SOX2 haploinsufficiency is associated with slow progressing hypothalamo-pituitary tumours

Abstract

SOX2 is an early developmental transcription factor and marker of stem cells that has recently been implicated in the development of the pituitary gland. Heterozygous SOX2 mutations have been described in patients with hypopituitarism and severe ocular abnormalities. In the majority of published cases, the pituitary gland is either small or normal in size. Here, we report two unrelated patients with SOX2 haploinsufficiency (a heterozygous gene deletion and a novel c.143TC>AA/p.F48X mutation) who developed nonprogressive pituitary tumors of early onset, suggesting a congenital etiology. The truncating mutation resulted in significant loss of function and impaired nuclear localization of the mutant protein, in addition to a failure to repress β-catenin transcriptional activity in vitro. This is the first indication that SOX2 haploinsufficiency is implicated in the generation of pituitary tumors with distinct clinical characteristics, possibly mediated via its effects on the Wnt signaling pathway.

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).