👤 Eva R Smit

Preterm infants, especially those born small for gestational age (SGA), are at risk of short-term and long-term health complications. Characterization of changes in circulating proteins postnatally in preterm infants may provide valuable fundamental insights into this population. Here, we investigated postnatal developmental patterns in preterm infants and explored protein signatures that deviate between SGA infants and appropriate for gestational age (AGA) infants using a mass spectrometry (MS)-based proteomics workflow. Longitudinal serum samples obtained at postnatal days 0, 3, 7, 14, and 28 from 67 preterm infants were analyzed using unbiased MS-based proteomics. 314 out of 833 quantified serum proteins change postnatally, including previously described age-related changes in immunoglobulins, hemoglobin subunits, and new developmental patterns, e.g. apolipoproteins (APOA4) and terminal complement cascade (C9) proteins. Limited differences between SGA and AGA infants were found at birth while longitudinal monitoring revealed 69 deviating proteins, including insulin-sensitizing hormone adiponectin, platelet proteins, and 24 proteins with an annotated function in the immune response. This study shows the potential of MS-based serum profiling in defining circulating protein trajectories in the preterm infant population and its ability to identify longitudinal alterations in protein levels associated with SGA. Postnatal changes of circulating proteins in preterm infants have not fully been elucidated but may contribute to development of health complications. Mass spectrometry-based analysis is an attractive approach to study circulating proteins in preterm infants with limited material. Longitudinal plasma profiling reveals postnatal developmental-related patterns in preterm infants (314/833 proteins) including previously described changes, but also previously unreported proteins. Longitudinal monitoring revealed an immune response signature between SGA and AGA infants. This study highlights the importance of taking postnatal changes into account for translational studies in preterm infants. Show less

G-protein coupled receptor 146 (GPR146)-deficient mice exhibit a moderate 21 % reduction in plasma cholesterol. This is associated with decreased phosphorylation of ERK1/2 and reduced SREBP2 activity in the liver, which leads to lower VLDL secretion. Insight into the role of GPR146 in humans is however limited. We therefore set out to study rare genetic variants in GPR146 to improve our understanding of this new player in lipid metabolism. We used whole genome sequencing data from UK Biobank participants to search for rare coding variants in GPR146. We first carried out gene-based burden tests (using SAIGE-GENE-framework) and examined the association of individual variants with plasma cholesterol levels. One of the variants (P62L) was also studied using the Global Lipids Genetics Consortium (GLGC) data set and in a knock-in mouse model. We found that the combination of rare genetic variants identified in GPR146 is significantly associated with plasma cholesterol levels. Three rare variants, i.e. P62L, I129I, and A175T were individually associated with reduced plasma cholesterol. In the GLGC cohort, the P62L variant was associated with reductions in both HDL and LDL cholesterol. Follow-up experiments show lower plasma cholesterol levels in GPR146 This study shows that rare GPR146 gene variants are associated with lower plasma cholesterol levels in humans. One of these variants, P62L is associated with reductions of HDL cholesterol and LDL cholesterol in humans while the ortholog in mice confers a loss of GPR146 function leading to only reduced HDL cholesterol. How GPR146 affects HDL metabolism in humans and mice remains to be resolved. Show less

CLN3 Batten disease is a lethal pediatric neurodegenerative disease caused by mutations in the CLN3 gene. Typically, the disease manifests as vision loss in early childhood and progresses to neurological dysfunction and death in young adulthood. Most therapeutic developments have focused on treating the brain and may not protect against vision loss, which greatly affects quality of life. We have previously shown that a splice-switching antisense oligonucleotide (ASO) delivered to the central nervous system can reduce neurological disease burden in mouse models of CLN3 disease. Here, we apply a similar ASO approach for treating retinal dysfunction in a pig model of CLN3 Batten disease, which is more representative of human vision. A single intravitreal injection of ASO induces robust exon skipping in the retina for up to 12 months. The ASO treatment resulted in higher amplitudes on electroretinograms, suggesting mitigation of retinal dysfunction at early timepoints of disease. One ASO that efficiently induces exon skipping in vivo was well-tolerated and targets a region conserved in humans, making it a promising candidate for clinical translation. Our findings demonstrate the utility of an ASO-based approach to treat retinal dysfunction in CLN3 Batten disease and support broader ASO applications for treating ocular diseases. Show less

Atrial fibrillation (AF) is a prevalent and morbid abnormality of the heart rhythm with a strong genetic component. Here, we meta-analyzed genome and exome sequencing data from 36 studies that included 52,416 AF cases and 277,762 controls. In burden tests of rare coding variation, we identified novel associations between AF and the genes MYBPC3, LMNA, PKP2, FAM189A2 and KDM5B. We further identified associations between AF and rare structural variants owing to deletions in CTNNA3 and duplications of GATA4. We broadly replicated our findings in independent samples from MyCode, deCODE and UK Biobank. Finally, we found that CRISPR knockout of KDM5B in stem-cell-derived atrial cardiomyocytes led to a shortening of the action potential duration and widespread transcriptomic dysregulation of genes relevant to atrial homeostasis and conduction. Our results highlight the contribution of rare coding and structural variants to AF, including genetic links between AF and cardiomyopathies, and expand our understanding of the rare variant architecture for this common arrhythmia. Show less

The small intestine plays a central role in lipid metabolism, most notably the uptake of dietary fats that are packaged into chylomicrons and secreted into the circulation for utilisation by peripheral tissues. While microsomal triglyceride transfer protein (MTP) is known to play a key role in this pathway, the intracellular assembly, trafficking, and secretion of chylomicrons is incompletely understood. Using human transcriptome datasets to find genes co-regulated with MTTP, we identified ERICH4 as a top hit. The gene encodes for glutamate-rich protein 4, a protein of unknown function. REACTOME gene-function prediction tools indicated that ERICH4 is involved in intestinal lipid metabolism. In addition, GWAS data point to a strong relationship between ERICH4 and plasma lipids. To validate ERICH4 as a lipid gene, we generated whole-body Erich4 knockout (Erich4 Despite prediction tools indicating ERICH4 as a strong candidate gene in intestinal lipid metabolism, we here show that ERICH4 does not play a role in intestinal lipid metabolism in mice. It remains to be established whether ERICH4 plays a role in human lipid metabolism. Show less

The developmental and epileptic encephalopathies (DEE) are a heterogeneous group of rare neurodevelopmental disorders, characterised by early-onset seizures that are often intractable, electroencephalographic abnormalities, and developmental delay or regression. There is a paucity of data from sub-Saharan Africa on the genetic basis of DEE. The aim of this study was to investigate the genetic background of DEE using targeted next generation sequencing (NGS) analysis in a tertiary pediatric neurology outpatient department at Tygerberg Hospital, South Africa. In addition, we assessed the value of the genetic results to the parents and managing physicians. A prospective cohort study of 41 consecutive children with DEE (onset before 3 years of age) that were recruited over a 2-year period (2019-2021). Pre- and post-test genetic counselling were offered to all study participants. The results were categorized as either: positive (pathogenic/likely pathogenic variant identified), inconclusive (variant(s) of unknown significance identified), or negative (no variants identified). Result interpretation and careful matching of the variant to the clinical phenotype was performed. Subsequently, questionnaires were administered to both the physicians and the parents. A genetic underlying cause for DEE was identified in 18 of 41 children (diagnostic yield 43.9%). Variants in SCN1A (n=7), KANSL1 (n=2), KCNQ2 (n=2) and CDKL5 (n=2) were identified in more than one patient. Rarer genes included IQSEC2, SMC1A and STXBP1. All of the identified pathogenic variants fully explained and matched the respective phenotypic description of the patient at the time of clinical diagnosis. In 26% of patients the genetic result facilitated precision medicine management changes to anti-seizure medication. Both parents and physicians expressed benefit of genetic testing in patients with DEE. Targeted NGS analysis proved an efficient diagnostic tool in detection of a genetic cause of DEE in a large proportion of South African children. The 43.9% diagnostic yield is similar to previously reported international pediatric cohorts. Additionally, the genetic findings proved useful for targeted therapeutic decision-making and accurate genetic counseling. Show less

The glucose-dependent insulinotropic polypeptide (GIP) is a 42-residue metabolic hormone that is actively being targeted for its regulatory role of glycemia and energy balance. Limited structural data of its receptor has made ligand design tedious. This study investigates the structure and function of the GIP receptor (GIPR), using a homology model based on the GLP-1 receptor. Molecular dynamics combined with in vitro mutational data were used to pinpoint residues involved in ligand binding and/or receptor activation. Significant differences in binding mode were identified for the naturally occurring agonists GIP(1-30)NH Show less

The impact of cholesteryl ester transfer protein (CETP) on atherosclerosis is highly debated. This study aimed to investigate the associations between plasma CETP or CETP genotypes and carotid intima-media thickness (cIMT) and the influence of high-density lipoprotein cholesterol (HDL-C) on these associations. Plasma CETP and HDL-C concentrations were measured in 552 subjects free of any pharmacological treatment from the IMPROVE cohort, which includes 3711 European subjects at high cardiovascular risk. CETP single-nucleotide polymorphisms (SNPs) and cIMT measures (cIMT Show less

Enzymatic cleavage of endogenous peptides is a commonly used principle to initiate, modulate and terminate action for instance among cytokines and peptide hormones. The incretin hormones, glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1), and the related hormone glucagon-like peptide-2 (GLP-2) are all rapidly N-terminally truncated with severe loss of intrinsic activity. The most abundant circulating form of full length GIP(1-42) is GIP(3-42) (a dipeptidyl peptidase-4 (DPP-4) product). GIP(1-30)NH Show less

In patients with type 2 diabetes mellitus (T2DM), the insulinotropic action of the GIP system is desensitized, whereas this is not the case for the GLP-1 system. This has raised an interesting discussion of whether GIP agonists or antagonists are most suitable for future treatment of T2DM together with GLP-1-based therapies. Homozygous carriers of the GIP receptor (GIPR) variant, [E354Q], display lower bone mineral density, increased bone fracture risk and slightly increased blood glucose. Here, we present an in-depth molecular pharmacological phenotyping of GIPR-[E354Q]. In silico modelling suggested similar interaction of the endogenous agonist GIP(1-42) to [E354Q] as to GIPR wt. This was supported by homologous competition binding in COS-7 cells revealing GIPR wt-like affinities of GIP(1-42) with K Show less

Cholesteryl ester transfer protein (CETP) is mainly expressed by Kupffer cells in the liver. A reduction of hepatic triglyceride content (HTGC) by pioglitazone or caloric restriction is accompanied by a decrease in circulating CETP. Since GLP-1 analogues also reduce HTGC, we assessed whether liraglutide decreases CETP. Furthermore, we investigated the association between HTGC and CETP in a population-based cohort. In a placebo-controlled trial, 50 patients with type 2 diabetes were randomly assigned to treatment with liraglutide or placebo added to standard care. In this trial and in 1,611 participants of the Netherlands Epidemiology of Obesity (NEO) study, we measured HTGC and circulating CETP by proton magnetic resonance spectroscopy and ELISA, respectively. The HTGC was decreased in the liraglutide group (-6.3%; 95%CI of difference [-9.5, -3.0]) but also in the placebo group (-4.0%; 95%CI[-6.0, -2.0]), without between-group differences. CETP was not decreased by liraglutide (-0.05 µg/mL; 95%CI[-0.13, 0.04]) or placebo (-0.04 µg/mL; 95%CI[-0.12, 0.04]). No association was present between HTGC and CETP at baseline (β: 0.002 µg/mL per %TG, 95%CI[-0.005, 0.009]) and between the changes after treatment with liraglutide (β: 0.003 µg/mL per %TG, 95%CI[-0.010, 0.017]) or placebo (β: 0.006 µg/mL per %TG, 95%CI[-0.012,0.024]). Also, in the cohort n o association between HTGC and CETP was present (β: -0.001 µg/mL per SD TG, 95%CI[-0.005, 0.003]). A reduction of HTGC after treatment with liraglutide or placebo does not decrease circulating CETP. Also, no association between HTGC and CETP was present in a large cohort. These findings indicate that circulating CETP is not determined by HTGC.Clinical Trial Registration: Clinicaltrials.gov (NCT01761318). Show less

The Wnt/β-catenin signaling pathway is important for multiple developmental processes and tissue maintenance in adults. Consequently, deregulated signaling is involved in a range of human diseases including cancer and developmental defects. A better understanding of the intricate regulatory mechanism and effect of physiological (active) and pathophysiological (hyperactive) WNT signaling is important for predicting treatment response and developing novel therapies. The constitutively expressed CTNNB1 (commonly and hereafter referred to as β-catenin) is degraded by a destruction complex, composed of amongst others AXIN1 and GSK3. The destruction complex is inhibited during active WNT signaling, leading to β-catenin stabilization and induction of β-catenin/TCF target genes. In this study we investigated the mechanism and effect of β-catenin stabilization during active and hyperactive WNT signaling in a combined in silico and in vitro approach. We constructed a Petri net model of Wnt/β-catenin signaling including main players from the plasma membrane (WNT ligands and receptors), cytoplasmic effectors and the downstream negative feedback target gene AXIN2. We validated that our model can be used to simulate both active (WNT stimulation) and hyperactive (GSK3 inhibition) signaling by comparing our simulation and experimental data. We used this experimentally validated model to get further insights into the effect of the negative feedback regulator AXIN2 upon WNT stimulation and observed an attenuated β-catenin stabilization. We furthermore simulated the effect of APC inactivating mutations, yielding a stabilization of β-catenin levels comparable to the Wnt-pathway activities observed in colorectal and breast cancer. Our model can be used for further investigation and viable predictions of the role of Wnt/β-catenin signaling in oncogenesis and development. Show less

We performed a comprehensive quantitative proteomics study on human hippocampus tissue involving all Braak stages to assess changes in protein abundance over the various stages of Alzheimer's disease (AD). Hippocampal subareas CA1 and subiculum of 40 cases were isolated using laser capture microdissection and analyzed using mass spectrometry. Immunoblotting and immunohistochemistry were used for validation. Over the Braak stages, an altered expression was found for 372 proteins including changes in levels of extracellular matrix components, and in calcium-dependent signaling proteins. Early changes were observed in levels of proteins related to cytoskeletal dynamics and synaptic components including an increase in RIMS1 and GRIK4. Several synaptic proteins, such as BSN, LIN7A, DLG2, -3, and -4, exhibit an early-up, late-down expression pattern. This study provides new insight into AD-dependent changes in protein levels in the hippocampus during AD pathology, identifying potential novel therapeutic targets and biomarkers. Show less

Fetal akinesia deformation sequence (FADS) refers to a clinically and genetically heterogeneous group of disorders with congenital malformations related to impaired fetal movement. FADS can result from mutations in CHRNG, CHRNA1, CHRND, DOK7 and RAPSN; however, these genes only account for a minority of cases. Here we identify MUSK as a novel cause of lethal FADS. Fourteen affected fetuses from a Dutch genetic isolate were traced back to common ancestors 11 generations ago. Homozygosity mapping in two fetuses revealed MUSK as a candidate gene. All tested cases carried an identical homozygous variant c.1724T>C; p.(Ile575Thr) in the intracellular domain of MUSK. The carrier frequency in the genetic isolate was 8%, exclusively found in heterozygous carriers. Consistent with the established role of MUSK as a tyrosine kinase that orchestrates neuromuscular synaptogenesis, the fetal myopathy was accompanied by impaired acetylcholine receptor clustering and reduced tyrosine kinase activity at motor nerve endings. A functional assay in myocytes derived from human fetuses confirmed that the variant blocks MUSK-dependent motor endplate formation. Taken together, the results strongly support a causal role of this founder mutation in MUSK, further expanding the gene set associated with FADS and offering new opportunities for prenatal genetic testing. Show less

Very low calorie diets (VLCD) with and without exercise programs lead to major metabolic improvements in obese type 2 diabetes patients. The mechanisms underlying these improvements have so far not been elucidated fully. To further investigate the mechanisms of a VLCD with or without exercise and to uncover possible biomarkers associated with these interventions, blood samples were collected from 27 obese type 2 diabetes patients before and after a 16-week VLCD (Modifast ∼ 450 kcal/day). Thirteen of these patients followed an exercise program in addition to the VCLD. Plasma was obtained from 27 lean and 27 obese controls as well. Proteomic analysis was performed using mass spectrometry (MS) and targeted multiple reaction monitoring (MRM) and a large scale isobaric tags for relative and absolute quantitation (iTRAQ) approach. After the 16-week VLCD, there was a significant decrease in body weight and HbA1c in all patients, without differences between the two intervention groups. Targeted MRM analysis revealed differences in several proteins, which could be divided in diabetes-associated (fibrinogen, transthyretin), obesity-associated (complement C3), and diet-associated markers (apolipoproteins, especially apolipoprotein A-IV). To further investigate the effects of exercise, large scale iTRAQ analysis was performed. However, no proteins were found showing an exercise effect. Thus, in this study, specific proteins were found to be differentially expressed in type 2 diabetes patients versus controls and before and after a VLCD. These proteins are potential disease state and intervention specific biomarkers. Controlled-Trials.com ISRCTN76920690. Show less

Major depressive disorder (MDD) is a common complex disorder with a partly genetic etiology. We conducted a genome-wide association study of the MDD2000+ sample (2431 cases, 3673 screened controls and >1 M imputed single-nucleotide polymorphisms (SNPs)). No SNPs achieved genome-wide significance either in the MDD2000+ study, or in meta-analysis with two other studies totaling 5763 cases and 6901 controls. These results imply that common variants of intermediate or large effect do not have main effects in the genetic architecture of MDD. Suggestive but notable results were (a) gene-based tests suggesting roles for adenylate cyclase 3 (ADCY3, 2p23.3) and galanin (GAL, 11q13.3); published functional evidence relates both of these to MDD and serotonergic signaling; (b) support for the bipolar disorder risk variant SNP rs1006737 in CACNA1C (P=0.020, odds ratio=1.10); and (c) lack of support for rs2251219, a SNP identified in a meta-analysis of affective disorder studies (P=0.51). We estimate that sample sizes 1.8- to 2.4-fold greater are needed for association studies of MDD compared with those for schizophrenia to detect variants that explain the same proportion of total variance in liability. Larger study cohorts characterized for genetic and environmental risk factors accumulated prospectively are likely to be needed to dissect more fully the etiology of MDD. Show less

Concentrations of liver enzymes in plasma are widely used as indicators of liver disease. We carried out a genome-wide association study in 61,089 individuals, identifying 42 loci associated with concentrations of liver enzymes in plasma, of which 32 are new associations (P = 10(-8) to P = 10(-190)). We used functional genomic approaches including metabonomic profiling and gene expression analyses to identify probable candidate genes at these regions. We identified 69 candidate genes, including genes involved in biliary transport (ATP8B1 and ABCB11), glucose, carbohydrate and lipid metabolism (FADS1, FADS2, GCKR, JMJD1C, HNF1A, MLXIPL, PNPLA3, PPP1R3B, SLC2A2 and TRIB1), glycoprotein biosynthesis and cell surface glycobiology (ABO, ASGR1, FUT2, GPLD1 and ST3GAL4), inflammation and immunity (CD276, CDH6, GCKR, HNF1A, HPR, ITGA1, RORA and STAT4) and glutathione metabolism (GSTT1, GSTT2 and GGT), as well as several genes of uncertain or unknown function (including ABHD12, EFHD1, EFNA1, EPHA2, MICAL3 and ZNF827). Our results provide new insight into genetic mechanisms and pathways influencing markers of liver function. Show less

Obesity is globally prevalent and highly heritable, but its underlying genetic factors remain largely elusive. To identify genetic loci for obesity susceptibility, we examined associations between body mass index and ∼ 2.8 million SNPs in up to 123,865 individuals with targeted follow up of 42 SNPs in up to 125,931 additional individuals. We confirmed 14 known obesity susceptibility loci and identified 18 new loci associated with body mass index (P < 5 × 10⁻⁸), one of which includes a copy number variant near GPRC5B. Some loci (at MC4R, POMC, SH2B1 and BDNF) map near key hypothalamic regulators of energy balance, and one of these loci is near GIPR, an incretin receptor. Furthermore, genes in other newly associated loci may provide new insights into human body weight regulation. Show less

Recent genome-wide association (GWA) studies of lipids have been conducted in samples ascertained for other phenotypes, particularly diabetes. Here we report the first GWA analysis of loci affecting total cholesterol (TC), low-density lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) cholesterol and triglycerides sampled randomly from 16 population-based cohorts and genotyped using mainly the Illumina HumanHap300-Duo platform. Our study included a total of 17,797-22,562 persons, aged 18-104 years and from geographic regions spanning from the Nordic countries to Southern Europe. We established 22 loci associated with serum lipid levels at a genome-wide significance level (P < 5 x 10(-8)), including 16 loci that were identified by previous GWA studies. The six newly identified loci in our cohort samples are ABCG5 (TC, P = 1.5 x 10(-11); LDL, P = 2.6 x 10(-10)), TMEM57 (TC, P = 5.4 x 10(-10)), CTCF-PRMT8 region (HDL, P = 8.3 x 10(-16)), DNAH11 (LDL, P = 6.1 x 10(-9)), FADS3-FADS2 (TC, P = 1.5 x 10(-10); LDL, P = 4.4 x 10(-13)) and MADD-FOLH1 region (HDL, P = 6 x 10(-11)). For three loci, effect sizes differed significantly by sex. Genetic risk scores based on lipid loci explain up to 4.8% of variation in lipids and were also associated with increased intima media thickness (P = 0.001) and coronary heart disease incidence (P = 0.04). The genetic risk score improves the screening of high-risk groups of dyslipidemia over classical risk factors. Show less