Expanding the spectrum of mutations in GH1 and GHRHR: genetic screening in a large cohort of patients with congenital isolated growth hormone deficiency.

CONTEXT: It is estimated that 3-30% of cases with isolated GH deficiency (IGHD) have a genetic etiology, with a number of mutations being reported in GH1 and GHRHR. The aim of our study was to genetically characterize a cohort of patients with congenital IGHD and analyze their characteristics. PATIENTS AND METHODS: A total of 224 patients (190 pedigrees) with IGHD and a eutopic posterior pituitary were screened for mutations in GH1 and GHRHR. To explore the possibility of an association of GH1 abnormalities with multiple pituitary hormone deficiencies, we have screened 62 patients with either multiple pituitary hormone deficiencies (42 pedigrees), or IGHD with an ectopic posterior pituitary (21 pedigrees). RESULTS: Mutations in GH1 and GHRHR were identified in 41 patients from 21 pedigrees (11.1%), with a higher prevalence in familial cases (38.6%). These included previously described and novel mutations in GH1 (C182X, G120V, R178H, IVS3+4nt, a>t) and GHRHR (W273S, R94L, R162W). Autosomal dominant, type II IGHD was the commonest form (52.4%), followed by type IB (42.8%) and type IA (4.8%). Patients with type II IGHD had highly variable phenotypes. There was no difference in the endocrinology or magnetic resonance imaging appearance between patients with and without mutations, although those with mutations presented with more significant growth failure (height, -4.7 +/- 1.6 SDS vs. -3.4 +/- 1.7 SDS) (P = 0.001). There was no apparent difference between patients with mutations in GH1 and GHRHR. CONCLUSIONS: IGHD patients with severe growth failure and a positive family history should be screened for genetic mutations; the evolving endocrinopathy observed in some of these patients suggests the need for long-term follow-up.

Congenital hypopituitarism: clinical, molecular and neuroradiological correlates.

OBJECTIVE: Recent studies have suggested that mutations in genes encoding several hypothalamo-pituitary (H-P) transcription factors result in hypopituitarism [isolated GH deficiency (IGHD) and combined pituitary hormone deficiency (CPHD)], which may in turn be related to the neuroanatomy revealed by magnetic resonance (MR) imaging. Although studies have focused on patients with either optic nerve hypoplasia (ONH) or isolated hypopituitarism with normal optic nerves, few studies have compared the two groups. We aimed to relate the clinical phenotype of a large cohort (n = 170) of children with congenital hypopituitarism including septo-optic dysplasia (SOD) attending a single centre to the neuroradiological and genetic findings. DESIGN: Clinical, biochemical, MR imaging and molecular data were analysed retrospectively in 170 patients with or 'at-risk' (with ONH) of hypopituitarism to determine predictors of hypopituitarism. RESULTS: The presence of ONH was significantly associated with an absent septum pellucidum [odds ratio (OR) 31.5, 95% confidence intervals (CI) 7.3-136.6, P < 0.001], an abnormal corpus callosum (OR 10.5, 95% CI 3.8-28.6, P < 0.001) and stalk abnormalities (OR 2.3, 95% CI 1.2-4.2, P = 0.009). The risk of hypopituitarism was 27.2 times greater in patients with an undescended posterior pituitary (95% CI 3.6-205.1, P < 0.001). Anterior pituitary hypoplasia (OR 3.1, 95% CI 1.3-7.0, P = 0.006) and an absent pituitary stalk (P < 0.001) were also significantly associated with hypopituitarism. With respect to the type or severity of hypopituitarism, CPHD was more often associated with an abnormal corpus callosum (OR 6.1, 95% CI 1.4-27.4, P = 0.008) and stalk abnormalities (OR 2.8, 95% CI 1.3-6.1, P = 0.006). Male to female ratio was significantly greater in patients with normal optic nerves (3.3:1) as compared with those with ONH (1.2:1). The prevalence of diabetes insipidus, thyrotrophin and ACTH deficiencies was significantly greater in patients with ONH as compared with 'idiopathic' hypopituitarism. Mutations in pituitary transcription factors and genes regulating GH secretion were rare (5/170) in this cohort of patients with sporadic hypopituitarism. CONCLUSION: Our data suggest that individuals presenting with ONH are at high risk for neuroradiologic and endocrine abnormalities. The neuroradiologic features are predictive not only of the presence, but also of the type, of hypopituitarism. The association of midline abnormalities with hypopituitarism in this cohort suggests a common developmental origin for these features, the aetiology of which remains unidentified in the majority of cases.

Role of gonadotropin-releasing hormone and human chorionic gonadotropin stimulation tests in differentiating patients with hypogonadotropic hypogonadism from those with constitutional delay of growth and puberty.

BACKGROUND: Delayed puberty can be due to either constitutional delay of growth and puberty (CDGP) or hypogonadotropic hypogonadism (HH). Differentiating between the two using current testing can be difficult. We assessed the utility of a GnRH test in combination with a 3-d and 19-d human chorionic gonadotropin (HCG) test to discriminate between the two conditions. METHODS: We performed a retrospective analysis of 43 boys with pubertal delay who required pubertal induction with testosterone. All were followed through puberty; 29 were subsequently diagnosed with CDGP and 14 with HH. A standard GnRH test (2.5 microg/kg) was undertaken and was followed by a short [3 d; n = 38 (13 HH, 25 CDGP)] or extended [19 d; n = 31 (12 HH, 19 CDGP)] HCG stimulation test, or both [n = 27 (11 HH, 16 CDGP)]. Receiver operating characteristic analysis was performed to assess the performance of the tests. RESULTS: Peak testosterone concentrations to both 3-d and 19-d HCG tests were significantly lower in patients with HH compared with CDGP. The 19-d test performed better than the 3-d test, and a combination of the LHRH, 3-d and 19 d HCG test [peak LH cutoff, 2.8 U/liter; peak 3-d testosterone cutoff, 1.04 microg/liter (3.6 nmol/liter); peak 19-d testosterone cutoff, 2.75 microg/liter (9.5 nmol/liter)] gave a sensitivity and a specificity of 100%. CONCLUSIONS: Our data suggest that a GnRH test in combination with both a 3-d and 19-d HCG test may aid in differentiating between CDGP and HH.

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.

The role of growth hormone in determining birth size and early postnatal growth, using congenital growth hormone deficiency (GHD) as a model.

OBJECTIVE: The role of GH in early human growth is unclear. Congenital GH deficiency (CGHD) provides a useful tool to explore this putative role. We have assessed the effects of CGHD on birth size and early postnatal growth, and the further impact of the presence of additional pituitary hormone deficiencies and midline brain defects on these parameters. DESIGN, PATIENTS AND MEASUREMENTS: Weight, length and BMI expressed as standard deviation scores (SDS), over the first two years of life, were retrospectively compared in 44 GH-deficient children (M:F 26 : 18). Thirty-eight of 44 patients underwent GH provocation testing and all patients had neuro-imaging of the brain. The patients were divided into three groups of increasing phenotypic complexity {group A [n = 12, isolated GHD, no midline defects], group B [n = 10, combined pituitary hormone deficiency (CPHD); no midline defects], group C (n = 22, CPHD with midline defects)}. RESULTS: Mean birth weight, length and BMI SDS were -0.4, -0.9 and +0.1 SDS, respectively. The differences were significant for weight (P = 0.03) and BMI (P = 0.003), but not length (P = 0.3) SDS, between groups A and C. Of the three groups, group A had a lower weight and BMI SDS than group C. The prevalence of postnatal complications (n = 25) was significantly different in the three groups [group A (8%), group B (80%), group C (73%); P < 0.001] and particularly between patients with isolated GH deficiency (IGHD) (group A) and CPHD (groups B and C; P < 0.0001). No patients in group A presented with neonatal hypoglycaemia as compared with 70% of those in group B and 59% in group C (P = 0.001). A reduced length SDS was observed in all patients within 6 months of birth and the reduction was greatest in group B (P = 0.03). Group C remained significantly (P < 0.05) heavier at 12, 18 and 24 months compared to group A. BMI SDS was significantly (P < 0.05) greater at all study points in CPHD patients (groups B and C) as compared with IGHD. Serum GH concentrations at testing did not correlate significantly with birth length (r = -0.08, P = 0.7), birth weight (r = -0.08, P = 0.6) or the age at induction of GH treatment (r = 0.12, P = 0.5). There were no significant differences between peak serum GH concentrations in patients in groups A (7.8 +/- 6.3 mU/l), B (3.9 +/- 4.8 mU/l) or C (8.7 +/- 5.4 mU/l). CONCLUSIONS: Length, weight and BMI data from our study groups suggest that GH per se has a minimal effect on intrauterine growth but a significant effect during the infancy period. Early growth may also be influenced by the complexity of the hypopituitary phenotype reflected by the presence of additional pituitary hormone deficiencies and midline forebrain defects.

Novel mutations within the POU1F1 gene associated with variable combined pituitary hormone deficiency.

CONTEXT: Mutations within the gene encoding the pituitary-specific transcription factor POU1F1 are associated with combined pituitary hormone deficiency (CPHD). Most of the affected individuals manifest GH, prolactin, and TSH deficiency. OBJECTIVE: We have now screened 129 individuals with CPHD and isolated GH deficiency for mutations within POU1F1. RESULTS: Causative mutations were identified in 10 of 129 individuals (7.8%). Of these, five patients harbored the dominant negative R271W mutation, which is a well-recognized mutational hot spot. We have also identified a second frequently occurring mutation, E230K, which appears to be common in Maltese patients. Additionally, we describe two novel mutations within POU1F1, an insertion of a single base pair (ins778A) and a missense mutation (R172Q). Functional studies have revealed that POU1F1 (E230K) is associated with a reduction in transactivation, although DNA-binding affinity is similar to the wild-type protein. On the other hand, POU1F1 (R172Q) is associated with a reduction in DNA binding and transactivation, whereas POU1F1 (ins778A) is associated with loss of DNA binding and a reduction in transactivation. CONCLUSIONS: Our data suggest that the phenotype associated with POU1F1 mutations may be more variable, with the occasional preservation of TSH secretion. Additionally, our data revealed POU1F1 mutations in three patients who were diagnosed as having ACTH deficiency but who, on further evaluation, were found to have normal cortisol secretion. Hence, elucidation of the genotype led to further evaluation of the phenotype, with the cessation of cortisol replacement that had been commenced unnecessarily. These data reflect the importance of mutational analysis in patients with CPHD.

Mutations within the transcription factor PROP1 are rare in a cohort of patients with sporadic combined pituitary hormone deficiency (CPHD).

OBJECTIVE: Mutations within the pituitary-specific paired-like homeobox gene PROP1 have been described in 50-100% of patients with familial combined pituitary hormone deficiency (CPHD). We screened a cohort of sporadic (n = 189) and familial (n = 44) patients with hypopituitarism (153 CPHD and 80 isolated hormone deficiencies) for mutations within the coding sequence of PROP1. DESIGN AND PATIENTS: Patients with congenital hypopituitarism were recruited from the London Centre for Paediatric Endocrinology as well as several national and international centres. The pituitary phenotype ranged from isolated growth hormone deficiency (IGHD) to panhypopituitarism. Clinical data, including endocrine and neuro-radiological studies were obtained from patient records, and DNA was collected and screened for mutations within PROP1 using PCR and single-stranded conformation polymorphism (SSCP) analysis. Positive results on SSCP were sequenced directly. RESULTS: The prevalence of PROP1 mutations in unselected sporadic cases of hypopituitarism was lower (1.1%) than in familial cases (29.5%). PROP1 mutations can be associated with a highly variable phenotype, and both pituitary hypoplasia and pituitary hyperplasia. We describe the waxing and waning of a pituitary mass over 20 months in association with a PROP1 mutation that is predicted to lead to complete loss of function. Additionally, we have identified a possible founder mutation in CPHD patients from the Indian subcontinent. CONCLUSIONS: PROP1 mutations are rare in sporadic cases of CPHD, although the prevalence rises if there is a positive family history or if the patients are carefully selected with respect to the endocrine and neuroradiological phenotype. There is considerable phenotypic variability in families with the same mutation, indicating the role of other genetic or environmental factors on phenotypic expression. Finally, the pituitary enlargement that is observed in patients with PROP1 mutations can wax and wane in size before eventual involution.

Over- and underdosage of SOX3 is associated with infundibular hypoplasia and hypopituitarism.

Duplications of Xq26-27 have been implicated in the etiology of X-linked hypopituitarism associated with mental retardation (MR). Additionally, an expansion of a polyalanine tract (by 11 alanines) within the transcription factor SOX3 (Xq27.1) has been reported in patients with growth hormone deficiency and variable learning difficulties. We report a submicroscopic duplication of Xq27.1, the smallest reported to date (685.6 kb), in two siblings with variable hypopituitarism, callosal abnormalities, anterior pituitary hypoplasia (APH), an ectopic posterior pituitary (EPP), and an absent infundibulum. This duplication contains SOX3 and sequences corresponding to two transcripts of unknown function; only Sox3 is expressed in the infundibulum in mice. Next, we identified a novel seven-alanine expansion within a polyalanine tract in SOX3 in a family with panhypopituitarism in three male siblings with an absent infundibulum, severe APH, and EPP. This mutation led to reduced transcriptional activity, with impaired nuclear localization of the mutant protein. We also identified a novel polymorphism (A43T) in SOX3 in another child with hypopituitarism. In contrast to findings in previous studies, there was no evidence of MR or learning difficulties in our patients. We conclude that both over- and underdosage of SOX3 are associated with similar phenotypes, consisting of infundibular hypoplasia and hypopituitarism but not necessarily MR.

An update on the biochemical diagnosis of congenital ACTH insufficiency.

OBJECTIVE: Congenital ACTH insufficiency due to disorders of the hypothalamo-pituitary axis is life threatening. However, the optimal method for establishing the diagnosis remains controversial. The standard Synacthen test (SST) is safe and easy to perform in infancy, but its performance has not been well evaluated in infants and children with ACTH insufficiency. DESIGN, PATIENTS AND MEASUREMENTS: We aimed to determine the value of the SST by comparing the 30-min SST cortisol concentration with physiological (spontaneous) cortisol secretion (2-hourly measurements for 24 h) in 28 patients (13 male; age range 0-5.1 years) with one or a combination of optic nerve (25) midline forebrain (17) and other pituitary hormone (17) abnormalities. Spontaneous cortisol secretion (mean < 145 nmol/l) was arbitrarily used as the 'gold-standard' in the diagnosis of ACTH insufficiency as it offered a reflection of endogenous cortisol mileu. A normal SST was defined as a 30-min serum cortisol > 540 nmol/l. RESULTS: The SST basal, 30-min and 60-min serum cortisol concentrations were significantly (P < 0.001) lower in patients with ACTH insufficiency (mean spontaneous cortisol < 145 nmol/l). 3/13 patients without and 12/15 patients with ACTH insufficiency failed the SST (specificity 76.9%; sensitivity 80%). Two out of three patients with ACTH insufficiency who passed the test were symptomatic with fatigue, poor growth and hypoglycaemia that resolved with hydrocortisone treatment. An 08:00 h serum cortisol cut-off of 175 nmol/l provided a sensitivity of 93.3% and a specificity of 92.3% as compared with spontaneous cortisol secretion. The 30-min increment and the 60-min SST cortisol concentrations although highly specific (100%) had reduced sensitivity (40% and 60%, respectively). CONCLUSIONS: A normal SST does not rule out ACTH insufficiency and measurement of physiological cortisol secretion may be required to confirm the diagnosis. A combination of 08:00 h serum cortisol > 175 nmol/l and a 30-min SST serum cortisol > 540 nmol/l to exclude ACTH insufficiency (sensitivity 69%, specificity 100%) would ensure that no child with ACTH insufficiency would be missed and hence would prevent serious consequences, although it may lead to over-diagnosis in some cases.

Is the thyrotropin-releasing hormone test necessary in the diagnosis of central hypothyroidism in children.

To determine the value of the TRH test, we analyzed the unstimulated serum T(4) and TSH concentrations in 54 children with central hypothyroidism. A TRH test was performed in 30 patients. Midline brain defects (septo-optic dysplasia, 28; holoprosencephaly, 2) and combined pituitary hormone deficiencies were present in 30 and 52 patients, respectively. The mean serum free T(4), total T(4), and basal TSH concentrations were 0.6 ng/dl, 4.0 microg/dl, and 2.8 microU/ml, respectively. Five patients demonstrated elevated basal serum TSH concentrations. A normal TRH test [increase (delta) in TSH, 4.5-17.8], based on data from 30 controls, was documented in 23.3% of patients. Brisk (deltaTSH, >17.8), absent/blunted (deltaTSH, <4.5), and delayed responses were documented in 16.7%, 30%, and 30% of patients, respectively. The mean age at diagnosis was 2.8 yr, with 8 patients evolving into TSH deficiency. It was not possible to differentiate patients as having pituitary or hypothalamic disease based solely on the TRH test results. Patients with septo-optic dysplasia were diagnosed earlier and had elevated basal serum TSH and PRL concentrations, diabetes insipidus, and evolving disease. Although full pituitary function assessment is mandatory to identify combined pituitary hormone deficiencies, a TRH test is not essential, and the diagnosis should be made by serial T(4) measurements.

The use of somatropin (recombinant growth hormone) in children of short stature.

The availability of somatropin [recombinant human growth hormone (GH)] has revolutionized the treatment of short stature resulting from GH deficiency. It is also widely used as an adjunct in the treatment of other disorders which do not fit the definition of classic GH deficiency, such as intrauterine growth restriction, Turner syndrome, healthy children with short stature and skeletal dysplasias. The widespread use and ready availability of GH treatment has prompted questions about its tolerability, rationality, and the psychological effects of long-term treatment, leading to several trials. Early treatment of GH deficiency will allow the child to reach his or her genetic potential, although there continues to be marked variability in the criteria used to diagnose the deficiency, and in the treatment schedule, especially during puberty. Treatment has also been shown to have a beneficial effect on growth in children with chronic renal failure, with no adverse effects on the renal function. There are, however, no long-term data to determine final height, or randomized controlled studies to justify routine use of GH in conditions such as intrauterine growth restriction. It remains controversial in conditions such as Turner syndrome and achondroplasia, where the response to treatment is only moderate. Healthy children with short stature have not been shown to have a psychological disadvantage, again proving difficult to justify prolonged GH treatment for idiopathic short stature. Meticulous monitoring, long-term follow-up to adult or near-adult final height, and well-defined endpoints of treatment need to be better clarified. The metabolic effects of treatment on the patient's lipid profile, bone mineral density, and muscle mass need careful documentation, especially with the high doses used in an already susceptible population such as low birthweight children and those with Turner syndrome. Lastly, the psychosocial impact of GH treatment, financial implications, and cost efficacy of treatment in an ever-increasing list of indications should be taken into consideration for rationalizing its use in future.