A lymphoblast model for IDH2 gain-of-function activity in d-2-hydroxyglutaric aciduria type II: Novel avenues for biochemical and therapeutic studies

• Published in: Biochimica et biophysica acta 2011-11, Vol.1812 (11), p.1380-1384
• Main Authors: Kranendijk, M., Salomons, G.S., Gibson, K.M., Van Schaftingen, E., Jakobs, C., Struys, E.A.
• Format: Article
• Language: English
• Subjects: 2-hydroxyglutarate; Brain Diseases, Metabolic, Inborn - enzymology; Brain Diseases, Metabolic, Inborn - genetics; Brain Diseases, Metabolic, Inborn - therapy; Case-Control Studies; Cells, Cultured; Chromatography, Liquid; D-2-hydroxyglutaric aciduria; Enzyme assay; Enzyme Inhibitors - pharmacology; Glutarates - metabolism; Humans; IDH2 R140Q gain-of-function; Isocitrate Dehydrogenase - genetics; Isocitrate Dehydrogenase - metabolism; Isocitrate dehydrogenase 2; Ketoglutaric Acids - metabolism; Lymphoblast model; Lymphocytes - drug effects; Lymphocytes - enzymology; Lymphocytes - pathology; Mutation - genetics; Sensitivity and Specificity; Tandem Mass Spectrometry
• ISSN: 0925-4439; 0006-3002; 1879-260X
• DOI: 10.1016/j.bbadis.2011.08.006

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- 2H 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 (14.4 nmol h −1 mg protein −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. ► IDH2 R140Q gain-of-function enzyme assay developed in lymphoblasts with UPLC-MS/MS. ► 8 times increased IDH2 R140Q reaction rate is found in D-2-HGA type II vs. controls. ► Intracellular D-2-HG levels are increased 140 times in D-2-HGA type II lymphoblasts. ► IDH2 R140Q reaction rate was inhibited with oxaloacetate reducing D-2-HG production. ► Lymphoblast model creates novel avenues for biochemical and therapeutic studies.