NM_001122740.1:c.1613A>G (p.Asp538Gly) in ESR1 is a missense variant in exon 9 encoding a substitution in helix 12 of the ligand-binding domain. This variant is absent from gnomAD v2.1, v4.1, and gnomAD-Canada population databases, satisfying PM2 (moderate).¹ The variant is located at a statistically significant mutational hotspot within helix 12 of the ESR1 ligand-binding domain, a critical functional domain, satisfying PM1 (moderate).² Multiple independent functional studies demonstrate that p.Asp538Gly confers constitutive, ligand-independent transcriptional activity, promotes coactivator recruitment in the absence of estrogen, and confers resistance to antiestrogen therapies. Evidence includes luciferase reporter assays, co-immunoprecipitation, X-ray crystallography, HDX-MS, and cell proliferation assays across multiple laboratories, satisfying PS3 (strong).³ The REVEL meta-predictor score of 0.914 supports a deleterious effect, satisfying PP3 (supporting).⁴ Caveat: all functional evidence derives from somatic cancer studies of acquired endocrine resistance; no germline-specific functional or clinical evidence is available. The variant has not been reported as a germline pathogenic variant. Classification should be interpreted with the understanding that this variant's clinical significance in a germline context is uncertain. PVS1 is not applicable as this is a missense variant. PS1, PS2, PS4, PS5, PM5, PM6, PP1, PP4, PP5, BA1, BS1, BS2, BS3, BS4, BP1, BP2, BP4, BP5, BP6, and BP7 are not met or not applicable.