👤 Kathrin Dittrich

Apolipoprotein standardization in multiple calibration laboratories requires equivalent results to value assign matrix-based reference and external quality assurance materials. A multiplexed LC-MS/MS-based reference measurement procedure (RMP) has been developed for serum apolipoproteins apo(a), apoA-I, apoB, apoC-I, apoC-II, apoC-III, and apoE. This study evaluates the transferability of the RMP between 3 calibration labs and determines the between-laboratory imprecision. Six periodic ring trial surveys were held. The study protocol, calibrators, internal standards, quality controls (QCs), and clinical samples (CSs) were shared among the laboratories. Intra-laboratory imprecision and inter-peptide comparisons evaluated intra-laboratory performance, while inter-laboratory imprecision evaluated equivalence between the calibration labs. Precision of the common bilevel QC monitored the level of harmonization over time. Intra-laboratory imprecision fulfilled predefined analytical performance, defined as repeatability <50% of the maximum allowable uncertainty (MAU) at minimal criteria. Median interlaboratory variation (CVbl) was 3.71%, 3.33%, 7.38%, 6.74%, 3.88%, and 3.90% for apoA-I, apoB, apoC-I, apoC-II, apoC-III, and apoE, respectively. For apo(a), CVbl was concentration (x) dependent following 206.32×x-0.899%. In QC samples, the average imprecision for all apolipoproteins decreased from 6.0% and 18.1% for QC1 and QC2, to 5.2% and 9.5%, indicating improvement of analytical performance of the network over time. This study shows the feasibility of transferring the multiplex apo LC-MS/MS-based RMP between laboratories. Predefined performance specifications were fulfilled for all seven apolipoproteins. Ongoing round-robin studies will ensure stable performance of the calibration labs required to accomplish an accurate value-base for apolipoprotein certification of commercial reagents. Show less

Carbohydrate response element-binding protein (ChREBP) is a transcription factor activated by glucose metabolites that orchestrates the expression of genes involved in glycolysis, de novo lipogenesis, and ATP homeostasis. Inadequate ChREBP activity impairs the cellular adaptations to glucose exposure and in humans associates with dyslipidemia, fatty liver disease, and type 2 diabetes. ChREBP activity is regulated by cytosolic-nuclear translocation involving its low-glucose inhibitory domain (LID). Whether this domain is targeted by post-translational lysine acetylation is unknown. Here we report a novel LID acetylation site that controls activity and protein interactions of ChREBP. Mutation of this residue increased glucose-induced activity and target gene expression of ChREBP. Mechanistically, mutant ChREBP protein showed more nuclear localization and enhanced genomic binding to a target promoter. Interactions with proteins that exhibit differential binding upon glucose exposure were attenuated by the mutation, demonstrating the importance of the LID in the formation of the protein interactome. Particularly interactions with 14-3-3 proteins, factors that regulate cytosolic/nuclear trafficking of ChREBP, were reduced, whereas interactions with proteins of the nucleosome remodeling deacetylase complex (NuRD) were increased. These molecular insights may shape new therapeutic strategies to target ChREBP activity and counteract metabolic diseases. Show less

In this study we aimed to establish the genetic cause of a myriad of cardiovascular defects prevalent in individuals from a genetically isolated population, who were found to share a common ancestor in 1728. Trio genome sequencing was carried out in an index patient with critical congenital heart disease (CHD); family members had either exome or Sanger sequencing. To confirm enrichment, we performed a gene-based association test and meta-analysis in two independent validation cohorts: one with 2685 CHD cases versus 4370 . These controls were also ancestry-matched (same as FTAA controls), and the other with 326 cases with familial thoracic aortic aneurysms (FTAA) and dissections versus 570 ancestry-matched controls. Functional consequences of identified variants were evaluated using expression studies. We identified a loss-of-function variant in the Notch target transcription factor-encoding gene HEY2. The homozygous state (n = 3) causes life-threatening congenital heart defects, while 80% of heterozygous carriers (n = 20) had cardiovascular defects, mainly CHD and FTAA of the ascending aorta. We confirm enrichment of rare risk variants in HEY2 functional domains after meta-analysis (MetaSKAT p = 0.018). Furthermore, we show that several identified variants lead to dysregulation of repression by HEY2. A homozygous germline loss-of-function variant in HEY2 leads to critical CHD. The majority of heterozygotes show a myriad of cardiovascular defects. Show less

Dyslipidemia is a major risk factor for atherosclerotic cardiovascular disease (ASCVD). As key regulators of lipoprotein metabolism, apolipoproteins (apos) are discussed as vascular risk factors. This study aimed to analyze associations of major plasma apos with coronary artery disease (CAD), peripheral artery disease (PAD) and carotid artery plaque (CAP) to elucidate their diagnostic potential in risk assessment. ApoA-I, apoA-II, apoA-IV, apoB-100, apoC-I, apoC-III, apoE, and apoJ were simultaneously quantified in 3 μL EDTA-plasma by LC-MS/MS in a case-control subgroup of the Leipziger LIFE-Heart Study (N = 911). Confounder analysis with demographic, clinical covariates and serum lipids, cardiac, inflammatory, and hepatic markers were performed. Apos were associated with CAD, CAP, and PAD in a multivariate regression model. Fasting and statin therapy showed strongest effects on apo concentrations. Inverse correlations of HDL-related apos A-I, A-II, A-IV, and C-I were observed for troponin T and interleukin 6. Concentrations of apos A-II, B-100, C-I, and E were decreased under statin therapy. After adjustment for influencing factors and related lipids, only apoB-100 (odds ratio per one SD [OR], 1.39; 95% confidence interval [CI], 1.05-1.84) was independently associated with CAD while apoA-IV (OR, 0.74; 95% CI 0.58-0.95) indicated PAD. ApoB-100 (OR, 1.55; 95% CI, 1.18-2.04), apoC-III (OR, 1.30; 95% CI, 1.06-1.58), and apoE (OR, 1.34; 95% CI, 1.13-1.58) were associated with CAP. Triglyceride rich lipoproteins (TRLs) associated apos A-IV, B-100, C-III, and E are independently associated with stable ASCVD, providing further evidence for a potential role of TRLs in atherogenesis. Show less

Recent genome-wide association studies identified novel candidate genes for fasting and 2 h blood glucose and insulin levels in adults. We investigated the role of four of these loci (ADCY5, GIPR, GCKR and VPS13C) in early impairment of glucose and insulin metabolism in children. We genotyped four variants (rs2877716; rs1260326; rs10423928; rs17271305) in 638 Caucasian children with detailed metabolic testing including an oGTT and assessed associations with measures of glucose and insulin metabolism (including fasting blood glucose, insulin levels and insulin sensitivity/secretion indices) by linear regression analyses adjusted for age, sex, BMI-SDS and pubertal stage. The major allele (C) of rs2877716 (ADCY5) was nominally associated with decreased fasting plasma insulin (P = 0.008), peak insulin (P = 0.009) and increased QUICKI (P = 0.016) and Matsuda insulin sensitivity index (P = 0.013). rs17271305 (VPS13C) was nominally associated with 2 h blood glucose (P = 0.009), but not with any of the insulin or insulin sensitivity parameters. We found no association of the GIPR and GCKR variants with parameters of glucose and insulin metabolism. None of the variants correlated with anthropometric traits such as height, WHR or BMI-SDS, which excluded potential underlying associations with obesity. Our data on obese children indicate effects of genetic variation within ADCY5 in early impairment of insulin metabolism and VPS13C in early impairment of blood glucose homeostasis. Show less