Community Research and Development Information Service - CORDIS

Periodic Report Summary 2 - TRANSCARD (Translating disease to cardiovascular health)

Project Context and Objectives:
TransCard is a collaborative effort between 2 industrial and 4 academic partners in 4 European countries, i.e. Switzerland (UZH and Nebion; lead small medium enterprise), Germany (PolyQuant; second small medium enterprise), Denmark (REGIONH) and The Netherlands (UMCG, AMC) from which the work is coordinated.
After three years, the TransCard consortium is extending studies into the most promising candidate genes that have been selected at the start of the project.
One of these genes is COMMD1 as it was just discovered that this protein is required for normal trafficking of the low-density lipoprotein (LDLR) to the membrane of hepatocytes, which in turn controls the levels of circulating low-density lipoprotein (LDL) cholesterol. Additional investigations have been published in 2016 which showed that COMMD1 acts in concert with members of the CCC complex and others. The function of these protein complexes in endosomal cargo sorting (such as LDLR) is evolutionary conserved and is shown to control plasma cholesterol concentration in mice, dogs and humans. Further genetic epidemiological studies into the relation between COMMD1, lipid traits, and clinical endpoints of ischemic disease by REGIONH were unfortunately hampered by the lack of powerful genetic tools but alternative approaches to study the genetics of this family of proteins and its binding partners are being explored. Ongoing studies indicate that we have multiple tools to study the endosomal sorting of lipoprotein receptors which control plasma lipoprotein levels. These studies are supported by targeted proteomic analyses which were made possible through PolyQuant and the UMCG that provides the mass spectroscopy expertise and machinery. The ongoing basic research studies are anticipated to increase our understanding of cholesterol and lipid homeostasis at the cellular and systemic level.
LRP1 was a second gene that was picked up for study at the start of TransCard. It concerns a long-established gene which is thus far almost exclusively studied in mice and in cell culture. Through a collaborative effort between AMC, UMCG, we show that functional mutations in LRP1 in humans are likely causally related to low high HDL cholesterol. While LRP1, a member of the LDLR protein family, is mostly known for its function in the clearance of remnant (triglyceride-rich) lipoproteins, these investigations show that one of the other ligands of LRP1 affects the expression of major players in HDL metabolism, i.e. ABCA1 and SRB1). REGIONH has corroborated the finding that LRP1 is associated with HDL cholesterol and also shows an association with triglycerides in a large genetic epidemiological study.
Early on, TransCard’s lead small medium enterprise, NEBION, identified Gene 1 (in collaboration with UMCG) as an interesting novel lead gene through in silico analyses. Soon after our discovery, Gene 1 was also identified through genome wide association studies to be associated with total cholesterol levels. Studying whole body knock-out mice by UMCG shows interesting lipid and other phenotypes while REGIONH showed that rare variation at the respective gene locus is associated with the major protein component of LDL. In view of the promising data thus far we will continue our studies in both humans and mice.
STAP1 was identified by the AMC as a novel candidate gene for familial hypercholesterolemia in several families. Interestingly, this gene is expected to exert a function in cells of the immune system (B cells). If we can prove that there exists a causal link between loss of STAP1 and hypercholesterolemia, this would be a breakthrough. In the AMC, blood products of affected individuals and unaffected family members are currently studied. Genetic studies of STAP1 by REGIONH were hampered by lack of powerful genetic tools. The UMCG has generated Stap1 knock-out mice but the first results are negative. We are currently challenging these mice with an inflammatory diet as well as a hepatic knock down of the LDLR.
In 2016, TransCard has also started studies into the role of two novel candidate Genes 2 and 3. Gene 2 was identified by NEBION through additional mRNA co-expression analyses while gene 3 was identified through literature studies. For both genes, the UMCG is generating whole body knock-out mice. This has been successful for gene 3, while for gene 2, we have not yet obtained homozygous knock-out mice. Preliminary studies show that complete loss of gene 3 is associated with increased cholesterol levels. We are currently investigating whether gene 3 products are affected in a rare human disorder that is studied within the UMCG.
REGIONH furthermore identified (in collaboration with UMCG) Gene 4 as candidate gene for further study.
Through studies in families, the AMC identified Gene 5 as a gene involved in accelerated development of atherosclerosis. Preliminary findings suggest a role of its gene product in vessel integrity. In addition, AMC has collected precious materials of patients with various dyslipidemias and premature atherosclerosis which will be studied at the gene, mRNA, protein and metabolome levels.
In a drug screen for genes that control the transport of lipoprotein across in cells by UZH, Genes 6 and 7 were identified and validated as players in this important phenomenon in atherogenesis. Studies in mice are being considered. Finally, we look forward to the result of a massive whole genome RNAi based screening effort for genes that are involved in the uptake of HDL and LDL in a hepatoma cell line by the same partner. This screening will undoubtedly reveal new insights that will become important to TransCard.
The new projects will need quick validation steps in view of only two years till the end of the project. We have therefore requested for prolongation of TransCard by six months.
NEBION has improved their power to identify new genes after curating additional publicly available mRNA datasets (incuding RNAseq) and is continuously developing tools to prioritize genes for further study.
Our studies will also benefit from other technical improvements in TransCard. The use of CRISPR/Cas9 to quickly generate whole body knockout mouse models has recently been extended by the possibility to quickly study the impact of liver specific ablation of novel candidate genes. The latter will be important in that it renders outcome in a 3 month time period. Further support comes from targeted proteomics studies, enabled by UMCG and PolyQuant, that will provide rapid insight into changes in multiple proteins involved in cholesterol and beyond. In addition, REGIONH will have expanded their repertoire of variants in candidate genes through further sequencing efforts. This will help to better select and prioritize genes of potential interest for large scale studies in humans.
TransCard is well underway with studies in multiple candidate genes with proven or yet anticipated roles in the regulation of plasma lipids and lipoproteins. Most of our work is directed to get better insight into the molecular aetiology of genotype-phenotype relations. It is too early to speculate whether this knowledge can be directly used to develop pharmaceutical intervention strategies but of all genes studied thus far, Gene 1 is likely most targetable of all.

Project Results:
UMCG: Several mouse models that have been or are being characterized to study their role in lipid metabolism: These are liver-specific knockout models for COMMD1, COMMD6, COMMD9 and WASH1, and whole body knock-out models for GPR146, STAP1, TMEM97, and SMLR1. Phenotyping of these mice includes monitoring lipid levels following e.g. challenges with different diets. In addition, oral fat tolerance tests, possible effects on VLDL production and catabolism and changes in glucose metabolism are or will be studied.
UMCG has also developed a mouse model that enables very quick monitoring of hepatic ablation of a gene of interest (2 months). It concerns mice expressing Cas9 only in hepatocytes. Candidate genes will be knocked out through delivery of guidance RNAs to the liver through intravenous injection of adenovirus. This model has been successfully used to verify the impact on CCDC22 on plasma LDLc levels.
UMCG & Polyquant: Assays were further developed to simultaneously quantify multiple proteins in cells/tissue homogenates and blood products (serum, plasma). These targeted proteomic studies have been made possible through the generation of QconCats by Polyquant and the development of assays with mass spectrometry equipment at the UMCG. These studies mostly entailed the effects of hepatic specific depletion of candidate genes (see above) on established and new players in lipid homeostasis in murine (liver) cells. In WP6 we describe how this technique has helped to better understand the trafficking of important lipoprotein receptors like LDLR to the plasma membrane in cells.
In addition, we have set up targeted proteomic assays for whole plasma of human origin where we have focused on factors that are involved in the lipolysis of triglycerides. With the current protocols, we are ready to run targeted proteomics in relatively large sets (n=200) of samples of human origin.
NEBION: continued its efforts to expand the content of its curated gene expression database, improve its methodology for target discovery, and provided training and support to the consortium members for the use of its analysis tools.
REGIONH Thirteen genes selected by the Transcard consortium were studied in the Copenhagen City Heart Study. APOA4, was included in this list as a result of a collaboration with the UMCG. The possible roles of all 13 genes in lipid metabolism were assessed through studying (rare) genetic variation. As prime targets for the consortium, COMMD1 and, GPR146 were studied in more detail.
AMC: has collected clinical data, DNA, plasma, and cells from patients with extreme forms of dyslipidemia and from patients suffering from premature atherosclerosis (PAS). We focused on optimisation of cellular experiments to quantify the role of LDLR mediated uptake in order to enable future studies in readily available fibroblasts from patients with unexplained extreme plasma LDL-C levels (“FH4”). Moreover, we conducted a number of genetic studies, ranging from genome wide association studies (focussing on the differential LDL-C effects of currently available lipid lowering therapies) to candidate, exome and genome sequencing (both in subjects with FH4 and PAS).
UZH: executed the challenging task to identify cellular regulators of lipoprotein endocytosis by using image-based drug and RNA interference screening for uptake of high-density lipoprotein (HDL) and LDL by endothelial cells and hepatocytes, respectively.

Potential Impact:
UMCG: We expect that our studies in mice with attenuated CCC/WASH/retromer function will help basic understanding of endosomal trafficking of major lipoproteins to the cell membrane. Since the presence of the reappearance of these receptors at the cell membrane has a direct impact on circulating levels of LDL and other lipoproteins, it may provide interesting insights for therapeutic intervention. Insightful in this regard is the presence of compounds that induces the level and function of the retromer component VPS35. Preliminary genetic studies furthermore show that one of the involved genes may be very suitable for genetic epidemiological studies by REGIONH. Such a study will provide insight how trafficking of LDLR to the cell membrane affects LDLc levels and risk of ischemic disease in the general population.
Of the other mouse models developed and characterized thus far, we expect that studies into GPR146, SMLR1 and TMEM97 will reveal interesting results. GPR146: we expect that through collaboration with REGIONH, we can provide evidence that natural GPR146 gene variation has a direct impact on ApoCIII, LRP1 and SCD1 protein levels (preliminary evidence from targeted proteomics as well as Western blotting studies). In addition we expect that REGIONH will provide evidence that GPR146 affects risk of ischemic heart disease in the general population (see below for more detail). SMLR1: since it is co-expressed prominently (R2 of 0.7) with one of the foremost important apolipoproteins in both men and mice, i.e. apoCIII, we are confident that ablation of this gene (no information in the public domain) is going to provide novel insight. TMEM97: Our preliminary data show that complete TMEM97 ablation significantly increases cholesterol levels in mice. The evolutionary conservation of this gene is very high as exemplified by the near absence of variation in the ExAC database (exome data of >60.000 individuasl) and the fact that REGIONH did not identify any variant in the first 190 individuals at the extreme ends of the LDLc distribution. It will be of great interest to understand how this SREBP regulated gene (co-regulated with all genes involved in de novo cholesterol synthesis) and the encoded protein - that directly interacts with NPC1 - affects cholesterol homeostasis.
Targeted proteomics assays have been set up and tested well in homogenates of cells and tissues as well as in plasma while untargeted proteomics studies are currently being developed. We expect that in the next two years, studies in endogenous cells (peripheral blood cells, fibroblasts, and urine derived hepatocytes), and plasma of patients (AMC; see below) will help our search for novel candidate genes for human dyslipidemia and premature atherosclerosis. In addition, we expect to find novel biomarkers for hyper and hypocholesterolemia in samples of individuals with extreme dyslipidemias (selected from the Lifelines prospective population cohorts).
Finally, the mouse model for quick validation of the expected roles of candidate genes in lipid homeostasis (see paragraph 1.2) will be very helpful to study new candidate genes that will be identified shortly by UZH and those that will be put forward by NEBION.
NEBION: expects its current integration of additional expression data, in particular RNA-seq data, will open new opportunities for target discovery. Not only more protein-coding genes were measured with the new technologies, but also non-coding transcripts are measured which can be used as surrogate markers or constitute companion diagnostics signatures. The extension of NEBION’s tools and database content continuously improves in-silico target discovery and the predictive power of the analysis. Creating user-friendly online tools will allow biologists with little bioinformatics training to perform complex and large-scale queries efficiently, without relying on external bioinformatics support.
REGIONH: expects that extending the 1% high/low LDLc groups will lead to a better selection and prioritization of the genes. The selection of the genes based on the detected variants will be far better with 2% high/low LDLc groups, this extra 1% will lead to more variants detected in each group and will give a clearer picture about the expected phenotype. More, in depth, following up work has been carried out recently on the effects of variation at the GPR146 locus on different cholesterol-related phenotypes and endpoints and the full results from the human populations available in REGIONH are expected to be available soon. APOA4, has hardly been studied in the field is of interest to TransCard, and will be studied further. Studies in our cohorts might need to be confirmed through understanding the physiological role of this gene in functional studies by other partners. At the same time, using some of the new bioinformatics tools that might indicate the effects of the detected variants on the expression level of the genes might push the study towards better prioritization. Data inferred from the studied variants/genes on the different available endpoints in our human populations could be of a high value, and source of information to the pharmaceutical companies to target those genes. In collaboration with UMCG, AMC and UZH, REGIONH will evaluate and confirm their findings in our human populations.
AMC: anticipates identifying novel pathways involved in lipid metabolism by combining the generated genetics data from cases and families with extreme forms of dyslipidemia with the results from planned cellular and proteomic studies. We will make use of the protein concatamer developed by our colleagues from UMCG/Polyquant to assess the absolute and relative abundance of lipid associated proteins in cases (10 patients with FH4, 5 patients with extreme Tg levels) and normolipidemic controls. The dynamics will be studied by comparing the proteomic profiles in patients prior and on statin therapy and in subjects with mutations in established lipid genes. Moreover, lipidomics will also be studied in the same individuals.
UZH: expects the siRNA screen to find novel regulators of HDL and LDL uptake into cells. In line with the overall goal of Transcard such regulators are potential targets for the treatment of dyslipidemias and preventive measures towards cardiovascular disease. The identification of VEGF as a regulator of HDL and LDL transport through the endothelium may have relevance beyond CVD: VEGF is a driver of angiogenesis and as such an established target of inhibitory therapies of several cancers as well as retina diseases, notably age-related macula degeneration and diabetic retinopathy. The identification of VEGF as a regulator of transendothelial transport of LDL and HDL raise the possibility of diagnostic or perhaps also therapeutic exploitation. In fact, partner UZH started to investigate the role of apoAI and apoB as well as SR-BI in tissue of Clear Renal Cell Carcinoma (CRCC) patients as prognostic biomarkers.

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