Table 1. Endocrine investigations in patients carrying SOX2 heterozygous mutations Patient 1 (Ht SOX2 deletion) Patient 2 (c.143TC>AA, p.F48X) 1a - 1.40 1.41 270.7 6.84 13.70 - 0.19 0. 31 25.60 - 1b 8.98 1.68 291 0.60 22.51 - 0.10 0. 47 9.16 - normal range 4.5-12.5 0.9-1.9 0.4-4.5 96-502 - 5-25 - 1.24 - 48 1.42 - 18.05 27.80 0.20 1.73 normal range 0.7-1.94 51-303 0.80-3.90 - 5-25 - Total T4 (μg/dl) FT4 (ng/dl) TSH (mU/L) IGF-1 (μg/L) IGFBP3 (mg/L) Random GH (ng/ml) Basal cortisol (μg/dl) Peak cortisol (μg/dl) Basal LH (U/L) Basal FSH (U/L) Estradiol (pg/ml) Basal testosterone (μg/L) Testosterone at 3–week HCG test (μg/L) pubertal delay. She was the second child of nonconsanguineous parents born at term with a birth weight of –1.0 standard devia- tion scor (SDS), and had severely impaired language development and delayed motor milestones. At presentation, she was prepubertal (Tanner staging 1) with a height of 144.8 cm (–3.12 SDS). Basal endocrine investigations demonstrated undetectable estradiol with low basal gonadotropins and a ﬂat luteinising hormone (LH) and follicle stimulating hormone (FSH) response to GnRH stimula- tion conﬁrming a diagnosis of HH (Table 1). Magnetic resonance imaging (MRI) revealed a sellar tumor with a cystic component, extending into the suprasellar area (Fig. 1A), without evidence of compression syndrome. Hormone replacement treatment was de- clined and at the age of 24 years, she went on to develop spontaneous but incomplete puberty (breast Tanner stage 2). Repeat MRI at that stage as well as sequential MR imaging over a period of 10 years did not show any signiﬁcant change in the size or morphology of the tu- mor (Fig. 1B). There was no evidence of development of additional pituitary hormone deﬁciencies (Table 1) and the patient has been managed conservatively. Case 2 is a male infant, diagnosed at birth with bilateral anoph- thalmia, who was referred to the pediatric endocrine department at the age of 17 months for investigation of micropenis. He was the ﬁrst child of nonconsanguineous parents, born at term with a birth weight of 2.83 kg (–1.7 SDS). At the time of presentation, his weight was 8.6 kg (–2.59 SDS) with a length of 74.8 cm (–2.24 SDS). He had a stretched penile length of 2.5 cm and a hypoplastic scrotum with testes of 0.5–1 ml palpable high in the scrotal sacs. Endocrine investigations revealed a normal FT4 of 1.24 ng/dl (16 pmol/L, nor- mal range 9–25 pmol/L), peak cortisol to synacthen of 27.8 μg/dl (769 nmol/L) with a low IGF1 48 μg/L (normal range 51–303 μg/L). A 3-week hCG test demonstrated a testosterone response that was consistent with HH (1.73 μg/L, 6.0 nmol/L). MRI at that point re- vealed a pituitary mass with a cystic component extending to the suprasellar area (Fig. 1D). Review of an MRI scan performed in the neonatal period, which was thought to have been normal apart from absent prechiasmatic optic nerves, revealed that the mass had been present at that stage. Subsequent imaging demonstrated a modest increase in size between the age of 17 and 32 months (Fig. 1E). Based on the auxological data and the low IGF-1, he commenced treatment with rhGH (0.025 mg/kg/day) and he is under regular clinical and neuroradiological follow-up. In both cases, there was no family history of eye abnormalities, pubertal delay, or infertility. DNA analysis using array CGH identiﬁed a heterozygous 731-kb deletion on Chr3q26 encompassing SOX2 in patient 1 (Fig. 1C); this deletion was not identiﬁed in the unaffected mother, but paternal DNA was unavailable. Patient 2 was heterozygous for a mutation (c.143TC>AA) resulting in the substitution of phenylalanine at po- sition 48 by a stop codon, predicted to generate a truncated protein lacking most of the HMG domain and the C-terminal domain of SOX2 (Fig. 1F). DNA from either parent was unavailable. To characterize the functional consequences of the p.F48X trun- cated protein, we ﬁrst analyzed the cellular localization of this mu- tant protein in HEK293T cells (Fig. 1G). Anti-FLAG immunostain- ing of cells transfected with wild-type FLAG-SOX2 revealed normal nuclear localization of the protein. However, FLAG-p.F48X protein was detected mainly in the cytoplasm, suggesting a failure of the mu- tant protein to concentrate in the nucleus. This is not unexpected as this mutation introduces a stop codon prior to the HMG DNA- binding domain, which contains the nuclear localization signals. Next, we assessed the transcriptional activity of the p.F48X mu- tant protein on the murine Hesx1 promoter, which contains SOX- binding sites and has been previously shown to be regulated by SOX2 [Eroshkin et al., 2002] (Fig. 1H). Transfection of plasmids expressing wild-type SOX2 led to a 3.31-fold (±0.44 SD) increase in luciferase expression levels relative to cells transfected with empty vector. In contrast, expression of the p.F48X mutant protein re- sulted in only a 1.59-fold (±0.36 SD) activation of the basal reporter activity (t-test, P = 0.0006). Similar effects have been reported for other SOX2 mutant proteins previously identiﬁed in patients with hypopituitarism and eye defects [Kelberman et al., 2006, 2008]. Finally, we investigated the ability of the p.F48X mutant protein to antagonize the β-catenin-mediated activation of the TOPFLASH reporter [Korinek et al., 1997] (Fig. 1I). Transfection of the con- stitutively active p.S33Y mutant β-catenin resulted in an 89.7-fold (±4.6 SD) activation of the basal TOPFLASH reporter activity. This transactivation of the reporter was signiﬁcantly reduced when cells were cotransfected with plasmids expressing wild-type SOX2 (2.8- fold, ±0.34 SD, t-test, P < 0.0001). In contrast, cotransfection of a construct expressing the p.F48X mutant SOX2 failed to suppress the β-catenin-mediated activation of TOPFLASH compared with the wild-type SOX2 (72.3-fold, ±4.91 SD, t-test, P = 0.0002). These differences were not due to impaired synthesis of SOX2 protein, as conﬁrmed by western blot using whole-cell extracts following trans- fection with wild-type and p.F48X FLAG-SOX2 (Fig 1J). Together, these experiments demonstrate that the p.F48X mutant protein is severely impaired in its transactivation activity as well as its ability to antagonise β-catenin-mediated transcriptional activation. The possible oncogenic potential of SOX2 has been demonstrated in breast cancer [Chen et al., 2008] and its expression is upregulated in up to 23% of squamous cell lung cancers [Bass et al., 2009] as well HUMAN MUTATION, Vol. 32, No. 12, 1376–1380, 2011 1377