Genetic basis of growth hormone deficiency: An update for pediatric endocrinologists

INTRODUCTION

The pituitary gland regulates major body systems by the production of multiple key hormones. The anterior pituitary has five functional cell types: somatotropes that secrete growth hormone (GH), lactotropes that secrete prolactin (PRL), thyrotropes that secrete thyroid-stimulating hormone (TSH), gonadotropes that secrete luteinizing hormone (LH) and follicle-stimulating hormone (FSH), and corticotropes that secrete adrenocorticotropic hormone (ACTH). Arginine vasopressin (AVP) is synthesized in the magnocellular neurons of the hypothalamus and is secreted by the posterior pituitary. GH deficiency (GHD) is the most common anterior pituitary deficiency. It may occur as an isolated deficiency (IGHD) or in combination with deficiencies of one or more other pituitary hormones (multiple pituitary hormone deficiency [MPHD]). Both IGHD and MPHD may be congenital or acquired as a result of tumors, irradiation, pituitary surgery, or infection. IGHD has a birth prevalence of 1/4,000–10,000 live births,^[1] whereas that of congenital MPHD is 10/100,000 live births.^[2]

In 1981, for the first time, shortly after the human GH gene, growth hormone 1 (GH1) was characterized, a homozygous deletion in the gene was detected and described as a cause of familial IGHD.^[3] Further, in 1996, the role of GH releasing hormone receptor gene (GHRHR) mutation as the second molecular basis for recessively inherited IGHD was reported.^[4] Over the past three decades, our understanding of the genetic basis of GHD has considerably expanded. Further, the availability of next-generation sequencing (NGS) has helped in the identification of variants in cohorts of patients with GHD. Similar advances have occurred in the sphere of GH resistance or insensitivity (GHI). The condition now not only includes the most severe phenotype caused by receptor mutations, first described by Laron in 1966, but also milder phenotypes caused by genes involved in post-receptor signaling.^[5] This review focuses on the genetic basis of congenital forms of GHD, in the context of clinical evaluation and management.

CLINICAL FEATURES AND DIAGNOSIS OF GHD

Clinical features vary with the age of presentation. Birth weight and length are often normal. A history of breech delivery, neonatal hypoglycemia due to deficiencies of GH and ACTH, and micropenis on examination may be clues in early life. Prolonged neonatal jaundice and nystagmus are common associations of septo-optic dysplasia (SOD). In severe congenital GHD, growth failure becomes apparent in the latter half of infancy, with significant height deficits < –3 standard deviation score (SDS) by the age of 3–4 years. However, the more common presentation of GHD is that of short stature that becomes increasingly marked during childhood years, with height falling below the expected parental target. Other clinical features include midline defects, midfacial hypoplasia, and truncal obesity. Beyond the neonatal period, the sequence of onset of hormonal deficiencies is variable, for example, GHD followed by TSH deficiency in early years, and delayed puberty due to gonadotropin-releasing hormone (GnRH) deficiency follows in later years. ACTH deficiency may evolve variably throughout childhood.

Median age at diagnosis of GHD in children is around 8 years.^[6] In a birth cohort study, the mean cumulative incidence of a diagnosis of GHD increased by more than 10-fold from that at an age of 4 years (4/100,000) to 8 years (55/100,000).^[7] When suspected clinically, GHD should be further evaluated by dynamic stimulation tests using various agents such as clonidine, arginine, glucagon, L-dopa, or insulin. Two “failed” tests, where peak GH levels are below 7 ng/mL, are considered confirmatory for GHD. Insulin-like growth factor-1 (IGF-1) is a useful biomarker and is often low in children with GHD; however, an IGF-1 level in the reference range does not exclude GHD. In a setting of additional pituitary hormone deficiencies and magnetic resonance imaging (MRI) abnormalities in newborns and young infants, GHD may be diagnosed based on low random GH levels, without requiring a GH stimulation test.^[8]

Besides auxological criteria and biochemistry, pituitary neuroimaging is a key investigation. Pituitary findings seen in association with GHD could be confined to the anterior pituitary, such as small or absent anterior pituitary (anterior pituitary hypoplasia [APH]), or those related to the stalk and posterior pituitary, such as pituitary stalk interruption syndrome (PSIS) and ectopic posterior pituitary (EPP). MRI abnormalities are common in 83% of IGHD, and all cases of MPHD had MRI abnormalities in a study.^[9] The basis of various findings on neuroimaging can be better understood by considering pituitary neurodevelopment as described in the section on the genetic basis of GHD below. Although neuroradiology is valuable in evaluation, it cannot be used on its own to diagnose GHD without supporting anthropometric and biochemical parameters.

CASE SCENARIOS

GENETIC BASIS OF GHD

Rathke’s pouch which originates in the oral ectoderm, develops into the anterior pituitary and subsequently forms connections with the neurohypophysis of hypothalamic origin. The pituitary stalk, an extension of the hypothalamus, consists of both portal vascular channels that transport hypothalamic hormones to the anterior pituitary, and axons of hypothalamic origin that regulate neuroendocrine function of the posterior pituitary. Synchronized expression of several common transcription factors and their signaling molecules in the developing hypothalamus and Rathke’s pouch are essential for the normal structural and functional development of the pituitary gland. Further, the development of the forebrain, hypothalamus, and pituitary is genetically linked by common transcription factors and signaling molecules.^[10] This explains why eye and craniofacial abnormalities, along with MRI abnormalities in the suprasellar region as well as in the optic nerves, optic chiasm, and corpus callosum, are associated with some forms of GHD [Figure 1].

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Figure 1:

Illustration of the GHRH-GH-IGF-1 axis with examples of genetic mutations associated with GHD at various levels. The hypothalamus secretes GHRH, which acts on the pituitary GHRH receptor to stimulate growth hormone secretion. Growth hormone acting though it’s receptors on peripheral tissues produces IGF-1. A number of genes are associated with early development of both pituitary and hypothalamus, whereas, genes such as GHRHR, POU1F1 and GH1 have predominant function in the pituitary gland. Genes involved in further steps of the axis such the GH receptor gene and those involved with IGF-1 synthesis, circulation and action are associated with growth hormone insensitivity. GH: Growth hormone, GHD: Growth hormone deficiency, GHRH: Growth hormone-releasing hormone, IGF-1: Insulin-like growth factor-1, GHRHR: Growth hormone releasing hormone receptor.

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SOD (presence of two of the following three features: septum pellucidum abnormalities, optic nerve hypoplasia, and pituitary hormonal deficiencies) is associated with mutations in transcription factors homeobox expressed in ES cells 1 (HESX1), sex determining region Y-box (SOX2, SOX3) and orthodenticle homeobox 2 (OTX2).^[11] Though there is broad overlap in the MRI findings among various genetic causes, findings confined to the pituitary (e.g., APH) are more common in IGHD, whereas EPP and PSIS are more frequently observed in MPHD.^[12,13]

The physiology of GH axis further explains links to the genetic basis of GHD. GH is secreted by pituitary somatotropes in response to stimulation by the hypothalamic regulating hormone, GH-releasing hormone (GHRH). It is a 191-amino acid single-chain polypeptide coded by GH1 gene located on chromosome 17 (q22-24). In target tissues, binding of GH to the extracellular domain of GH receptor triggers the JAK/STAT (Janus kinase/signal transducers and activator of transcription proteins) pathway leading to transcription and IGF-1 production. IGF-1 circulates as a complex with IGF-binding proteins (IGFBPs) and acid-labile subunit (ALS). Acting by both autocrine and paracrine pathways, on binding to its receptors, IGF-1 stimulates the PI3K (phosphatidylinositide-3 kinase) and mTOR (mammalian target of rapamycin) pathways.

Variants in a number of genes and transcription factors involved in the synthesis and secretion of GH have been identified in children with GHD, whereas variants in GHR and post-receptor signaling of its effector molecule, IGF-1, are associated with GHI [Figure 1].

GENETIC CAUSES OF MPHD

Mutations of genes involved in hypothalamic and pituitary development are associated with both IGHD and MPHD [Tables 1 and 2, Figure 2]. A certain pattern of affected hormones may be seen, for example, POU1F1 mutations cause GH, TSH, and PRL deficiencies, whereas in PROP1 mutations, LH and FSH are also affected.^[14] However, ACTH deficiency has been reported to develop throughout childhood and later years in PROP1 mutations.^[15] Further, the presence of specific clinical features such as restriction of neck movements (LHX3 as in Case 3), eye abnormalities (OTX2),^[16] and polydactyly (GLI2) together with MRI findings increases the likelihood of certain groups of genetic variants [Tables 1 and 2].^[17-25]

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Figure 2:

Genetic mutations grouped by predominant clinical presentation and MRI findings. Approach to identifying the genetic etiology of GHD. (A) Genes are grouped based on the clinical phenotype, certain groups of genes are associated primarily with short stature, while others are linked to syndromic features identified on clinical examination. Asterisks (*) indicate the pattern of pituitary hormone deficiencies seen with specific mutations, and selected distinctive clinical features are provided in parentheses. (B) Genes are grouped according to characteristic patterns observed on pituitary MRI. GHD: Growth hormone deficiency, MRI: Magnetic resource imaging.

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GENETIC CAUSES OF IGHD

IGHD is familial in 30% of cases^[26] and was classified as types I, II, and III based on inheritance patterns and phenotype in 1994.^[27]

IGHD type 1a is recessive and caused by a complete lack of GH production due to homozygous GH1 deletion. There is an early onset, severe growth failure (from 6 months of life with height >4.5 SDS below mean). Anti-GH antibodies limit response to rhGH therapy. IGHD type 1b was previously described to have autosomal recessive inheritance, with a milder phenotype than 1a, with good response to rhGH therapy. It is caused by homozygous or compound heterozygous mutations in the GH1 gene.

IGHD type II, which has a dominant inheritance, is caused by splice site mutations, particularly in intron 3 of GH1. The resultant 17.5 kDa GH isoform has been shown to reduce the secretion of wild-type GH (wt-GH) in cell cultures.^[28] This also accounts for the likelihood of progression to deficiencies of other pituitary hormones, despite the genetic locus being in GH1, explaining the course in the illustrative Case 2.^[29] Diagnostically, children present with variable degrees of familial short stature with low IGF-1 and IGF binding protein-3 (IGF-BP3) levels. Individuals have shown a good response to rhGH therapy. Pharmacological modulation in animal models using butyrate has been shown to influence splicing, with improved output of wt-GH.^[30]

IGHD type III, caused by mutations in the BTK gene, has an X-linked recessive inheritance and is associated with agammaglobulinemia

IGHD type IV has now been described in individuals with homozygous mutations in the GHRHR. Recently, a homozygous nonsense mutation in exon 3 of the growth-hormone-releasing hormone receptor gene GHRHR (MIM#139191) was described in a family with severe short stature and GHD.^[31]

With further understanding of pathways involved in abnormalities of the hypothalamus, pituitary, and forebrain in animal models, new genetic variations are increasingly being identified in patients with GHD, for example, TCF7L1, a regulator of the WNT/β-catenin signaling pathway.^[32] Further, genes involved in non-endocrine systems have been identified, such as IFT172, in which mutations cause a syndrome suggestive of a ciliopathy with pituitary hypoplasia and EPP. Mutations in PCSK1 gene, which is involved in enteroendocrine function known to cause diarrhea and malabsorption, may be associated with MPHD.^[33]

GENETIC BASIS OF IGF-1 DEFICIENCY

Laron syndrome or “classic GH insensitivity” is a rare condition resulting from mutations in the GH receptor gene, which results in severe short stature. Milder phenotypes of GHI due to several other monogenic causes are being recognized.^[5] For example, mutations in signal transducer and activator of transcription (STAT5B), IGF1 gene as well ALS have been identified as causes of GH insensitivity [Figure 1].

APPLICABILITY OF GENETIC TESTING IN CLINICAL PRACTICE

The use of genetic studies in clinical practice has been reported by several groups in India. GH1 deletions were studied using polymerase chain reaction in a cohort of 30 children with IGHD. Those who had a deletion (40%) were significantly shorter and had very low peak GH levels on stimulation testing.^[34] In another study from India, GH1 and GHRHR were studied using Sanger sequencing in 116 children with IGHD, with a 21% positivity rate; the rate of GHRHR variants being higher.^[35] In a mixed IGHD and MPHD cohort from Western India, we reported 30% (16/53) mutation-positive cases using a panel consisting of GH1, GHRHR, LHX3, LHX4, and PROP1, POU1F1, and HESX1. GH1 and GHRHR accounted for most (14/16) of the mutation-positive cases.^[36] These studies show that GH1 and GHRHR together constitute a high number of mutation-positive cases in Indian cohorts where testing was done in routine clinical practice. Overall prevalence of mutation-positive cases in MPHD is low (<20–30%), as in a study, only 2 out of 27 MPHD patients had a positive mutation using a panel consisting of PROP1, POU1, and HESX1.^[37] The positivity rate remained low (15.3%) in a larger study involving 170 patients using a 67-gene panel.^[38]

Genetic testing may guide monitoring in patients with GHD. Progression from IGHD to MPHD has been reported to be greater in those with an identified genetic change.^[39] According to current understanding, response to GH therapy is not significantly different in those with or without identified mutations as such.^[36] In this study, those with homozygous GH1 deletions had a poor response to GH therapy, a well-characterized aspect of this genetic change.

Genotype-phenotype correlation is not well established in children with GHD, but certain features may help the clinician consider certain groups of genes [Figure 2]. The pattern of affected hormones, in combination with pituitary MRI findings, may suggest a genetic etiology in MPHD. External eye abnormalities such as anophthalmia or microphthalmia are associated with OTX2, whereas nystagmus and visual impairment may be associated with genes involved in SOD. Further clues, such as cleft lip/palate, polydactyly, and dysmorphic facies, could point to certain gene defects. GH1 and GHRHR are common mutations in familial IGHD, whereas biallelic PROP1 is the most common cause of familial MPHD. Screening for PROP1 mutation using Sanger sequencing in patients with MPHD and an intact posterior pituitary was proposed as a cost-effective approach in ethnic populations where this mutation is common.^[15]

Clinical exome sequencing is available for use in India and abroad. The ability to study a number of target genes in a single study is advantageous; however, costs are considerably higher for families with financial constraints. However, a genetic diagnosis may be particularly beneficial in circumstances such as:^[40]

1. When a GH stimulation test is not considered safe (as in young infants, with hypoglycemia, and those with comorbidities),

2. Establishing a molecular diagnosis in GHD with syndromic features,

3. Cases with suboptimal response to rhGH therapy,

4. Identifying a familial mutation for diagnosis or for the prediction of recurrence risk and

5. Antenatal testing is recommended when there is a family history.

CONCLUSION

Recent advances in molecular genetics have widened our understanding of the genetic associations of congenital IGHD and MPHD. Extensive descriptions of clinical and radiological features have helped characterize the genetic variants in broad phenotype-based groups. While GH1 and GHRHR mutations remain the most commonly identified causes in IGHD, a growing number of genes encoding transcription factors and intracellular signaling molecules are now recognized, particularly in syndromic and MPHD cases. Genetic testing, though not essential in the diagnostic pathway, is valuable in selected scenarios – especially where syndromic features are present, diagnostic uncertainty exists, or there is a need for family counseling. Whole-exome sequencing or NGS has made it possible to identify newer genes and genetic variations in the research setting. The yield of a positive mutation remains low despite recent advances in genetic diagnosis, but the pre-test probability of identifying a mutation is higher in familial cases, those with a severe phenotype, and in MPHD. Despite considerable advances in genetics, auxological parameters, biochemistry, and neuroimaging continue to remain the cornerstones of diagnosis in clinical practice.