Reasoning
PS3 is met at strong strength. Multiple independent, variant-specific functional studies evaluated IDH1 p.Arg132His with appropriate wild-type or control comparisons and complementary systems. Purified-enzyme and cellular assays showed loss of native isocitrate-dependent activity together with neomorphic NADPH-dependent alpha-ketoglutarate reduction and production of R-2-hydroxyglutarate (PMID:19935646). Independent glioma-cell and xenograft experiments demonstrated altered enzymatic activity, increased D-2-hydroxyglutarate, reduced proliferation, and an in-vivo phenotype (PMID:21446021). Conditional knock-in mouse studies showed approximately tenfold increased serum 2-hydroxyglutarate, progenitor expansion, splenomegaly, anemia, extramedullary hematopoiesis, and epigenetic abnormalities (PMID:22763442). Human erythroleukemia and murine progenitor-cell models reproduced increased 2-hydroxyglutarate, cytokine-independent growth, and impaired differentiation, with rescue when the metabolite pathway was reduced (PMID:23393090). Isogenic human astrocyte experiments additionally showed mutant-specific 2-hydroxyglutarate production and reproducible methylome remodeling (PMID:22343889). These orthogonal biochemical, cellular, and in-vivo results directly support a damaging neomorphic effect of the exact R132H variant; no generic numeric strength threshold was supplied beyond the governing ACMG/AMP definition, so the original PS3 strong category is retained rather than upgraded.
Rule
Well-established in vitro or in vivo functional studies supportive of a damaging effect on the gene or gene product.
Evidence considered
- Dang et al. 2009 (PMID:19935646), exact IDH1 c.395G>A/R132H: purified recombinant enzyme and glioblastoma-cell assays showed impaired native isocitrate activity, novel NADPH-dependent alpha-ketoglutarate reduction, and 2-hydroxyglutarate accumulation; wild-type controls did not show appreciable 2-hydroxyglutarate.
- Bralten et al. 2011 (PMID:21446021), exact IDH1 R132H: stable U87, U138, and HEK cell models plus purified-enzyme assays and intracranial SCID-mouse xenografts showed altered enzymatic activity, increased D-2-hydroxyglutarate, reduced proliferation, and an in-vivo phenotype.
- Sasaki et al. 2012 (PMID:22763442), exact IDH1 R132H conditional knock-in mice: serum 2-hydroxyglutarate was elevated approximately tenfold, with progenitor expansion, splenomegaly, anemia, extramedullary hematopoiesis, and DNA/histone hypermethylation.
- Kraus et al. 2013 (PMID:23393090), exact IDH1 R132H in TF-1 and murine progenitor cells: increased intracellular R-2-hydroxyglutarate, cytokine-independent growth after 12–16 days, and impaired differentiation were observed, with pathway-specific reversibility.
- Turcan et al. 2012 (PMID:22343889), exact IDH1 R132H in isogenic human astrocytes: mutant but not wild-type IDH1 produced 2-hydroxyglutarate and progressively induced the glioma-CIMP-like hypermethylator phenotype.
- The evidence is variant-specific and includes biochemical, cellular, and in-vivo assays with comparator controls; none is a validated clinical diagnostic-laboratory assay.
Still missing
- The available studies are research assays rather than validated, reproducible clinical diagnostic-laboratory assays; this supports PS3 but does not justify an upgrade beyond strong.
Sources