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

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.

SOX2 is a member of the sex-determining region of the Y chromosome–related (SRY-related) high-mobility group (HMG) box (SOX) family of transcription factors, encoded by 20 genes in humans and mice, each of which carries a 79–amino acid HMG box DNA-binding domain similar to that of SRY as well as domains implicated in transcriptional regulation (1, 2). Based on HMG box homology, they are grouped into different subfamilies. SOX1, SOX2, and SOX3 belong to the B1 subfamily and are expressed in various phases of embryonic development and cell differentiation, where they play critical roles in embryogenesis (3, 4). All 3 mark neuroepithelial progenitors and stem cells from the earliest stages of development, and there is a strong, but not absolute, tendency for them to be downregulated as cells differentiate.

In the mouse, Sox2 RNA is first detected in cells at the morula stage (2.5 dpc) and then in the inner cell mass of the blastocyst (3.5 dpc). After implantation the gene is expressed throughout the epiblast and extraembryonic ectoderm, the multipotent precursors of all embryonic and trophoblastic cell types, and targeted mutagenesis has shown that Sox2 plays a crucial role in both these regions (5). After gastrulation Sox2 expression within the embryo becomes largely restricted to the presumptive neuroectoderm, and by 9.5 dpc it is expressed throughout the brain, neural tube, sensory placodes, branchial arches, and gut endoderm (6). Later, Sox2 is expressed in neural stem cells residing in the ventricular zone of the developing neural tube and in their immediate progeny throughout embryogenesis and into adulthood, where it continues to mark stem cells and neural progenitors in the subventricular layer of the lateral ventricles and the subgranular layer of the hippocampus/dentate gyrus. It is also maintained in some differentiated cell types within the CNS.

Sox2^βgeo heterozygous mice appear relatively normal but show a reduction in size and male fertility, the extent of which is correlated with genetic background (5). Sox2^–/– embryos are unaffected at blastocyst stages, but fail to survive shortly after implantation (5). By introducing a regulatory mutation in Sox2 (Sox2^ΔENH) in addition to the Sox2^βgeo null allele, Ferri et al. accessed some later functions of SOX2 in the CNS (7). Compound heterozygosity for both alleles reduced levels of Sox2 expression to 25–30% of wild-type levels within specific regions of the CNS. This resulted in a reduction both in the number of pups being born and in their postnatal survival, with surviving mice showing severe abnormalities including growth retardation (although this was usually compensated by 6 weeks of age), dystonic reactions, epileptic spikes in the cortex and hippocampus, and circling behavior. These data support a role for SOX2 in neural progenitors, as has been suggested from studies in other model systems (3), as well as highlight its importance for the maintenance of specific neuronal populations.

Like its murine counterpart, the human SOX2 gene is a single-exon gene encoding a 317–amino acid protein containing an N-terminal domain of unknown function, a DNA-binding HMG domain, and a C-terminal transcriptional activation domain. Heterozygous mutations within SOX2 in humans have to date been associated with bilateral anophthalmia and severe microphthalmia (8–12). Additional phenotypic abnormalities include developmental delay, learning difficulties, esophageal atresia, sensorineural hearing loss, and genital abnormalities. To date, 12 heterozygous mutations have been identified in 14 patients, all characterized by bilateral eye disease. All these mutations — 5 nonsense, 4 frameshift, 1 deletion, and 2 missense — occurred de novo. It has been speculated that the mutations are associated with loss of function, but detailed functional studies have not yet been performed.

Given the reduced size and impaired male fertility in Sox2^βgeo heterozygotes, we investigated the hypothalamo-pituitary axis in these animals. We found that they manifested a variable hypopituitary phenotype affecting the size and shape of the pituitary gland and causing a generalized reduction in hormone content, including a significant lowering of growth hormone (GH), luteinizing hormone (LH), ACTH, and thyroid-stimulating hormone (TSH), which would explain many aspects of their phenotype. We also report the phenotypes associated with 3 recently identified heterozygous frameshift mutations and 1 nonsense mutation that we believe to be novel, in addition to 1 previously described heterozygous frameshift mutation and 1 nonsense mutation within SOX2 in 6 patients with anophthalmia/microphthalmia. Surprisingly, all of the affected patients manifested hypogonadotropic hypogonadism (HH), which resulted in the absence of puberty in all patients and genital abnormalities in males. We show that the 4 SOX2 mutations we believe to be novel as well as the 3 previously described mutations (8, 9, 11) led to varying degrees of loss of transcriptional activity, revealing that haploinsufficiency for SOX2 is likely to be the cause of the human phenotypes. There were differences in the range of other tissues affected in mice and humans; however, a common feature was the prevalence of pituitary endocrine deficits, implying a critical role for SOX2 in the development and function of the hypothalamo-pituitary axis in both species.

Sox2 is required for normal murine pituitary development

In mice, homozygous deletion of Sox2 results in peri-implantation lethality, preventing studies of its later roles, while Sox2 heterozygous animals appear to be mostly unaffected. Some of the heterozygotes, however, are smaller than their wild-type littermates, and fertility problems are common in males (5). Moreover, since the related Soxb1 gene, Sox3, has recently been shown to be necessary during development of the hypothalamic-pituitary axis (13), we explored whether there was a similar involvement for Sox2.

There was a moderate reduction of pituitary LH and GH content in 2-month-old Sox2 heterozygotes compared with wild-type littermates, which was statistically significant in males but not in females (Figure 1A). Clearly, the LH deficit contributed to the fertility problems observed in males (5), while the reduction in GH was consistent with the moderate and variable reduction in size observed in the mutants. We considered the possibility that feeding difficulties could also be responsible, since esophageal atresia has been observed in some SOX2 human patients (patient 1; see Methods) (12), and Sox2 is expressed in the embryonic gut endoderm (5, 12). However, comparison of the esophagus at 14.5 dpc in heterozygous mutants (n = 5) and wild-type littermates showed no abnormalities.

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).

Deficiencies in GH and LH may be of either hypothalamic or pituitary etiology. The regulation of pituitary hormone secretion by hypothalamic tropic factors does not become prominent until after birth, so we also examined embryonic pituitaries at 18.5 dpc (Figure 1A) and found a significant reduction in GH content in both male and female Sox2 heterozygous embryos (LH contents at this stage were below levels detectable by RIA). We also studied pituitary somatotropes (GH) and gonadotropes (LH) by immunohistochemistry at 3 months of age (Figure 1, B and C) and at 18.5 dpc (Figure 1, D and E). In the adult, the somatotrope and gonadotrope content did not appear strikingly reduced in the mutant sections (Figure 1, B and C). However, we noted the presence of some morphological defects: extra clefts were sometimes observed in the mutants (arrows in Figure 1, B and C). Moreover, the region between the 2 clefts that histologically resembled the intermediate lobe was aberrantly positive for both somatotropes and gonadotropes. At 18.5 dpc, not only were morphological defects observed that were similar to those seen in the adults, immunohistochemistry suggested that somatotropes and gonadotropes were reduced in number in the mutants (Figure 1, D and E). We therefore counted the number of somatotropes and found that they were significantly reduced in Sox2 heterozygous mutants (wild-type, 2,969.5 ± 456.4, n = 4; Sox2 heterozygotes, 1,230 ± 248.7, n = 4; P = 0.0155). Although the number of gonadotropes was also reduced (wild-type, 559.5 ± 54.2, n = 4; Sox2 heterozygotes, 378.5 ± 54.2, n = 4; P = 0.0534), this difference did not reach statistical significance. The anterior lobe itself was reduced in size in some mutants (Figure 1E), and the reduction in the number of endocrine cells may therefore reflect a general reduction in gland size. We then compared ratios between the number of somatotropes and the surface area of the anterior lobe. Using this approach, we found no significant difference between wild-type and Sox2 heterozygous pituitaries (wild-type, 0.0106 ± 0.0017, n = 4; Sox2 heterozygotes, 0.0064 ± 0.0004, n = 4; P = 0.525). These data strongly suggest that the reduction in somatotropes (and gonadotropes) is not cell type specific, but rather reflects a general reduction in gland size, and this alone could explain the GH deficit at 18.5 dpc. Consistent with a pan-pituitary defect, other hormones (e.g., TSH and prolactin [PRL]) were also found at reduced levels in some adult pituitaries (data not shown).

It should also be noted that about one-third of heterozygous mutant animals are lost between birth and weaning (A. Avilion and R. Lovell-Badge, unpublished observations), and therefore the Sox2 heterozygous adult population reflects surviving, mildly affected mutants. In agreement with this, the number of endocrine cells did not seem to be affected in the adults (Figure 1, B and C). Moreover, no significant difference in ACTH content was observed between wild-type and Sox2 heterozygous adult pituitaries, whereas a significant reduction in ACTH was observed in Sox2 heterozygotes at P7 (wild-type, 33.6 ± 2.4, n = 8; Sox2 heterozygotes, 22.5 ± 3.1, n = 6; P = 0.01), suggesting a loss of ACTH-deficient Sox2 heterozygous pups. However, the GH deficit was comparable between 18.5-dpc and adult mice (Figure 1A) and was also observed at P7 (wild-type, 1.2 ± 0.1; Sox2 heterozygotes, 0.72 ± 0.1; P = 0.004). These data suggest that in the adult, if the gland is itself less affected, then defects in the hypothalamus, where Sox2 expression is maintained (our unpublished observations), may partially explain the occurrence of comparable deficits.

These results suggested that Sox2 heterozygous deletion affects the development of the pituitary gland and possibly hypothalamic secretion. Hence we examined Sox2 expression during early development of the hypothalamo-pituitary axis (Figure 2). At 11.5 dpc, Sox2 was expressed both in the presumptive hypothalamus and in the primordium of the anterior pituitary, Rathke’s pouch (Figure 2A). Later on, as cell differentiation occurs, Sox2 expression is mostly confined to the proliferative zone, around the Rathke’s pouch lumen (14), and in the adult gland, expression is maintained in some non-endocrine cell types (T. Fauquier and K. Rizzoti, unpublished observations). We then examined Rathke’s pouch development in Sox2 heterozygous embryos at 12.5 dpc (Figure 2, B and C). In one-third of the embryos (n = 9) we observed the presence of a bifurcated Rathke’s pouch (Figure 2C), which may explain the extra cleft that we observed in some adult mutants (arrow, Figure 2E).

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.

Sox2 ^{β geo} heterozygous males show impaired fertility

On an inbred 129 background, fertility in Sox2 heterozygous males was clearly impaired. Fertile outbred males were therefore selected for and used to maintain the colony, resulting in a decreased penetrance of the infertility phenotype. However morphological analysis on outbred Sox2 heterozygous males showed that these animals occasionally had smaller testes with sperm blockage in the seminiferous tubules (Figure 3). In these tubules, somatic cells also looked abnormal. These blockages may affect epididymal sperm count, which in some mutants was found to be approximately 50% that of wild-type animals. However, these changes should not affect fertility. The infertility is more likely a consequence of a primary defect in the developing sperm that compromises their motility and/or their ability to fertilize (A. Avilion and R. Lovell-Badge, unpublished observations). The fertility of heterozygous females, on the other hand, was not significantly reduced (A. Avilion, unpublished observations).

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).

Mutation analysis of human SOX2

Of the cohort of 235 individuals screened for variations in the SOX2 gene, 6 unrelated individuals were found to have heterozygous mutations in SOX2 coding regions predicted to result in the production of a truncated mutant protein (Figure 4). Two mutations were single-base deletions (c.387delC and c.479delA), one was a single-base insertion (c.60insG), and another affected individual had a deletion of 20 nucleotides (c.70del20; Figure 4, A and B). We believe three of these mutations are novel, while the fourth (c.70del20) was recently described in an 11-month-old girl with bilateral anophthalmia, rudimentary optic nerves, and partial agenesis of the corpus callosum (11). All of these mutations gave rise to a frameshift in the coding sequence. We also identified 2 nonsense mutations: what we believe to be a novel insertion at nucleotide 480 (c.480insA) resulting in the amino acid change Y160X, as well as an individual with the mutation Q177X (c.529C→T) that has been observed previously in 2 unrelated individuals (Figure 4B and ref. 8). Sequencing the parental DNAs of each of these patients (data not shown) revealed they had a normal SOX2 sequence, indicating that all 6 mutations identified occurred de novo. Figure 4C shows the extent of the predicted truncations in the mutant proteins compared with wild-type SOX2, the 2 shortest of which (c.60insG and c.70del20) would result in proteins lacking the DNA-binding HMG domain. The other 4 mutations retain the HMG domain but would be predicted to lack variable portions of the transactivation domain.

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.

From the rest of the cohort, 2 individuals were found to harbor heterozygous nonsynonymous amino acid variants. One of these was a G→A transition at nucleotide c.571, resulting in a substitution of a threonine for an alanine at codon 191 (A191T). A191T is conserved among human, mouse, and chicken, but is substituted by threonine in the zebrafish. The other nucleotide change (c.389G→C) resulted in a glycine to alanine substitution at residue 130 (G130A), which is highly conserved among mouse, human, zebrafish, and chicken. This individual also had a synonymous c.387G→C change 2 nucleotides upstream (G129G; Figure 4B). In both cases, these variants were inherited from the affected patients’ respective fathers (data not shown), who were phenotypically normal, although the variants were not identified in 100 control chromosomes. Three other synonymous variants were also identified, A151A, N187N, and L278L; the latter was observed in 2 unrelated individuals in this cohort and has also been previously reported (9). Finally, a common single nucleotide polymorphism in the 3′ untranslated region (976G→A) was detected with a frequency of approximately 5%, consistent with previous observations (8).

Since SOX2 is a single exon gene, it is highly unlikely that the mutations described would lead to nonsense-mediated decay (NMD), because intronless genes do not undergo NMD (15–18). We therefore proceeded to study the functional consequences of the sequence variants.

Functional analysis of mutant SOX2 proteins

Cellular localization. Given that efficient transport from the cytoplasm to the nucleus plays a key role in the function of SOX proteins as transcription factors (19) and that SOX2 is known to be regulated at this level (ref. 5 and our unpublished observations), we investigated the localization of wild-type and mutant SOX2 proteins within the cell. Wild-type SOX2 was primarily localized to the nucleus of the cell, with little cytoplasmic staining (Figure 5A). In contrast, neither mutation c.60insG nor mutation c.70del20 (both lacking the HMG domain) showed nuclear localization. The other SOX2 constructs tested showed similar results to wild-type SOX2, with localization principally within the cell nucleus (Figure 5A). The c.479delA mutation behaved in the same way as the Y160X mutation (data not shown). Although the previously described L97P missense mutation lies in the HMG domain, it does not disrupt the previously identified nuclear localization signal sequences at the N and C termini of the HMG box (20–22), and the protein was correctly targeted to the nucleus in this assay.

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.

DNA binding. In order to assess the ability of the mutant proteins to bind DNA, wild-type and mutant SOX2 proteins were synthesized by in vitro transcription/translation. EMSA performed with these proteins demonstrated the ability of wild-type SOX2 to bind specifically to a radiolabeled consensus SOX sequence DNA probe (Figure 5B). Both c.60insG and c.70del20 mutations generated proteins unable to bind to DNA, as expected, given that they completely lack the DNA-binding HMG domain. The other 4 mutations (c.387delC, c.479delA [data not shown], Y160X, and Q177X) produced truncations downstream of the HMG domain, and all retained the ability to bind DNA, in complexes that migrated faster than wild-type SOX2 due to their smaller size. The L97P mutation (9) showed complete loss of DNA binding compared with the 2 missense variants identified in this study (G130A and A191T), which bound to DNA with a similar affinity to wild-type SOX2 in this assay (Figure 5B).

Reporter assays. We next tested the ability of the mutant proteins to activate transcription of a luciferase reporter construct downstream of the proximal promoter of the Hesx1 gene that contains binding sites for SOX proteins and can be activated in a dose-dependent manner (23). As shown in Figure 5C, wild-type SOX2 activated transcription with a 20-fold increase in reporter expression. The mutant constructs c.60insG, c.70del20, and L97P failed to show any activation, as expected, given their inability to bind DNA (Figure 5B). The other 4 truncating mutant constructs (c.387delC, c.479delA [data not shown], Y160X, and Q177X) showed minimal activation compared with the wild-type construct, with their residual activity correlating with the length of the mutant protein. Q177X, which retained the longest fragment of the transactivation domain (see Figure 4C), showed a 7-fold increase in activation, but was clearly less effective than the wild-type SOX2. Increasing concentrations of the missense variants, G130A and A191T, reached levels of activation similar to wild-type SOX2 in this assay. These differences were not due to impaired synthesis of SOX2 proteins, as confirmed by Western blot using whole-cell extracts following transfection (data not shown). Cotransfection of wild-type SOX2 with mutated constructs (1:1 ratio) showed no evidence for a dominant-negative effect of mutant proteins on the ability of wild-type SOX2 to activate transcription in this assay (data not shown).

We have shown that the transcription factor SOX2, expressed in neuroepithelial progenitors and stem cells from the earliest stages, is implicated in the normal development of the brain and the pituitary gland in addition to that of the eyes and inner ear. The pituitary gland is a midline structure consisting of the anterior, intermediate, and posterior lobes of varying sizes and complexities in different species. Hypopituitarism may result in isolated hormone deficiency (e.g., GH, TSH, PRL, ACTH, LH and follicle-stimulating hormone [FSH]), combined pituitary hormone deficiency (CPHD) with or without involvement of the posterior pituitary, or CPHD with other congenital anomalies such as optic nerve hypoplasia, midline forebrain abnormalities (septo-optic dysplasia; SOD), and cerebellar abnormalities. The condition is associated with significant morbidity and, if undetected or inadequately treated, mortality. Its incidence is reported as ranging from 1 in 3,000 in the case of isolated GH deficiency to 1 in 50,000 for SOD, with a 2:1 male preponderance.

Several transcription factors are essential for normal pituitary development, and mutations within a number of these have been described in association with pituitary disease in humans (24, 25). Among these, Sox3/SOX3 was the first member of this gene family to be implicated in X-linked hypopituitarism in both mice and humans (13, 23, 26). It is now clear that infundibular and pituitary development is critically sensitive to dosage of SOX3, with both duplications of SOX3 and loss-of-function mutations being associated with anterior pituitary hypoplasia (APH), infundibular hypoplasia, an undescended/ectopic posterior pituitary, and anterior pituitary endocrine deficits (23). Given the growth and fertility defects in Sox2 mutant mice, and given that expression domains of SOX proteins frequently overlap, we suspected that SOX2 might also be involved in pituitary development. This was indeed the case, although there were important similarities and differences in phenotypic consequences and penetrance in humans and mice.

One obvious difference is in eye development. SOX2 haploinsufficiency in humans is associated with bilateral anophthalmia/microphthalmia, whereas there are no ocular abnormalities in Sox2^βgeo heterozygous mice (5, 8). However, this is more likely due to a difference in sensitivity between the species than to a novel role for the protein in humans. Indeed it has very recently been shown in mice that further reduction of Sox2 levels (<40% of wild type) does result in a dose-dependent series of eye malformations closely related to those of the human condition (27).

In contrast, both species show abnormalities and hypoplasia of the pituitary, the details of which are different. All of our patients with SOX2 mutations showed evidence of marked gonadotropin deficiency, but while 4 of the 6 patients (patients 1, 4, 5, and 6) were short (heights less than –2 standard deviation score [SDS]), only 2 showed evidence of blunted GH secretion (patients 5 and 6). Patient 5 in fact demonstrated a normal GH response to provocation on 2 occasions, and although the overnight GH secretory profile was suboptimal, this could simply reflect a lack of sex steroid, given that the IGF-1 and IGF-binding protein 3 (IGFBP3) concentrations have been documented as being normal (see Table 1 and “Patient phenotypes associated with de novo SOX2 mutations” in Methods). Moreover, the 2 patients that had attained final height were within the normal range (NR; patient 2, –0.1 SDS; patient 5, –1.2 SDS). It is unusual to observe a normal GH response to provocation in the face of abnormal gonadotropin secretion and pituitary hypoplasia in patients with hypopituitarism (24). Additionally, the corticotrope, thyrotrope, and lactotrope axes appeared to be clinically normal despite the structural hypoplasia of the gland. In contrast, heterozygous loss of function of Sox2 in the mouse was associated with a reduction in somatotrope number and GH content, as part of a general reduction in pituitary size, as early as 18.5 dpc. Moreover, in the adult, we found a general reduction of all hormones assayed. Pubertal development was not consistently affected in the mouse, but as a significant proportion of Sox2 heterozygotes are lost postnatally, this effect may be masked. We found that some adult Sox2^βgeo heterozygotes were small, while others were normal, but still GH deficient (data not shown). While GH deficiency could explain the growth retardation, other aspects of the phenotype could be responsible, such as CNS defects. Some growth retardation was also observed in Sox2^ΔENH/Sox2^βgeo compound heterozygotes (7).

Table 1

Endocrine phenotype in patients with SOX2 mutations

One possible explanation for the differences between the human and murine phenotypes might be the degree of redundancy that exists among members of the SoxB1 family, the extent and location of which may not be identical in both species. The phenotype resulting from mutations in each of the 3 SoxB1 genes is often limited to those sites at which only the mutant gene is expressed. For example, Sox1 mutant mice show lens defects beginning at the time when Sox2 is turned off and lack olfactory tubercles, an area of the CNS where Sox1 is uniquely expressed (28, 29). Both Sox2 and Sox3 are expressed in the hypothalamo-pituitary region; however, in mice, Sox3 is expressed at a particularly high level in the infundibulum, and its expression in this region is also likely to be critical in humans, as reflected by the phenotype of infundibular hypoplasia in patients with abnormal amounts of SOX3 (23). In the mouse, the Sox2^βgeo heterozygotes demonstrate similar defects in pituitary morphology to Sox3 null embryos (13), although the phenotype is both less severe and less penetrant, presumably because 1 normal copy of Sox2 remains in the former. As Sox3 is not expressed in Rathke’s pouch, it has been suggested that the morphological defects may originate from changes in the overlying infundibulum (13). A similar mechanism might be operating for Sox2, but unlike Sox3, this is also prominently expressed in Rathke’s pouch, so a simpler explanation in this case could be a “pouch-autonomous” defect, not compensated for by redundancy from other Sox genes. The reduction in somatotrope numbers associated with anterior lobe hypoplasia at 18.5 dpc supports a pouch-autonomous function for Sox2. However, conditional deletion of the genes singly and in combination will be necessary to unravel this issue further. While endocrine deficits are present in both Sox2 and Sox3 mutant mice, they differ significantly. In contrast to Sox2 heterozygotes, GH content is unaffected at 18.5 dpc in Sox3 mutants (13). This suggests primary pituitary deficits in the former, while the adult endocrine deficits in Sox3 null animals are more consistent with a hypothalamic contribution to the phenotype. Moreover, the deficits in LH are not as profound in the latter.

It is conceivable that dysregulation of hypothalamic secretion associated with SOX2 mutations could lead to secondary hypoplasia of the anterior pituitary. This would have to be more severe for gonadotropin-releasing hormone (GnRH) in humans than in mice. Given the difficulties in measuring GnRH content in the mouse and the complexity of the GH-releasing hormone–GH (GHRH-GH) axis, it would be difficult to establish the effect of Sox2 mutations on the hypothalamus at this stage, and further studies — possibly using conditional transgenesis — would be required to study these effects in greater detail. It is also possible, though we believe it is unlikely, that a primary abnormality in the formation of Rathke’s pouch selectively affects the gonadotrope lineage in humans heterozygous for mutations in SOX2. On the other hand, it must be remembered that SOX2 is expressed widely in the CNS, so the phenotype of HH might be due to a loss of function earlier in development. HH is known to be caused by genetic defects in GnRH neuronal migration (KAL1 and FGFR1 mutations; ref. 30), and by mutations in the GnRH receptor (GNRHR) or in the G protein–coupled receptor GPR54 (30–32). A role for SOX2 in determining normal numbers of GnRH neurons and/or neuronal migration cannot be excluded. In this context, it is interesting that defects in olfactory tubercle development in Sox1 mutant mice are due to a requirement for the protein in postmitotic neuronal precursors for their correct migration and differentiation (29).

A direct effect of Sox2 deficiency in germ cells, where the gene is known to be expressed (5), could also contribute to the hypogonadal phenotype. The sperm blockage observed in mice could reflect a local “stem cell maintenance” role for SOX2, in parallel with its role in the CNS (for review see ref. 3). In its absence, germ cells would no longer persist but differentiate, resulting in tubules filled by sperm, as we observed. To resolve this issue and understand the potential requirement for SOX2 at different levels in the hypothalamo-pituitary-gonadal axis, conditional deletion of the gene exclusively in each compartment will be necessary. Hence, an effect of SOX2 mutations at the additional level of the gonads cannot be completely excluded, given the coexistence of HH. This could possibly explain the complete lack of testosterone response to human chorionic gonadotropin (HCG) stimulation in patients 3 and 5 (Table 1).

It is also possible that SOX2 participates directly in the regulation of genes such as Hesx1, Prop1, and Aes, all of which show similar phenotypes when mutated; however, heterozygosity for Sox2 may not be sufficient to reveal any significant reduction in expression of these putative target genes.

The endocrine status of patients 1–6 and 8 has been carefully evaluated over the last 12 months, and to date no evolution of other hormonal deficiencies has been documented. It is important to note, however, that further hormone deficiencies may evolve in patients with mutations in transcription factors that are implicated in pituitary development (33–35), and hence careful long-term follow-up is mandatory in these patients.

In addition to the endocrine abnormalities identified in our patients, we also identified a number of neurological abnormalities. Many of these could be explained by the widespread expression of SOX2 in the developing and postnatal brain. In the mouse, significant expression is maintained in the differentiating areas of the thalamus and medial-dorsal striatum and septum during embryogenesis and postnatally (7). The CNS defects observed in our patients included abnormalities of the mesial temporal structures including the hippocampi (patients 1–4 and 6), partial agenesis or hypoplasia of the corpus callosum (patients 1, 3, 4, and 6), and hypothalamic hamartomata (patients 1 and 3). Similar anatomical defects were also previously noted in Sox2^ΔENH/Sox2^βgeo compound heterozygous mice (7); moreover, Sisodiya et al. recently reported hippocampal and parahippocampal malformations in 4 previously published patients with SOX2 mutations, 2 of which had a history of epilepsy (36). None of the patients we report here with SOX2 mutations presented with a history of epilepsy, and no association has been found between clinical epilepsy phenotypes and SOX2 variation (36). However, mesial temporal structure abnormalities, particularly hippocampal malformations, appeared to be a common finding in association with SOX2 mutations. The presence of hypothalamic hamartoma in 2 of our patients is interesting, and, given that SOX2 is expressed in the developing hypothalamus, our data suggest that the gene may be implicated in some patients with a hamartoma. Usually, a hamartoma is a benign condition that is associated with precocious puberty and/or gelastic seizures. If it was not for the other neurological defects, the hamartomata may well have remained undetected in these 2 patients, given the gonadotropin deficiency.

All of our patients showed evidence of learning difficulties. This was probably to be expected given the widespread expression of SOX2 in the CNS. Also of note, 3 of our patients had marked motor deficits in the form of spastic diplegia or quadriparesis (patients 1, 3, and 4), and this might reflect the critical nature of Sox2 expression in the pyramidal cells of the cerebral cortex (7). One of our patients had evidence of sensorineural deafness (patient 6), similar to that in the patient described by Hagstrom et al. (10). Recently, regulatory mutations affecting Sox2 expression in the murine inner ear, where it is the sole Soxb1 gene to be expressed, have been associated with inner ear malformations and, in the most extreme case, complete absence of the sensory epithelium (37). Heterozygous mice are normal, however, again suggesting variable correspondence between human and mouse phenotypes.

The association between anophthalmia and esophageal atresia is well known (anophthalmia-esophageal atresia-genitalia syndrome, OMIM 600992; refs. 38–40), and mutations in SOX2 have been described in a number of these cases (12). While one of our patients manifested esophageal atresia (patient 1), the other 5 did not; moreover we failed to find evidence for esophageal abnormalities in heterozygous Sox2^βgeo mice despite the expression of Sox2 in the gut endoderm (refs. 6, 12 and our unpublished observations).

It is noteworthy that the incidence of loss-of-function mutations in the coding region of SOX2 in our small cohort of patients with anophthalmia/microphthalmia was quite high (approximately 50%), whereas no mutations were identified in patients with hypopituitarism only or in patients with optic nerve hypoplasia. This indicates that the eye is particularly sensitive to a reduction in levels of SOX2, perhaps more so than the pituitary. Previous studies identified mutations in 4 of 102 patients with anophthalmia, microphthalmia, and coloboma (8). The majority of the mutations identified in this and other studies are nonsense or frameshift mutations, with only 2 previously reported missense mutations, both of which occurred de novo (8, 12). The 2 missense changes identified in patients 7 and 8 were in both cases inherited from their unaffected fathers. The difference in phenotype between these 2 individuals (CNS abnormalities with optic nerve hypoplasia in patient 7, and CNS abnormalities, optic nerve hypoplasia, and pituitary hypoplasia in patient 8) and the 6 harboring truncating mutations (severe CNS and ocular abnormalities with HH; Table 2), in addition to the absence of any obvious functional compromise, suggests that they may be assumed to be neutral variants, although it is interesting to note that the changes were not identified in 100 control chromosomes in our studies, nor in a previously reported screen of 176 control chromosomes (8). It is, however, important to consider the possibility that the 2 missense variants affect some hitherto uncharacterized function of SOX2 and that the mutations may be variably penetrant. Mutation in another gene implicated in CNS and pituitary development may lead to digenic inheritance, which would account for the lack of a phenotype in the parent carrying the SOX2 variant. We have screened HESX1 for mutations in patients 7 and 8, but failed to find a mutation in this gene.

Table 2

Clinical phenotype in patients with SOX2 mutations

Our data are consistent with previous findings showing that the HMG box of SOX2 is critical for its interaction with DNA (41, 42) and that 2 nuclear localization signals that lie at both the N (bipartite; KRP, RRK) and C (PRRK) termini of the HMG box in the SRY and SOX9 genes (20–22) — and which are highly conserved in SOX2 — are critical for correct nuclear translocation. As expected, truncated proteins lacking the HMG domain were associated with impairment of nuclear localization, DNA binding, and transcriptional activation, while mutant proteins retaining the HMG domain preserved DNA binding and correct nuclear transport; their residual transcriptional activation also correlated with the extent of the remaining C-terminal activation domain. We took the opportunity to assess the functional impact of 1 of only 2 missense mutations identified in SOX2 hitherto, L97P (9). This mutation of a highly conserved residue in the HMG box did not impair nuclear localization, but led to loss of DNA binding and transcriptional activation. There was, however, no obvious phenotype-genotype correlation in our patients (Table 3), suggesting that all the mutations simply compromised overall function and that the various affected tissues were sensitive both to levels of SOX2 and to other genetic or environmental factors.

Table 3

Summary of molecular and clinical features associated with SOX2 mutations

In conclusion, we have demonstrated for the first time to our knowledge that in addition to bilateral eye disease, SOX2 mutations are commonly associated with pituitary hypoplasia and endocrine deficits. The endocrine phenotype is unusual in that the gonadotropin deficiency is profound, whereas GH secretion appears to be relatively spared. Our data suggest that haploinsufficiency of SOX2, as opposed to a dominant-negative effect, is the most likely cause of the phenotypes. Finally, SOX2 is the second member of this gene family to be implicated in the development and normal function of the human hypothalamo-pituitary axis, with an important role in establishing normal reproductive development. Timely diagnosis of sex steroid deficiency in these individuals with a multitude of problems would lead to prompt treatment with the prevention of long-term morbidity, for example osteoporosis with attendant bone fractures.

We would like to thank all the pediatricians and patients who participated in the study. This study was supported by grants from the Medical Research Council (MRC) UK (to M.T. Dattani and D. Kelberman). Research at the Institute of Child Health and the Great Ormond Street Hospital for Children NHS Trust benefits from research-and-development funding from the NHS executive. We are grateful to J.P. Martinez-Barbera for providing us with the pCMV/SV-Flag plasmid and for technical support and to Teddy Fauquier for helpful discussions.

Address correspondence to: Mehul T. Dattani, London Centre for Paediatric Endocrinology, BEM Unit, Institute of Child Health and Great Ormond Street Hospital for Children, 30 Guilford Street, London WC1N 1EH, United Kingdom. Phone: 44-20-7905-2657; Fax: 44-20-7404-6191; E-mail: mdattani@ich.ucl.ac.uk .

Nonstandard abbreviations used: APH, anterior pituitary hypoplasia; FSH, follicle-stimulating hormone; GH, growth hormone; GHRH, GH-releasing hormone; GnRH, gonadotropin-releasing hormone; HCG, human chorionic gonadotropin; HH, hypogonadotropic hypogonadism; HMG, high-mobility group; IGFBP3, IGF-binding protein 3; LH, luteinizing hormone; NR, normal range; PRL, prolactin; rhGH, recombinant human GH; SDS, standard deviation score; SOD, septo-optic dysplasia; SOX, SRY-related HMG box; TSH, thyroid-stimulating hormone; TRH, thyrotropin-releasing hormone.

Conflict of interest: The authors have declared that no conflict of interest exists.

Reference information: J. Clin. Invest.116:2442–2455 (2006). doi:10.1172/JCI28658.

Daniel Kelberman and Karine Rizzoti contributed equally to this manuscript.

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