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

We performed an initial assessment of 24 missense <i>POT1</i>variants 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 <i>POT1</i> 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 <i>POT1</i> variant classification.