In silico and in vitro mutational analysis to investigate the structure and pathogenicity of POT1 missense variants

We performed an initial assessment of 24 missense POT1variants using variant effect predictors (VEPs) followed by an extensive molecular dynamics (MD) simulations of 14 selected variants: N-terminal: I115T, Y223C, D224N, M251V, H266Q, and S270N, C-terminal: A364E, P371T, T416A, S421L, A532P, L538V, P601L, and Q623H for the unbound state and bound state with telomeric ssDNA (N-terminal) or TPP1–PBD (C-terminal). Wet-lab validations using electrophoretic mobility shift assay (EMSA), telomere dysfunction-induced foci (TIF) formation analysis and coimmunoprecipitation were done to further explore the functional impacts. Initial assessment included VEPs, FoldX, and I-Mutant2.0 followed by MD analysis, which revealed structural instability and reduction in associated binding efficiency in D224N, H266Q, and S270N. EMSA showed remarkable reduction of POT1-telomere oligo binding in D224N, H266Q, and S270N. Through TIF formation analysis, H266Q and P371T exhibited the highest number of co-localized foci compared to other mutant and WT cell lines. Also, POT1–(FLAG)-TPP1 coimmunoprecipitation suggested a reduction in POT1–TPP1 binding in all C-terminal mutants, most notably in P601L and Q623H. Our study demonstrated a comprehensive workflow to characterize missense POT1 variants of uncertain significance and hence proposing this workflow to be considered in the American College of Medical Genetics and Genomics (ACMG) criteria for missense POT1 variant classification.