Figure 1. Identiﬁcation and characterization of novel SOX2 heterozygous mutations in association with slow-growing sellar tumors. (A and B) Midline sagittal (left) and coronal (right) MRI scans of patient 1 at 18 years (A) and 28 years of age (B) revealing a large mass in the location of the pituitary gland (arrows). (C) Array-CGH proﬁle from patient 1 showing the ratio of probes, each represented by a single dot plotted as a function of chromosome position; loss of copy number of a probe shifts the ratio to the left showing deletion of SOX2. (D and E) Midline sagittal (left) and coronal (right) MRI scans of patient 2 at 17 months (D) and at 32 months of age (E) showing a large mass in the location of the pituitary gland (arrows). (F) Sequence electropherogram showing a two-base substitution c.143_144TT>AA (p.F48X) in patient 2. Nucleotide numbering reﬂects cDNA with +1 corresponding to the A of the ATG translation initiation codon in the reference sequence. (G) Overexpression of FLAG-tagged SOX2 p.F48X in HEK293T cells results in impaired localization of the protein whereas FLAG-tagged wild type SOX2 localizes in the nucleus. Nuclei are counterstained with DAPI. (H) Luciferase assay using the Hesx1 minimal promoter demonstrates impaired activation of the promoter through expression of SOX2 p.F48X (t-test, P = 0.0006). (I) Luciferase assay using the TOPFLASH reporter reveals that expression of the SOX2 p.F48X mutant protein does not result in repression of Wnt signaling, activated by expression of a stable mutant form of β-catenin (p.S33Y) (t-test, P = 0.0002), whereas expression of wild-type SOX2 represses this activation (t-test, P < 0.0001). (J) Western blot analysis showing similar expression levels of both wild-type (WT) and p.F48X proteins. Although the intensity of the signal obtained with the α-FLAG antibody is not comparable, this is due to unequal loading of protein extracts in the gel, as shown by the loading control (α-β-Actin). 1378 HUMAN MUTATION, Vol. 32, No. 12, 1376–1380, 2011