The pituitary gland, located within the sella turcica at the base of the brain is the master regulator of growth, puberty, metabolism, response to stress, reproduction and lactation(54). The pituitary gland consists of three lobes-the anterior, intermediate and the posterior lobes. The anterior and the intermediate lobe (involutes in the adult) comprise of the adenohypophysis and the posterior lobe comprises of the neurohypophysis(55). The adenohypophysis or the anterior part of the pituitary gland secrete six different hormones produced from five different cell types: growth hormone(GH) from somatotrophs, thyroid stimulating hormone (TSH) or thyrotrophin from the thyrotrophs, adrenocorticotropic hormone(ACTH) from corticotrophs, prolactin(PRL) from lactotrophs, follicular stimulating hormone(FSH) and luteinizing hormone(LH) from gonadotrophs(56). The intermediate lobe consists of melanotrophs that secrete pro-opiomelanocortin (POMC), which is a precursor to melanocyte-stimulating hormone (MSH) and endorphins(56). Within the posterior lobe, arginine vasopressin(AVP) and oxytocin are secreted which are synthesised magnocellular neurones of the paraventricular and supra-optic nuclei within the hypothalamus(56). Secretory and inhibitory peptides that regulate the secretion of hormones from the anterior pituitary gland are produced from the hypothalamus. The infundibulum or the pituitary stalk not only carries the neural tracts from the hypothalamus to the posterior pituitary gland but also carries the portal blood delivering the hypothalamic regulating hormones to the anterior pituitary gland(56).
2.2.1 Embryology and Development of Pituitary Gland
The oral ectoderm gives rise to the formation of the anterior and intermediate lobes of the pituitary while the posterior pituitary is derived from the neural ectoderm(56). The development of the pituitary gland has been extensively studied in mouse which occurs in a sequential, well defined and coordinated manner that involves the formation of the pituitary placode, rudimentary Rathke’s pouch, definitive Rathke’s pouch and the mature pituitary gland(57). The developmental cascade of murine pituitary gland reflects that of humans(57). At embryonic stage, E7.5 in the mouse, the roof of the oral ectoderm thickens and gives rise to the pituitary placode, which by E9.0, invaginates to form the rudimentary Rathke’s pouch that then forms into the definitive Rathke’s pouch by E10.5(58). The Rathke’s pouch then gives rise to the anterior and the intermediate lobes of the pituitary gland(54). The neural ectoderm evaginates at the base of the ventral diencephalon to give rise to the infundibulum and the posterior pituitary(54). At E12.5, there is separation of Rathke’s pouch from the oral ectoderm(54). The various stages of pituitary gland development in rodent are depicted in figure 2.1 (54). The hormone-secreting progenitor cells proliferate between E12.5 and E15.5 followed by spatial and temporal differentiation of the various cell types(54), starting from the thyrotrophs and corticotrophs, followed by the somatotrophs, gonadotrophs and lactotrophs that secrete their respective hormones(59, 60).
Figure 2.1: Stages in mouse pituitary gland development. (a) Oral ectoderm. (b) Rudimentary pouch. (c) Definitive pouch. (d) Adult pituitary gland. I-Infundibulum; NP-neural plate; N-notochord; PP-pituitary placode; OM-oral membrane; H-heart; F-Forebrain; MB-midbrain; HB-hindbrain; RP-Rathke’s pouch; AN-anterior neural pore; O-oral cavity; PL-posterior lobe; OC-optic chiasm; P-pontine flexure; PO-pons; IL-intermediate lobe; AL-anterior lobe; DI-diencephalon; SC-sphenoid cartilage (55).
220.127.116.11 Early Developmental Genes and Signals in Pituitary Development
The development of pituitary gland is orchestrated by a series of well-co-ordinated cascade of morphogenetic signalling molecules and transcription factors that play a vital role in the process of organ commitment, cell proliferation, patterning and differentiation(55). The formation of the rudimentary Rathke’s pouch(RP), is initiated by the signalling molecules such as Bone Morphogenetic Protein 4(Bmp4) and thyroid transcription factor(Ttf1) from the ventral diencephalon combined with the Sonic Hedgehog (Shh) from the oral ectoderm(61). The invagination of the RP and further pituitary progenitor cell proliferation is directed by the co-ordination of Fibroblast growth factor signalling (Fgf8 and Fgf10) and Wnt5a along with early transcription factors such as Gli1, Gli2, Lhx3, Ptx1 and Ptx2(61).The expression of Hesx1 and Prop1(prophet of Pit-1) in RP leads to differentiation of specific pituitary cell types.Prop1 in-turn induces the expression of Pou1f1, leading to the terminal differentiation of somatotrophs, lactotrophs and thyrotrophs(62). The gonadotrophs differentiation is induced by the expression of Gata2 and steroidogenic factor 1(sf1)(62). The differentiation of corticotrophs is regulated by the expression of Tbx19 from the POMC-producing cells(62).
The development of pituitary is a carefully co-ordinated complex process which requires the expression of the signalling molecules during critical periods of development. The genes that are expressed early are not only involved in the organ commitment but also play an important role in the activation and expression of downstream signalling molecules that has a specific role in the differentiation of progenitor cells(55).The differentiation of various pituitary cell types from the various transcription factors is shown in the figure 2.2(55).
Figure 2.2: Pituitary signalling cascade and differentiation of pituitary cell types(55)
The understanding of human pituitary disease has been significantly enhanced by studying the effects of induced mutations and gene knock outs in the murine models. This has led to the identification of mutations in a number of genes that give rise to the phenotype of hypopituitarism in humans. Mutations in transcription factors such as Hesx1, Lhx3, Lhx4, Prop1, Pou1f1, Pitx2 and Tbx19 have been implicated to cause hypopituitarism in both the mice and the humans(56). Hypopituitarism is the deficiency of one or more hormones secreted by the pituitary gland. Congenital hypopituitarism comprises of a spectrum of disorders with variable phenotypes that can range in severity, from isolated hormone deficiency [isolated growth hormone deficiency being the most common] to combined pituitary hormone deficiency (CPHD) when two or more pituitary hormones are deficient(56). Congenital hypopituitarism may present as part of a syndrome with abnormalities in structures that share a common embryological origin with the pituitary gland(56).
The mutations in the known transcription factors implicated in the etiology of syndromic and non-syndromic hypopituitarism are described below:
2.3 SYNDROMIC FORMS OF HYPOPITUITARISM
2.3.1 Septo-Optic Dysplasia
De Morsier syndrome or septo-optic dysplasia(SOD) is a rare condition with a reported incidence of 1 in 10,000 newborns and is equally prevalent in both sex(63). SOD is heterogeneous congenital anomaly and is characterised by the presence of at least two of the three features: hypopituitarism (one or more pituitary hormone deficiency), hypoplasia of the optic nerve and defects of the midline structures of the forebrain such as absent septum pellucidum or agenesis of corpus callosum(63). While the presence of all the three features occurs only in about 30% of the patients, hypopituitarism and midline brain abnormalities can be a feature in 62% and 60% of the patients respectively(54). SOD has multifactorial etiologies such as genetic, viral infections, alcohol, drugs, vascular and environmental teratogens. Mutations in HESX1, SOX2 and SOX3 have been implicated in SOD(64, 65). However, the proportion of patients with SOD having mutations in these genes are very small, implying that there may be other unidentified genetic factors that may account for SOD(64, 65).
Any disruption occurring during the development of forebrain and the pituitary between 3-6 weeks of gestation can account for SOD(54). Disturbances in visual axis, squint or nystagmus secondary to unilateral or bilateral(majority) optic nerve hypoplasia can be the presenting feature of SOD(65). The pituitary hormone deficiencies may not be always present in patients with SOD but may evolve later in life, thus requiring life-long follow-up. The commonest pituitary hormone deficiency is GH followed by TSH, ACTH and gonadotropins(65). Posterior pituitary involvement and diabetes insipidus are rare. There also may be associated neurological manifestations such as cerebellar hypoplasia, schizencephaly, seizures and global developmental delay(66).
2.3.2 HESX1 mutations and SOD
HESX1 is critical transcription factor that has a central role in the early differentiation and determination of the forebrain and the pituitary gland.(54) Expression studies during mouse embryogenesis show that Hesx1 is one of the earliest markers of the pituitary primordium, first expressed in the anterior midline visceral endoderm and its continued expression in this area plays a vital role in the development of the forebrain, ventral diencephalon, anterior pituitary and the presumptive hypothalamus(54). Evidence from murine studies show that targeted disruption of Hesx1 causes forebrain abnormalities, micropthalmia and absent optic vesicles, the features which are in line with SOD in humans(67). The first homozygous missense mutation (Arg160Cys) was found in the homeobox of HESX1 in two siblings with SOD with optic nerve hypoplasia, absent corpus callosum and septum pellucidum, an ectopic/undescended posterior pituitary and anterior pituitary hypoplasia with hypopituitarism(67). Subsequently homozygous and heterozygous mutations have been reported to cause variable phenotype ranging from IGHD to SOD. Only minority of patients with SOD (<1%) have HESX1 mutations, implying that there are potential unidentified genes contributing to this complex disorder(68, 69).
2.3.3 SOX2 and SOX3 Mutations
The expression of SOX2 and SOX3 are important for the differentiation of progenitor stem cells and pituitary development(70, 71). SOX2 mutations have been described in association with anterior pituitary hypoplasia, hypogonadotropic hypogonadism, hippocampal and corpus callosum abnormalities(72, 73). SOX3 causes X linked hypopituitarism in males and may consist of abnormalities of corpus callosum, hypoplasia of the infundibulum, isolated or combined hormonal deficiencies(74, 75). The phenotype may be associated with learning difficulties(76).
2.3.4 Holoprocencephaly (HPE)
HPE is a heterogeneous condition with multifactorial etiology resulting from the abnormal cleavage of the forebrain and may be associated with pituitary, corpus callosum, nasal and ocular abnormalities(77). The most common endocrine manifestation in HPE is cranial diabetes insipidus(54). The transcription factor GLI2 mediates the signal from the Sonic Hedgehog(SHH) signalling pathway, the mutation of which has been implicated in the development of HPE sequence(78). GLI2 mutations have been associated with hypopituitarism, partial agenesis of corpus callosum, single central maxillary incisor, post axial polydactyly and single nares(54).
2.3.5 LHX3 mutations: Hypopitutarism with Spine Abnormalities
Lhx3 or the LIM homeobox is an important early development gene detected in the Rathke’s pouch and the developing nervous system, the continuous expression of which helps in the proliferation of gonadotrophs, thyrotrophs, somatotrophs and lactotrophs(79). Homozygous targeted disruption of this gene from mouse causes pituitary aplasia and results in their death, shortly after birth(80). Patients with homozygous LHX3 mutations have deficiencies of GH, PRL, TSH, LH,FSH and also sometimes ACTH deficiencies with small to an enlarged anterior pituitary with a lesion suggestive of microadenoma(81, 82). There also can be an association of short rigid cervical spine with limitation of neck movement and sensorineural hearing loss(82-84).
2.3.6 LHX4 mutations
The expression of Lhx4 is closely related to that of Lhx3(85). The expression pattern of Lhx4 is found throughout the invaginating Rathke’s pouch at very early mouse embryonic stage but by E15.5 its expression is predominantly found in the anterior lobe of the pituitary(54). Targeted Lhx4 deletion in mice results in hypoplastic pituitary due to reduction in cell numbers(57). Experiments involving Lhx3 and Lhx4 gene dosage in murine models demonstrate that a single allele of Lhx3 or Lhx4 is sufficient for the formation of definitive Rathke’s Pouch(57). With targeted deletion of Lhx3 and Lhx4 in mouse, the progression from rudimentary to definitive Rathke’s pouch does not occur(57). Variable degrees of hypopituitarism involving GH, ACTH, TSH and gonadotropin deficiencies with ectopic posterior pituitary, hypoplastic sella, cerebellar abnormalities and Chiari malformation have been reported in patients with heterozygous LHX4 mutations(82, 86, 87). LHX4 is required for the activation and expression of GH1, therefore LHX4 mutations result in short stature due to GH deficiency(88).
2.3.7 OTX2 mutations
OTX2 is another important transcription factor required for the formation of forebrain and its maintenance(89, 90). In humans, mutations in OTX2 have been described in patients with hypopituitarism and bilateral anophthalmia with a normal or small anterior pituitary and an ectopic posterior pituitary. OTX2 mutations can also cause hypopituitarism without associated ocular abnormalities(91). Among the anophthalmia/micropthalmia syndromes, 2-3% of the underlying genetic aetiology is due to a mutation in OTX2(92, 93).
2.3.8 Axenfield-Rieger syndrome (PITX2 mutations)
In the mouse, Pitx2 is initially expressed in the oral ectoderm and then in the Rathke’s pouch. It is also expressed in the mesenchyme near the optic eminence, forelimbs and the domains of the abdominal cavity(56). It is required at multiple stages of the pituitary development after the commitment of the definitive Rathke’s pouch. Targeted Pitx2 mutations in mice causes hypopituitarism with hypoplastic anterior pituitary(94). Heterozygous mutations in PITX2 (OMIM 601542 also known as RIEG1) are implicated in Axenfield-Rieger syndrome, an autosomal dominant condition that comprises of malformation of the anterior segment of eye, dental hypoplasia, protuberant umbilicus and brain abnormalities(95, 96). Pituitary abnormalities due to PITX2 mutation in humans are yet to be described.
2.4 NON-SYNDROMIC FORMS OF HYPOPITUITARISM
2.4.1 PROP1 Mutations
In the mouse embryo, the transcription factor Prop1 is expressed within the Rathke’s pouch from E10, in a region overlapping the Hesx1 expression domain(54). The expression of Prop1 starts to decrease after E12 and disappears at E15.5(97). This decline in Prop1 expression is important as overexpression can result in delay in the differentiation of gonadotrophs leading to transient gonadotrophin deficiency and delayed puberty(54). Prop1 regulates the expression of Hesx1 and Pou1f1 by acting as transcriptional repressor of the former and transcriptional activator for the latter(98). Therefore the appropriate and timely suppression of Prop1 is important for Pou1f1 determination and in the establishment of other cell lineages(99). Naturally occurring Prop1 mutations in Ames dwarf mice cause GH,PRL,TSH and gonadotrophin deficiencies due to failure in determination of Pou1f1 lineage which determines the differentiation of somatotrophs, lactotrophs and thyrotrophs (54). In humans, PROP1 is located at chromosome 5q and consists of three exons and a protein product of 226 amino acids(55). The most common mutations in PROP1 are recessive mutations associated with GH, TSH, PRL and gonadotropin deficiencies(100). ACTH deficiency is uncommon but have been reported in patients with PROP1 mutations(101). The degree of TSH and gonadotropin deficiencies are variable. The gonadotropin deficiency may range from micropenis, undescended testes, delayed puberty and infertility(100).
2.4.2 POU1F1(PIT1) Mutations
POUIFI (previously PIT-1), is a transcription factor that is expressed at E14.5 during the mouse pituitary development and persists throughout the postnatal period and in the adult pituitary(54). The differentiation of somatotrophs, lactotrophs and thyrotrophs depends on the Pou1f1 expression(54). POU1F1 mutations in humans are mostly autosomal recessive but dominant mutations have been described(102). The phenotype consists of GH and PRL deficiencies in early life and TSH deficiency occurring in late childhood with normal sized or small anterior pituitary and a normal posterior pituitary with no midline abnormalities(103).
2.4.3 TBX19 mutations (OMIM 604614. Previously known as TPIT)
TBX19 belongs to T-box family of transcription factors and is expressed from E11.5 within the developing anterior pituitary and the ventral diencephalon in mouse embryos(104). In the adult mouse pituitary, TBX19 expression is detected in corticotrophs in anterior lobe that express POMC and in the melanotrophs forming the intermediate lobe(105). TBX19 mutations are thus associated with isolated ACTH deficiency(106). Homozygous and compound heterozygous mutations in TBX19 have been described implying a recessive mode of inheritance(106). ACTH deficiency can present in the neonatal period with severe hypoglycaemia, jaundice or seizures(107).
2.5 ISOLATED HORMONE DEFICIENCY DUE TO MUTATION IN SPECIFIC CELL TYPE
In addition to mutations in the developmental genes, causing combined pituitary hormonal deficiencies, there are genetic mutations that contribute to isolated hormone deficiencies. These are discussed below:
2.5.1 GH1 Mutations
Growth hormone is encoded by GH1 gene, the mutations of which is estimated in up to 12.5% of patients with isolated GH deficiency although the true incidence is difficult to ascertain owing to geographical and ethnic differences (108). Isolated GH deficiency(IGHD) due to GH1 mutations consists of three types: Type 1a is the severest form due to the deletion of GH1 gene in which patients will not have any detectable serum GH and respond well to recombinant GH but some patients develop anti-GH neutralizing antibodies(109). Type 1b IGHD is due to homozygous splice site mutation in GH1 and are characterized by low but detectable GH after provocative stimulation(109). Type 2 IGHD is the common form and is due to splice site and missense mutations in GH1 and is an autosomal dominant condition. Type 3 IGHD, an X-linked recessive disorder is associated with X-linked agammaglobulinemia (109).
A mutation leading to the absence of a disulphide bridge in GH1 results in the decreased binding of GH to its receptor and subsequent downstream signalling, causing a high serum GH levels with low serum IGF-1 concentration leading to short stature(110).Patients with IGHD can also develop additional pituitary hormonal deficiencies later in life and therefore require life-long monitoring. In addition to GH1 mutations, several other mutations in genes encoding GHRHR, TSH, LH and FSH have been known to cause specific hormonal deficiencies(111-113).
2.6 CLINICAL MANIFESTATIONS OF HYPOPITUITARISM
Establishing the diagnosis of congenital hypopituitarism is important as untreated hypopituitarism can result in serious morbidities such as global developmental delay due to prolonged undetected hypoglycaemia and untreated hypothyroidism, significant reduction in final height and morbidities related to water and electrolyte imbalance.
The clinical spectrum of hypopituitarism is dependent on the combination of hormonal deficits. The clinical manifestations may be non-specific in the neonatal period such as poor feeding, temperature instability, jitteriness, poor weight gain and prolonged jaundice(114). Early diagnosis in this age group can be challenging due to factors such as prematurity, associated neonatal comorbidities, lack of data appropriate for gestation and immaturity of hypothalamic-pituitary axis(54). Neonates can also present with associated developmental defects such as ocular, midline, genital abnormalities or syndromes associated with hypopituitarism. Presence of fixed squint, nystagmus in a neonate can be due to underlying optic nerve hypoplasia which can be isolated or a part of the spectrum of septo optic dysplasia. Such patients will need life- long follow up and assessment of endocrine function, even if their initial endocrine investigations are normal(115). Congenital hypopituitarism can be life threatening in neonates due to an underlying ACTH deficiency and may present as sepsis, seizures or conjugated hyperbilirubinaemia (116).
Growth failure can occur in infancy in severe GHD(117). Male neonates may present with micropenis or undescended testes as the penile growth is dependent on normal LH secretion during the second and third trimester(55). Diabetes insipidus can present as polyuria, polydipsia but may be masked in patients with ACTH deficiency as cortisol is essential to excrete the water load. Hydrocortisone replacement may unmask diabetes insipidus and hence vigilance is essential in patients with suspected hypopituitarism with or without midline defects when being treated with hydrocortisone therapy for cortisol deficiency.
2.7 INVESTIGATIONS IN HYPOPITUITARISM
The investigations of hypopituitarism comprise of stimulation or provocative tests to check the adequacy of the hypothalamic-pituitary axis(HPA) and neuroradiology.
2.7.1 ACTH Deficiency
In neonates, ACTH deficiency can be life threatening. It is however challenging to assess the intactness of the hypothalamic-pituitary-adrenal axis as the circadian rhythm of cortisol is not well established during the first six months of life(118). The usefulness of baseline cortisol samples in the morning or evening are limited in this age group. Multiple cortisol measurements during the various points during the day and night can be challenging and may not reveal definitive information on the integrity of the axis. Hypoglycaemia induced cortisol stimulation is contraindicated in the age group but standard synacthen test is safe and easy to perform in this age group. However the standard synacthen test is limited by sensitivity of 80% and therefore false negative results are a possibility despite ACTH deficiency(116). In older children, after the establishment of circadian rhythm , 08:00 am cortisol of 175nmol/L or more combined with a 30 minute stimulated level of more than 540nmol/L on a standard synacthen test has a sensitivity of 69% and specificity of 100% in excluding ACTH deficiency(116).
2.7.2 TSH Deficiency
TSH deficiency is normally characterised by a low or inappropriately normal TSH combined with a low serum free thyroxine. Isolated TSH deficiency is rare but can occur in combination of other pituitary hormone deficiencies(119). Provocative test such as thyrotropin releasing hormone(TRH) test is generally not required to establish the diagnosis of central hypothyroidism(120).
2.7.3 GH Deficiency
Severe GH deficiency can present in the neonatal period as hypoglycaemia. The provocative tests to assess the hypothalamic-pituitary-growth axis are contraindicated in children less than one year of age. The diagnosis of GH deficiency in this age group is normally suggested by low plasma GH concentration in response to spontaneous hypoglycaemia, low plasma IGF-1 and/or the presence of additional hormonal deficiencies(121).
2.7.4 Gonadotropin Deficiency
Males with gonadotropin deficiency can present with micropenis with or without undescended testes. The physiological postnatal surge in LH, FSH and testosterone is detected up to 6 months in males and about 2 years in females(122). A combination of GnRH (gonadotropin releasing hormone) and hCG (human chorionic gonadotropin) stimulation tests are useful in the earlier detection of hypogonadotropic hypogonadism(HH) in infants where a low baseline gonadotropin concentrations (LH & FSH) and blunted response to stimulation with GnRH are found(123).
2.7.5 ADH Deficiency (Diabetes Insipidus(DI))
Early morning paired serum and urine osmolality, hypernatraemia along with symptoms such as polyuria, weight loss may be helpful in the diagnosis as water deprivation test can be dangerous in this age group. The symptoms of DI can be masked in patients with ACTH deficiency as cortisol is important for water excretion from the kidneys.
2.7.6 The Role of Neuroradiology
In infants with suspected or diagnosed hypopituitarism, MRI of the pituitary and brain can help to assess the size of anterior pituitary, the location of posterior pituitary, the morphologies of infundibulum, corpus callosum, septum pellucidum, optic nerves, optic chiasma and associated brain abnormalities(124). The neuro radiological abnormalities are usually related to the severity or the evolution of hypopituitarism(125). In patients with ectopic posterior pituitary, the risk of hypopituitarism is much greater than patients with normally positioned pituitary. Neonates with optic nerve hypoplasia and small anterior pituitary will require a lifelong follow-up despite their initial normal endocrine function as hypopituitarism may evolve over a period of time (125).
2.8 MANAGEMENT OF CONGENITAL HYPOPITUITARISM
The management of congenital hypopituitarism is multidisciplinary and requires a lifelong follow-up. The management should not only focus on optimizing the hormone replacement but also to monitor carefully for the evolvement of potential hormonal deficiencies in the future. In syndromic forms of hypopituitarism, it is also vital to address the wider issues such as visual and neuro-developmental issues and offer appropriate genetic counselling(54).
While replacing hormones in suspected combined pituitary hormone deficiency, it is important to assess the adequacy of cortisol secretion and replacing with hydrocortisone when the cortisol secretion is suboptimal before commencing on thyroxine(54). As mentioned above, patients on cortisol replacement should be carefully monitored for symptoms of DI.
Overall, the mainstay of treatment of congenital hypopituitarism is to identify the existing and evolving hormonal deficits optimizing their replacement.