The phenotypic spectrum associated with OTX2 mutations in humans.

Objective: The transcription factor OTX2 is implicated in ocular, craniofacial, and pituitary development. Design: We aimed to establish the contribution of OTX2 mutations in congenital hypopituitarism patients with/without eye abnormalities, study functional consequences, and establish OTX2 expression in the human brain, with a view to investigate the mechanism of action. Methods: We screened patients from the UK (n = 103), international centres (n = 24), and Brazil (n = 282); 145 were within the septo-optic dysplasia spectrum, and 264 had no eye phenotype. Transactivation ability of OTX2 variants was analysed in murine hypothalamic GT1-7 neurons. In situ hybridization was performed on human embryonic brain sections. Genetically engineered mice were generated with a series of C-terminal OTX2 variants. Results: Two chromosomal deletions and six haploinsufficient mutations were identified in individuals with eye abnormalities; an affected relative of one patient harboured the same mutation without an ocular phenotype. OTX2 truncations led to significant transactivation reduction. A missense variant was identified in another patient without eye abnormalities; however, studies revealed it was most likely not causative. In the mouse, truncations proximal to aa219 caused anophthalmia, while distal truncations and the missense variant were tolerated. During human embryogenesis, OTX2 was expressed in the posterior pituitary, retina, ear, thalamus, choroid plexus, and partially in the hypothalamus, but not in the anterior pituitary. Conclusions: OTX2 mutations are rarely associated with hypopituitarism in isolation without eye abnormalities, and may be variably penetrant, even within the same pedigree. Our data suggest that the endocrine phenotypes in patients with OTX2 mutations are of hypothalamic origin.

Activating mutations in BRAF disrupt the hypothalamo-pituitary axis leading to hypopituitarism in mice and humans.

Germline mutations in BRAF and other components of the MAPK pathway are associated with the congenital syndromes collectively known as RASopathies. Here, we report the association of Septo-Optic Dysplasia (SOD) including hypopituitarism and Cardio-Facio-Cutaneous (CFC) syndrome in patients harbouring mutations in BRAF. Phosphoproteomic analyses demonstrate that these genetic variants are gain-of-function mutations leading to activation of the MAPK pathway. Activation of the MAPK pathway by conditional expression of the BrafV600E/+ allele, or the knock-in BrafQ241R/+ allele (corresponding to the most frequent human CFC-causing mutation, BRAF p.Q257R), leads to abnormal cell lineage determination and terminal differentiation of hormone-producing cells, causing hypopituitarism. Expression of the BrafV600E/+ allele in embryonic pituitary progenitors leads to an increased expression of cell cycle inhibitors, cell growth arrest and apoptosis, but not tumour formation. Our findings show a critical role of BRAF in hypothalamo-pituitary-axis development both in mouse and human and implicate mutations found in RASopathies as a cause of endocrine deficiencies in humans.

Otx2b mutant zebrafish have pituitary, eye and mandible defects that model mammalian disease.

Combined pituitary hormone deficiency (CPHD) is a genetically heterogeneous disorder caused by mutations in over 30 genes. The loss-of-function mutations in many of these genes, including orthodenticle homeobox 2 (OTX2), can present with a broad range of clinical symptoms, which provides a challenge for predicting phenotype from genotype. Another challenge in human genetics is functional evaluation of rare genetic variants that are predicted to be deleterious. Zebrafish are an excellent vertebrate model for evaluating gene function and disease pathogenesis, especially because large numbers of progeny can be obtained, overcoming the challenge of individual variation. To clarify the utility of zebrafish for the analysis of CPHD-related genes, we analyzed the effect of OTX2 loss of function in zebrafish. The otx2b gene is expressed in the developing hypothalamus, and otx2bhu3625/hu3625 fish exhibit multiple defects in the development of head structures and are not viable past 10 days post fertilization (dpf). Otx2bhu3625/hu3625 fish have a small hypothalamus and low expression of pituitary growth hormone and prolactin (prl). The gills of otx2bhu3625/hu3625 fish have weak sodium influx, consistent with the role of prolactin in osmoregulation. The otx2bhu3625/hu3625 eyes are microphthalmic with colobomas, which may underlie the inability of the mutant fish to find food. The small pituitary and eyes are associated with reduced cell proliferation and increased apoptosis evident at 3 and 5 dpf, respectively. These observations establish the zebrafish as a useful tool for the analysis of CPHD genes with variable and complex phenotypes.

ZBTB20 is required for anterior pituitary development and lactotrope specification.

The anterior pituitary harbours five distinct hormone-producing cell types, and their cellular differentiation is a highly regulated and coordinated process. Here we show that ZBTB20 is essential for anterior pituitary development and lactotrope specification in mice. In anterior pituitary, ZBTB20 is highly expressed by all the mature endocrine cell types, and to some less extent by somatolactotropes, the precursors of prolactin (PRL)-producing lactotropes. Disruption of Zbtb20 leads to anterior pituitary hypoplasia, hypopituitary dwarfism and a complete loss of mature lactotropes. In ZBTB20-null mice, although lactotrope lineage commitment is normally initiated, somatolactotropes exhibit profound defects in lineage specification and expansion. Furthermore, endogenous ZBTB20 protein binds to Prl promoter, and its knockdown decreases PRL expression and secretion in a lactotrope cell line MMQ. In addition, ZBTB20 overexpression enhances the transcriptional activity of Prl promoter in vitro. In conclusion, our findings point to ZBTB20 as a critical regulator of anterior pituitary development and lactotrope specification.

Heterozygous defects in PAX6 gene and congenital hypopituitarism.

BACKGROUND: The prevalence of congenital hypopituitarism (CH) attributable to known transcription factor mutations appears to be rare and other causative genes for CH remain to be identified. Due to the sporadic occurrence of CH, de novo chromosomal rearrangements could be one of the molecular mechanisms participating in its etiology, especially in syndromic cases. OBJECTIVE: To identify the role of copy number variations (CNVs) in the etiology of CH and to identify novel genes implicated in CH. SUBJECTS AND METHODS: We enrolled 88 (syndromic: 30; non-syndromic: 58) Japanese CH patients. We performed an array comparative genomic hybridization screening in the 30 syndromic CH patients. For all the 88 patients, we analyzed PAX6 by PCR-based sequencing. RESULTS: We identified one heterozygous 310-kb deletion of the PAX6 enhancer region in one patient showing isolated GH deficiency (IGHD), cleft palate, and optic disc cupping. We also identified one heterozygous 6.5-Mb deletion encompassing OTX2 in a patient with bilateral anophthalmia and multiple pituitary hormone deficiency. We identified a novel PAX6 mutation, namely p.N116S in one non-syndromic CH patient showing IGHD. The p.N116S PAX6 was associated with an impairment of the transactivation capacities of the PAX6-binding elements. CONCLUSIONS: This study showed that heterozygous PAX6 mutations are associated with CH patients. PAX6 mutations may be associated with diverse clinical features ranging from severely impaired ocular and pituitary development to apparently normal phenotype. Overall, this study identified causative CNVs with a possible role in the etiology of CH in <10% of syndromic CH patients.

Genetic aspects of hypothalamic and pituitary gland development.

Hypothalamo-pituitary development during embryogenesis is a highly complex process involving the interaction of a network of spatiotemporally regulated signaling molecules and transcription factors. Mutations in any of the genes encoding these components can lead to congenital hypopituitarism, which is often associated with a wide spectrum of defects affecting craniofacial/midline development. In turn, these defects can be incompatible with life, or lead to disorders encompassing holoprosencephaly (HPE) and cleft palate, and septo-optic dysplasia (SOD). In recent years, there has been increasing evidence of an overlapping genotype between this spectrum of disorders and Kallmann syndrome (KS), defined as the association of hypogonadotropic hypogonadism (HH) and anosmia. This is consistent with the known phenotypic overlap between these disorders and opens a new avenue of identifying novel genetic causes of the hypopituitarism spectrum. This chapter reviews the genetic and molecular events leading to the successful development of the hypothalamo-pituitary axis during embryogenesis, and focuses on genes in which variations/mutations occur, leading to congenital hypopituitarism and associated defects.

ARNT2 mutation causes hypopituitarism, post-natal microcephaly, visual and renal anomalies.

We describe a previously unreported syndrome characterized by secondary (post-natal) microcephaly with fronto-temporal lobe hypoplasia, multiple pituitary hormone deficiency, seizures, severe visual impairment and abnormalities of the kidneys and urinary tract in a highly consanguineous family with six affected children. Homozygosity mapping and exome sequencing revealed a novel homozygous frameshift mutation in the basic helix-loop-helix transcription factor gene ARNT2 (c.1373_1374dupTC) in affected individuals. This mutation results in absence of detectable levels of ARNT2 transcript and protein from patient fibroblasts compared with controls, consistent with nonsense-mediated decay of the mutant transcript and loss of ARNT2 function. We also show expression of ARNT2 within the central nervous system, including the hypothalamus, as well as the renal tract during human embryonic development. The progressive neurological abnormalities, congenital hypopituitarism and post-retinal visual pathway dysfunction in affected individuals demonstrates for the first time the essential role of ARNT2 in the development of the hypothalamo-pituitary axis, post-natal brain growth, and visual and renal function in humans.

Neurofibromin regulates somatic growth through the hypothalamic-pituitary axis.

To study the role of the neurofibromatosis-1 (NF1) gene in mammalian brain development, we recently generated mice in which Nf1 gene inactivation occurs in neuroglial progenitor cells using the brain lipid binding protein (BLBP) promoter. We found that Nf1(BLBP)CKO mice exhibit significantly reduced body weights and anterior pituitary gland sizes. We further demonstrate that the small anterior pituitary size reflects loss of neurofibromin expression in the hypothalamus, leading to reduced growth hormone releasing hormone, pituitary growth hormone (GH) and liver insulin-like growth factor-1 (IGF1) production. Since neurofibromin both negatively regulates Ras activity and positively modulates cAMP levels, we examined the signaling pathway responsible for these abnormalities. While BLBP-mediated expression of an activated Ras molecule did not recapitulate the body weight and hypothalamic/pituitary defects, treatment of Nf1(BLBP)CKO mice with rolipram to increase cAMP levels resulted in a partial restoration of the body weight phenotype. Furthermore, conditional expression of the Ras regulatory GAP domain of neurofibromin also did not rescue the body weight or Igf1 mRNA defects in Nf1(BLBP)CKO mice. Collectively, these data demonstrate a critical role for neurofibromin in hypothalamic-pituitary axis function and provide further insights into the short stature and GH deficits seen in children with NF1.

Developmental disorders of the hypothalamus and pituitary gland associated with congenital hypopituitarism.

The pituitary gland is a complex organ secreting six hormones from five different cell types. It is the end product of a carefully orchestrated pattern of expression of signalling molecules and transcription factors. Naturally occurring and transgenic murine models have demonstrated a role for many of these molecules in the aetiology of congenital hypopituitarism. These include the transcription factors HESX1, PROP1, POU1F1, LHX3, LHX4, PITX1, PITX2, SOX2 and SOX3. The expression pattern of these transcription factors dictates the phenotype that results when the gene encoding the relevant transcription factor is mutated. The highly variable phenotype may consist of isolated hypopituitarism or more complex disorders such as septo-optic dysplasia and holoprosencephaly. However, the overall incidence of mutations in known transcription factors in patients with hypopituitarism is low, indicating that many genes remain to be identified; characterization of these will further elucidate the pathogenesis of this complex condition and also shed light on normal pituitary development and function.

Hypothalamic and pituitary development: novel insights into the aetiology.

The anterior pituitary gland is a central regulator of growth, reproduction and homeostasis, and is the end-product of a carefully orchestrated pattern of expression of signalling molecules and transcription factors leading to the development of this complex organ secreting six hormones from five different cell types. Naturally occurring and transgenic murine models have demonstrated a role for many of these molecules in the aetiology of combined pituitary hormone deficiency (CPHD). These include the transcription factors HESX1, PROP1, POU1F1, LHX3, LHX4, TBX19, SOX2 and SOX3. The expression pattern of these transcription factors dictates the phenotype that results when the gene encoding the relevant transcription factor is mutated. The highly variable phenotype may consist of isolated hypopituitarism, or more complex disorders such as septo-optic dysplasia and holoprosencephaly. Since mutations in any one transcription factor are uncommon, and since the overall incidence of mutations in known transcription factors is low in patients with CPHD, it is clear that many genes remain to be identified, and the characterization of these will further elucidate the pathogenesis of these complex conditions and also shed light on normal pituitary development.

The role of transcription factors implicated in anterior pituitary development in the aetiology of congenital hypopituitarism.

The anterior pituitary gland is a central regulator of growth, reproduction and homeostasis, and is the end-product of a carefully orchestrated pattern of expression of signalling molecules and transcription factors leading to the development of this complex organ secreting six hormones from five different cell types. Naturally occurring and transgenic murine models have demonstrated a role for many of these molecules in the aetiology of combined pituitary hormone deficiency (CPHD). These include the transcription factors HESX1, PROP1, POU1FI, LHX3, LHX4, TBX19 (TPIT), SOX3 and SOX2. The expression pattern of these transcription factors, their interaction with co-factors and their impact on target genes dictate the phenotype that results when the gene encoding the relevant transcription factor is mutated. The highly variable phenotype may consist of isolated hypopituitarism, or more complex disorders such as septo-optic dysplasia (SOD) and holoprosencephaly. Since mutations in any one transcription factor are uncommon, and since the overall incidence of mutations in known transcription factors is low in patients with CPHD, it is clear that many genes remain to be identified, and characterization of these will further elucidate the pathogenesis of these complex conditions, and also shed light on normal pituitary development.

A previously unidentified amino-terminal domain regulates transcriptional activity of wild-type and disease-associated human GLI2.

Zinc finger-containing Gli proteins mediate responsiveness to Hedgehog (Hh) signaling, with Gli2 acting as the major transcriptional activator in this pathway in mice. The discovery of disease-associated mutations points to a critical role for GLI2 in human Hh signaling as well. Here, we show that human GLI2 contains previously undescribed 5' sequence, extending the amino-terminus an additional 328 amino acids. In vitro, transcriptional activity of full-length GLI2 is up to 30 times lower than that of GLI2DeltaN (previously thought to represent the entire GLI2 protein), revealing the presence of an amino-terminal repressor domain in the full-length protein. GLI2DeltaN also exhibits potent transcriptional activity in vivo: overexpression in mouse skin leads to the formation of Hh-independent epithelial downgrowths resembling basal cell carcinomas, which in humans are associated with constitutive Hh signaling. The discovery of this additional, functionally relevant GLI2 sequence led us to re-examine several pathogenic human GLI2 mutants, now containing the entire amino-terminal domain. On the basis of the functional domains affected by the mutations, mutant GLI2 proteins exhibited either loss-of-function or dominant-negative activity. Moreover, deletion of the amino-terminus abrogated dominant-negative activity of mutant GLI2, revealing that this domain is required for transcriptional repressor activity of pathogenic GLI2. Our results establish the presence of an amino-terminal transcriptional repressor domain that plays a critical role in modulating the function of wild-type GLI2 and is essential for dominant-negative activity of a GLI2 mutant associated with human disease.

Array comparative genomic hybridisation analysis of boys with X linked hypopituitarism identifies a 3.9 Mb duplicated critical region at Xq27 containing SOX3.

INTRODUCTION: Array comparative genomic hybridisation (array CGH) is a powerful method that detects alteration of gene copy number with greater resolution and efficiency than traditional methods. However, its ability to detect disease causing duplications in constitutional genomic DNA has not been shown. We developed an array CGH assay for X linked hypopituitarism, which is associated with duplication of Xq26-q27. METHODS: We generated custom BAC/PAC arrays that spanned the 7.3 Mb critical region at Xq26.1-q27.3, and used them to search for duplications in three previously uncharacterised families with X linked hypopituitarism. RESULTS: Validation experiments clearly identified Xq26-q27 duplications that we had previously mapped by fluorescence in situ hybridisation. Array CGH analysis of novel XH families identified three different Xq26-q27 duplications, which together refine the critical region to a 3.9 Mb interval at Xq27.2-q27.3. Expression analysis of six orthologous mouse genes from this region revealed that the transcription factor Sox3 is expressed at 11.5 and 12.5 days after conception in the infundibulum of the developing pituitary and the presumptive hypothalamus. DISCUSSION: Array CGH is a robust and sensitive method for identifying X chromosome duplications. The existence of different, overlapping Xq duplications in five kindreds indicates that X linked hypopituitarism is caused by increased gene dosage. Interestingly, all X linked hypopituitarism duplications contain SOX3. As mutation of this gene in human beings and mice results in hypopituitarism, we hypothesise that increased dosage of Sox3 causes perturbation of pituitary and hypothalamic development and may be the causative mechanism for X linked hypopituitarism.

Magnetic resonance imaging of the hypothalamus-pituitary unit in childrensuspected of hypopituitarism: who, how and when toinvestigate.

The magnetic resonance (MR) identification of pituitary hyperintensity in the posterior part of the sella has been the most striking recent finding contributing to the diagnosis of "idiopathic" and permanent GH deficiency (GHD). Moreover, advancements in DNA technology have shed new light on the study of the genetic causes of hypopituitarism. Abnormalities in two genes, the GH-N encoding the GH and the GHRH receptor (GHRH-R), have been identified, while mutations in five other gene-encoding transcription factors such as Pit-1, Prop-1, Hesx-1, Lhx-3 and Lhx-4 involved in anterior pituitary development, have also been described. MR imaging shows marked differences in pituitary morphology indicating different GHD etiologies and different prognoses. Ectopic posterior pituitary is a specific marker of permanent GHD. These patients do not have Pit-1, Prop-1, or Lhx-3 mutations and should be carefully monitored for evolving pituitary hormone defects, though they do not require GH re-evaluation in adulthood; selected cases may have Hesx-1 or Lhx-4 mutations. MR evidence of normal or small anterior pituitary gland, enlarged empty sella, pituitary hyperplasia and/or intrasellar or suprasellar mass when associated with combined pituitary hormone deficiency call for molecular analysis of Pit-1, Prop-1, Hesx-1, or Lhx-3. Limitation of neck rotation and Chiari-I malformation may suggest Lhx-3 or Lhx-4 mutations (exceedingly rare). In "idiopathic" isolated GHD, evidence of normal anterior or small anterior pituitary size with normal location of posterior pituitary and normal connection between the hypothalamus and pituitary gland is suggestive of "transitory" or false positive GHD; patients with such characteristics should be re-evaluated well before reaching adult height. In selected cases, anterior pituitary height that is 2 SD below age-adjusted normal pituitary height could be suggestive of GHRH-R gene defect; it is worth pointing out that normal pituitary MR together with severe GHD has been observed, though rarely, in subjects with a genetic origin of GHD.

Prolactin replacement must be continuous and initiated prior to 21 d of age to maintain hypothalamic dopaminergic neurons in hypopituitary mice.

The prolactin (PRL) deficit in mice homozygous for the spontaneous Ames dwarf (df) mutation coincides with a marked reduction in the number of PRL-regulating tuberoinfundibular dopaminergic (TIDA) neurons. The TIDA deficit develops after 14 21 d postnatally and may be prevented by PRL replacement initiated at 12, but not at 60, d of age. The present study was designed to define further the developmental period during which PRL can prevent the deficit in the number of TIDA neurons in df/df mice, as well as to evaluate whether exposure to PRL neonatally affects the response to PRL by TIDA neurons in later development. To address the first aim, litters of df/df and normal (DF/df) mice were treated daily with ovine PRL (50 microg intraperitoneally), starting at 12, 21, or 30 d of age. To address the second aim, DF/df and df/df animals treated with PRL for 30 d starting at 12 d of age were subjected to PRL withdrawal (15 d of saline vehicle treatment), followed by PRL retreatment. All brains were evaluated using both catecholamine histofluorescence and tyrosine hydroxylase (TH) immunocytochemistry. Total numbers of TH-immunostained cells were counted in area A12 (TIDA neurons) and in A13 (medial zona incerta). Qualitatively, catecholamine fluorescence in A12 perikarya and terminals in df/df mice was enhanced by PRL treatment initiated at 12 or 21, but not at 30, d of age. TH immunostaining intensity was enhanced in all df/df PRL-treated groups, compared with saline treatment. However, total numbers of TH-positive TIDA neurons were reduced significantly in df/df mice treated with PRL beginning at 21 or 30 d, as well as with saline at 12 d, compared with similarly treated DF/df groups and with df/df animals treated with PRL beginning at 12 d (p < 0.01 for all comparisons). Among dwarf mice treated with PRL beginning at 12 d, followed by PRL withdrawal, the numbers of TH-positive TIDA neurons were greater than those of saline-treated dwarfs, but less than those in DF/df mice (p < 0.05 for both comparisons). In dwarfs retreated with PRL after withdrawal, the TIDA population was also smaller than that in normal animals (p < 0.05), although it was larger than in vehicle-treated dwarfs of the same age (p < 0.05).

Combined pituitary hormone deficiency caused by a novel mutation of a highly conserved residue (F88S) in the homeodomain of PROP-1.

Mutations in the pituitary-specific paired-like homeodomain transcription factor, PROP-1, result in combined pituitary hormone deficiency. We studied a Brazilian girl, offspring of first cousins, who presented with short stature and deficiencies of GH, TSH, PRL, LH, and FSH. Her cortisol response to hypoglycemia was determined at age 4.9, 10.7, and 14.1 yr and remained normal. Magnetic resonance imaging at the age of 9 yr revealed an anterior pituitary lobe of diminished height (3 mm; normal, 4.5 +/- 0.6), but radiography revealed a sella turcica volume above the normal mean. Direct sequencing of the PROP-1 gene revealed homozygosity for a novel 263T>C transition that results in the replacement of a highly conserved phenylalanine by serine at codon 88 (F88S). F88 constitutes the hydrophobic core of the first helix of the homeodomain of PROP-1, and the substitution by the polar residue serine is expected to alter the secondary structure and impair binding of the mutated PROP-1 to DNA target sequences. The F88S mutation (which corresponds to murine F85S) was introduced into the murine Prop-1 complementary DNA and its consequences on DNA binding and trans-activation were assessed in vitro. In contrast to wild-type Prop-1, the F88S mutant showed no significant DNA binding to a PRDQ9 Prop-1 response element in gel shift assays. Transcriptional activation of a luciferase reporter gene containing a PRDQ9 site upstream of a simian virus 40 promoter was reduced to approximately 34% compared with that of wild-type Prop-1 in transiently transfected TSA-201 human embryonic kidney cells. The F88S mutation further expands the repertoire of mutations in PROP-1.

A dominant phenocopy of hypopituitarism in transgenic mice resulting from central nervous system synthesis of human growth hormone.

We have produced a line of transgenic mice in which expression of human GH has been detected only in the cerebral cortex. Both male and female transgenic mice are growth inhibited with respect to their nontransgenic littermates. Mouse GH mRNA and insulin-like growth factor-I mRNA levels in the pituitary and liver, respectively, are reduced, and circulating insulin-like growth factor-I levels are lower in these mice. Within the hypothalamus somatostatin mRNA levels are increased and GH-releasing factor mRNA levels are reduced compared to those in nontransgenic littermates. We suggest that the growth retardation in these mice is a consequence of the ectopic human GH disturbing the normal controls that regulate mouse GH synthesis and release from the pituitary. These mice provide a resource for analysis of the regulation of GH production and demonstrate that a dominant phenocopy can be made by producing transgenic mice that have local production of an extra-cellular hormone.

Isolated deficiency of tyrosine hydroxylase immunoreactivity in tuberoinfundibular neurons in pituitary prolactin-deficient Snell dwarf mice.

Light microscopic morphology and relative numbers of tyrosine hydroxylase (TH)-immunoreactive neurons were assessed in hypothalamus of hypopituitary Snell dwarf mice, compared with normal mice of the same strain. Qualitatively, a specific deficit in staining of tuberoinfundibular neurons was noted in dwarf hypothalamus; TH-positive terminals were absent in dwarf median eminence, and morphology of axons in this region was aberrant. Qualitative observations were supported by quantitative assessment of hypothalamic dopaminergic neuron groups. Dorsal and intermediate TH-positive cells numbered 80% of those normal mouse brain; tuberoinfundibular (A12) TH-positive neurons in dwarf hypothalamus were 2% of those in normal mouse brain. The results imply that absence of pituitary hormone feedback impedes both structural and biochemical development of hypophysiotropic hypothalamic neurons.

Multiple pituitary hormone deficiencies in eight siblings of one Jewish Moroccan family.

In a Jewish Moroccan inbred family, 8 of 12 siblings were found to have multiple deficiencies of pituitary hormones, including GH, TSH and gonadotrophins. The parents showed no deficiency and are in good health, as are the other 4 siblings. The investigations carried out indicate that in this family the etiology is hereditary in nature, probably being autosomal recessive, with the defect located in the pituitary gland.