Figure 2.

Analysis of CDKN1B gene structure, protein domains and conservation, and effects of variants on subcellular localization. (A) The CDKN1B gene (12p13.1) includes 3 exons, 2 of which (exons 1 and 2) are translated into a 198 amino acid protein. Exon 1 includes the 5′ regulatory region that contains a glucocorticoid response element. In addition to multiple phosphorylation sites scattered throughout the protein (yellow rectangles) the following features have been identified in CDKN1B (22.1 kDa): a CDK inhibition domain (residues 25-93) (51), a nuclear export signal (NES, 32-45) (52), a scatter domain (118-158) (44), and a nuclear localization signal (NLS,152-168) (53). The position of the variants found in our patients has been annotated in the figure. (B) Both the CDKN1B frameshift variant p.Q107Rfs*12 and the wild-type sequence were detected in cDNA from variant carriers (proband and three family members); a wild-type chromatogram is presented for comparison. Using qPCR, CDKN1B was expressed at similar levels in variant carriers and in 1 family member not carrying the variant. (C) The human CDKN1B protein was annealed with orthologs from multiple species using the UniProt BLAST function. The 3 missense variants identified in our patients affect moderately (I119) or highly conserved residues (E126 and D136) localized within the scatter domain. (D) Subcellular localization of CDKN1B variants overexpressed in AtT20 cells. An anti-HA antibody was used to detect the HA-tagged CDKN1B proteins and nuclei were counterstained with DAPI. Images were obtained using the Texas Red (560/40 nm) and DAPI (360/40 nm) filters. Magnification for all images: 60×.