 11.7 (4.5) years; 1,199 relatives, mean age (SD) 10.1 (4.4) years). In total, 480 (25%) of all children had elevated Lp(a) concentrations (≥30 mg/dL or ≥75 nmol/L). Both opportunistic (732 indexes) and systematic (316 indexes) cascade screening identified relatives with elevated Lp(a). The NNS was of 3.7 (95% CI 3.3-4.3) for the systematic approach and 4.1 (95% CI 3.8-4.6) for the opportunistic approach. In the FH subgroup, NNS were 3.9 (95% CI 3.4-4.5) and 4.3 (95% CI 3.9-4.8), respectively. Our findings suggest that cascade screening using children as index cases is an effective strategy to identify asymptomatic relatives at risk. Cardiovascular risk assessment should include Lp(a), especially in patients with FH who face an even higher cardiovascular risk. Until effective therapies become available, management should focus on modifiable risk factors. Show less

Atherosclerotic CVD is a major cause of death in individuals with type 1 diabetes mellitus (T1DM). However, conventional risk factors do not fully account for the increased risk. This study aimed to investigate whether serum proteins associate with diabetes status and the occurrence of CVD in T1DM. We used isotope dilution-MS/MS to quantify 28 serum proteins in 228 subjects participating in the prospective Coronary Artery Calcification in Type 1 Diabetes study. We used linear regression to analyze the association between serum protein levels and T1DM status using 47 healthy controls and 134 T1DM patients without CVD and Cox proportional hazards regression to assess their prediction for incident CVD by a case-cohort study using a subcohort of 145 T1DM subjects and a total of 47 CVD events. Of the 28 serum proteins studied, five of them-alpha-2-macroglobulin (A2M), apolipoprotein A-IV, apolipoprotein L1, insulin-like growth factor 2, and phospholipid transfer protein-were significantly associated with T1DM status, with A2M being 1.6-fold higher in T1DM. After adjusting for potential confounders, A2M independently predicted incident CVD, with a mean hazard ratio of 3.3 and 95% CI of 1.8-6.1. In our study, A2M showed the largest increase in serum levels when comparing patients with T1DM to control subjects. A2M also predicted incident CVD, suggesting that it could serve as both a marker and possibly a mediator of atherosclerosis in T1DM. These findings emphasize the importance of specific serum proteins in assessing and managing CVD risk in T1DM. Show less

The chimeric cytokine IC7Fc conveys the metabolic signaling properties of the glycoprotein 130 receptor cytokines interleukin-6 and ciliary neurotrophic factor via membrane-bound signaling. IC7Fc was previously shown to slow the progression of type 2 diabetes mellitus, and here, we demonstrate its effect on atherosclerotic development. In APOE*3-Leiden.CETP mice, an atherosclerosis-prone model with a humanized lipoprotein metabolism, IC7Fc markedly lowered plasma triglyceride and total cholesterol levels. This was mechanistically explained by an inhibition of de novo lipogenesis in the liver, increased synthesis of bile acids from cholesterol, and down-regulated apolipoprotein B synthesis, which resulted in decreased cholesterol secretion in very low-density lipoprotein particles. As a consequence, IC7Fc treatment considerably reduced atherosclerotic lesion formation and vascular inflammation compared with current antihyperlipidemic therapy. In conclusion, IC7Fc is a promising pharmacological treatment for cardiometabolic diseases targeting hyperlipidemia and inflammation. Show less

Hypertrophic cardiomyopathy (HCM) is an important cause of morbidity and mortality with both monogenic and polygenic components. Here, we report results from a large genome-wide association study and multitrait analysis including 5,900 HCM cases, 68,359 controls and 36,083 UK Biobank participants with cardiac magnetic resonance imaging. We identified 70 loci (50 novel) associated with HCM and 62 loci (20 novel) associated with relevant left ventricular traits. Among the prioritized genes in the HCM loci, we identify a novel HCM disease gene, SVIL, which encodes the actin-binding protein supervillin, showing that rare truncating SVIL variants confer a roughly tenfold increased risk of HCM. Mendelian randomization analyses support a causal role of increased left ventricular contractility in both obstructive and nonobstructive forms of HCM, suggesting common disease mechanisms and anticipating shared response to therapy. Taken together, these findings increase our understanding of the genetic basis of HCM, with potential implications for disease management. Show less

Using proteomics and complexome profiling, we evaluated in a year-long study longitudinal variations in the plasma proteome of kidney failure patients, prior to and after a kidney transplantation. The post-transplant period was complicated by bacterial infections, resulting in dramatic changes in the proteome, attributed to an acute phase response (APR). As positive acute phase proteins (APPs), being elevated upon inflammation, we observed the well-described C-reactive protein and Serum Amyloid A (SAA), but also Fibrinogen, Haptoglobin, Leucine-rich alpha-2-glycoprotein, Lipopolysaccharide-binding protein, Alpha-1-antitrypsin, Alpha-1-antichymotrypsin, S100, and CD14. As negative APPs, being downregulated upon inflammation, we identified the well-documented Serotransferrin and Transthyretin, but added Kallistatin, Heparin cofactor 2, and interalpha-trypsin inhibitor heavy chain H1 and H2 (ITIH1, ITIH2). For the patient with the most severe APR, we performed plasma complexome profiling by SEC-LC-MS on all longitudinal samples. We observed that several plasma proteins displaying alike concentration patterns coelute and form macromolecular complexes. By complexome profiling, we expose how SAA1 and SAA2 become incorporated into high-density lipid particles, replacing largely Apolipoprotein (APO)A1 and APOA4. Overall, our data highlight that the combination of in-depth longitudinal plasma proteome and complexome profiling can shed further light on correlated variations in the abundance of several plasma proteins upon inflammatory events. Show less

We interviewed 188 carriers (57.4% male; aged 43.0±15.0 years) on exercise participation since the age of 10 years. The exercise was quantified as the metabolic equivalent of task-h/wk before the presentation. MCE was defined as a composite of malignant ventricular arrhythmia (sustained ventricular tachycardia/fibrillation), heart failure (heart failure hospitalizations or transplantation), and septal reduction therapy. Static and dynamic exercises were defined per the Bethesda classification. Associations of exercise with MCE and cardiomyopathy penetrance were adjusted for sex and assessed using Cox regression. Overall, 43 (22.9%) subjects experienced MCE and 139 (73.9%) were diagnosed with cardiomyopathy. No association was found between overall physical activity and high-static activity with MCE ( Overall exercise participation does not generally increase the risk of adverse events among Show less

Combined glucose-dependent insulinotropic polypeptide receptor (GIPR) and glucagon-like peptide-1 receptor (GLP1R) agonism is superior to single GLP1R agonism with respect to glycemic control and weight loss in obese patients with or without type 2 diabetes. As insulin resistance and obesity are strong risk factors for nonalcoholic fatty liver disease (NAFLD), in the current study we investigated the effects of combined GIPR/GLP1R agonism on NAFLD development. Male APOE∗3-Leiden.CETP mice, a humanized model for diabetic dyslipidemia and NAFLD when fed a high-fat high-cholesterol diet, received subcutaneous injections with either vehicle, a GIPR agonist, a GLP1R agonist, or both agonists combined every other day. GIPR and GLP1R agonism reduced body weight and additively lowered fasting plasma levels of glucose, triglycerides and total cholesterol. Strikingly, we report an additive reduction in hepatic steatosis as evidenced by lower hepatic lipid content and NAFLD scores. Underlying the lipid-lowering effects were a reduced food intake and intestinal lipid absorption and an increased uptake of glucose and triglyceride-derived fatty acids by energy-combusting brown adipose tissue. Combined GIPR/GLP1R agonism also attenuated hepatic inflammation as evidenced by a decreased number of monocyte-derived Kupffer cells and a reduced expression of inflammatory markers. Together, the reduced hepatic steatosis and inflammation coincided with lowered markers of liver injury. We interpretate that GIPR and GLP1R agonism additively attenuate hepatic steatosis, lower hepatic inflammation, ameliorate liver injury, together preventing NAFLD development in humanized APOE∗3-Leiden.CETP mice. We anticipate that combined GIPR/GLP1R agonism is a promising strategy to attenuate NAFLD progression in humans. This work was supported by a grant from the Netherlands CardioVascular Research Initiative: the Dutch Heart Foundation, Dutch Federation of University Medical Centers, the Netherlands Organization for Health Research and Development, and the Royal Netherlands Academy of Sciences [CVON-GENIUS-II] to P.C.N.R., a Lilly Research Award Program [LRAP] Award to P.C.N.R. and S.K., a Dutch Heart Foundation [2017T016] grant to S.K., and an NWO-VENI grant [09150161910073] to M.R.B.; J.F.D.B. is supported by the Nutrition and Health initiative of the University of Groningen; Z.Y. is supported by a full-time PhD scholarship from the China Scholarship Council (201806850094 to Z.Y.). Show less

Combined agonism of the glucose-dependent insulinotropic polypeptide receptor (GIPR) and the glucagon-like peptide-1 receptor (GLP1R) is superior to single GLP1R agonism in terms of glycemic control and lowering body weight in individuals with obesity and with or without type 2 diabetes mellitus. As both GIPR and GLP1R signaling have also been implicated in improving inflammatory responses and lipid handling, two crucial players in atherosclerosis development, here we aimed to investigate the effects of combined GIPR/GLP1R agonism in APOE*3-Leiden.CETP mice, a well-established mouse model for human-like lipoprotein metabolism and atherosclerosis development. Female APOE*3-Leiden.CETP mice were fed a Western-type diet (containing 16% fat and 0.15% cholesterol) to induce dyslipidemia, and received subcutaneous injections with either vehicle, a GIPR agonist (GIPFA-085), a GLP1R agonist (GLP-140) or both agonists. In the aortic root area, atherosclerosis development was assessed. Combined GIPR/GLP1R agonism attenuated the development of severe atherosclerotic lesions, while single treatments only showed non-significant improvements. Mechanistically, combined GIPR/GLP1R agonism decreased markers of systemic low-grade inflammation. In addition, combined GIPR/GLP1R agonism markedly lowered plasma triglyceride (TG) levels as explained by reduced hepatic very-low-density lipoprotein (VLDL)-TG production as well as increased TG-derived fatty acid uptake by brown and white adipose tissue which was coupled to enhanced hepatic uptake of core VLDL remnants. Combined GIPR/GLP1R agonism attenuates atherosclerosis severity by diminishing inflammation and increasing VLDL turnover. We anticipate that combined GIPR/GLP1R agonism is a promising strategy to lower cardiometabolic risk in humans. Show less

The prevalence of obesity is rising, creating an urgent need for efficacious therapies. Recent clinical trials show that tirzepatide, a dual agonist of receptors for the incretin hormones glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), yields more weight loss than selective GLP-1 receptor (GLP-1R) agonists. Incretin receptors in the central nervous system (CNS) may contribute to these effects. Yet exactly how each receptor regulates body weight from within the CNS is not clearly understood. It remains especially unclear how GIP receptor (GIPR) signalling contributes to the effects of tirzepatide because both stimulation and inhibition of CNS GIPRs yield weight loss in preclinical models. We summarise current knowledge on CNS incretin receptor pharmacology to provide insight into the potential mechanisms of action of dual GIPR/GLP-1R agonists, with tirzepatide as the exemplar. In addition, we discuss recent developments in incretin-based dual- and tri-agonism for inducing weight loss in obese individuals. Show less

Liver X receptor (LXR) agonism has theoretical potential for treating NAFLD/NASH, but synthetic agonists induce hyperlipidemia in preclinical models. Desmosterol, which is converted by Δ24-dehydrocholesterol reductase (DHCR24) into cholesterol, is a potent endogenous LXR agonist with anti-inflammatory properties. We aimed to investigate the effects of DHCR24 inhibition on NAFLD/NASH development. Here, by using APOE*3-Leiden. CETP mice, a well-established translational model that develops diet-induced human-like NAFLD/NASH characteristics, we report that SH42, a published DHCR24 inhibitor, markedly increases desmosterol levels in liver and plasma, reduces hepatic lipid content and the steatosis score, and decreases plasma fatty acid and cholesteryl ester concentrations. Flow cytometry showed that SH42 decreases liver inflammation by preventing Kupffer cell activation and monocyte infiltration. LXRα deficiency completely abolishes these beneficial effects of SH42. Together, the inhibition of DHCR24 by SH42 prevents diet-induced hepatic steatosis and inflammation in a strictly LXRα-dependent manner without causing hyperlipidemia. Finally, we also showed that SH42 treatment decreased liver collagen content and plasma alanine transaminase levels in an established NAFLD model. In conclusion, we anticipate that pharmacological DHCR24 inhibition may represent a novel therapeutic strategy for treatment of NAFLD/NASH. Show less

Hypertrophic cardiomyopathy (HCM) is a relatively common genetic heart disease characterised by myocardial hypertrophy. HCM can cause outflow tract obstruction, sudden cardiac death and heart failure, but severity is highly variable. In this exploratory cross-sectional study, circulating acylcarnitines were assessed as potential biomarkers in 124 MYBPC3 founder variant carriers (59 with severe HCM, 26 with mild HCM and 39 phenotype-negative [G + P-]). Elastic net logistic regression identified eight acylcarnitines associated with HCM severity. C3, C4, C6-DC, C8:1, C16, C18 and C18:2 were significantly increased in severe HCM compared to G + P-, and C3, C6-DC, C8:1 and C18 in mild HCM compared to G + P-. In multivariable linear regression, C6-DC and C8:1 correlated to log-transformed maximum wall thickness (coefficient 5.01, p = 0.005 and coefficient 0.803, p = 0.007, respectively), and C6-DC to log-transformed ejection fraction (coefficient -2.50, p = 0.004). Acylcarnitines seem promising biomarkers for HCM severity, however prospective studies are required to determine their prognostic value. Show less

The intestinal hormones glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-2 (GLP-2) are key regulators of postprandial bone turnover in humans. We hypothesized that GIP and GLP-2 co-administration would provide stronger effect on bone turnover than administration of the hormones separately, and tested this using subcutaneous injections of GIP and GLP-2 alone or in combination in humans. Guided by these findings, we designed series of GIPR-GLP-2R co-agonists as template for new osteoporosis treatment. The clinical experiment was a randomized cross-over design including 10 healthy men administered subcutaneous injections of GIP and GLP-2 alone or in combination. The GIPR-GLP-2R co-agonists were characterized in terms of binding and activation profiles on human and rodent GIP and GLP-2 receptors, and their pharmacokinetic (PK) profiles were improved by dipeptidyl peptidase-4 protection and site-directed lipidation. Co-administration of GIP and GLP-2 in humans resulted in an additive reduction in bone resorption superior to each hormone individually. The GIPR-GLP-2R co-agonists, designed by combining regions of importance for cognate receptor activation, obtained similar efficacies as the two native hormones and nanomolar potencies on both human receptors. The PK-improved co-agonists maintained receptor activity along with their prolonged half-lives. Finally, we found that the GIPR-GLP-2R co-agonists optimized toward the human receptors for bone remodeling are not feasible for use in rodent models. The successful development of potent and efficacious GIPR-GLP-2R co-agonists, combined with the improved effect on bone metabolism in humans by co-administration, support these co-agonists as a future osteoporosis treatment. Show less

Within the human population, considerable variability exists between individuals in their susceptibility to develop obesity and dyslipidemia. In humans, this is thought to be caused by both genetic and environmental variation. APOE*3-Leiden.CETP mice, as part of an inbred mouse model in which mice develop the metabolic syndrome upon being fed a high-fat high-cholesterol diet, show large inter-individual variation in the parameters of the metabolic syndrome, despite a lack of genetic and environmental variation. In the present study, we set out to resolve what mechanisms could underlie this variation. We used measurements of glucose and lipid metabolism from a six-month longitudinal study on the development of the metabolic syndrome. Mice were classified as mice with either high plasma triglyceride (responders) or low plasma triglyceride (non-responders) at the baseline. Subsequently, we fitted the data to a dynamic computational model of whole-body glucose and lipid metabolism (MINGLeD) by making use of a hybrid modelling method called Adaptations in Parameter Trajectories (ADAPT). ADAPT integrates longitudinal data, and predicts how the parameters of the model must change through time in order to comply with the data and model constraints. To explain the phenotypic variation in plasma triglycerides, the ADAPT analysis suggested a decreased cholesterol absorption, higher energy expenditure and increased fecal fatty acid excretion in non-responders. While decreased cholesterol absorption and higher energy expenditure could not be confirmed, the experimental validation demonstrated that the non-responders were indeed characterized by increased fecal fatty acid excretion. Furthermore, the amount of fatty acids excreted strongly correlated with bile acid excretion, in particular deoxycholate. Since bile acids play an important role in the solubilization of lipids in the intestine, these results suggest that variation in bile acid homeostasis may in part drive the phenotypic variation in the APOE*3-Leiden.CETP mice. Show less

Migraine affects over a billion individuals worldwide but its genetic underpinning remains largely unknown. Here, we performed a genome-wide association study of 102,084 migraine cases and 771,257 controls and identified 123 loci, of which 86 are previously unknown. These loci provide an opportunity to evaluate shared and distinct genetic components in the two main migraine subtypes: migraine with aura and migraine without aura. Stratification of the risk loci using 29,679 cases with subtype information indicated three risk variants that seem specific for migraine with aura (in HMOX2, CACNA1A and MPPED2), two that seem specific for migraine without aura (near SPINK2 and near FECH) and nine that increase susceptibility for migraine regardless of subtype. The new risk loci include genes encoding recent migraine-specific drug targets, namely calcitonin gene-related peptide (CALCA/CALCB) and serotonin 1F receptor (HTR1F). Overall, genomic annotations among migraine-associated variants were enriched in both vascular and central nervous system tissue/cell types, supporting unequivocally that neurovascular mechanisms underlie migraine pathophysiology. Show less

Glucose-dependent insulinotropic polypeptide (GIP) is best known as an incretin hormone that is secreted from K-cells of the proximal intestine, but evidence also implicates a role for GIP in regulating lipid metabolism and adiposity. It is well-established that GIP receptor knockout (GIPR KO) mice are resistant to diet-induced obesity; however, the factors mediating this effect remain unresolved. Accordingly, we aimed to elucidate the mechanisms leading to adiposity resistance in GIPR KO mice with a focus on whole-body energy balance and lipid metabolism in adipose tissues. Studies were conducted in age-matched male GIPR KO and wild-type (WT) mice fed a high-fat diet for 10 weeks. GIPR KO mice gained less body weight and fat mass compared to WT littermates, and this was associated with increased energy expenditure but no differences in food intake or fecal energy loss. Upon an oral lipid challenge, fatty acid storage in inguinal adipose tissue was significantly increased in GIPR KO compared with WT mice. This was not related to differential expression of lipoprotein lipase in adipose tissue. Adipose tissue lipolysis was increased in GIPR KO compared with WT mice, particularly following β-adrenergic stimulation, and could explain why GIPR KO mice gain less adipose tissue despite increased rates of fatty acid storage in inguinal adipose tissue. Taken together, these results suggest that the GIPR is required for normal maintenance of body weight and adipose tissue mass by regulating energy expenditure and lipolysis. Show less

The heart muscle diseases hypertrophic (HCM) and dilated (DCM) cardiomyopathies are leading causes of sudden death and heart failure in young, otherwise healthy, individuals. We conducted genome-wide association studies and multi-trait analyses in HCM (1,733 cases), DCM (5,521 cases) and nine left ventricular (LV) traits (19,260 UK Biobank participants with structurally normal hearts). We identified 16 loci associated with HCM, 13 with DCM and 23 with LV traits. We show strong genetic correlations between LV traits and cardiomyopathies, with opposing effects in HCM and DCM. Two-sample Mendelian randomization supports a causal association linking increased LV contractility with HCM risk. A polygenic risk score explains a significant portion of phenotypic variability in carriers of HCM-causing rare variants. Our findings thus provide evidence that polygenic risk score may account for variability in Mendelian diseases. More broadly, we provide insights into how genetic pathways may lead to distinct disorders through opposing genetic effects. Show less

Hypertrophic cardiomyopathy (HCM) is the most prevalent monogenic heart disease, commonly caused by truncating variants in the MYBPC3 gene. HCM is an important cause of sudden cardiac death; however, overall prognosis is good and penetrance in genotype-positive individuals is incomplete. The underlying mechanisms are poorly understood and risk stratification remains limited. To create a nationwide cohort of carriers of truncating MYBPC3 variants for identification of predictive biomarkers for HCM development and progression. In the multicentre, observational BIO FOr CARe (Identification of BIOmarkers of hypertrophic cardiomyopathy development and progression in Dutch MYBPC3 FOunder variant CARriers) cohort, carriers of the c.2373dupG, c.2827C > T, c.2864₂₈₆₅delCT and c.3776delA MYBPC3 variants are included and prospectively undergo longitudinal blood collection. Clinical data are collected from first presentation onwards. The primary outcome constitutes a composite endpoint of HCM progression (maximum wall thickness ≥ 20 mm, septal reduction therapy, heart failure occurrence, sustained ventricular arrhythmia and sudden cardiac death). So far, 250 subjects (median age 54.9 years (interquartile range 43.3, 66.6), 54.8% male) have been included. HCM was diagnosed in 169 subjects and dilated cardiomyopathy in 4. The primary outcome was met in 115 subjects. Blood samples were collected from 131 subjects. BIO FOr CARe is a genetically homogeneous, phenotypically heterogeneous cohort incorporating a clinical data registry and longitudinal blood collection. This provides a unique opportunity to study biomarkers for HCM development and prognosis. The established infrastructure can be extended to study other genetic variants. Other centres are invited to join our consortium. Show less

Single-stranded DNA (ssDNA) containing four guanine repeats can form G-quadruplex (G4) structures. While cellular proteins and small molecules can bind G4s, it has been difficult to broadly assess their DNA-binding specificity. Here, we use custom DNA microarrays to examine the binding specificities of proteins, small molecules, and antibodies across ∼15,000 potential G4 structures. Molecules used include fluorescently labeled pyridostatin (Cy5-PDS, a small molecule), BG4 (Cy5-BG4, a G4-specific antibody), and eight proteins (GST-tagged nucleolin, IGF2, CNBP, FANCJ, PIF1, BLM, DHX36, and WRN). Cy5-PDS and Cy5-BG4 selectively bind sequences known to form G4s, confirming their formation on the microarrays. Cy5-PDS binding decreased when G4 formation was inhibited using lithium or when ssDNA features on the microarray were made double-stranded. Similar conditions inhibited the binding of all other molecules except for CNBP and PIF1. We report that proteins have different G4-binding preferences suggesting unique cellular functions. Finally, competition experiments are used to assess the binding specificity of an unlabeled small molecule, revealing the structural features in the G4 required to achieve selectivity. These data demonstrate that the microarray platform can be used to assess the binding preferences of molecules to G4s on a broad scale, helping to understand the properties that govern molecular recognition. Show less

Genomic analyses of chronic lymphocytic leukemia (CLL) identified somatic mutations and associations of clonal diversity with adverse outcomes. Clonal evolution likely has therapeutic implications but its dynamic is less well studied. We studied clonal composition and prognostic value of seven recurrently mutated driver genes using targeted next-generation sequencing in 643 CLL patients and found higher frequencies of mutations in TP53 (35 vs. 12%, p < 0.001) and SF3B1 (20 vs. 11%, p < 0.05) and increased number of (sub)clonal (p < 0.0001) mutations in treated patients. We next performed an in-depth evaluation of clonal evolution on untreated CLL patients (50 "progressors" and 17 matched "non-progressors") using a 404 gene-sequencing panel and identified novel mutated genes such as AXIN1, SDHA, SUZ12, and FOXO3. Progressors carried more mutations at initial presentation (2.5 vs. 1, p < 0.0001). Mutations in specific genes were associated with increased (SF3B1, ATM, and FBXW7) or decreased progression risk (AXIN1 and MYD88). Mutations affecting specific signaling pathways, such as Notch and MAP kinase pathway were enriched in progressive relative to non-progressive patients. These data extend earlier findings that specific genomic alterations and diversity of subclones are associated with disease progression and persistence of disease in CLL and identify novel recurrently mutated genes and associated outcomes. Show less

Vaccinia virus is a promising viral vaccine and gene delivery candidate and has historically been used as a model to study poxvirus-host cell interactions. We employed a genome-wide insertional mutagenesis approach in human haploid cells to identify host factors crucial for vaccinia virus infection. A library of mutagenized HAP1 cells was exposed to modified vaccinia virus Ankara (MVA). Deep-sequencing analysis of virus-resistant cells identified host factors involved in heparan sulfate synthesis, Golgi organization, and vesicular protein trafficking. We validated EXT1, TM9SF2, and TMED10 (TMP21/p23/p24δ) as important host factors for vaccinia virus infection. The critical roles of EXT1 in heparan sulfate synthesis and vaccinia virus infection were confirmed. TM9SF2 was validated as a player mediating heparan sulfate expression, explaining its contribution to vaccinia virus infection. In addition, TMED10 was found to be crucial for virus-induced plasma membrane blebbing and phosphatidylserine-induced macropinocytosis, presumably by regulating the cell surface expression of the TAM receptor Axl. Show less

It is well established that, besides facilitating lipid absorption, bile acids act as signaling molecules that modulate glucose and lipid metabolism. Bile acid metabolism, in turn, is controlled by several nutrient-sensitive transcription factors. Altered intrahepatic glucose signaling in type 2 diabetes associates with perturbed bile acid synthesis. We aimed to characterize the regulatory role of the primary intracellular metabolite of glucose, glucose-6-phosphate (G6P), on bile acid metabolism. Hepatic gene expression patterns and bile acid composition were analyzed in mice that accumulate G6P in the liver, that is, liver-specific glucose-6-phosphatase knockout (L-G6pc Show less

Multiple single-nucleotide polymorphisms (SNPs) conferring susceptibility to osteoarthritis (OA) mark imbalanced expression of positional genes in articular cartilage, reflected by unequally expressed alleles among heterozygotes (allelic imbalance [AI]). We undertook this study to explore the articular cartilage transcriptome from OA patients for AI events to identify putative disease-driving genetic variation. AI was assessed in 42 preserved and 5 lesioned OA cartilage samples (from the Research Arthritis and Articular Cartilage study) for which RNA sequencing data were available. The count fraction of the alternative alleles among the alternative and reference alleles together (φ) was determined for heterozygous individuals. A meta-analysis was performed to generate a meta-φ and P value for each SNP with a false discovery rate (FDR) correction for multiple comparisons. To further validate AI events, we explored them as a function of multiple additional OA features. We observed a total of 2,070 SNPs that consistently marked AI of 1,031 unique genes in articular cartilage. Of these genes, 49 were found to be significantly differentially expressed (fold change <0.5 or >2, FDR <0.05) between preserved and paired lesioned cartilage, and 18 had previously been reported to confer susceptibility to OA and/or related phenotypes. Moreover, we identified notable highly significant AI SNPs in the CRLF1, WWP2, and RPS3 genes that were related to multiple OA features. We present a framework and resulting data set for researchers in the OA research field to probe for disease-relevant genetic variation that affects gene expression in pivotal disease-affected tissue. This likely includes putative novel compelling OA risk genes such as CRLF1, WWP2, and RPS3. Show less

The ST-segment and adjacent T-wave (ST-T wave) amplitudes of the electrocardiogram are quantitative characteristics of cardiac repolarization. Repolarization abnormalities have been linked to ventricular arrhythmias and sudden cardiac death. We performed the first genome-wide association meta-analysis of ST-T-wave amplitudes in up to 37 977 individuals identifying 71 robust genotype-phenotype associations clustered within 28 independent loci. Fifty-four genes were prioritized as candidates underlying the phenotypes, including genes with established roles in the cardiac repolarization phase (SCN5A/SCN10A, KCND3, KCNB1, NOS1AP and HEY2) and others with as yet undefined cardiac function. These associations may provide insights in the spatiotemporal contribution of genetic variation influencing cardiac repolarization and provide novel leads for future functional follow-up. Show less

Diabetic cardiomyopathy is a myocardial disease triggered by impaired insulin signalling, increased fatty acid uptake and diminished glucose utilisation. Liver X receptors (LXRs) are key transcriptional regulators of metabolic homeostasis. However, their effect in the diabetic heart is largely unknown. We cloned murine Lxrα (also known as Nr1h3) behind the α-myosin heavy chain (αMhc; also known as Myh6) promoter to create transgenic (Lxrα-Tg) mice and transgene-negative littermates (wild-type [WT]). A mouse model of type 2 diabetes was induced by a high-fat diet (HFD, 60% energy from fat) over 16 weeks and compared with a low-fat diet (10% energy from fat). A mouse model of type 1 diabetes was induced via streptozotocin injection over 12 weeks. HFD manifested comparable increases in body weight, plasma triacylglycerol and insulin resistance per OGTT in Lxrα-Tg and WT mice. HFD significantly increased left ventricular weight by 21% in WT hearts, but only by 5% in Lxrα-Tg. To elucidate metabolic effects in the heart, microPET (positron emission tomography) imaging revealed that cardiac glucose uptake was increased by 1.4-fold in WT mice on an HFD, but further augmented by 1.7-fold in Lxrα-Tg hearts, in part through 5' adenosine monophosphate-activated protein kinase (AMPK) phosphorylation and restoration of glucose transporter 4 (GLUT4). By contrast, streptozotocin-induced ablation of insulin signalling diminished cardiac glucose uptake levels and caused cardiac dysfunction, indicating that insulin may be important in LXRα-mediated glucose uptake. Chromatin immunoprecipitation assays identified natriuretic peptides, atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP), as potential direct targets of cardiac LXRα overexpression. Cardiac-specific LXRα overexpression ameliorates the progression of HFD-induced left ventricular hypertrophy in association with increased glucose reliance and natriuretic peptide signalling during the early phase of diabetic cardiomyopathy. These findings implicate a potential protective role for LXR in targeting metabolic disturbances underlying diabetes. Show less

Pathological cardiac hypertrophy is characterized by a shift in metabolic substrate utilization from fatty acids to glucose, but the molecular events underlying the metabolic remodeling remain poorly understood. Here, we investigated the role of liver X receptors (LXRs), which are key regulators of glucose and lipid metabolism, in cardiac hypertrophic pathogenesis. Using a transgenic approach in mice, we show that overexpression of LXRα acts to protect the heart against hypertrophy, fibrosis, and dysfunction. Gene expression profiling studies revealed that genes regulating metabolic pathways were differentially expressed in hearts with elevated LXRα. Functionally, LXRα overexpression in isolated cardiomyocytes and murine hearts markedly enhanced the capacity for myocardial glucose uptake following hypertrophic stress. Conversely, this adaptive response was diminished in LXRα-deficient mice. Transcriptional changes induced by LXRα overexpression promoted energy-independent utilization of glucose via the hexosamine biosynthesis pathway, resulting in O-GlcNAc modification of GATA4 and Mef2c and the induction of cytoprotective natriuretic peptide expression. Our results identify LXRα as a key cardiac transcriptional regulator that helps orchestrate an adaptive metabolic response to chronic cardiac stress, and suggest that modulating LXRα may provide a unique opportunity for intervening in myocyte metabolism. Show less

Liver X receptors (LXRs) transcriptionally regulate inflammation, metabolism, and immunity. Synthetic LXR agonists have been evaluated for their efficacy in the cardiovascular system; however, they elicit prolipogenic side effects which substantially limit their therapeutic use. AZ876 is a novel high-affinity LXR agonist. Herein, we aimed to determine the cardioprotective potential of LXR activation with AZ876. Cardiac hypertrophy was induced in C57Bl6/J mice via transverse aortic constriction (TAC) for 6 weeks. During this period, mice received chow supplemented or not with AZ876 (20 µmol/kg/day). In murine hearts, LXRα protein expression was up-regulated ∼7-fold in response to TAC. LXR activation with AZ876 attenuated this increase, and significantly reduced TAC-induced increases in heart weight, myocardial fibrosis, and cardiac dysfunction without affecting blood pressure. At the molecular level, AZ876 suppressed up-regulation of hypertrophy- and fibrosis-related genes, and further inhibited prohypertrophic and profibrotic transforming growth factor β (TGFβ)-Smad2/3 signalling. In isolated cardiac myocytes and fibroblasts, immunocytochemistry confirmed nuclear expression of LXRα in both these cell types. In cardiomyocytes, phenylephrine-stimulated cellular hypertrophy was significantly decreased in AZ876-treated cells. In cardiac fibroblasts, AZ876 prevented TGFβ- and angiotensin II-induced fibroblast collagen synthesis, and inhibited up-regulation of the myofibroblastic marker, α-smooth muscle actin. Plasma triglycerides and liver weight were unaltered following AZ876 treatment. AZ876 activation of LXR protects from adverse cardiac remodelling in pathological pressure overload, independently of blood pressure. LXR may thus represent a putative molecular target for antihypertrophic and antifibrotic therapies in heart failure prevention. Show less

We analysed single nucleotide polymorphisms (SNPs) tagging the genetic variability of six candidate genes (ATF6, FABP1, LPIN2, LPIN3, MLXIPL and MTTP) involved in the regulation of hepatic lipid metabolism, an important regulatory site of energy balance for associations with body mass index (BMI) and changes in weight and waist circumference. We also investigated effect modification by sex and dietary intake. Data of 6,287 individuals participating in the European prospective investigation into cancer and nutrition were included in the analyses. Data on weight and waist circumference were followed up for 6.9 ± 2.5 years. Association of 69 tagSNPs with baseline BMI and annual changes in weight as well as waist circumference were investigated using linear regression analysis. Interactions with sex, GI and intake of carbohydrates, fat as well as saturated, monounsaturated and polyunsaturated fatty acids were examined by including multiplicative SNP-covariate terms into the regression model. Neither baseline BMI nor annual weight or waist circumference changes were significantly associated with variation in the selected genes in the entire study population after correction for multiple testing. One SNP (rs1164) in LPIN2 appeared to be significantly interacting with sex (p = 0.0003) and was associated with greater annual weight gain in men (56.8 ± 23.7 g/year per allele, p = 0.02) than in women (-25.5 ± 19.8 g/year per allele, p = 0.2). With respect to gene-nutrient interaction, we could not detect any significant interactions when accounting for multiple testing. Therefore, out of our six candidate genes, LPIN2 may be considered as a candidate for further studies. Show less

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. 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. 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. 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. Show less

Several candidate gene studies on the metabolic syndrome (MetS) have been conducted. However, for most single nucleotide polymorphisms (SNPs) no systematic review on their association with MetS exists. A systematic electronic literature search was conducted until the 2nd of June 2010, using HuGE Navigator. English language articles were selected. Only genes of which at least one SNP-MetS association was studied in an accumulative total population ≥ 4000 subjects were included. Meta-analyses were conducted on SNPs with three or more studies available in a generally healthy population. In total 88 studies on 25 genes were reviewed. Additionally, for nine SNPs in seven genes (GNB3, PPARG, TCF7L2, APOA5, APOC3, APOE, CETP) a meta-analysis was conducted. The minor allele of rs9939609 (FTO), rs7903146 (TCF7L2), C56G (APOA5), T1131C (APOA5), C482T (APOC3), C455T (APOC3) and 174G>C (IL6) were more prevalent in subjects with MetS, whereas the minor allele of Taq-1B (CETP) was less prevalent in subjects with the MetS. After having systematically reviewed the most studied SNP-MetS associations, we found evidence for an association with the MetS for eight SNPs, mostly located in genes involved in lipid metabolism. Show less