Figure 2

Segregation, mutational analysis, and functional consequences of a novel SMS variant. A. Pedigree of the family of the propositi. Affected males are shown by black squares. B. Sanger sequencing chromatograms showing the segregation of the SMS mutation NM_004595.4:c.443A > G from the carrier mother to the affected boys. The unaffected father did not have this mutation. C. Conservation of the p.Gln148 (p.Q148) residue across species. D. Drawing of the human SMS protein crystal complexed with spermidine and 5-methylthioadenosine. The mutated amino acid (Gln148) is highlighted in yellow [Mac PyMOL [23]]. E-J. Immunofluorescent detection of SMS protein subcellular distribution in unaffected (E, F), Patient II-1 (G, H) and Patient II-3 (I, J) skin fibroblasts. SMS protein is shown in red and the nucleus is shown in blue. K. Immunoblot of skin fibroblast lysates showing reduced SMS protein levels in the patients (II-1, II-3) compared to an unaffected control (cnt). Tubulin is shown as a loading control. L. Graph showing steady state SMS protein levels in the patient and control fibroblasts relative to ß-tubulin levels. The data are based on 3 independent experiments for each cell line. M. Graph quantifying immunoblot detected steady state SMS protein levels in the cytoplasm and nuclei of patient and control fibroblasts. The cytoplasmic expression was normalized to β-tubulin expression and the nuclear expression to p84 expression. The data are based on 2 independent experiments for each cell line. N. SMS enzyme activity (spermidine d8 peak per hour) in lymphoblasts of unaffected individuals (Cnt), a cohort of 4 individuals with SRS (SRS) and patient II-1, * p < 0.05.