Whole genome sequence analysis of platelet traits in the NHLBI Trans-Omics for Precision Medicine (TOPMed) initiative

Platelets play a key role in thrombosis and hemostasis. Platelet count (PLT) and mean platelet volume (MPV) are highly heritable quantitative traits, with hundreds of genetic signals previously identified, mostly in European ancestry populations. We here utilize whole genome sequencing (WGS) from NHLBI's Trans-Omics for Precision Medicine initiative (TOPMed) in a large multi-ethnic sample to further explore common and rare variation contributing to PLT (n = 61 200) and MPV (n = 23 485). We identified and replicated secondary signals at MPL (rs532784633) and PECAM1 (rs73345162), both more common in African ancestry populations. We also observed rare variation in Mendelian platelet-related disorder genes influencing variation in platelet traits in TOPMed cohorts (not enriched for blood disorders). For example, association of GP9 with lower PLT and higher MPV was partly driven by a pathogenic Bernard-Soulier syndrome variant (rs5030764, p.Asn61Ser), and the signals at TUBB1 and CD36 were partly driven by loss of function variants not annotated as pathogenic in ClinVar (rs199948010 and rs571975065). However, residual signal remained for these gene-based signals after adjusting for lead variants, suggesting that additional variants in Mendelian genes with impacts in general population cohorts remain to be identified. Gene-based signals were also identified at several genome-wide association study identified loci for genes not annotated for Mendelian platelet disorders (PTPRH, TET2, CHEK2), with somatic variation driving the result at TET2. These results highlight the value of WGS in populations of diverse genetic ancestry to identify novel regulatory and coding signals, even for well-studied traits like platelet traits

Whole-genome sequencing in diverse subjects identifies genetic correlates of leukocyte traits: The NHLBI TOPMed program

Many common and rare variants associated with hematologic traits have been discovered through imputation on large-scale reference panels. However, the majority of genome-wide association studies (GWASs) have been conducted in Europeans, and determining causal variants has proved challenging. We performed a GWAS of total leukocyte, neutrophil, lymphocyte, monocyte, eosinophil, and basophil counts generated from 109,563,748 variants in the autosomes and the X chromosome in the Trans-Omics for Precision Medicine (TOPMed) program, which included data from 61,802 individuals of diverse ancestry. We discovered and replicated 7 leukocyte trait associations, including (1) the association between a chromosome X, pseudo-autosomal region (PAR), noncoding variant located between cytokine receptor genes (CSF2RA and CLRF2) and lower eosinophil count; and (2) associations between single variants found predominantly among African Americans at the S1PR3 (9q22.1) and HBB (11p15.4) loci and monocyte and lymphocyte counts, respectively. We further provide evidence indicating that the newly discovered eosinophil-lowering chromosome X PAR variant might be associated with reduced susceptibility to common allergic diseases such as atopic dermatitis and asthma. Additionally, we found a burden of very rare FLT3 (13q12.2) variants associated with monocyte counts. Together, these results emphasize the utility of whole-genome sequencing in diverse samples in identifying associations missed by European-ancestry-driven GWASs

Molecular metabolism underlying hypertriglyceridemia: From bench to bedside

This thesis explores the underlying biochemical mechanisms that explain the hypertriglyceridemic phenotype in type 2 diabetes mellitus (T2DM), an important factor for the increased risk for cardiovascular disorders. Two recently identified proteins involved in TG metabolism are central in this thesis: glycosylphosphatidyl inositol HDL binding protein (GPIHBP1) and sulfatase 2 (SULF2). Various in vivo studies both in human and mice model, and in vitro experiments using hepatic and endothelial cell lines were used. GPIHBP1 is essential for LPL-mediated lipolysis where triglycerides are degraded to free fatty acids. We show that mutations in GPIHBP1 leads to loss of function of the protein, leads to hypertriglyceridemia and may be the underlying cause of high TG in T2DM. Here, we tested and show that GPIHBP1 expression in adipose tissue is associated with insulin resistance and leads to reduced LPL-mediated lipolysis. Furthermore, we also show that in general population, a genetic risk score is associated with CVD risk independent of metabolic syndrome, which is also associated with CVD risk. SULF2 is a protein that interferes with the degree of sulfation of the heparan sulfate proteoglycans involved in the hepatic clearance of TG-rich lipoproteins. Here we show that genetic variant in SULF2 is associated with less SULF2 protein and an improved clearance of TG and thus support the interest for SULF2 as a target for intervention in T2DM. Furthermore, we also show in in vitro experiments that SULF2 expression and secretion is upregulated in insulin resistance state via the AKT2/IRS2 pathway

Quantification of the BRI1 receptor in planta

In plants, green fluorescent protein (GFP) is routinely used to determine the subcellular location of fusion proteins. Here, we show that confocal imaging can be employed to approximate the number of GFP-labeled protein molecules present in living Arabidopsis (Arabidopsis thaliana) root cells. The technique involves calibration with soluble GFP to provide a usable protein concentration range within the confocal volume of the microscope. As a proof of principle, we quantified the Brassinosteroid Insensitive1 (BRI1) receptor fused to GFP, under control of its own promoter. The number of BRI1-GFP molecules per root epidermal cell ranges from 22,000 in the meristem and 130,000 in the elongation zone to 80,000 in the maturation zone, indicating that up to 6-fold differences in BRI1 receptor content exist. In contrast, when taking into account differences in cell size, BRI1-GFP receptor density in the plasma membrane is kept constant at 12 receptors µm-2 in all cells throughout the meristem and elongation zone. Only the quiescent center and columella cells deviate from this pattern and have 5 to 6 receptors µm-2. Remarkably, root cell sensitivity toward brassinosteroids appears to coincide with uniform meristem receptor densit

Quantification of the BRI1 receptor in planta

In plants, green fluorescent protein (GFP) is routinely used to determine the subcellular location of fusion proteins. Here, we show that confocal imaging can be employed to approximate the number of GFP-labeled protein molecules present in living Arabidopsis (Arabidopsis thaliana) root cells. The technique involves calibration with soluble GFP to provide a usable protein concentration range within the confocal volume of the microscope. As a proof of principle, we quantified the Brassinosteroid Insensitive1 (BRI1) receptor fused to GFP, under control of its own promoter. The number of BRI1-GFP molecules per root epidermal cell ranges from 22,000 in the meristem and 130,000 in the elongation zone to 80,000 in the maturation zone, indicating that up to 6-fold differences in BRI1 receptor content exist. In contrast, when taking into account differences in cell size, BRI1-GFP receptor density in the plasma membrane is kept constant at 12 receptors µm-2 in all cells throughout the meristem and elongation zone. Only the quiescent center and columella cells deviate from this pattern and have 5 to 6 receptors µm-2. Remarkably, root cell sensitivity toward brassinosteroids appears to coincide with uniform meristem receptor densit

SULF2 Strongly Prediposes to Fasting and Postprandial Triglycerides in Patients with Obesity and Type 2 Diabetes Mellitus

Cancer Signaling networks and Molecular Therapeutic