Reasoning
The exact AKT1 NM_001382430.1:c.49G>A (p.Glu17Lys) variant has been evaluated in multiple orthogonal functional systems with relevant controls. In NIH 3T3 cells, E17K produced approximately fourfold higher kinase activity than wild type, increased downstream phosphorylation, constitutive plasma-membrane localization, and cellular transformation; an in vivo mouse assay induced leukemia in 6/10 animals. Independent biochemical and live-cell studies showed a 7.7-fold increase in PI(3,4,5)P3 affinity, conversion to PI(4,5)P2 binding from an unmeasurable baseline to 130 ± 30 nM, an 18-fold slower dissociation rate, and constitutive plasma-membrane targeting. Patient-derived Proteus-syndrome tissues and mutant cell lines showed increased AKT phosphorylation, and a separate murine transformation model showed increased transformation, AKT signaling, survival, and resistance to imatinib-induced apoptosis, while also documenting that the mutation alone did not transform primary bone-marrow cells. The concordant biochemical, cellular, tissue, and in vivo results directly demonstrate an altered and activating AKT1 product effect for this exact variant, satisfying the generic PS3 definition. The evidence is strong rather than moderate because the variant-specific effect is reproducible across several independent assay types and includes physiologically relevant tissue and in vivo observations; no BS3-supportive result was identified.
Rule
Well-established in vitro or in vivo functional studies supportive of a damaging effect on the gene or gene product.
Evidence considered
- PMID:17611497, Results: exact AKT1 c.49G>A/p.E17K increased phosphorylation and produced approximately fourfold higher kinase activity than wild type in NIH 3T3 cells, constitutive membrane localization, Rat1-cell transformation, and leukemia in 6/10 mice; tumor sequencing included matched normal or white-blood-cell controls.
- PMID:18954143, Results and Table 2: isolated human AKT1 PH-domain assays showed 7.7-fold higher PI(3,4,5)P3 affinity, new PI(4,5)P2 binding at 130 ± 30 nM from an unmeasurable wild-type baseline, an 18-fold slower dissociation rate, and constitutive plasma-membrane localization in live NIH 3T3 cells.
- PMID:20440266, Results: exact E17K increased v-Abl transformation efficiency, AKT phosphorylation and kinase activity, membrane localization, BCL2/BAD survival signaling, and resistance to imatinib-induced apoptosis in murine cell models; the paper also reports that E17K alone did not transform primary bone-marrow cells.
- PMID:21793738, Results: exact AKT1 c.49G>A/p.Glu17Lys was detected across affected Proteus-syndrome tissues and mutant cell lines; mutant cells showed increased AKT phosphorylation at Ser473 and Thr308, with affected-tissue cell lines showing increased Ser473 phosphorylation compared with controls.
Sources