Low-Cost High-Throughput Genotyping for Diagnosing Familial Hypercholesterolemia

BACKGROUND: Familial hypercholesterolemia (FH) is a common but underdiagnosed genetic disorder characterized by high low-density lipoprotein cholesterol levels and premature cardiovascular disease. Current sequencing methods to diagnose FH are expensive and time-consuming. In this study, we evaluated the accuracy of a low-cost, high-throughput genotyping array for diagnosing FH. METHODS: An Illumina Global Screening Array was customized to include probes for 636 variants, previously classified as FH-causing variants. First, its theoretical coverage was assessed in all FH variant carriers diagnosed through next-generation sequencing between 2016 and 2022 in the Netherlands (n=1772). Next, the performance of the array was validated in another sample of FH variant carriers previously identified in the Dutch FH cascade screening program (n=1268). RESULTS: The theoretical coverage of the array for FH-causing variants was 91.3%. Validation of the array was assessed in a sample of 1268 carriers of whom 1015 carried a variant in LDLR, 250 in APOB, and 3 in PCSK9. The overall sensitivity was 94.7% and increased to 98.2% after excluding participants with variants not included in the array design. Copy number variation analysis yielded a 89.4% sensitivity. In 18 carriers, the array identified a total of 19 additional FH-causing variants. Subsequent DNA analysis confirmed 5 of the additionally identified variants, yielding a false-positive result in 16 subjects (1.3%).CONCLUSIONS: The FH genotyping array is a promising tool for genetically diagnosing FH at low costs and has the potential to greatly increase accessibility to genetic testing for FH. Continuous customization of the array will further improve its performance.</p

Experimental Conditions That Influence the Utility of 2′7′-Dichlorodihydrofluorescein Diacetate (DCFH2-DA) as a Fluorogenic Biosensor for Mitochondrial Redox Status

Oxidative stress has been causally linked to various diseases. Electron transport chain (ETC) inhibitors such as rotenone and antimycin A are frequently used in model systems to study oxidative stress. Oxidative stress that is provoked by ETC inhibitors can be visualized using the fluorogenic probe 2′,7′-dichlorodihydrofluorescein-diacetate (DCFH2-DA). Non-fluorescent DCFH2-DA crosses the plasma membrane, is deacetylated to 2′,7′-dichlorodihydrofluorescein (DCFH2) by esterases, and is oxidized to its fluorescent form 2′,7′-dichlorofluorescein (DCF) by intracellular ROS. DCF fluorescence can, therefore, be used as a semi-quantitative measure of general oxidative stress. However, the use of DCFH2-DA is complicated by various protocol-related factors that mediate DCFH2-to-DCF conversion independently of the degree of oxidative stress. This study therefore analyzed the influence of ancillary factors on DCF formation in the context of ETC inhibitors. It was found that ETC inhibitors trigger DCF formation in cell-free experiments when they are co-dissolved with DCFH2-DA. Moreover, the extent of DCF formation depended on the type of culture medium that was used, the pH of the assay system, the presence of fetal calf serum, and the final DCFH2-DA solvent concentration. Conclusively, experiments with DCFH2-DA should not discount the influence of protocol-related factors such as medium and mitochondrial inhibitors (and possibly other compounds) on the DCFH2-DA-DCF reaction and proper controls should always be built into the assay protocol

Taking One Step Back in Familial Hypercholesterolemia:STAP1 Does Not Alter Plasma LDL (Low-Density Lipoprotein) Cholesterol in Mice and Humans

International audienceSTAP1, encoding for STAP1 (signal transducing adaptor family member 1), has been reported as a candidate gene associated with familial hypercholesterolemia. Unlike established familial hypercholesterolemia genes, expression of STAP1 is absent in liver but mainly observed in immune cells. In this study, we set out to validate STAP1 as a familial hypercholesterolemia gene. Approach and Results: A whole-body Stap1 knockout mouse model (Stap1 -/ - ) was generated and characterized, without showing changes in plasma lipid levels compared with controls. In follow-up studies, bone marrow from Stap1 -/ - mice was transplanted to Ldlr -/ - mice, which did not show significant changes in plasma lipid levels or atherosclerotic lesions. To functionally assess whether STAP1 expression in B cells can affect hepatic function, HepG2 cells were cocultured with peripheral blood mononuclear cells isolated from heterozygotes carriers of STAP1 variants and controls. The peripheral blood mononuclear cells from STAP1 variant carriers and controls showed similar LDLR mRNA and protein levels. Also, LDL (low-density lipoprotein) uptake by HepG2 cells did not differ upon coculturing with peripheral blood mononuclear cells isolated from either STAP1 variant carriers or controls. In addition, plasma lipid profiles of 39 carriers and 71 family controls showed no differences in plasma LDL cholesterol, HDL (high-density lipoprotein) cholesterol, triglycerides, and lipoprotein(a) levels. Similarly, B-cell populations did not differ in a group of 10 STAP1 variant carriers and 10 age- and sex-matched controls. Furthermore, recent data from UK Biobank do not show association between STAP1 rare gene variants and LDL cholesterol

Transintestinal cholesterol excretion in humans

PURPOSE OF REVIEW: To discuss recent insights into the measurement and cellular basis of transintestinal cholesterol excretion (TICE) in humans and to explore TICE as a therapeutic target for increasing reverse cholesterol transport. RECENT FINDINGS: TICE is the net effect of cholesterol excretion by the enterocyte into the intestinal lumen and is the balance between input and output fluxes through the enterocytes. These fluxes are: cholesterol excretion into the intestinal lumen mainly via ATP-binding cassette (ABC) G5/8, cholesterol absorption from the intestine by Niemann-Pick C1 like protein 1, the uptake of plasma lipoproteins by enterocytes at the basolateral membrane, and the excretion of cholesterol in chylomicrons into the lymph. Multiple studies have shown that TICE contributes to fecal neutral sterol (FNS) excretion in humans. TICE can be targeted with plant sterols, liver X receptor agonists, bile acids, ezetimibe, and proprotein convertase subtilisin/kexin type 9 inhibitors. SUMMARY: TICE contributes significantly to FNS excretion in humans, independently of the biliary pathway. Knowledge about its underlying cellular mechanisms surges through in-vivo and in-vitro studies in mice and humans. TICE might be an interesting therapeutic target for increasing cholesterol disposal with the feces. Albeit multiple therapeutic options are available, studies showing clinical benefit are still needed

Novel lipid modifying drugs to lower LDL cholesterol

Purpose of review Statins have long been the cornerstone for the prevention of cardiovascular disease (CVD). However, because of perceived adverse effects and insufficient efficacy in certain groups of patients, considerable interest exists in the search for alternatives to lower LDL-cholesterol (LDL-C), and the recent approvals of proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors underlines the success of this quest. Here, we give an updated overview on the most recent developments in the area of LDL-C lowering agents. Recent findings The clinical effects of the PCSK9 inhibitors are promising, especially now that the FOURIER and SPIRE programmes are published. Most cholesterylester-transfer protein inhibitors, however, except anacetrapib, have been discontinued because of either toxicity or lack of efficacy in large cardiovascular outcome trials. Other agents - like mipomersen, lomitapide, ETC-1002, and gemcabene - aim to lower LDL-C in different ways than solely through the LDL receptor, opening up possibilities for treating patients not responding to conventional therapies. New discoveries are also being made at the DNA and RNA level, with mipomersen being the first approved therapy based on RNA intervention in the United States for homozygous familial hypercholesterolemia. Summary Recent years have witnessed a new beginning for cholesterol-lowering compounds. With increased knowledge of lipid metabolism a score of new therapeutic targets has been identified. Mechanisms for modulation of those targets are also becoming more diverse while statins remain the backbone of CVD prevention, the new alternatives, such as PCSK9 monoclonals will probably play an important additional role in treatment of patients at risk for CV

Ethnicity, lipids and cardiovascular disease

The prevalence of cardiovascular disease differs among ethnic groups and along geographic boundaries. At present, most of the projected increase in mortality from cardiovascular disease occurs in sub-Saharan African, Chinese and Southeast Asian populations. Ethnic disparities in the prevalence of cardiovascular disease coincide with quantitative and qualitative differences in risk factors for cardiovascular disease. High plasma cholesterol is one of the most important preventable causes of ischemic heart disease. The current review summarizes recent evidence on ethnic differences in ischemic heart disease and its correlates with genetic and acquired differences in plasma lipid and lipoprotein levels. The nature of ethnic differences in plasma lipid levels, apolipoprotein L1 en lipoprotein(a) [Lp(a)] is outlined, and the effects of lipid-lowering therapy and future efforts and challenges regarding implementation are discussed. Ethnic differences in HDL-cholesterol (HDL-C), triglyceride levels and Lp(a) may impact ethnic differences in cardiovascular disease and result in higher residual risk during lipid-lowering therapy. Further efforts should be made to stimulate the use of statins in both high-income and low-income countries and study their effects in individuals with different ethnic background

The next generation of triglyceride-lowering drugs: will reducing apolipoprotein C-III or angiopoietin like protein 3 reduce cardiovascular disease?

PURPOSE OF REVIEW: Apolipoprotein C-III (ApoC-III) and angiopoietin like protein 3 (angptl3) have emerged as key regulators of triglyceride metabolism. Based on Mendelian randomisation studies, novel therapeutic strategies inhibiting these proteins using monoclonal antibodies or gene silencing techniques might reduce residual cardiovascular disease (CVD) risk in dyslipidemic patients. This article aims to review the role of apoC-III and angptl3 in triglyceride metabolism and combine early clinical evidence of CVD reducing potential of these new therapeutic targets. RECENT FINDINGS: Angptl3 inhibition by mAb or antisense therapy has recently completed phase I and II studies, respectively and demonstrate robust apolipoprotein B (apoB) lowering up to 46%. Volanesorsen is an antisense therapy approved for patients with extremely elevated plasma triglyceride levels in which it showed no consistent apoB reduction. However, the GalNAc-conjugated oligonucleotide showed moderate (up to ∼30%) apoB reduction in a phase 1/2a dose-finding study. SUMMARY: Angptl3 and apoC-III are novel targets in lipoprotein metabolism that reduce triglycerides when inhibited. The expected CVD risk reduction may be mediated through reduced triglyceride-rich lipoprotein particle number, reflected by apoB, rather than triglyceride reduction per se. Limited human evidence shows that apoC-III and angptl3 inhibition both potently lower triglycerides, but since angptl3 inhibition reduces apoB more robustly it may be expected to confer more favorable CVD risk reduction