title: A lymphoblast model for IDH2 gain-of-function activity in d-2-hydroxyglutaric aciduria type II: novel avenues for biochemical and therapeutic studies.
abstract: The recent discovery of heterozygous isocitrate dehydrogenase 2 (IDH2) mutations of residue Arg(140) to Gln(140) or Gly(140) (IDH2(wt/R140Q), IDH2(wt/R140G)) in d-2-hydroxyglutaric aciduria (D-2-HGA) has defined the primary genetic lesion in 50% of D-2-HGA patients, denoted type II. Overexpression studies with IDH1(R132H) and IDH2(R172K) mutations demonstrated that the enzymes acquired a new function, converting 2-ketoglutarate (2-KG) to d-2-hydroxyglutarate (D-2-HG), in lieu of the normal IDH reaction which reversibly converts isocitrate to 2-KG. To confirm the IDH2(wt/R140Q) gain-of-function in D-2-HGA type II, and to evaluate potential therapeutic strategies, we developed a specific and sensitive IDH2(wt/R140Q) enzyme assay in lymphoblasts. This assay determines gain-of-function activity which converts 2-KG to D-2-HG in homogenates of D-2-HGA type II lymphoblasts, and uses stable-isotope-labeled 2-keto[3,3,4,4-(2)H(4)]glutarate. The specificity and sensitivity of the assay are enhanced with chiral separation and detection of stable-isotope-labeled D-2-HG by ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Eleven potential inhibitors of IDH2(wt/R140Q) enzyme activity were evaluated with this procedure. The mean reaction rate in D-2-HGA type II lymphoblasts was 8-fold higher than that of controls and D-2-HGA type I cells (14.4nmolh(-1)mgprotein(-1) vs. 1.9), with a corresponding 140-fold increase in intracellular D-2-HG level. Optimal inhibition of IDH2(wt/R140Q) activity was obtained with oxaloacetate, which competitively inhibited IDH2(wt/R140Q) activity. Lymphoblast IDH2(wt/R140Q) showed long-term cell culture stability without loss of the heterozygous IDH2(wt/R140Q) mutation, underscoring the utility of the lymphoblast model for future biochemical and therapeutic studies.
authors: Department of Clinical Chemistry, VU University Medical Center, Amsterdam, The Netherlands.
authors: M
authors: M
authors: Kranendijk
authors: G S
authors: GS
authors: Salomons
authors: K M
authors: KM
authors: Gibson
authors: E
authors: E
authors: Van Schaftingen
authors: C
authors: C
authors: Jakobs
authors: E A
authors: EA
authors: Struys
mesh_terms: D020739:Brain Diseases, Metabolic, Inborn / Q000201:enzymology* / Q000235:genetics / Q000628:therapy; D016022:Case-Control Studies; D002478:Cells, Cultured; D002853:Chromatography, Liquid; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005977:Glutarates / Q000378:metabolism; D006801:Humans; D007521:Isocitrate Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D007656:Ketoglutaric Acids / Q000378:metabolism; D008214:Lymphocytes / Q000187:drug effects / Q000201:enzymology* / Q000473:pathology; D009154:Mutation / Q000235:genetics; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry
chemical_list: D004791:Enzyme Inhibitors; D005977:Glutarates; D007656:Ketoglutaric Acids; C019417:alpha-hydroxyglutarate; C538784:IDH2 protein, human; D007521:Isocitrate Dehydrogenase