Fig. 2: Missense variants.

A Schematic depiction of the PNKP protein with FHA in blue, Phosphatase and kinase domain in green and linker region in gray (based on Uniprot Q96T60 and [20]). Disease-associated missense variants are displayed toward the top, red shapes show (likely) pathogenic variants, yellow shapes show variants of unknown significance, and gray shapes show (likely) benign variants. The variants identified in the four individuals reported here are depicted as triangles (black labels), variants from the literature as dots (gray labels). The length of the segments corresponds to each variant’s CADD score. Gray dots downwards show homozygous missense variants from gnomAD, the dot size represents the logarithm of the allele count. AA positions 20, 441, and 462 are labeled for orientation. In the panel below, a generalized additive model shows the CADD values for all possible missense variants (red horizontal line = recommended cut-off [20]). Additional boxes show the affected regions of the in-frame deletions resulting from the synonymous variant c.498G>A in orange or the splicing variant c.1029+2T>C in pink. B, C Two representative views of the FHA domain crystal structure (based on 2BRF). AAs of missense variants are depicted in gray for benign variants (Pro20), in yellow for VUS (Gln50, Val51, Ala55, Gln66, and Leu104), and in red for pathogenic variants (Pro101). All missense variants classified as unknown significance or (likely) pathogenic affect conserved residues in beta sheets. D The substitution Pro101Leu alters the local interatomic interaction of the protein, generating important steric clashes at the same time it decreases molecule stability. The most right panel represents the ΔVibrational Entropy Energy derived from the Pro101Leu variant, showing a local rigidification of the protein. E The Leu104Pro variant generates the opposite effect observed for Pro101Leu as it introduces a proline in the core of the FHA domain, destabilizing the protein domain. The most right panel shows the ΔVibrational Entropy Energy with a local gain in flexibility.