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

作者：Kranendijk M; Salomons G S; Gibson K M; Van Schaftingen E; Jakobs C^*; Struys E A

来源：Biochimica et Biophysica Acta-Molecular Basis of Disease, 2011, 1812(11): 1380-1384.

DOI：10.1016/j.bbadis.2011.08.006

• D-2-hydroxyglutaric aciduria
• 2-hydroxyglutarate
• Isocitrate dehydrogenase 2
• IDH2 R140Q gain-of-function
• Enzyme assay
• Lymphoblast model

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 o-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/R14Q) 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 o-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/140Q) mutation, underscoring the utility of the lymphoblast model for future biochemical and therapeutic studies.

• 出版日期2011-11