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

Kranendijk, M;Salomons, G S;Gibson, K M;Van Schaftingen, Emile;Struys, E A;et.al.
(2011) Biochimica et Biophysica Acta : international journal of biochemistry and biophysics — Vol. 1812, n° 11, p. 1380-1384 (2011)

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Authors
  • Kranendijk, M
    Author
  • Salomons, G S
    Author
  • Gibson, K M
    Author
  • Van Schaftingen, EmileUCLouvain
    Author
  • Struys, E A
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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.
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Kranendijk, M., Salomons, G. S., Gibson, K. M., Van Schaftingen, E., Jakobs, C., & Struys, E. A. (2011). A lymphoblast model for IDH2 gain-of-function activity in d-2-hydroxyglutaric aciduria type II: novel avenues for biochemical and therapeutic studies. Biochimica et Biophysica Acta : international journal of biochemistry and biophysics, 1812(11), 1380-1384. https://doi.org/10.1016/j.bbadis.2011.08.006 (Original work published 2011)