(A) Pedigree of brothers affected by KS carrying a novel SEMA3E mutation. Circle denotes female; square denotes male; black square denotes affected male; arrows indicate the probands. (B) Sequence chromatograms of nucleotides 1849–1863 of the SEMA3E coding sequence in 2 brothers carrying a nucleotide substitution in exon 16 (left side, forward strand; right side, reverse strand; the positions of the C>T and corresponding G>A change are indicated with asterisks). (C) Diagram of the SEMA3E functional domains: SEMA, PSI (plexin/semaphorin/integrin), Ig (Ig-like, C2-type), and basic domains; their position and the position of the mutated amino acid residue within the protein sequence are indicated. (D) Alignment of partial protein sequences of vertebrate SEMA3E orthologs shows that the R619 residue is evolutionarily conserved in mammals (red), but not in chick or zebrafish (green). (E) Genomic evolutionary rate profiling of sequence constraint for the SEMA3E mutation in the 2 KS brothers using GERP++ analysis provided an RS score of 5.54, which is close to the maximum score of 6.18 for complete conservation across all mammalian species. (F) Computational models of a dimer of the Ig domain of WT and mutant SEMA3E. The model is based on the Robetta algorithm for comparative protein structure prediction. The highest-scoring dimer model is shown. The R619 residue and the R619C substitution are indicated by white arrowheads. (G) Schematic drawing illustrating the protein interaction between SEMA3E and PLXND1, including its structural domains — the SEMA and PSI domains, similar to those of SEMA3E — as well as the IPT (Ig-like, plexin, transcription factor) domains and the serine/threonine protein kinase catalytic domains 1 and 2 (SP1 and SP2).