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Identifying genetic variants associated with lower waist-to-hip ratio can reveal new therapeutic targets for abdominal obesity. We use exome sequences from 362,679 individuals to identify genes associated with waist-to-hip ratio adjusted for BMI (WHRadjBMI), a surrogate for abdominal fat that is causally linked to type 2 diabetes and coronary heart disease. Predicted loss of function (pLOF) variants in INHBE associate with lower WHRadjBMI and this association replicates in data from AMP-T2D-GENES. INHBE encodes a secreted protein, the hepatokine activin E. In vitro characterization of the most common INHBE pLOF variant in our study, indicates an in-frame deletion resulting in a 90% reduction in secreted protein levels. We detect associations with lower WHRadjBMI for variants in ACVR1C , encoding an activin receptor, further highlighting the involvement of activins in regulating fat distribution. These findings highlight activin E as a potential therapeutic target for abdominal obesity, a phenotype linked to cardiometabolic disease. Abdominal fat has been shown to increase cardiometabolic disease risk. In this study, the authors report that loss-of-function variants in the gene INHBE associate with lower BMI-adjusted waist-to-hip ratio, a surrogate measure of abdominal fat.

Fibrolamellar carcinoma (FLC) is a rare liver cancer that disproportionately affects adolescents and young adults. Currently, no standard of care is available and there remains a dire need for new therapeutics. Most patients harbor the fusion oncogene DNAJB1-PRKACA (DP fusion), but clinical inhibitors are not yet developed and it is critical to identify downstream mediators of FLC pathogenesis. Here, we identify long noncoding RNA LINC00473 among the most highly upregulated genes in FLC tumors and determine that it is strongly suppressed by RNAi-mediated inhibition of the DP fusion in FLC tumor epithelial cells. We show by loss- and gain-of-function studies that LINC00473 suppresses apoptosis, increases the expression of FLC marker genes, and promotes FLC growth in cell-based and in vivo disease models. Mechanistically, LINC00473 plays an important role in promoting glycolysis and altering mitochondrial activity. Specifically, LINC00473 knockdown leads to increased spare respiratory capacity, which indicates mitochondrial fitness. Overall, we propose that LINC00473 could be a viable target for this devastating disease.

Unlabelled Box Fibrinogen is a complex molecule comprised of two sets of Aα, Bβ, and γ chains. Fibrinogen deficiencies can lead to the development of bleeding or thromboembolic events. The objective of this study was to perform DNA sequence analysis of patients with clinical fibrinogen abnormalities, and to perform genotype‐phenotype correlations. DNA from 31 patients was sequenced to evaluate disease‐causing mutations in the three fibrinogen genes: FGA, FGB, and FGG. Clinical data were extracted from medical records or from consultation with referring hematologists. Fibrinogen antigen and functional (Clauss method) assays, as well as reptilase time (RT) and thrombin time (TT) were obtained for each patient. Molecular modeling was used to simulate the functional impact of specific missense variants on the overall protein structure. Seventeen mutations, including six novel mutations, were identified in the three fibrinogen genes. There was little correlation between genotype and phenotype. Molecular modeling predicted a substantial conformational change for a novel variant, FGG p.Ala289Asp, leading to a more rigid molecule in a region critical for polymerization and alignment of the fibrin monomers. This mutation is associated with both bleeding and clotting in the two affected individuals. Robust genotype‐phenotype correlations are difficult to establish for fibrinogen disorders. Molecular modeling might represent a valuable tool for understanding the function of certain missense fibrinogen mutations but those should be followed by functional studies. It is likely that genetic and environmental modifiers account for the incomplete penetrance and variable expressivity that characterize fibrinogen disorders.

Jorge Di Paola, Christopher Porter, Walter Kahr and colleagues report germline mutations in the transcriptional repressor gene ETV6 in three families with thrombocytopenia and elevated red blood cell volume. All three mutations affect the ability of ETV6 to repress transcription of a reporter construct, and the two protein-altering mutations affect megakaryocyte development. Some familial platelet disorders are associated with predisposition to leukemia, myelodysplastic syndrome (MDS) or dyserythropoietic anemia 1 , 2 . We identified a family with autosomal dominant thrombocytopenia, high erythrocyte mean corpuscular volume (MCV) and two occurrences of B cell–precursor acute lymphoblastic leukemia (ALL). Whole-exome sequencing identified a heterozygous single-nucleotide change in ETV6 ( et s variant 6), c.641C>T, encoding a p.Pro214Leu substitution in the central domain, segregating with thrombocytopenia and elevated MCV. A screen of 23 families with similar phenotypes identified 2 with ETV6 mutations. One family also had a mutation encoding p.Pro214Leu and one individual with ALL. The other family had a c.1252A>G transition producing a p.Arg418Gly substitution in the DNA-binding domain, with alternative splicing and exon skipping. Functional characterization of these mutations showed aberrant cellular localization of mutant and endogenous ETV6, decreased transcriptional repression and altered megakaryocyte maturation. Our findings underscore a key role for ETV6 in platelet formation and leukemia predisposition.

Non-alcoholic fatty liver disease (NAFLD) is a liver manifestation of metabolic syndrome, and is estimated to affect one billion individuals worldwide. An increased intake of a high-fat diet (HFD) and sugar-sweetened beverages are risk-factors for NAFLD development, but how their combined intake promotes progression to a more severe form of liver injury is unknown. Here we show that fructose metabolism via ketohexokinase (KHK) C isoform increases endoplasmic reticulum (ER) stress in a dose dependent fashion, so when fructose is coupled with a HFD intake it leads to unresolved ER stress. Conversely, a liver-specific knockdown of KHK in C57BL/6J male mice consuming fructose on a HFD is adequate to improve the NAFLD activity score and exert a profound effect on the hepatic transcriptome. Overexpression of KHK-C in cultured hepatocytes is sufficient to induce ER stress in fructose free media. Upregulation of KHK-C is also observed in genetically obesity ob/ob, db/db and lipodystrophic FIRKO male mice, whereas KHK knockdown in these mice improves metabolic function. Additionally, in over 100 inbred strains of male or female mice hepatic KHK expression correlates positively with adiposity, insulin resistance, and liver triglycerides. Similarly, in 241 human subjects and their controls, hepatic Khk expression is upregulated in early, but not late stages of NAFLD. In summary, we describe a novel role of KHK-C in triggering ER stress, which offers a mechanistic understanding of how the combined intake of fructose and a HFD propagates the development of metabolic complications.Competing Interest StatementAlnylam Pharmaceuticals, Inc., sponsored the research with grant funding and performed KHK knockdown experiment at their institution. JW, LN, HCT and KF are employees of Alnylam Pharmaceuticals. Experimental Pathology Laboratories, Inc., was hired by Alnylam to objectively grade liver histology. This work was also supported by NASPGHAN Foundation Young Investigator Award, Pediatric Scientist Development Program Award (HD000850) and COCVD Pilot and Feasibility Grant (GM127211) awarded to SS; K01DK128022 and UL1TR001998 to RNH; R01 DK067536 to RNK; R01DK099222, Department of Defense grant W81XWH2010392, & CCRF Endowed Scholar Award to SD and R01 DK117850, and R01 HL147883 grants to AJL. We acknowledge the support of instrumentation for the Orbitrap Eclipse Tribrid from the NCRR shared instrumentation grant 1S10 OD028654 to B.S. Rashmi Nemade, Ph.D. of BioMedText, Inc. helped edit the manuscript.

Whole exome sequencing on a family with autosomal dominant thrombocytopenia and two occurrences of acute lymphoblastic leukemia (ALL) of unknown cause identified a heterozygous single nucleotide change in the gene ETV6, encoding a p.Pro214Leu amino acid substitution. We screened families with the same phenotype and found mutations in ETV6 in two additional families: one with the identical p.Pro214Leu mutation and one with a p.Arg418Gly substitution. ETV6 is a transcriptional repressor that functions through self-oligomerization and is essential for hematopoietic stem cell proliferation and platelet production in mice. Our report was among the first to describe pathogenic germline mutations in ETV6. Functional characterization of the corresponding ETV6 protein mutations showed aberrant cytoplasmic localization, impaired transcriptional repression, and delayed megakaryocyte maturation. Furthermore, we found that mutant ETV6 protein oligomerizes with WT ETV6 and sequesters it from the nucleus suggesting a dominant negative mechanism. Platelet transcriptome analysis of patients with the p.P214L mutation revealed significant differential expression of 189 transcripts between patients and controls. Downregulated transcripts in patients with the P214L mutation were significantly enriched in transcripts encoding extracellular matrix and cytoskeletal proteins. Interestingly, maintenance of the extracellular matrix is essential for megakaryocyte migration in the bone marrow and plays a role in cancer progression. Furthermore, microtubule and cytoskeletal organization are essential for platelet production. Mouse models of Etv6 disruption, including one expressing the human P214L mutation, exhibit thrombocytopenia and defects in megakaryocyte maturation, similar to humans. My thesis project describes germline mutations in ETV6 that are associated with megakaryocyte differentiation and platelet production as well as ALL predisposition.

Fibrolamellar carcinoma (FLC) is a rare liver cancer that disproportionately affects adolescents and young adults. Currently, no standard of care is available and there remains a dire need for new therapeutics. Most patients harbor the fusion oncogene DNAJB1-PRKACA (DP fusion), but clinical inhibitors are not yet developed and it is critical to identify downstream mediators of FLC pathogenesis. Here, we identify long non-coding RNA LINC00473 among the most highly upregulated genes in FLC tumors and determine that it is strongly suppressed by RNAi-mediated inhibition of the DP fusion in FLC tumor epithelial cells. We show by loss- and gain-of-function studies that LINC00473 suppresses apoptosis, increases the expression of FLC marker genes, and promotes FLC growth in cell-based and in vivo models of disease. Mechanistically, LINC00473 plays an important role in promoting glycolysis and altering mitochondrial activity. Specifically, LINC00473 knockdown leads to increased spare respiratory capacity, an indicator of mitochondrial fitness. Overall, we propose that LINC00473 could be a viable target for this devastating disease. Fibrolamellar carcinoma (FLC) is a lethal liver cancer lacking effective therapeutic options. Ma et al. demonstrate that primate-specific RNA LINC00473 is enriched in tumor epithelial cells and functions to promote FLC growth and dysregulate cellular energetics, unveiling an important mechanism downstream of the fusion oncogene, DNAJB1-PRKACA, in FLC pathogenesis. LINC00473 is consistently elevated in primary FLC tumor tissue from different patient cohorts and in multiple disease models. DP fusion, the signature oncoprotein of FLC, drives LINC00473 expression. LINC00473 promotes FLC growth via anti-apoptotic function. LINC00473 modulates FLC energetics by promoting glycolysis and altering mitochondrial fitness.

Abstract Tyrosine phosphorylation of extracellular proteins is observed in cell cultures and in vivo, but little is known about the functional roles of tyrosine phosphorylation of extracellular proteins. Vertebrate Lonesome Kinase (VLK) is a broadly expressed secretory pathway tyrosine kinase present in platelet ɑ-granules. It is released from platelets upon activation and phosphorylates substrates extracellularly. Its role in platelet function, however, has not been previously studied. In human platelets, we identified phosphorylated tyrosines mapped to luminal or extracellular domains of transmembrane and secreted proteins implicated in the regulation of platelet activation. To determine the role of VLK in extracellular tyrosine phosphorylation and platelet function, we generated mice with a megakaryocyte/platelet-specific deficiency of VLK. Platelets from these mice are normal in abundance and morphology, but have dramatic changes in function both in vitro and in vivo. Resting and thrombin-stimulated VLK-deficient platelets demonstrate a significant decrease of several tyrosine phosphobands. Functional testing of VLK-deficient platelets shows decreased PAR4- and collagen-mediated platelet aggregation, but normal responses to ADP. Dense granule and α-granule release are reduced in these platelets. Furthermore, VLK-deficient platelets exhibit decreased PAR4-mediated Akt (S473) and Erk1/2(T202/Y204) phosphorylation, indicating altered proximal signaling. In vivo, mice lacking VLK in megakaryocytes/platelets demonstrate strongly reduced platelet accumulation and fibrin formation following laser-injury of cremaster arterioles compared to controls. These studies demonstrate that the secretory pathway tyrosine kinase VLK is critical for stimulus-dependent platelet activation and thrombus formation, providing the first evidence that a secreted protein kinase is required for normal platelet function. Competing Interest Statement The authors have declared no competing interest. Footnotes * Key points Platelets lacking secretory pathway tyrosine kinase VLK exhibit defective platelet aggregation, dense granule release, and α-granule release in vitro. Mice with a megakaryocyte/platelet-specific deficiency of VLK demonstrate reduced platelet accumulation and fibrin formation following arteriole damage, but have normal bleeding times.