Objective The severe forms of hypertriglyceridaemia (HTG) are caused by mutations in genes that lead to loss of function of lipoprotein lipase (LPL). In most patients with severe HTG (TG >10 mmol/L) it is a challenge to define the underlying cause. We investigated the molecular basis of severe HTG in patients referred to the Lipid Clinic at the Academic Medical Center Amsterdam. Methods The coding regions of LPL, APOC2, APOA5 and two novel genes, lipase maturation factor 1 (LMF1) and GPI-anchored HDL-binding protein 1 (GPIHBP1), were sequenced in 86 patients with type 1 and type 5 HTG and 327 controls. Results In 46 patients (54%) rare DNA sequence variants were identified, comprising variants in LPL (n=19), APOC2 (n=1), APOA5 (n=2), GPIHBP1 (n=3) and LMF1 (n=8). In 22 patients (26%) only common variants in LPL (p.Asp36Asn, p.Asn318Ser and p.Ser474Ter) and APOA5 (p.Ser19Trp) could be identified, whereas no mutations were found in 18 patients (21%). In vitro validation revealed that the mutations in LMF1 were not associated with compromised LPL function. Consistent with this, five of the eight LMF1 variants were also found in controls and therefore cannot account for the observed phenotype. Conclusion The prevalence of mutations in LPL was 34% and mostly restricted to patients with type 1 HTG. Mutations in GPIHBP1 (n=3), APOC2 (n=1) and APOA5 (n=2) were rare but the associated clinical phenotype was severe. Routine sequencing of candidate genes in severe HTG has improved our understanding of the molecular basis of this phenotype associated with acute pancreatitis, and may help to guide future individualized therapeutic strategies.

Objective-GPIHBP1 is an endothelial cell protein that binds lipoprotein lipase (LPL) and chylomicrons. Because GPIHBP1 deficiency causes chylomicronemia in mice, we sought to determine whether some cases of chylomicronemia in humans could be attributable to defective GPIHBP1 proteins. Methods and Results-Patients with severe hypertriglyceridemia (nϭ60, with plasma triglycerides above the 95th percentile for age and gender) were screened for mutations in GPIHBP1. A homozygous GPIHBP1 mutation (c.344AϾC) that changed a highly conserved glutamine at residue 115 to a proline (p.Q115P) was identified in a 33-year-old male with lifelong chylomicronemia. The patient had failure-to-thrive as a child but had no history of pancreatitis. He had no mutations in LPL, APOA5, or APOC2. The Q115P substitution did not affect the ability of GPIHBP1 to reach the cell surface. However, unlike wild-type GPIHBP1, GPIHBP1-Q115P lacked the ability to bind LPL or chylomicrons (d Ͻ 1.006 g/mL lipoproteins from Gpihbp1 Ϫ/Ϫ mice). Mouse GPIHBP1 with the corresponding mutation (Q114P) also could not bind LPL. Conclusions-A homozygous missense mutation in GPIHBP1 (Q115P) was identified in a patient with chylomicronemia.The mutation eliminated the ability of GPIHBP1 to bind LPL and chylomicrons, strongly suggesting that it caused the patient's chylomicronemia. See accompanying article on page 792Mice lacking GPIHBP1 manifest severe chylomicronemia, even on a low-fat chow diet, with plasma triglycerides Ͼ2000 mg/dL. 2 GPIHBP1 is found on the luminal surface of capillaries in heart, skeletal muscle, and adipose tissue, 2 where the lipolytic processing of triglyceride-rich lipoproteins occurs. 3 Transfection of a GPIHBP1 expression vector into CHO cells confers the ability to bind LPL, chylomicrons, as well as apo-AV-phospholipid disks. 2 The ability of GPIHBP1-expressing cells to bind LPL and chylomicrons suggested that GPIHBP1 might function as a platform for lipolysis on endothelial cells. 2 Two structural features of GPIHBP1 are important in the binding of LPL and chylomicrons. The first is an aminoterminal acidic domain, approximately 25 amino acids in length. Mutant GPIHBP1 proteins lacking all or part of the acidic domain are unable to bind LPL and chylomicrons. 4 The second is a Lymphocyte antigen 6 (Ly6) domain. Ly6 motifs, which contain either 8 or 10 cysteines with a characteristic spacing pattern, are found in a number of GPI-anchored proteins, for example CD59 and the urokinase-type plasminogen activator receptor (UPAR). 5 When the Ly6 domain of GPIHBP1 is replaced with the Ly6 domain from CD59, the chimeric protein reaches the cell surface but cannot bind LPL, even though the acidic domain of GPIHBP1 is intact. 4 All mammalian GPIHBP1 proteins share the acidic domain and the Ly6 domain (with 10 conserved cysteines). 6 The highest level of amino acid conservation lies within a portion of the Ly6 domain (residues 101 to 121 in human GPIHBP1, which contains the 6th and 7th cysteines of the Ly6 motif). 6 The finding of chylomicronemia...

Key Points• A novel gain-of-function mutation in factor V leading to increased levels of TFPI and bleeding was identified by whole exome sequencing.• Factor V Amsterdam (F5 C2588G) resembles the mutation (F5 A2350G) leading to East Texas bleeding disorder.We investigated a small Dutch family with a bleeding diathesis, prolonged prothrombin, and activated partial thromboplastin times, in whom no classifying diagnosis was made. The 2 affected relatives had severely decreased in vitro thrombin generation, and levels of tissue factor pathway inhibitor (TFPI) were strongly increased. To identify the genetic cause of the bleeding diathesis, we performed whole exome sequencing analysis of all living relatives. We found a novel gain-of-function mutation in the F5 gene (c.C2588G), which leads to an aberrant splicing of F5 and ultimately to a short factor V protein (missing 623 amino acids from the B domain), which we called factor V Amsterdam. Factor V Amsterdam binds to TFPI, prolonging its half-life and concentration. This is the second report of an association between a shorter form of factor V and increased TFPI levels, resulting in severely reduced thrombin generation and a bleeding tendency. (Blood. 2015; 125(11):1822-1825 IntroductionIn clinical practice, we occasionally encounter patients with a bleeding tendency and prolonged coagulation screening tests, in whom a classifying diagnosis cannot be made. Here, we describe our search toward elucidating the etiology of a bleeding tendency in a mother and her son. Study design Subjects and samplesThe pedigree of the family is depicted in Figure 1A. Detailed clinical features of the family are provided in supplemental Table 1, available on the Blood Web site. We collected blood from the mother (II:2) and son (III:1) with clinical manifestations of bleeding and 4 unaffected relatives. All participants provided written informed consent. Coagulation testing and exome sequencingCoagulation parameters were determined by standard methods. Thrombin generation was performed with calibrated automated thrombography. Genomic DNA was isolated from peripheral blood mononuclear cells. Direct exome sequencing of the TFPI and PROS1 genes was performed in the 2 patients and 1 unaffected relative. Whole exome sequencing data were generated for all relatives, and candidate mutations were confirmed by Sanger sequencing. Details of methods are available in supplemental Materials. Results and discussionThrombin generation was measured in plasma from individuals II:1 (unaffected husband), II:2 (proband), and III:1 (affected son). Thrombin generation initiated with 1 pM tissue factor was severely affected in the proband and son ( Figure 1B); this also occurred at higher concentrations of tissue factor (5 pM, Figure 1C; 20 pM, data not shown). Mixing patient plasma with normal plasma did not normalize thrombin generation, suggesting the presence of a circulating inhibitor (data not shown). Because lupus anticoagulant and deficiencies of antithrombin and protein C had been excluded in both pat...

Protein UFMylation, i.e., post‐translational modification with ubiquitin‐fold modifier 1 (UFM1), is essential for cellular and endoplasmic reticulum homeostasis. Despite its biological importance, we have a poor understanding of how UFM1 is conjugated onto substrates. Here, we use a rebuilding approach to define the minimal requirements of protein UFMylation. We find that the reported cognate E3 ligase UFL1 is inactive on its own and instead requires the adaptor protein UFBP1 to form an active E3 ligase complex. Structure predictions suggest the UFL1/UFBP1 complex to be made up of winged helix (WH) domain repeats. We show that UFL1/UFBP1 utilizes a scaffold‐type E3 ligase mechanism that activates the UFM1‐conjugating E2 enzyme, UFC1, for aminolysis. Further, we characterize a second adaptor protein CDK5RAP3 that binds to and forms an integral part of the ligase complex. Unexpectedly, we find that CDK5RAP3 inhibits UFL1/UFBP1 ligase activity in vitro. Results from reconstituting ribosome UFMylation suggest that CDK5RAP3 functions as a substrate adaptor that directs UFMylation to the ribosomal protein RPL26. In summary, our reconstitution approach reveals the biochemical basis of UFMylation and regulatory principles of this atypical E3 ligase complex.