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Developmental Endocrinology Research Group (D.K., J.C.A., M.T.D.), Clinical and Molecular Genetics Unit, and Medical Research Council-Wellcome Trust Human Developmental Biology Resource (S.C.P.d.C., D.G.), Neural Development Unit (J.P.M.-B.), Institute of Child Health, University College London, London WC1N 1EH, United Kingdom; National Genetics Reference Laboratory (Wessex) (S.H., J.A.C.), Salisbury District Hospital, Salisbury SP2 8BJ, United Kingdom; Departments of Cytogenetics (R.P.) and Ophthalmology (D.T.), Great Ormond Street Hospital for Children National Health Service Trust, London WC1N 3JH, United Kingdom; Department of Child Health (J.W.G.), Wales College of Medicine, Cardiff University, Cardiff, Wales CF10 3XQ, United Kingdom; Department of Biomedicine of Evolutive Age (L.C., M.F.F., R.F.), University of Bari, Bari 70121, Italy; and Divisions of Developmental Genetics (K.R., R.L.-B.) and Molecular Neuroendocrinology (I.C.A.F.R.), Medical Research Council National Institute for Medical Research, Mill Hill, London NW7 1AA, United Kingdom
Address all correspondence and requests for reprints to: Professor Mehul T. Dattani, Clinical and Molecular Genetics Unit, Institute of Child Health, 30 Guilford Street, London WC1N 1EH, United Kingdom. E-mail: mdattani{at}ich.ucl.ac.uk.
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Abstract |
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Objective: We have ascertained a further three patients with severe eye defects and pituitary abnormalities who were screened for mutations in SOX2. To provide further evidence of a direct role for SOX2 in hypothalamo-pituitary development, we have studied the expression of the gene in human embryonic tissues.
Results: All three patients harbored heterozygous SOX2 mutations: a deletion encompassing the entire gene, an intragenic deletion (c.70_89del), and a novel nonsense mutation (p.Q61X) within the DNA binding domain that results in impaired transactivation. We also show that human SOX2 can inhibit β-catenin-driven reporter gene expression in vitro, whereas mutant SOX2 proteins are unable to repress efficiently this activity. Furthermore, we show that SOX2 is expressed throughout the human brain, including the developing hypothalamus, as well as Rathke’s pouch, the developing anterior pituitary, and the eye.
Conclusions: Patients with SOX2 mutations often manifest the unusual phenotype of hypogonadotropic hypogonadism, with sparing of other pituitary hormones despite anterior pituitary hypoplasia. SOX2 expression patterns in human embryonic development support a direct involvement of the protein during development of tissues affected in these individuals. Given the critical role of Wnt-signaling in the development of most of these tissues, our data suggest that a failure to repress the Wnt-β-catenin pathway could be one of the underlying pathogenic mechanisms associated with loss-of-function mutations in SOX2.
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Introduction |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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Sox2–/– null embryos die shortly after implantation; however, Sox2 heterozygous mice appear relatively normal, but about one third show perinatal lethality, and others show a reduction in size and male fertility (5). We have recently demonstrated a role for Sox2 in the development of the anterior pituitary gland in the mouse. Analysis of heterozygous mutant mice showed a variable hypopituitary phenotype affecting the size and shape of the pituitary gland, and a significant lowering of pituitary GH, LH, ACTH, and TSH contents in affected mice (8).
Heterozygous mutations within SOX2 in humans have been associated with bilateral anophthalmia or severe microphthalmia, in addition to hypopituitarism characterized by anterior pituitary hypoplasia and gonadotropin deficiency (hypogonadotropic hypogonadism) with genital abnormalities in males (7, 8, 9). Additional forebrain defects may also include hypoplasia of the corpus callosum, hypothalamic hamartoma, and hippocampal malformation (8, 10), frequently associated with additional abnormalities, including esophageal atresia, sensorineural hearing loss, and learning difficulties.
We now report the endocrine phenotype in a further three patients with de novo heterozygous mutations in SOX2, including a novel mutation in the HMG box (p.Q61X), in addition to an individual with an interstitial deletion of chromosome 3q26-q27 encompassing the entire gene. We describe here for the first time the expression pattern of SOX2 during human embryonic development using in situ hybridization and immunohistochemistry. In addition, because the disruption of Wnt signaling and the normal function of β-catenin have been associated with pituitary defects in mice (11, 12), and because SOX proteins have been reported to interact with β-catenin (13, 14), we have used an in vitro reporter system to show that several of the naturally occurring mutations identified in patients impair the ability of human SOX2 to antagonize the transcriptional activity of β-catenin.
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Patients and Methods |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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)Patient 1 Patient 1 has previously been reported to show an unusual karyotype consisting of a de novo translocation with an associated interstitial deletion of chromosome 3q at the break point: 46,XX,del (3) (q26.33q28)t (3, 7) (q28;q21.1) (patient 2 in Ref. 15). This female patient was born to nonconsanguineous parents at term by a normal vaginal delivery with a birth weight of 1.42 kg [–5.68 SD score (SDS)]. She had right anophthalmia and left microphthalmia, and mild pulmonary stenosis. Global developmental delay was noted at the age of 15 months. A right ocular prosthesis was inserted and a corneal graft performed in the left eye. Visual evoked responses and an electroretinogram revealed good retinal and optic nerve function. Audiometry revealed normal hearing.
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). Subsequently, growth velocity was poor over the first 18 months (height 65.7 cm, –5 SDS; weight 5.54 kg, –4.36 SDS; height velocity –2 SDS between the ages of 0.5 and 1.55 yr). Investigations revealed a peak GH of 26.8 µg/liter (67.2 mU/liter), with a normal cortisol of 24 µg/dl (670 nmol/liter), normal prolactin [12.1 µg/liter (242 mU/liter)], and normal thyroid function [free T4 (FT4) 1.5 ng/dl (19.4 pmol/liter); peak TSH to TRH 23.5 mU/liter]. Gonadotropin deficiency could not be excluded (peak LH to LHRH 3.5 IU/liter, peak FSH to LHRH at 60 min of 7.8 IU/liter at 20 months age). Subsequently, her height velocity ranged from –1.93 to –2.7 SDS but with an IGF-I and its binding protein-3 concentrations of 227 ng/ml (+1 SDS) and 3.30 mg/liter (0 SDS), respectively. The proband is now aged 6.5 yr, and no endocrine treatment is required at present.
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Patient 3 Patient 3 is a 14.5-yr-old girl who is the second child of four offspring, born to unrelated parents at full term after a pregnancy complicated by uterine contractions in the fifth month that waned with tocolytic therapy that was maintained until delivery. The birth weight was 3.35 kg (–0.5 SDS) and birth length 50 cm (–0.38 SDS). She presented with bilateral anophthalmia. At 15 d of life, she underwent corneal transplantation, and brain MRI showed an arachnoid cyst in the suprasellar area, with right deflection of the pituitary pedunculus. She was referred to the endocrinologist at the age of 9.4 yr because of short stature (height 125.8 cm, –1.31 SDS; weight 24.2 kg, –1 SDS). Investigations performed at the age of 11.4 yr revealed normal thyroid function [FT4 1.01 ng/dl (13 pmol/liter), TSH 2.45 mU/liter], and a peak GH response to clonidine of 14.1 µg/liter (42.3 mU/liter) and to arginine stimulation of 10.2 µg/liter (30.6 mU/liter). The IGF-I concentration was within the NR (312 ng/ml; NR 97–699 ng/ml). Subsequent investigations at the age of 14.5 yr, at which stage she had no evidence of puberty (height –2.58 SDS, weight –0.34 SDS), revealed gonadotropin deficiency with low basal gonadotropin concentrations (LH undetectable, FSH 0.6 IU/liter) and poor responses to GnRH stimulation (peak LH 2.6 IU/liter; peak FSH 2.7 IU/liter). TRH testing performed at this time showed normal thyroid function with normal TSH (peak 12.3 mU/liter) and prolactin [peak 24.6 µg/liter (492 mU/liter)] responses, and a normal FT4 [1.3 ng/dl (16.8 pmol/liter); NR 0.8–1.8] concentration. At the last outpatient visit, no pubertal development had occurred, and estrogen replacement is currently being considered. There was no evidence of learning difficulties.
Mutation analysis
To detect chromosomal abnormalities, we used the Agilent 44k Human Genome CGH microarray (Agilent Technologies, Wokingham, UK). Genomic DNA from patient 1 and reference normal human male genomic DNA (Promega Corp., Madison, WI) were digested and labeled incorporating either 3 mM Cy5-deoxyuridine 5-triphosphate (test sample) or Cy3-deoxyuridine 5-triphosphate (male reference). Purified Cy5- and Cy3-labeled DNA was combined and mixed with human Cot-1 DNA (Invitrogen, Paisley, UK). Automated hybridization and washes were performed in an HS Pro 4800 Pro Hybridization Station (Tecan UK Ltd., Reading, UK). Slides were scanned using an Agilent microarray scanner (Agilent), and data analysis was performed using Agilent Analytics v3.3.27 software.
For intragenic mutations, the coding sequence of SOX2 was amplified from genomic DNA and directly sequenced as described elsewhere (8). Sequences were compared with SOX2 reference sequence (RefSeq NM_003106). Ethical committee approval was obtained from the Institute of Child Health/Great Ormond Street Hospital for Children Joint Research Ethics Committee. Informed written consent was obtained from the parents, and, where applicable, the patients before collection of samples and genomic analysis.
Plasmid constructs
PCR products comprising the entire SOX2 coding region from patient 2 were generated and cloned into pcDNA3.1(+) (Invitrogen) and pCMV/SV-Flag, containing an in-frame N-terminal FLAG epitope. Constructs containing wild-type SOX2, in addition to the mutations c.60_61insG, c.387_388delC, and p.Q177X, have been previously described (8). A clone containing the full-length human β-catenin coding sequence (CTNNB1; IMAGE Consortium CloneID 6151332) (16) was obtained from Geneservice Ltd. (Cambridge, UK). To generate a constitutively active phosphorylation mutant of β-catenin, the mutation c.96C>A (p.S33Y) (17) was introduced using the QuikChange Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA).
Luciferase assay experiments
SOX2 luciferase reporter assays were performed using Chinese hamster ovary cells as previously described (8). For the TOPFLASH assay (18), human embryonic kidney 293 cells were cotransfected with 20 ng TOPFLASH reporter, 30 ng β-catenin expression construct, and increasing amounts of wild-type SOX2 expression construct (1–70 ng). Luciferase activity was measured using a BMG FLUOstar Optima multiplate reader (BMG LABTECH Gmbh, Offenburg, Germany), and experiments were each repeated three times independently and performed in triplicate.
Cell localization studies
Chinese hamster ovary cells were transfected with 50 ng FLAG-tagged SOX2 expression construct (wild type or p.Q61X). After transfection, cells were washed, fixed, and immunostaining was performed as described elsewhere (8).
EMSA
SOX2 proteins were generated using the TNT Quick Coupled Transcription/Translation System (Promega). EMSAs were performed as described previously (8).
In situ hybridization
Analysis of human embryonic/fetal material was performed by the Medical Research Council-Wellcome Trust Human Developmental Biology Resource in situ hybridization service with full ethical approval from the Joint Great Ormond Street Hospital National Health Service Trust/Institute of Child Health Ethics Committee.
Human embryos/fetuses at selected stages were dissected and fixed in 4% paraformaldehyde, then dehydrated and embedded in paraffin wax. Sections of 7 µm were cut using a standard microtome and mounted on Superfrost Plus slides (BDH Laboratory Supplies, Poole, UK). In situ hybridization was performed essentially as described by Wilkinson (19) using digoxigenin incorporated riboprobes generated from a 697-bp fragment of the 3' untranslated region of human SOX2 and cloned into the pCR4-TOPO vector (Invitrogen). For antibody detection, slides were incubated with antidigoxigenin antibody conjugated with alkaline phosphatase (diluted 1:1000, containing 2% fetal calf serum). Expression patterns were visualized using the Nitro-Blue Tetrazolium Chloride/5-Bromo-4-Chloro-3'-Indolyphosphate p-Toluidine Salt system (Roche, Welwyn Garden City, UK). Sections were mounted in VectaMount (Vector Laboratories, Burlingame, CA) and analyzed using the Axioplan2 imaging system (Carl Zeiss, Inc., Thornwood, NY).
Immunohistochemistry
Human embryo and fetal sections were dewaxed and rehydrated. Antigen retrieval was performed by boiling for 10 min in 0.01 M citric acid buffer (pH 6.0). After cooling to room temperature, slides were transferred to Tris-buffered saline with Tween 20 (TBST) [0.5 M Tris Base, 9% NaCl (pH 7.6), 0.025% Triton] and blocked with 10% heat-inactivated rabbit serum (Sigma-Aldrich, St. Louis, MO) in TBST. Slides were incubated with goat anti-SOX2 antibody (1:500 dilution; Immune Systems Ltd., Paignton, UK) in TBST with 1% heat-inactivated rabbit serum, then washed and incubated with a polyclonal rabbit antigoat IgG biotinylated secondary antibody (1:100 dilution; Dako Corp., Carpinteria, CA). Biotin was detected using streptavidin-Alexa Fluor 555 conjugate in PBS (1:300 dilution; Molecular Probes, Inc., Eugene OR). Slides were mounted in Vectashield with DAPI (Vector Laboratories) and images captured on a Leica DC500 microscope using Fire Cam Software (Leica Microsystems GmbH, Wetzlar, Germany).
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Results |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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Fluorescent in situ hybridization analysis of patient 1 showed that SOX2 was deleted on the der(3) chromosome of the patient in all metaphase preparations examined (Fig. 2A). Subsequently, microarray comparative genomic hybridization analysis defined the extent of the deletion to approximately 7.8 Mb of chromosomal region 3q26.33-q27.3 (start: 181216930 bp; end: 189014508 bp) (Fig. 2B). The array CGH data showed no other significant copy number changes.
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), resulting in the nonsense mutation p.Q61X, which is predicted to result in a mutant protein truncated within the HMG domain. This mutation creates a BfaI restriction site that was used to genotype the parents of the affected individual, neither of whom carried the mutation (Fig. 2C). Patient 3 was found to harbor a deletion of 20 nucleotides upstream of the HMG domain (c.70_89del), which occurred de novo in this individual because it was not present in her unaffected parents (data not shown). Molecular analysis of SOX2 mutations
The single exon structure of SOX2 implies that nonsense-mediated decay mechanisms are unlikely to result in degradation of the mutant transcript (20, 21). Therefore, we sought to confirm the functional consequence of the p.Q61X mutation in vitro. Luciferase reporter assays using a reporter construct previously shown to be activated by SOX2 in vitro (8) showed that the mutant construct failed to activate this reporter (Fig. 2D) as a result of the complete loss of DNA binding, compared with wild-type SOX2 (Fig. 2E). We also investigated the localization of the mutant protein within the cell. Whereas the majority of wild-type SOX2 was localized within the cell nucleus, the p.Q61X mutant protein showed predominantly cytoplasmic staining (Fig. 2F). The c.70_89del mutation has previously been identified in other unrelated individuals and shown to result in the loss of function of the predicted protein product (8, 22, 23).
Previous studies have demonstrated that murine Sox2 is capable of interfering with the activity of a β-catenin responsive promoter containing TCF/LEF binding sites mediated by the COOH terminal portion of Sox2 outside of the DNA binding HMG domain (13). To investigate the ability of human SOX2 to repress β-catenin-TCF-mediated transcription, we performed a TOPFLASH reporter assay (18). Cotransfection of the reporter with an expression construct containing human β-catenin leads to increased activation of the reporter. Cotransfection with increasing amounts of wild-type human SOX2 led to a dose-dependent reduction in β-catenin induced activation, whereas SOX2 alone had no effect on transcriptional activation of the reporter (Fig. 3A). To determine the effect of various naturally occurring truncating mutations in SOX2, we cotransfected SOX2 expression constructs containing the mutations c.60_61insG, p.Q61X, c.387_388delC, and p.Q177X (c.529C>T); the latter two maintain an intact HMG domain and retain the ability to bind DNA in vitro (8). Products from all four of these mutations failed to repress β-catenin-mediated transcription (Fig. 3B). Repeating the experiments using a constitutively active β-catenin mutant construct (17) showed identical results to wild-type β-catenin (data not shown).
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To understand the consequences of SOX2 mutations directly involved in specific tissue abnormalities, it is important to establish its expression patterns during human development. To assess this, we analyzed available sections of normal human embryos from Carnegie Stage (CS) 14 through to fetal stage (F) 2 [
4.5- to 9-wk development (24)]. Expression of SOX2 mRNA and protein was observed throughout the central nervous system at all stages investigated. Strong expression is observed in the forebrain at CS16, which is maintained throughout embryonic development (Fig. 4, A–E), with SOX2 transcripts detected in the cortex and neuroepithelium of the lateral ventricles at Fs (data not shown). SOX2 expression is observed within Rathke’s pouch at CS16 (Fig. 4, A and B), and expression is maintained within the pouch and throughout anterior pituitary development, in addition to the overlying presumptive hypothalamus and generally within neural ectoderm. SOX2 transcripts and protein are also present within the cells lining the lumen of the maturing anterior gland during Fs (Fig. 4, C and D; data not shown). No expression was observed in the infundibulum or posterior pituitary.
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). In later stages of fetal development, SOX2 is expressed abundantly in the thalamus, and specific populations of SOX2 positive cells are detected throughout the hypothalamus, which may represent neuronal and/or glial cell populations (Fig. 4, F–H). SOX2 also shows significant expression along the entire length of the developing neural tube, in addition to the dorsal root ganglia in sections analyzed from CS14–CS20 (data not shown).
Expression of SOX2 was observed during eye development at all stages investigated between CS16 and F2. At CS16, low levels of SOX2 expression are detected in the developing lens vesicle (Fig. 4I) and proximally within the developing neural retina, with a boundary of expression at the distal region of the retina. By CS20, and during Fs thereafter, SOX2 expression is limited to the inner layer of the neural retina, and is also present in the optic nerve (Fig. 4J). Expression within the lens appears to be maintained at low levels as the lens differentiates, becoming undetectable by in situ hybridization after F2 (9-wk development; data not shown). SOX2 is also expressed within the cells lining the nasal epithelium where SOX2 continues to be detected during fetal development, in addition to the developing laryngopharynx extending ventrally at the point of division of the trachea and esophagus, as well as in the embryonic lung (Fig. 4, K and L; data not shown).
In situ hybridization and immunohistochemistry failed to detect the presence of SOX2 within the developing gonad from CS14 onwards. Using RT PCR analysis, we detected SOX2 transcripts in total RNA extracted from the developing gonads of both males, from 7 wk (the earliest material available) through to 12 wk, and females at 10-wk development (data not shown). Strong expression was observed in the epithelium lining the lumen of the stomach and gut endoderm. No expression was observed by in situ hybridization in the heart, kidneys, liver, adrenal glands, or pancreas at any stage investigated (data not shown).
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Discussion |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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Mice with a heterozygous disruption of Sox2 also display endocrine deficits and hypoplasia of the anterior pituitary; moreover, SOX2 is expressed in the developing hypothalamus and Rathke’s pouch in both mice and humans, suggesting a direct role for the gene in hypothalamo-pituitary development in both species. The phenotype observed in patients of anterior pituitary hypoplasia with isolated gonadotropin deficiency is unusual, particularly because other pituitary hormone axes appear to be clinically unaffected in the majority of patients, although a few patients do exhibit GH insufficiency. It is of interest to note that SOX2 expression is not uniform in the developing hypothalamus in humans (Fig. 4, G and H), suggesting that haploinsufficiency for SOX2 may affect only certain populations of glia, neuroendocrine neurons, their progenitors, or their afferent inputs. The report of a patient with isolated hypogonadotropic hypogonadism without pituitary hypoplasia on imaging (26) suggests that SOX2 might be involved independently at multiple levels during the development of the hypothalamo-pituitary-gonadal axis. Because mice with haploinsufficiency of Sox2 show more generalized pituitary deficits, the apparent selectivity for SOX2 mutations impairing the gonadotrope axis in human subjects remains intriguing.
The ocular phenotype associated with SOX2 mutations is fully penetrant in all patients described to date; however, this may be a result of ascertainment bias with patients selected for screening based on the manifestation of bilateral anophthalmia or microphthalmia. On the other hand, we have previously screened a cohort of 235 patients with varying degrees of hypopituitarism and eye defects, and only identified pathogenic mutations in those with bilateral anophthalmia/microphthalmia. More recently, we have not detected any coding sequence variations in SOX2 in a small cohort of patients (n = 20) with isolated hypogonadotropic hypogonadism (data not shown). The manifestation of hypogonadotropic hypogonadism with unilateral eye disease and no other obvious neurological defects in the patient described by Sato et al. (26) suggests that in humans, the hypothalamo-pituitary-gonadal axis may be more sensitive to SOX2 levels, or timing of expression, than other tissues. Consistent with this, we note that mice do not display an eye phenotype unless the level of Sox2 expression is reduced to levels less than 40% that of wild type (27), whereas heterozygous knockout mice do exhibit abnormal pituitary morphogenesis and endocrine deficits (8).
It is always difficult to assign quantitative loss of function to different mutations based on in vitro model assays on a single reporter target, but there is good reason to suppose that the effects we report on the β-catenin/Wnt signaling pathway could be relevant. Previous studies have shown that murine Sox2, as well as Xenopus XSox3, XSox17
, and XSox17β, are capable of associating with β-catenin and repressing the activity of a β-catenin responsive reporter (13, 14, 28). This inhibitory activity is mediated by the COOH terminal domain and is independent of either the HMG domain or the ability to bind DNA. We have shown that human SOX2 protein is also capable of inhibiting β-catenin-mediated reporter gene activation, and that several naturally occurring de novo mutations, producing truncated mutant proteins of varying length, were unable to repress this activity in vitro. Both the c.387_388delC and p.Q177X mutations retain the ability to bind DNA (8) but show impaired repression of β-catenin. This suggests that the ability of SOX2 to repress transcription in this assay is not a result of competition for binding sites on the reporter construct.
Wnt-β-catenin signaling pathways provide key signals for forebrain and pituitary development, and members of the Tcf/Lef family of transcription factors show distinct patterns of expression in the developing anterior pituitary gland. The formation of a complex between β-catenin and Prop1 mediates the attenuation of expression of the transcriptional repressor Hesx1, and simultaneous activation of the pituitary specific transcription factor Pou1f1 (Pit1) (11); both of these events are critical for the determination of specific hormone secreting cell types (29, 30, 31). Furthermore, premature activation of β-catenin signaling in the developing anterior pituitary in mice results in complete loss of the gland by E13.5 (11). Hesx1 is an interesting target in this context because it is also implicated in Wnt signaling pathways during forebrain development, demonstrated by the abnormal domain of Wnt1 expression and ectopic activation of Wnt/β-catenin targets in Hesx1 mutant embryos (12). Furthermore, the phenotype associated with mutations in HESX1 in humans is somewhat reminiscent of that in individuals with SOX2 mutations, including ocular abnormalities, anterior pituitary hypoplasia, and midline forebrain abnormalities (8, 32, 33, 34). Disruption of these interactions with β-catenin and consequent effects on the regulation of β-catenin target genes may be one mechanism by which loss-of-function mutations in SOX2 can result in such a pleiotropic phenotype.
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Acknowledgments |
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Footnotes |
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Disclosure Statement: The authors have nothing to disclose.
First Published Online February 19, 2008
Abbreviations: CS, Carnegie Stage; E, embryonic d; F, fetal stage; FT4, free T4; HMG, high mobility group; MRI, magnetic resonance imaging; NR, normal range; SDS, SD score; TBST, Tris-buffered saline with Tween 20.
Received October 22, 2007.
Accepted February 13, 2008.
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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