The NM_000546.6: c.490A>G variant in TP53 is a missense variant predicted to cause substitution of lysine by glutamic acid at amino acid 164 (p.Lys164Glu). This variant has been reported in 2 unrelated families meeting Classic/Revised Chompret criteria. Based on this evidence this variant scores 2 total points meeting the TP53 VCEP phenotype scoring criteria of 2-3.5 points. (PS4_Moderate; PMIDs: 33138793, 38050059). The variant has been reported to segregate with LFS-associated cancers in 3-4 meioses in 1 family (PP1; PMID: 38050059). At least one individual with this variant was found to have a variant allele fraction of 5-35%, which is a significant predictor of variant pathogenicity (PP4, PMID: 34906512, Internal lab contributors). This variant has an allele frequency of 6.195e-7 (1/1614174 alleles) across gnomAD v4.1.0 which is lower than the Clingen TP53 VCEP threshold (<0.00003) for PM2_Supporting and has no more than one allele per non-bottleneck subpopulation (PM2_Supporting). In vitro assays performed in yeast and/or human cell lines showed non-functional transactivation and loss of growth suppression activity indicating that this variant impacts protein function (PS3; PMIDs: 12826609, 30224644, 29979965). Computational predictor scores (BayesDel = 0.5572; Align GVGD = Class C55) are above recommended thresholds (BayesDel > 0.16 and an Align GVGD Class of > 15), evidence that correlates with impact to TP53 via protein change (PP3). This variant has 9 somatic occurrences for the same amino acid change in cancerhotspots.org (v2) sufficient to be defined as a mutational hotspot by the Clingen TP53 VCEP (2-9 somatic occurrences, PMID: 30311369) (PM1_Supporting). In summary, this variant meets the criteria to be classified as Pathogenic for Li Fraumeni syndrome based on the ACMG/AMP criteria applied, as specified by the ClinGen TP53 VCEP: PS4_Moderate, PP1, PM2_Supporting, PS3, PP3, PP4, PM1_Supporting. (Bayesian Points: 11; VCEP specifications version 2.3)