2.3.1 Development of the pituitary and genetic forms of hypopituitarism

Pituitary development occurs in distinct and sequential developmental steps, leading to the formation of a complex organ containing five different cell types secreting six different hormones. During this process the sequential temporal and spatial expression of a cascade of signalling molecules and transcription factors play a crucial role in organ commitment, cell proliferation, patterning, and terminal differentiation. Complex regulatory networks govern the process during which distinct cell types emerge from a common primordium. The mechanisms are not fully elucidated but it seems that opposing signalling gradients induce expression of interacting transcriptional regulators (activators or repressors) in overlapping patterns that act synergistically. Spontaneous or artificially induced mutations in the mouse and identification of mutations associated with human pituitary disease have contributed to defining the genetic cascades responsible for pituitary development.

The pituitary gland has a dual embryonic origin: the anterior and intermediate lobes are derived from the oral ectoderm whereas the posterior pituitary is derived from the neural ectoderm. The development of the pituitary gland has been studied extensively in the mouse and although relatively little is known about human pituitary development, it seems that it mirrors that in rodents (1) (Fig. 2.3.1.1).

The anterior pituitary develops from the hypophyseal or pituitary placode, one of the six cranial placodes that appear transiently as localized ectodermal thickenings in the prospective head of the developing embryo. The pituitary placode appears at embryonic day (E) 7.5 and is located ventrally in the midline of the anterior neural ridge and in continuity with the future hypothalamo-infundibular region, which is located posteriorly, in the rostral part of the neural plate. By E8.5 the neural tube has bent at the cephalic end and the placode appears as a thickening of the roof of the primitive oral cavity. At E9.0 the placode invaginates dorsally to form a rudimentary Rathke’s pouch, from which the anterior and intermediate lobes of the pituitary are derived. The definitive pouch is formed by E10.5, whereas the evagination of the neural ectoderm at the base of the developing diencephalon will give rise to the posterior pituitary. Between E10.5 and E12 the pouch epithelium continues to proliferate and separates from the underlying oral ectoderm at E12.5. The progenitors of the hormone-secreting cell types proliferate ventrally from the pouch between E12.5 and E15.5 and populate what will form the anterior lobe. The remnants of the dorsal portion of the pouch will form the intermediate lobe, whereas the lumen of the pouch remains as the pituitary cleft, separating the intermediate from the anterior lobe (2).

The development of the anterior pituitary gland is dependent upon a carefully orchestrated genetic cascade that then encodes extrinsic and intrinsic transcription factors and signalling molecules that are expressed in a temporally and spatially restricted manner (Fig. 2.3.1.2 and Fig. 2.3.1.3).

Extrinsic molecules from the ventral diencephalon (Bmp4, Fgf8, Fgf4, Nkx2.1, Wnt5α) as well as ventral signals from the oral ectoderm (Sonic hedgehog (Shh)), surrounding mesenchyme (Bmp2, Indian hedgehog IHH, chordin) and the pouch itself (Bmp2, Wnt4) create a network of signalling gradients, which is important for morphogenesis during early pituitary development (3, 4).

At least two sequential inductive signals from the diencephalon are required for the induction and formation of Rathke’s pouch (5). Bone morphogenetic protein 4 (Bmp4) is the earliest secreted signalling molecule detected at E8.5, followed by a second signal, fibroblast growth factor 8 (Fgf8). Fgf8 activates two key regulatory genes, LIM homoeobox 3 (Lhx3) and LIM homoeobox 4 (Lhx4), both of which are essential for subsequent development of the rudimentary pouch into a definitive pouch. Signalling from the ventral diencephalon is critical for normal anterior pituitary, e.g. murine mutations within the thyroid-specific enhancer binding protein (Ttf1 or Nkx2.1), only expressed in the presumptive ventral diencephalon, can cause severe defects in the development of not only the diencephalon but also the anterior pituitary gland.

At the early steps of pituitary development, Shh and its signalling pathway are also important for the patterning and morphogenesis of the gland as well as specification and expansion of ventral cell types. Shh null mice exhibit cyclopia and loss of midline structures of the brain. Shh binds to the transmembrane receptor Patched (PTC). This binding results in the release of the coreceptor Smoothened (SMO) and the activation of the downstream Gli transcription factors, which in turn act as activators (Gli1, Gli2) or repressors (Gli3). Shh is expressed in the ventral diencephalon and the oral ectoderm but its expression is excluded within Rathke’s pouch as soon as the pouch appears. Its expression is maintained throughout the ventral diencephalon until E14.9, when it disappears. However, its receptor (PTC) is expressed in Rathke’s pouch and Gli1-3 is expressed in the ventral diencephalon and the pouch (6). This pattern indicates that the developing gland can receive and respond to Shh signalling.

The Notch signalling pathway is an evolutionarily conserved mechanism implicated in many developmental processes. Molecules involved in Notch signalling (Jag1, Notch2, Notch3) and their downstream targets (Hes1) play critical roles in early steps of pituitary development. Notch signalling is required for maintaining expression of Prop1 which in turn is required for generation of the Pou1f1 (Pit-1) lineage. In the later phases of pituitary development, down-regulation of Notch signalling is necessary to permit terminal differentiation of the Pou1f1 cell lineage and maturation and proliferation of the GH-producing somatotrophs (7).

Lhx3 and Lhx4 are members of the LIM transcription factor family of homoeobox genes characterized by the presence of a unique cysteine/histidine-rich zinc-binding LIM domain (8). Lhx3 is one of the earliest transcription factors expressed in Rathke’s pouch (E9.5) and its expression is maintained forming a gradient of expression with higher levels being observed in the dorsal region. By E16.5, Lhx3 is expressed in the developing anterior and intermediate pituitary, but not in the posterior gland. Its expression persists throughout development and into adulthood. This highlights its importance for the establishment of hormone producing cell-types and may also play a role in the maintenance of some cell types in the mature pituitary. In addition, Lhx3 expression has also been detected in restricted regions of the central nervous system (CNS) and inner ear (9).

Lhx4 is closely related to Lhx3 and is also expressed in Rathke’s pouch at E9.5. However, expression of Lhx4 is restricted to the future anterior lobe, and is down-regulated at E15.5, therefore not persisting in the mature gland. Lhx4 is also expressed in specific fields in the developing hindbrain, cerebral cortex, and motor neurons of the spinal cord (10). Lhx3 null mice show early lethality and the anterior and intermediate lobes of the pituitary are lacking. Although Rathke’s pouch is initially formed, pituitary development is then arrested as there is failure to maintain Hesx1 expression and induce Pou1f1. There are some residual corticotrophs, but proopiomelanocortin (POMC)-expressing cells fail to proliferate, probably due to reduced expression of Tbx19 (T-Pit) (11). In Lhx4 null mice, Rathke’s pouch is formed and there is specification of all the anterior pituitary cell lines. However, their numbers are markedly reduced leading to anterior pituitary hypoplasia. Lhx3^–/–, Lhx4^–/– double mutant mice show a more severe phenotype than either single mutant, with an early arrest of pituitary development (1). This suggests that there is redundancy in their actions during pituitary development.

Hesx1 is a member of the paired-like class of homoeobox genes and one of the earliest markers of the pituitary primordium. During murine development Hesx1 is expressed early during gastrulation in a region that will become the forebrain and from E9.0 to 9.5 it is restricted to the ventral diencephalon and the developing Rathke’s pouch. From E12.5 its expression gradually disappears in a spatiotemporal sequence that corresponds to progressive pituitary cell differentiation and becomes undetectable by E15.5. Hesx1 is a transcriptional repressor and this activity is mediated by a conserved region in the N-terminal domain (the engrailed homology domain; eh-1) and the homoeodomain. The N-terminal domain binds TLE, a mammalian homologue of the Drosophila corepressor protein Groucho, whereas the homoeodomain interacts with the nuclear corepressor complex NCoR1/Sin3/HDAC, thus increasing Hesx1 repressor activity (12).

Lhx3 is important for maintaining Hesx1 expression, whereas Prop1/β-catenin is required for its repression (13). Down-regulation of Hesx1 is important for activation of other downstream genes such as Prop1 and the temporal regulation of their expression is critical for normal pituitary development. Prolonged expression of Hesx1 can block Prop1-dependent activation, whereas premature expression of Prop1 can block pituitary organogenesis.

The role of Hesx1 in pituitary development was elucidated by its targeted disruption in mice. Homozygous null animals had a reduction in the prospective forebrain tissue, absence of developing optic vesicles, optic cups, and olfactory placodes, markedly decreased head size, reduced telencephalic vesicles, severe microphthalmia, hypothalamic abnormalities, and abnormal morphogenesis of Rathke’s pouch. Although a small percentage (5%) of the most severely affected null mutants had complete lack of the pituitary, the majority had multiple oral ectodermal invaginations resulting in the apparent formation of multiple pituitary glands. The phenotype was variable and reminiscent of patients with septo-optic dysplasia (14).

In humans, homozygous and heterozygous mutations in HESX1 are associated with varying phenotypes characterized by isolated growth hormone deficiency, combined pituitary hormone deficiency, and septo-optic dysplasia.

The SOX family of transcription factors is characterized by the presence of a 79 amino acid high mobility group (HMG) DNA-binding domain which is similar to the HMG domain of the mammalian sex determining gene SRY. More than 20 SOX proteins and their genes have been identified and classified into eight groups, A–H. SOX3 was among the first of the SOX genes to be identified, and along with SOX1 and SOX2, belongs to the SOXB1 group, which exhibits the highest degree of similarity to SRY (15, 16).

During pituitary development SOX3 is expressed in the ventral diencephalon and infundibulum, but not in Rathke’s pouch. Targeted disruption of Sox3 in mice results in mutants with a variable phenotype, including craniofacial abnormalities, midline defects, and reduction in size and fertility. Mutant mice have variable endocrine deficits, including reduced growth hormone, luteinizing hormone, follicle-stimulating hormone (FSH), and thyroid-stimulating hormone, which correlates to body weight. The pituitary gland has an abnormal morphology with a hypoplastic anterior lobe and presence of additional abnormal clefts. In Sox3 mutants, Rathke’s pouch is bifurcated and the evagination of the infundibulum is less pronounced (17).

In the mouse, Sox2 expression is first detected before gastrulation at E2.5 at the morula stage. Following gastrulation, it is restricted to the presumptive neuroectoderm and by E9.5 it is expressed throughout the brain, CNS, sensory placodes, branchial arches, gut endoderm, the oesophagus, and the trachea. Homozygous loss of Sox2 results in peri-implantation lethality, whereas Sox2 heterozygous mice appear relatively normal but show a reduction in size and male fertility. Further studies that have resulted in the reduction of Sox2 expression levels below 40%, compared with normal levels, result in anophthalmia in the affected mutants (18). This highlights the fact that Sox2 function is dose dependent. Given the observation of growth retardation and reduced fertility, the role of Sox2 in murine pituitary development has been studied in detail. Sox2 expression is detected in the infundibulum and Rathke’s pouch at E11.5 but, as cell differentiation occurs, expression is confined to proliferative zones. In heterozygous mutant mice the morphogenesis of the gland was abnormal with bifurcation of Rathke’s pouch in a third of mutants at E12.5 and subsequent extra clefts in some of the adult pituitaries. Embryonic pituitaries at E18.5 were smaller and had significantly reduced numbers of somatotrophs and gonadotrophs, with reduced growth hormone content. Evaluation of hormonal content in 3-month-old heterozygotes showed that there was moderate reduction in growth hormone and luteinizing hormone, which was significant for males, whereas there was evidence that corticotrophs, lactotrophs, and thyrotrophs were also affected (19). In humans, mutations in SOX2 and SOX3 lead to variable hypopituitarism, as is described in the next section.

Terminal differentiation of cells in the anterior pituitary is the result of complex interactions between extrinsic signalling molecules and transcription factors (Lhx3, Lhx4, Gata2, Isl1, Prop1, Pou1f1). Differentiated hormone-producing pituitary cells emerge sequentially, at distinct positions in the anterior pituitary. Corticotrophs expressing POMC are the first to appear (E12.5), followed by thyrotrophs (E14.5), somatotrophs (E15.5), lactotrophs (E16.5), and finally gonadotrophs at around E16.5 (20, 21) (Fig. 2.3.1.4). Among the number of transcription factors involved, Prop1 and Pou1f1 are best characterized in terms of function in both humans and mice.

Prop1 (Prophet of Pit1) is a pituitary-specific paired-like homoeodomain transcription factor initially detected in the dorsal portion of Rathke’s pouch at E10–10.5. Its expression peaks at E12 and becomes undetectable by E15.5. Prop1 is both a transcriptional activator and repressor. Depending on associated cofactors, the β-catenin/Prop1 complex is important for activation of Pou1f1(Pit1) and repression of Hesx1. Temporal regulation of Prop1 expression is important for normal pituitary development. Premature expression of Prop1 in Rathke’s pouch leads to agenesis of the anterior pituitary, probably by repressing Hesx1 (13).

The Ames dwarf (df) mouse has a naturally occurring mutation in Prop1 that results in an eightfold reduction in DNA-binding activity compared with wild-type protein. Analysis of these animals showed that Prop1 is important for the determination of the three Pou1f1-dependent cell types and is also required for the generation of gonadotrophs. Homozygous Ames df mice exhibit severe proportional dwarfism, hypothyroidism, and infertility, and the emerging anterior pituitary gland is reduced in size by about 50% displaying an abnormal looping appearance. The adult Ames df mouse exhibits growth hormone, TSH, and prolactin deficiency resulting from a severe reduction of somatotroph, lactotroph, and caudomedial thyrotroph lineages. In addition, they exhibit reduced gonadotrophin expression correlating with low plasma luteinizing hormone and FSH concentrations (22).

Pou1f1 (Pit-1) is a pituitary specific transcription factor which belongs to the POU-homoeodomain family (23). It is expressed late during pituitary development (E13.5) and its expression persists throughout adulthood. Autoregulation of Pou1f1 is required to sustain its expression, once it has reached a critical threshold. The role of Pou1f1 in pituitary development has been elucidated by the study of two naturally occurring murine models, the Snell and Jackson df mice. In the Snell df mouse a recessive point mutation results in absence of somatotrophs, lactotrophs, and thyrotrophs. A similar phenotype results in the Jackson df mouse that harbours a recessive null mutation due to rearrangement of Pou1f1.

Pou1f1 is important for: terminal differentiation and expansion of somatotrophs, lactotrophs, and thyrotrophs in the intermediate caudomedial field; repression of gonadotroph cell fate; and transcriptional regulation of genes encoding the hormones produced by the above cell types (GH1, PRL, TSHβ, GHRHR) (24).

The emergence of the gonadotroph cell lineage does not depend on Pou1f1. Gonadotrophs arise in the most ventral part of the anterior pituitary and are the last cells to differentiate. A number of transcription factors have been shown to determine the gonadotroph cell fate, including GATA2, SF1, Egr1, Pitx1, Pitx2, Prop1, and Otx1. The result is terminal cell differentiation and expression of the markers LHβ, FSFβ, and GnRHR.

In the most ventral aspect of the anterior pituitary high levels of GATA2 restrict Pou1f1 expression. In the absence of Pou1f1, GATA2 induces transcription factors that will determine gonadotroph differentiation, including SF1, P-Frk, and Isl-1. Conversely, in the dorsal aspect the absence of GATA2 is critical for the differentiation of the Pou1f1-positive cells (somatotrophs and lactotrophs); this induced gradient of GATA2 expression determines gonadotroph and thyrotroph cell lineages (25).

Steroidogenic factor 1 (SF1) is expressed in the gonadotrophs as well as in the developing gonads, adrenal glands and the ventromedial hypothalamus. It is a zinc-finger nuclear receptor that regulates a number of genes involved in sex determination, steroidogenesis, and reproduction, including αGSU, LHβ, FSHβ, and GnRHR. In the developing pituitary, GATA2 is capable of inducing SF1 expression in gonadotrophs (E13.5). SF1-knockout mice exhibit adrenal and gonadal agenesis, male-to-female sex reversal, ablation of the ventromedial hypothalamic nucleus and selective loss of gonadotrophin, αGSU and Gnrhr expression.

Pituitary specific inactivation of SF1 results in mice with hypoplastic gonads, a dramatic decrease in pituitary gonadotropin expression, and failure to develop normal secondary sexual characteristics, while the adrenal glands and hypothalamus are unaffected. In these models, expression of LHβ and FSHβ can be restored by high-dose gonadotropin-releasing hormone (GnRH), demonstrating that SF1 is necessary for maturation of gonadotrophs but not cell fate specification (26).

The function of gonadotrophs in the anterior pituitary is under the control of hypothalamic GnRH; it is synthesized by neurons in the preoptic region, which project axons to the median eminence, where they secrete GnRH. Neuroendocrine GnRH cells arise from the olfactory placode. It has been shown that Pax6 is required for the generation of GnRH neurons, as a mouse strain with mutation in Pax6 shows failure to develop both optical and olfactory placodes. Following their generation, GnRH cells migrate along the olfactory nerve pathway across the cribriform plate, towards the olfactory bulb and their final position in the hypothalamus (27). In humans, it is estimated that migration of the GnRH cells begins during the sixth week of gestation. An increasing number of genes are implicated in the migration and maturation of GnRH neurons (i.e. KAL1, FGFR1, FGF8, PROK2, PROKR2, Kiss-1, GPR54, leptin, CHD7, TAC3, TACR3). Their role is highlighted by mutations found in cases of isolated hypogonadotropic hypogonadism, as is mentioned later as well as in the relevant chapter.

Corticotrophs producing adrenocorticotropic hormone (ACTH) are the first cell type to reach terminal differentiation. However, relatively little is known about the factors that determine the specification of corticotrophs and melanotrophs and the control of POMC expression (28). Tbx19 (T-Pit) is a member of the T-box transcription factors. During mouse pituitary development, Tbx19 is expressed at E11.5, in the most ventral region of Rathke’s pouch, in corticotrophs and melanotrophs; along with Pitx1, Tbx19 activates the POMC promoter (29).

Congenital hypopituitarism encompasses a group of different disorders and may manifest as an isolated hormone deficiency, or alternatively several pituitary hormone axes may be defective resulting in combined pituitary hormone deficiency (CPHD). Isolated hormone deficiencies include isolated growth hormone deficiency (IGHD), ACTH deficiency, gonadotropin deficiency (hypogonadotropic hypogonadism), TSH deficiency or central diabetes insipidus. Combined pituitary hormone deficiencies may occur in isolation or be associated with extra-pituitary defects such as optic nerve hypoplasia or midline forebrain abnormalities.

An increasing number of genes are implicated in the aetiology of congenital hypopituitarism (Table 2.3.1.1). Although mouse models have enhanced our understanding of the genetic basis of hypopituitarism in humans, the correlation with disease phenotypes is variable. In general, mutations in genes involved in early development and patterning of the forebrain and pituitary tend to result in syndromic forms of hypopituitarism in association with extrapituitary defects and midline abnormalities. On the other hand, mutations in genes encoding specific hormone subunits or required for specification of particular cell types give rise to isolated pituitary hormone deficiencies (24, 30, 31).

The majority of cases of combined pituitary hormone deficiencies have no identified genetic aetiology. Among the genetic causes, a number of genes have been implicated, which result in (1) nonsyndromic combined pituitary hormone deficiencies (PROP1, POU1F1) or (2) syndromic combined pituitary hormone deficiencies in association with ocular defects, midline abnormalities or other features. The timing and combination of pituitary hormone deficiencies, neuroimaging, and associated features may guide the diagnosis. In many cases, however, the phenotype is variable and overlapping. Table 2.3.1.2 compares the phenotype in patients with hypopituitarism as a result of mutations in some of these genes.

PROP1 lies on chromosome 5q and consists of three exons encoding a protein of 226 amino acids. Recessive mutations in PROP1 are the commonest cause of CPHD, identified in approximately 50% of familial cases. In sporadic cases, however, the incidence is much lower (32). More than 20 mutations have been reported in PROP1. The most frequent (50–72%) is a 2 bp deletion (GA or AG) among three tandem GA repeats (296-GAGAGAG-302) within exon 2. This results in a frame shift at codon 109 and generates a truncated protein (S109X) which disrupts DNA-binding and transcriptional activation. The mutation has been detected in multiple unrelated families and represents a mutational hot spot; along with the 150delA mutation it accounts for approximately 97% of all mutations in PROP1.

The first reported mutations in PROP1 were in members of four unrelated pedigrees with growth hormone, TSH, prolactin, luteinizing hormone, and FSH deficiencies (33). In patients with mutations in PROP1, the timing and severity of hormonal deficiencies is variable. In general, deficiency in growth hormone, TSH, and prolactin is milder in patients with mutations in PROP1 rather than in POU1F1. Most patients present with early-onset growth hormone deficiency, however, normal growth in early childhood and normal final height has been reported in an untreated patient with a PROP1 mutation. The TSH deficiency varies and may not be present from birth. Although PROP1 is essential for the differentiation of gonadotrophs in fetal life, the spectrum of gonadotrophin deficiency is highly variable. It ranges from presentation with microphallus and undescended testes, hypogonadism with lack of puberty, to spontaneous pubertal development with subsequent arrest, and infertility. This variation in timing and severity of gonadotrophin deficiency suggests that PROP1 is required for maintenance or differentiation of gonadotrophs, rather than the cell fate determination. Individuals with mutations in PROP1 exhibit normal ACTH and cortisol concentrations in early life but often demonstrate an evolving cortisol deficiency associated with increasing age, although it has also been described in a 7-year-old patient. The underlying mechanism for cortisol deficiency is unknown, especially as PROP1 is not expressed in corticotrophs, but appears to be required for maintenance of the corticotroph population (34–36).

Most patients with mutations in PROP1 have a small or normal anterior pituitary, with normal pituitary stalk and posterior lobe. However, in some cases, an enlarged anterior pituitary has also been reported. Longitudinal analyses of anterior pituitary size have revealed that a significant number of patients demonstrate pituitary enlargement in early childhood, which can wax and wane in size, with subsequent involution in older patients. This pituitary enlargement consists of a mass lesion between the anterior and posterior lobes, possibly originating from the intermediate lobe (37).

POUIFI is on chromosome 3p11 and consists of six exons encoding a 291 amino acid protein. The first mutation within POUIFI was identified in a child with growth hormone, prolactin, and profound TSH deficiency. To date, the majority of identified mutations are recessive, although a number of heterozygous mutations have also been reported. Among them, the dominant R271W seems to be a mutational ‘hot spot’. Functional analysis suggests that some mutations disrupt DNA binding whereas others disrupt transcriptional activation or other properties such as autoregulation (38). Patients with POUIF1 mutations present with growth hormone, TSH, and prolactin deficiency, however, the spectrum of hormone deficiencies varies. Growth hormone and prolactin deficiencies present early in life, whereas TSH deficiency can present later in childhood, or TSH secretion may even be preserved. The anterior pituitary may be small or normal with no other extrapituitary or midline abnormalities (39).

Mutations in LHX3 and LHX4 are rare causes of hypopituitarism. LHX3 is located on chromosome 9q34. Homozygous mutations in LHX3 have been described in patients with growth hormone, prolactin, TSH, and luteinizing hormone/FSH deficiencies (40). Although ACTH secretion has been reported to be usually spared, there has been a recent report of ACTH deficiency in patients with LHX3 mutations. In addition to combined pituitary hormone deficiencies, patients present with a short rigid cervical spine with limited head rotation and trunk movement (41). Recently, sensorineural deafness of varying severity has been reported in association with homozygous loss of LHX3 (42). Pituitary morphology is also variable, ranging from a small to a markedly enlarged anterior pituitary, whereas a hypointense lesion with a ‘microadenoma’ has also been described.

LHX4 extends over 45 kb on chromosome 1q25. Heterozygous mutations within LHX4 have been described in patients with growth hormone deficiency and variable additional endocrine deficits and extrapituitary abnormalities. The first reported patient presented with growth hormone, TSH, and ACTH deficiency (43). The anterior pituitary was hypoplastic with an ectopic posterior pituitary and absent stalk. However, other affected patients from the same family presented with isolated growth hormone deficiency and normal posterior pituitary. Additional manifestations included a poorly formed sella and pointed cerebellar tonsils. Since then, patients with variable hypopituitarism, with or without an ectopic posterior pituitary and Chiari malformation have been reported (44, 45)

Septo-optic dysplasia is defined by any combination of optic nerve hypoplasia, midline forebrain defects (i.e. agenesis of the corpus callosum, absent septum pellucidum), and pituitary hypoplasia with variable hypopituitarism. It is a highly heterogeneous condition with a reported incidence of 1:10 000, and although it is generally sporadic, familial cases have been described. Approximately 30% of patients with septo-optic dysplasia manifest the complete clinical triad, 62% have some degree of hypopituitarism, and 60% have an absent septum pellucidum. Optic nerve hypoplasia may be unilateral (12%) or bilateral (88%) and may be the first presenting feature, with the later onset of endocrine dysfunction. In rare cases the eye abnormalities may be more severe (microphthalmia, anophthalmia). Neurological manifestations are common in patients with septo-optic dysplasia (75–80%) and range from focal deficits to global developmental delay.

Endocrine abnormalities vary from isolated growth hormone deficiency to panhypopituitarism. It is worth noting, however, that the endocrinopathy may be evolving with a progressive loss of endocrine function over time. The commonest endocrine defect is growth hormone deficiency followed by TSH and ACTH deficiency, whereas gonadotropin secretion may be retained. Either sexual precocity or failure to develop in puberty may occur and it has been noted that in children with septo-optic dysplasia, commencement of growth hormone treatment may be associated with accelerated pubertal maturation. In addition, abnormal hypothalamic neuroanatomy or function and diabetes insipidus may occur (46).

Genetic and environmental factors have been implicated in the aetiology of septo-optic dysplasia, including viral infections, vascular or degenerative disorders, and antenatal exposure to alcohol and drugs. The condition presents more commonly in children born to younger mothers and clusters in geographical areas with a high frequency of teenage pregnancies. As forebrain and pituitary development are closely linked and occur as early as 3–6 weeks’ gestation in the human embryo, any insult at this critical stage of development could account for the features of septo-optic dysplasia.

HESX1, SOX2, and SOX3 have all been implicated in the aetiology of SOD and its variants (46). HESX1 is located on chromosome 3p21.1-3p21.2; its coding region consists of four exons and spans 1.7 kb. Autosomal dominant and recessive mutations have been described in a number of patients, resulting in variable phenotype, without clear genotype–phenotype correlation (Table 2.3.1.3). The overall frequency of HESX1 mutations in septo-optic dysplasia is low (approximately 1%) suggesting that mutations in other genes may contribute to this complex disorder (14, 47).

Heterozygous de novo mutations in SOX2 have been reported in patients with bilateral anophthalmia or severe microphthalmia and additional abnormalities (developmental delay, learning difficulties, oesophageal atresia, and genital abnormalities) (48). Kelberman et al. first described in detail the pituitary phenotype in six patients with heterozygous loss of function mutations in SOX2, which comprised bilateral eye abnormalities, anterior pituitary hypoplasia and hypogonadotropic hypogonadism (HH) (49). Patients with SOX2 mutations may present with forebrain abnormalities and associated developmental disorders (Table 2.3.1.4). They are at high risk of developing HH, even if it is not manifest at diagnosis, and long-term follow-up is recommended (49).

A number of pedigrees have been described with X-linked hypopituitarism involving duplications of Xq26-q27, encompassing the SOX3 gene (50). The phenotype comprises variable learning difficulties and hypopituitarism associated with anterior pituitary hypoplasia, infundibular hypoplasia, and an ectopic posterior pituitary, with variable abnormalities of the corpus callosum. Further implication of SOX3 in hypopituitarism comes from the identification of affected patients with expansion of a polyalanine (PA) tract within the gene. In this case, as well, the phenotype is variable. PA expansion by 11 residues has been reported to be associated with isolated growth hormone deficiency, short stature, learning difficulties, and facial abnormalities in some, but not all, patients (51). However, expansion of the tract by seven alanine residues has been associated with panhypopituitarism, anterior pituitary hypoplasia, a hypoplastic infundibulum, and an ectopic/undescended posterior pituitary, but no evidence of learning difficulties or facial abnormalities. It has been demonstrated that +7PA results in partial loss of function of the mutant protein, possibly due to impaired nuclear localization (52).

Holoprosencephaly (HPE) is characterized by abnormal separation of the midline structures of the brain. The phenotype is highly variable and associated with a number of midline defects, including nasal and ocular defects, abnormalities of the olfactory nerves and bulbs, hypothalamus, and pituitary gland. Other associated features may be partial agenesis of the corpus callosum, single central incisor, and postaxial polydactyly. The most common pituitary abnormality is diabetes insipidus, although anterior pituitary hormone deficiencies have also been described. Both environmental and genetic factors have been implicated in its aetiology. Mutations in components of the SHH pathway have been described in association with HPE. They include mutations in SHH (7q36), GLI2 (2q14), and PTC (9q22.3). Mutations in SHH and PTC result in variable phenotypes that range from alobar HPE to normal individuals (53). Recently, heterozygous mutations in GLI2 have reported in patients with variable craniofacial abnormalities, abnormal pituitary morphology (absent pituitary, hypoplasia) and function (isolated growth hormone deficiency or panhypopituitarism) (54).

Heterozygous mutations in OTX2 have been implicated in the aetiology of a small percentage (2–3%) of anophthalmia/microphthalmia in humans. Recent reports have implicated OTX2 in the aetiology of hypopituitarism (55); three mutations have been reported in four patients with variable hypopituitarism and MRI findings (Table 2.3.1.5). During normal pituitary development OTX2 is required for anterior neural plate induction and appears to regulate the expression of HESX1. The mutant proteins reported so far exhibit absent or reduced transcriptional activation of their putative target promoters.

Mutations in PITX2 in humans are associated with Rieger’s syndrome, an autosomal dominant heterogeneous condition (56). Abnormalities include malformations of the anterior chamber of the eye, dental hypoplasia, a protuberant umbilicus, and learning difficulties. In some patients reduced growth hormone concentrations and a small sella turcica have been noted, but the significance of these observations remains unclear.

Congenital isolated growth hormone deficiency has a reported prevalence of 1:4000- 1:10 000 livebirths. Although most cases are sporadic, a genetic aetiology is suggested in 3–30% of cases. Congenital isolated growth hormone deficiency may result from mutations in the genes encoding growth hormone (GH1) or growth hormone-releasing hormone receptor (GHRHR). In addition, isolated growth hormone deficiency may result from mutations within the genes encoding the transcription factors SOX3 and HESX1, or it may be the presenting symptom in some cases of combined pituitary hormone deficiencies. So far, no mutations in GHRH have as yet been described.

GH1 is located on chromosome 17q23, within a cluster of five related genes that include human chorionic somatomammotropic hormone pseudogene 1 (CSHP1), human chorionic somatomammotropic hormone 1 (CSH1), GH2, and human chorionic somatomammotropic hormone 2 (CSH2). GH1 consists of five exons, encoding a mature molecule of 22 kDa that represents 85–90% of circulating growth hormone. Alternative splicing of mRNA generates a 20 kDa form of growth hormone that accounts for 10–15% of circulating growth hormone. Its expression is regulated by a proximal promoter and by a locus control region (LCR) located 15–32 kb upstream of the gene, which confers pituitary-specific, high-level expression of human growth hormone. Both the proximal promoter and LCR contain binding sites for the pituitary-specific transcription factor Pou1f1. GHRHR consists of 13 exons spanning approximately 15 kb, mapped to chromosome 7p15. GHRHR is a 423 amino acid G-protein-coupled receptor that contains seven transmembrane domains. Expression of GHRHR is up-regulated by POU1F1 and is required for proliferation of somatotrophs.

There are four distinct types of congenital isolated growth hormone deficiency (57) (Table 2.3.1.6). Patients with isolated growth hormone deficiency type IA present with early and profound growth failure and undetectable or extremely low growth hormone concentrations on provocation. They develop antibodies to growth hormone treatment, resulting in a markedly decreased final height as an adult. The majority of these patients have large homozygous deletions within GH1, ranging from 6.7 to 45 kb. However, microdeletions leading to an altered reading frame, premature termination of translation, and a truncated protein have also been described.

Congenital isolated growth hormone deficiency type IB is also associated with a prenatal onset of growth hormone deficiency, but is milder than type IA, with detectable concentrations of growth hormone after provocation testing. It is also autosomal recessive and results from homozygous mutations in GH1 or GHRHR. The first reported cases of GHRHR mutations were described in patients from the Indian subcontinent. Since then, a number of mutations have been reported, including missense, nonsense, and splice site mutations. Patients with mutations in GHRHR present with severe growth failure and proportionate dwarfism, but only minimal facial hypoplasia and no hypoglycaemia or microphallus. Pubertal delay has also been reported. They have low growth hormone, insulin-like growth factor 1 (IGF-1) and anterior pituitary hypoplasia on MRI.

Isolated growth hormone deficiency type II is inherited in an autosomal dominant manner. The patients present with short stature and respond well to exogenous human growth hormone (hGH) treatment with no formation of antibodies. Isolated growth hormone deficiency type II is most commonly the result of splice site mutations in intron 3 (IVS3) within the GH1 gene, although missense mutations and mutations in the exon splice enhancer within exon 3 of the GH1 have also been implicated in its aetiology. The phenotype associated with these mutations is highly variable and evolution of endocrinopathy over time has been described (58). In most cases, mutations result in aberrant splicing, skipping of exon 3, and generation of a 17.5 kDa molecule which lacks amino acids 32–71. This molecule has a dominant negative effect preventing secretion of normal wild-type 22 kDa GH with a consequent deleterious effect on pituitary somatotrophs. Analysis of different mutations identified in IGHD type II showed different mechanisms of secretory pathophysiology at a cellular level (59). This might be caused by differences in folding or aggregation, processes that are necessary for sorting, packaging, or secretion through the regulated secretory pathway. In addition, invasion by activated macrophages lead to significant bystander cell damage, which in time may compromise the other cell lineages. Treatment with recombinant hGH may suppress the growth hormone-releasing hormone drive and hence production of the mutant 17.5 kDa protein, although it is unclear whether the evolution of the phenotype can be prevented.

Isolated growth hormone deficiency type III is inherited as an X-linked disorder and in addition to growth hormone deficiency patients may also present with agammaglobulinaemia. In these cases no abnormalities have been documented within the GH1 gene and the mechanism for the phenotype is unknown. Expansion of the PA tract within SOX3 has been described in association with X-linked learning difficulties and growth hormone deficiency, as described earlier in this chapter.

Central hypothyroidism has a reported prevalence of 1:50 000 livebirths. It is a rare disorder characterized by insufficient TSH secretion resulting in low concentrations of thyroid hormones (60). Familial cases have been reported, although the condition may also be sporadic. The first homozygous nonsense mutation in exon 2 of the TSH-subunit gene has been reported in three children with congenital TSH-deficient hypothyroidism within two related Greek families. Inactivating mutations in the TRH receptor gene have also been described as a cause for isolated central hypothyroidism. Patients present with absence of TSH and prolactin responses to Thyrotropin-releasing hormone (TRH). Central hypothyroidism is generally milder than primary hypothyroidism and neonates may present with nonspecific symptoms such as lethargy, poor feeding, failure to thrive, prolonged hyperbilirubinaemia, and cold intolerance.

Congenital isolated ACTH deficiency is rare and is more commonly associated with other pituitary hormone deficiencies. The clinical features are poorly defined and patients usually present in the neonatal period with nonspecific symptoms (poor feeding, failure to thrive, hypoglycaemia) or more acute signs of adrenal insufficiency (vascular collapse, shock). Abnormalities in salt excretion are unusual, as aldosterone secretion is largely controlled by the renin–angiotensin system.

Only a few cases of isolated ACTH deficiency have been reported to date; these can be due to mutations in POMC and TBX19 (T-PIT). Patients with homozygous or compound heterozygous mutations in POMC present with early-onset isolated ACTH deficiency, obesity, and red hair due to the lack of MSH production (61). TBX19 is located on chromosome 1q23-24, and encodes the transcription factor TPIT. Mutations in this gene are the principal molecular cause of congenital neonatal isolated ACTH deficiency. Recessive mutations result in severe ACTH deficiency, profound hypoglycemia associated with seizures and prolonged cholestatic jaundice (62). Neonatal deaths have been reported in up to 25% of families with TBX19 mutations, suggesting that isolated ACTH deficiency may be an underestimated cause of neonatal death. Patients with TBX19 mutations present with very low basal plasma ACTH and cortisol levels, with no significant ACTH response to corticotropin-releasing hormone (63).

Mutations in PC1 are rare and lead to ACTH deficiency in association with hypogonadotropic hypogonadism and a complex phenotype. A compound heterozygous mutation in PC1 has been described in a female patient with extreme early-onset obesity and ACTH deficiency. In addition she presented with hypogonadotropic hypogonadism, defective processing of other prohormones and type 1 diabetes mellitus. PC1 mutations were also reported in a child with isolated ACTH deficiency, red hair, and severe enteropathy.

Isolated hypogonadotropic hypogonadism may be sporadic or inherited in an autosomal dominant, autosomal recessive, or X-linked manner. As the maturation and migration of GnRH and olfactory neurons are closely linked during development, it is not surprising that isolated hypogonadotropic hypogonadism may be associated with abnormal smell (anosmia/hyposmia).

Kallmann’s syndrome consists of the association between isolated hypogonadotropic hypogonadism and anosmia, with approximately 75% of patients demonstrating agenesis of the olfactory bulbs on neuroimaging. It is a clinically heterogeneous condition, with a reported prevalence of 1:10 000 in males and 1: 50 000 in females. Mutations in five genes (KAL1, FGFR1, FGF8, PROKR2, and PROK2) account for about 30% of cases of Kallmann’s syndrome, indicating that other genes are also implicated in its aetiology (64). Mutations in KAL1are responsible for the X-linked form of Kallmann’s syndrome. The gene is located on chromosome Xp22.3 and encodes the extracellular matrix glycoprotein anosmin-1, which has a role in the control of the migratory process of the GnRH neurons, although the molecular mechanisms of this action are not fully elucidated. In addition to isolated hypogonadotropic hypogonadism, patients with KAL1 mutations may present with unilateral renal agenesis (30%), bimanual synkinesia (75%), sensorineural hearing loss, midline defects, and high arched palate (65).

Mutations in the receptor for fibroblast growth factor 1 (FGFR1) and in fibroblast growth factor 8 (FGF8) account for the autosomal dominant form of Kallmann’s syndrome. To date, more than 40 mutations have been reported in FGFR1 (10% of patients with Kallmann’s syndrome) and six in FGF8 (two of which are in association with FGFR1 mutations). The phenotype of the autosomal dominant form of KS is characterized by variable penetrance. Mutations in FGFR1 have been reported in association with complete absence of puberty, normal reproductive function, isolated anosmia or even normosmic isolated hypogonadotropic hypogonadism. Cleft lip and palate, agenesis of the corpus callosum, dental agenesis, skeletal abnormalities, and absent nasal cartilage may be associated features, whereas only two patients with bimanual synkinesia have been reported so far. Similarly, loss of function mutations in FGF8 have been recently reported both in association with Kallmann’s syndrome, normosmic hypogonadotropic hypogonadism, and variable degrees of GnRH deficiency. Their manifestation ranged from absent puberty to reproductive failure after completion of sexual maturation; cleft lip and palate, skeletal defects, and hearing loss have also been described in association with FGF8 mutations (66). There is evidence from animal models that FGF signalling is important for the GnRH cell specification, migration, and survival; this requirement at multiple levels may account for the wide spectrum of clinical phenotypes.

In addition, homozygous, heterozygous, or compound heterozygous mutations in prokineticin2 (PROK2) and prokineticin-2 receptor (PROKR2) have recently been identified in patients with Kallmann’s syndrome (MIM 612370) (9%). In animal models, Prok2^–/– mice exhibit dysgenesis of the olfactory bulb, decreased numbers of GnRH neurons, and infertility. In addition, in vitro experiments have demonstrated that missense mutations have deleterious effect on prokineticin signalling. However, many mutations have also been found in apparently unaffected individuals, thus raising questions about their significance (67). The heterogeneity of Kallmann’s syndrome does not allow for correlation between genotype and phenotype. However, it seems that patients with KAL1 mutations have severe and permanent hypogonadotropic hypogonadism, whereas those with mutations in FGFR1, FGF8, PROKR2, and PROK2 have greater variability.

Patients with Kallmann’s syndrome (MIM 214800) may have features which are also part of CHARGE syndrome (deafness, dysmorphic ears, hypoplasia or aplasia of the semicircular canals). Conversely anosmia or hyposmia have been noted in cases of CHARGE. These observations prompted the screening of CDH7 (chromodomain helicase DNA-binding protein-7), mutations of which have been identified in almost 70% of patients with CHARGE syndrome. So far heterozygous mutations in CDH7 have been reported in a small number of patients with Kallmann’s syndrome (three patients) or sporadic normosmic HH (four patients) (68). Animal studies have demonstrated high levels of CDH7 expression in the olfactory placode and in a pattern consistent with its involvement in the migratory pathway of GnRH neurons. However, its role remains to be established.

Hypogonadotropic hypogonadism in association with normal olfaction has been reported in association with mutations in the GnRH receptor (GnRHR), GPR54/Kisspeptin, LHβ, and FSHβ. As mentioned before, mutations in FGFR1 and FGF8 have also been reported in association with normosmic hypogonadotropic hypogonadism. Mutations in DAX-1 and leptin result in hypogonadotropic hypogonadism with a complex phenotype, whereas recently identified genes (TAC3, TAC3R) expand the spectrum of genetic changes in hypogonadotropic hypogonadism.

Approximately 20 homozygous or compound heterozygous mutations in GnRHR have been described. The gene, located on 4q13.2-2, encodes a 328 amino acid G-protein-coupled receptor. Mutations result in variable phenotypes that range from complete hypogonadism with undescended testes and presentation at birth to mild pubertal delay (65).

Hypogonadotropic hypogonadism may also result from mutations in the G-protein-coupled receptor GPR54 and its endogenous ligands, kisspeptins. Kisspeptins are products of the KiSS1 gene, derived after post-translational modification of kisspeptin-1.The longest of these peptides, kisspeptin-54, is also known as metastatin. GnRH neurons express Gpr54 and, in turn, KiSS1 expression has been detected in the arcuate and periventricular nuclei of the hypothalamus. Mice lacking GPR54 exhibit hypogonadism, but GnRH neurons migrate normally and have normal GnRH content. The role of Kiss1/GPR54 in the reproductive axis and pubertal timing is complex. Kisspeptins have a direct effect on GnRH neurons and their central administration results in GnRH release and LH secretion in vivo. In 2003, two independent groups reported deletions and inactivating mutations in GPR54 in patients with HH. Since then, loss-of function mutations described in GPR54 account for almost 5% of cases with normosmic HH. In these cases patients present with variable phenotypes that ranges from partial to severe hypogonadism (69). Recently, homozygous loss of function mutations in neurokinin-B (TAC3) and its receptor (TACR3) have been reported in eight patients from consanguineous families. They were in the second decade of life, or older, and demonstrated failure of pubertal progression. Neurokinin-B is expressed in hypothalamic neurons that also express kisspeptin; therefore, it is postulated that its function may affect the hypothalamic release of GnRH (70).

DAX1 (dosage-sensitive sex reversal, adrenal hypoplasia congenita critical region on the X chromosome) mutations in humans cause hypogonadotropic hypogonadism and adrenal hypoplasia congenita, which can result in severe neonatal adrenal crises. The condition is inherited as an X-linked disorder. DAX1 is a transcription factor that is expressed in several tissues, including the hypothalamus and pituitary, and interacts with SF1. Duplications of DAX1 result in persistent müllerian structures and XY sex reversal suggesting that the gene acts in a dosage-sensitive manner (71).

Leptin is secreted from adipocytes and, apart from its role in regulating nutrition, it appears to play an important role in several neuroendocrine functions by acting at a hypothalamic level. Congenital leptin deficiency, secondary to mutations in leptin or its receptor, are associated with obesity, marked hyperphagia, metabolic abnormalities, and hypogonadotropic hypogonadism (72). There is evidence that treatment with leptin results in significant weight loss and normalization of nocturnal luteinizing hormone secretion.

Central diabetes insipidus is commonly due to acquired disorders. Congenital central diabetes insipidus is rare and it may be a feature of midline disorders (septo-optic dysplasia, HSE) or due to mutations in genes involved in the secretion of arginine vasopressin (AVP). A number of mutations have been described in the gene that encodes the AVP preprohormone, prepro-AVP-NPII (arginine vasopressin-neurophysin II), resulting in autosomal dominant diabetes insipidus (73). The gene is located on chromosome 20 and consists of three exons. Exon 1 encodes the signal peptide of the preprohormone and AVP, exon 3 encodes the glycoprotein copeptin, whereas the carrier protein NPII is encoded by all three exons. In this rare familial disorder of AVP secretion, patients present usually in the first 10 years of life, whilst neonatal manifestations are uncommon. This suggests that the pathophysiology of familial central diabetes insipidus involves progressive postnatal degeneration of AVP-producing magnocellular neurons. More than 50 different mutations have been identified so far, mainly affecting amino acid residues important for the proper folding and/or dimerization of the NP moiety of the AVP pro-hormone. The proposed mechanism is that the mutant allele exerts a dominant negative effect; the misfolded mutant hormone precursor is accumulated in the endoplasmic reticulum resulting in progressive toxic damage of the vasopressin neurons and the clinical manifestation of diabetes insipidus.

Central diabetes insipidus is also a feature of Wolfram’s syndrome, a rare recessive disorder characterised by diabetes mellitus, diabetes insipidus, optic atrophy, sensorineural hearing loss, and progressive neurodegeneration. The gene, WFS1, is located on 4p16.1 and encodes wolframin. The protein is localized in the endoplasmic reticulum and is a component of the ‘misfolded protein/stress response’ machinery. In the brain, WFS1 expression has been detected in selected neurons in the hippocampus, amygdala, and olfactory tubercle.

Pituitary development depends on complex regulatory networks and the sequential expression of transcription factors and signalling molecules in a space- and time-specific manner. An ever-increasing number of genes are implicated in this process. Spontaneous or artificially induced mutations in the mouse and identification of mutations associated with human pituitary disease contribute to defining the genetic cascades responsible for pituitary development.