FIG. 2.

Analysis of SOX2 mutations. A, SOX2 gene deletion in patient 1. Metaphase chromosomes were hybridized with BAC clone RP11–43F17 containing SOX2 (red signal; arrowhead) and a chromosome 3 centromeric clone (green signal). The normal chromosome 3 shows both red and green signals. The der(3) shows only a green signal indicating that one copy of SOX2 is deleted. B, Array-CGH profile from patient 1. The chromosome 3 ideogram (left) and enlargement of the deleted region (right) show the ratio of probes (each represented by a single dot) plotted as a function of chromosomal position; loss of copy number of a probe shifts the ratio to the left. C, Electropherogram showing the c.181C>T transition (p.Q61X) in patient 2 (arrow). BfaI digestion of PCR products showing that the heterozygous mutation occurred de novo in patient 2 (filled circle) and is not present in the unaffected parents or in a normal control individual [wild type (WT)]. M, 100-bp DNA ladder. D–F, Functional effects of the p.Q61X mutation. D, The p.Q61X mutation failed to activate the reporter showing similar levels of activation to empty expression vector compared with wild-type SOX2. E, EMSA of wild-type and p.Q61X SOX2 proteins shows that the mutant SOX2 is unable to bind DNA. F, The p.Q61X mutation results in impaired nuclear localization of the mutant protein compared with wild-type SOX2, which stains predominantly within the nucleus. DAPI, 4′,6-diamidino-2-phenylindole; IVT, in vitro translated empty expression vector.