Mutations within Sox2/SOX2 are associated with abnormalities in the hypothalamo-pituitary-gonadal axis in mice and humans

Abstract

The transcription factor SOX2 is expressed most notably in the developing CNS and placodes, where it plays critical roles in embryogenesis. Heterozygous de novo mutations in SOX2 have previously been associated with bilateral anophthalmia/microphthalmia, developmental delay, short stature, and male genital tract abnormalities. Here we investigated the role of Sox2 in murine pituitary development. Mice heterozygous for a targeted disruption of Sox2 did not manifest eye defects, but showed abnormal anterior pituitary development with reduced levels of growth hormone, luteinizing hormone, and thyroid-stimulating hormone. Consequently, we identified 8 individuals (from a cohort of 235 patients) with heterozygous sequence variations in SOX2. Six of these were de novo mutations, predicted to result in truncated protein products, that exhibited partial or complete loss of function (DNA binding, nuclear translocation, or transactivation). Clinical evaluation revealed that, in addition to bilateral eye defects, SOX2 mutations were associated with anterior pituitary hypoplasia and hypogonadotropic hypogonadism, variable defects affecting the corpus callosum and mesial temporal structures, hypothalamic hamartoma, sensorineural hearing loss, and esophageal atresia. Our data show that SOX2 is necessary for the normal development and function of the hypothalamo-pituitary and reproductive axes in both humans and mice.

Figure 1. Pituitary hormone levels and immunohistochemistry in Sox2 heterozygous mice.

(A) GH and LH RIAs on pituitary protein extracts from 6 wild-type (XY^+/+) and 5 Sox2 heterozygous (XY^+/–) 2-month-old littermates (GH, P = 0.004; LH, P = 0.002) and 10 wild-type and 9 Sox2 heterozygous 18.5 dpc littermates (P = 0.002). Both GH and LH were affected; GH deficiency appeared before birth. (B–E) Immunohistochemistry for GH (B and D) and LH (C and E) on pituitary sections from 3-month-old (B and C) and 18.5 dpc (D and E) wild-type and Sox2 mutant littermates. Ant, anterior lobe; Int, intermediate lobe; Post, posterior lobe. Note the presence of extra clefts in the adult sections (arrows in B and C). Staining was clearly reduced in heterozygotes at 18.5 dpc (D and E). Scale bar: 0.3 mm (B and C); 0.05 mm (D and E).

Figure 2. Sox2 expression and abnormal morphogenesis of the pituitary in Sox2 heterozygotes.

(A) Sagittal section of an 11.5-dpc wild-type embryo hybridized to Sox2, showing expression in both the CNS and Rathke’s pouch. (B and C) Sagittal sections of 12.5 dpc wild-type (B) and Sox2 heterozygous (C) embryos, showing bifurcation of the pouch in the mutant embryo. (D and E) Pituitary transverse sections of 5-week-old wild-type (D) and Sox2 heterozygous mice (E). Note the presence of an extra cleft in the Sox2 heterozygous pituitary (arrow). HYP, presumptive hypothalamus; RP, Rathke’s pouch. Scale bars: 0.1 mm.

Figure 3. Testes morphology of Sox2^+/– males.

(A) Testis of an 11-month-old wild-type mouse. (B) Histological analysis of the testis shown in A. All the different stages of germ cells are present within the seminiferous tubules. (C) Testis of an 11-month-old Sox2^+/– mouse showing reduced size and white opaque patches, indicative of sperm blockage. This was confirmed by histological analysis (D), where aberrant tubules were seen that contained very large numbers of mature sperm only, instead of a range of spermatogenic stages (arrow). The somatic cells also looked abnormal in these tubules. Scale bars: 1,260 μm (A and C); 55 μm (B); 42 μm (D).

Figure 4. Mutation analysis of SOX2.

(A) Schematic diagram of the single exon of SOX2 showing the position of the HMG domain and transactivation domain. The position of sequence changes identified in the cohort reported here are shown with their relative positions within the gene; frameshift and nonsense mutations are shown in black, nonsynonymous changes in blue, and synonymous variants and a single nucleotide polymorphism in the 3′ untranslated region in gray. (B) Electropherograms of the frameshift, nonsense, and nonsynonymous coding variants with the position of each mutation marked by an arrow, demonstrating that each mutation is heterozygous. Also shown is a sequence trace from a cloned PCR product from patient 2 with the c.70del20 mutation showing the extent of the deletion by alignment with wild-type SOX2 sequence. Note patient 7 with the G130A variant had an additional heterozygous synonymous sequence change 2 bp upstream (c.387C→G). “N” denotes heterozygous peaks in the electropherogram. (C) Alignment of predicted SOX2 mutant proteins with part of the wild-type SOX2 protein sequence showing the extent of the truncations. The mutations c.60insG and c.70del20 were predicted to produce proteins that completely remove the HMG box (underlined). Amino acids shown in blue result from frameshifts and do not align to the wild-type SOX2 sequence. Asterisk indicates the position of L97.

Figure 5. Functional analysis of SOX2 mutants.

(A) Loss of the HMG box results in impaired nuclear localization. Cells were transfected with 50 ng plasmid construct containing wild-type or mutant SOX2 fused to an N-terminal FLAG epitope, fixed, and stained with anti-FLAG antibody; nuclei were counterstained with DAPI. Wild-type SOX2 showed predominantly nuclear staining. Mutant proteins lacking the HMG box (c.60insG and c.70del20) showed a majority of staining in the cytoplasm and impaired nuclear localization. Mutant proteins retaining the HMG box showed staining similar to wild-type. (B) SOX2 mutants show variable DNA binding. Identical amounts of in vitro translated wild-type (lanes 3 and 10) or individual variant SOX2 proteins (lanes 4–8 and 11–13) were incubated with a radiolabeled DNA probe. Specific binding of SOX2 to the probe was demonstrated by duplicate experiments using in vitro translated empty vector (lane 2) or 100 times excess cold probe (lane 9). (C) SOX2 mutants show impaired transcriptional activation. Expression constructs containing wild-type or variant SOX2 (10–50 ng) were cotransfected with 20 ng luciferase reporter construct containing part of the proximal promoter of HESX1. Wild-type SOX2 led to dose-dependent activation of the reporter (up to 20-fold activation), whereas mutant truncated SOX2 proteins showed impaired activation correlating with the extent of the truncation (dashed arrow). c.60insG, c.70del20, and L97P showed similar levels of activation to empty expression vector. In all experiments described here, c.479delA showed identical results to Y160X (not shown). Results are mean ± SD of 3 independent experiments performed in triplicate.

Figure 6. MRI scans on patients with SOX2 mutations.

(A and B) Sagittal and coronal sections from patient 1 showing APH with relatively normal posterior pituitary (pp) and infundibulum (i), a hypothalamic hamartoma (h) and a misshapen abnormal hippocampus (hi). (C and D) Sagittal sections from patient 3 showing APH and a hypothalamic hamartoma. (E) Coronal section in this patient revealed complete absence of the left optic nerve. r.on, right optic nerve. (F) Sagittal section in patient 4 showing hypoplasia of the anterior pituitary (ap) and the splenium of the corpus callosum (cc). The posterior pituitary and infundibulum were normal.