ENDOCRINE TODAY October 2007
Growth Hormone Disorders: Molecular Genomics and Clinical Implications

Download and mail the CME quiz

| |

---

Introduction Understanding the molecular basis of growth hormone disorders David R. Brown, MD, FACE Overview of IGF-I Ron G. Rosenfeld, MD, FACE Clinical and molecular aspects of IGF-I deficiency and resistance Mitchell E. Geffner, MD and Mark A. Sperling, MD Genomic regulation in the induction of puberty David R. Brown, MD, FACE Growth hormone treatment information for nurses Gaye Madigan, APRN, BC and Jan A. Penn, BSN, RN

Introduction

The diagnosis of growth hormone disorders was previously based on nonspecific criteria and poorly reproducible diagnostic testing, becoming a catchall for many conditions and including patients with undefined causalities of idiopathic short stature. More recently, a greater understanding of the specific genetic defects for diseases that result in growth deficiencies has enabled endocrinologists to make more specific diagnoses and develop appropriate individualized management protocols for patients in infancy through the onset of puberty and into adulthood. Pediatric endocrine nurses play a vital role in explaining these treatment strategies to patients and their families, ensuring their compliance with appropriate regimens to address their conditions.

Vindico Medical Education conducted symposia at the Pediatric Endocrine Nurses Society annual meeting in April, the Pediatric Academic Society meeting in May and the Endocrine Society meeting in June to bring together leading experts to discuss the latest developments in the understanding of growth hormone disorder. This monograph is based on the proceedings of those symposia.

Alan J. Garber, MD, PhD
Chief Medical Editor
Endocrine Today

David R. Brown, MD, FACE, Course Director, is a Staff Physician at Children’s Hospital and Clinics of Minnesota and a Clinical Professor of pediatrics at the University of Minnesota in Minneapolis, Minnesota. Ron G. Rosenfeld, MD, FACE, is a Professor of Pediatrics at Stanford University in Palo Alto, California. Mitchell E. Geffner, MD, is a Professor of Pediatrics at Children’s Hospital of Los Angeles in Los Angeles, California. Mark A. Sperling, MD, is a Professor of Pediatrics at Children’s Hospital of Pittsburgh in Pittsburgh, Pennsylvania. Gaye Madigan, APRN, BC, is a Nurse Practitioner in Pediatric Diabetes and Endocrinology at Monmouth Medical Center in Long Branch, New Jersey. Jan A. Penn, BSN, RN, is a Nurse Specialist and Clinical Research Study Coordinator at Pediatric Endocrinology and Metabolism and a Staff Nurse, Special Diagnostics Center at Children’s Hospital and Clinics of Minnesota in Minneapolis, Minnesota.

Understanding the molecular basis of growth hormone disorders
David R. Brown, MD, FACE

Pediatric endocrinology is progressively developing a foundation in genomics, as the discipline becomes synonymous with molecular medicine. This will confer a biological legitimacy in creating a molecular-based specialty. The result will be an increased number of diagnostic entities as a consequence of enhanced diagnostic specificity. This has been most demonstrable in the area of growth disorders, where diagnostic methodologies and expanding therapeutic options have been established on genomic principles and recombinant DNA technologies. In the recent past, diagnoses were based on problematic growth hormone assays and the availability of a single therapeutic option, the administration of biosynthetic growth hormone. No practical methodologies were available to identify specific defects in the growth hormone signaling pathway nor were target-based therapies for patients who failed to respond optimally to growth hormone treatment.

“Today’s endocrinologists are equipped to understand the underlying mechanisms of specific growth disorders and facilitate the development of new biopharmaceuticals based on recombinant technologies.” — David R. Brown, MD, FACE

Enhanced diagnostic capabilities utilizing molecular genomic-based procedures have allowed the description and identification of discrete mutations and deletions encompassing enumerable causalities of growth hormone disorders. Today’s endocrinologists are equipped to understand the underlying mechanisms of specific growth disorders and facilitate the development of new biopharmaceuticals based on recombinant technologies. Consequently, growth hormone disorders are better understood and the number of growth hormone-based conditions classified as idiopathic short stature is being reduced, concurrent with the availability of multiple treatment options. Genomic diagnosis and targeted therapies are the evolving paradigm. Growth hormone has earned its place as a relevant treatment method; however, management of idiopathic short stature is entering an IGF-centric era and new options and combined GH/IGF-I approaches will expand the endocrine treatment arsenal and offer alternative choices in the best interests of patients.

Growth hormone-related genomic transcription factors

Approximately 32 identified genomic factors are involved in the growth hormone and related pubertal pathways (Table). At the hypothalamic level, multiple transcription factors that regulate pituitary ontogeny and the induction of trophic hormone synthesis are active. Pituitary transcription factors further regulate pituicyte differentiation and growth hormone synthesis. Growth hormone target cells such as the hepatocyte and chondrocyte are sites of action for the growth hormone signal pathway factors, which modulate message transduction from the growth hormone receptor and activation of transcription of the ternary complex components. Additional transcription factors are identified which facilitate growth hormone mediated effects in peripheral sites affecting processes such as collagen biosynthesis and fibroblast differentiation. In addition, discrete genomic factors are known to regulate the GnRH neuron, initiating puberty and facilitating feedback regulation of gonadotropin synthesis and secretion.

Table: Growth-Related Genomic Transcription Factors

The growth hormone pathway

Genomic transcription factors are involved in the GH/IGF-I signal pathway. There are nine discrete steps in this pathway for which a molecular understanding exists. These include control of growth hormone synthesis and secretion; the transport of growth hormone in the circulation; growth hormone action in the liver and interaction with its receptor; intracellular signaling events; the production of the ternary complex, which includes insulin-like growth factor (IGF-I), binding protein 3 (IGFBP-3) and acid labile subunit (ALS); the release of IGF-I into the circulation; the transport of IGF-I to target sites; IGF-I interaction with the type I receptor; and the combined effect of growth hormone and IGF-I with chondrocytes at the epiphyseal growth plate. The three primary sites of action for these genomic transcription factors are the pituitary, liver and bone. The pituitary gland is associated with the initiation and completion of growth hormone synthesis. Activation of the growth hormone signal pathway occurs initially in the liver, and the corresponding chrondrocyte pathway mediates the growth process at the epiphyses in bone.

Receptor activation and phosphorylation

Recent studies by Waters and colleagues¹ have demonstrated that receptor dimerization occurs within the external domain of the growth hormone receptor, suggesting possible conformational effects that may alter growth hormone binding and provide another level of regulation as well as an additional target site for abnormalities blocking growth hormone signal transduction and resulting in defects of growth hormone resistance analogous to classic Laron syndrome.

In eukaryotes, the primary mechanism for informational signaling is phosphorylation. Dimerization of the hepatic GH receptor and subsequent binding of the GH ligand activates the signaling pathway by transduction from the external receptor domain to the cytoplasmic receptor elements, which in turn initiate tyrosine phosphorylation of the intracellular Janus Kinase (JAK-2), enlisting the participation of a STAT (Signal Transduction Activator of Transcription) pathway. Once a particular STAT is recruited to a JAK-2 receptors it undergoes phosphorylation resulting in two STAT proteins forming a dimer. Each STAT has an SH-2 domain, which recognizes phosphorylated tyrosine residues in certain sequences and is therefore a basis for specificity. Consequently, the specific STAT recruited may determine which genes are subsequently activated. Dimerized STAT-5b must localize to the nucleus, identify specific genomic DNA and activate the transcriptional gene sequences that encode for IGF-1, IGFBP-3 and ALS.

STAT transcription activation

The mechanisms by which STAT-5b is able to locate and identify, as well as activate transcription of the ternary complex genes, are only partially understood. Some specificity is confirmed by the particular SH-2 domain because the nuclear binding sites may be different for individual STATs. Promoter docking sites for STATs are primarily based on allosteric relationships between specific amino acids and nucleotides. Stereoconformational changes in DNA result from reciprocal hydrogen bonding. Strong covalent bonds occur most selectively between purine nucleotides and arginine-containing peptide sequences and result in rotations of 35º to 40° along the DNA helical axis. Such conformational changes open the genome and facilitate DNA transcription. We may anticipate the identification of growth disorders, which are the result of STAT-mediated transcriptional defects that occur as the result of failure of docking site recognition, inappropriate docking and failure to initiate conformational changes within the genome.

An educational opportunity

This monograph is designed to clarify the differences between IGF-I deficiency and IGF-I resistance and assist clinicians in understanding the molecular basis of GH and IGF-1 disorders. The initial article by Ron G. Rosenfeld, MD, provides an overview of growth hormone and IGF-I signal pathways. The following article, authored by Mark A. Sperling, MD, and Mitchell E. Geffner, MD, highlights the clinical and molecular aspects of IGF-I deficiency and resistance. My article will outline the genomic regulation of the onset of puberty. Finally, Gaye Madigan, APRN, BC, and Jan A. Penn, BSN, RN, will provide practical tips for nurse practitioners who work with growth hormone disorders.

Reference

1. Waters MJ, Hoang HN, Fairlie DP, Pelekanos RA, Brown RJ. New insights into growth hormone action. J Mol Endocrinol. 2006; 36:1-7.

Overview of IGF-I
Ron G. Rosenfeld, MD, FACE

Modern treatment of growth hormone (GH) disorders has evolved from processes unique to the field of health care. Historically, the fundamental axiom of medicine has been that appropriate diagnosis dictates appropriate treatment. In this manner, the algorithm of medical practice consists of first identifying the particular ailment and then selecting the appropriate treatment modality. However, beginning in the early 1960s, availability of growth hormone treatment resulted in a reversal of this model. For patients with short stature, the available treatment dictated the diagnosis of GH disorders. This was caused by the early development of growth hormone as a treatment that preceded any GH radioassay or diagnostic tool that could identify the molecular basis of growth hormone deficiency. Physicians were treating children with growth hormone before they had an understanding of the underlying mechanisms causing growth hormone deficiency.

This reversal in practice led to the emergence of nonspecific terminology used to describe a poorly understood condition. Terms such as partial GH deficiency, neurosecretory defect, bioinactive GH, non-GH disorder short stature and idiopathic short stature were used to describe a malfunction within the GH signal cascade. Although these terms fail to capture the underlying pathophysiology of GH disorders, they can be found today in pediatric endocrinology textbooks. Although our understanding of this disorder has improved over the past 40 years, a need exists for a new diagnostic paradigm for GH disorders.

The GH signal cascade

Many of the recent advances for patients with GH disorders have been based on a deeper understanding of the events leading to healthy growth (Figure 1). Growth is initiated by GH secretion by means of numerous pituitary transcription factors.¹ This leads to activation of the GH receptor, which is a member of the cytokine receptor superfamily. This receptor, unlike the insulin or insulin-like growth factor-I (IGF-I) receptor, has no intrinsic kinase capability and therefore cannot phosphorylate an intracellular signal. To complete the process, the GH receptor recruits the cytoplasmic protein Janus kinase 2 (JAK-2). ² JAK-2 binds to the GH receptor, autophosphorylates and acts as a docking site for a second cytosolic protein named signal transducer and activator of transcription 5b (STAT-5b). STAT-5b is phosphorylated in the receptor and then dissociates, dimerizes into the cytoplasm, binds to DNA and initiates transcription of genes for a number of proteins including IGF-I, IGF-I-binding protein-3 (IGFBP-3) and acid labile subunit (ALS). This event initiates a post-receptor signaling cascade and leads to the expression of IGF-I and IGFIBP-3.

Currently, researchers have identified at least 10 discrete molecular defects that will result in IGF-I deficiency. These defects are located in the extracellular and intracellular cellular domain of the IGF-I gene and include deletions or mutations of the IGF-I gene (classical Laron syndrome) or IGF-I receptor, miscommunications within the post-receptor signaling cascade or JAK-2/STAT-5b system, mutations of the carrier proteins for IGF-I such as ALS and inactivating mutations of the IGF-I gene. The following cases discuss documented mutations and deletions that have occurred in various stages of the GH/IGF-I signal cascade.

Figure 1: Normal Growth: The GH/IGF-I Axis Figure 1. The sequence of events that result in normal growth. Source: Ron G. Rosenfeld, MD, FACE

Deletion of the IGF-I gene

To date, only one case of a complete IGF-I gene deletion has been reported.³ The patient was born at 37 weeks’ gestation; his birth weight was 1.4 kg (3.9 standard deviation [SD] below the mean) and he displayed symmetric growth retardation. Severe growth failure continued throughout infancy and childhood. The patient demonstrated profound bilateral sensorineural deafness, delayed motor development, microcephaly and micrognathia. Treatment with GH was initiated from age 11 to age 12.7 with no effect. His peak serum GH concentration was 94 ng/mL and, at age 14, his serum IGF-I concentration was 0.05 U/mL (normal range, 0.48-2.8 U/mL).

Molecular studies revealed that the patient was homozygous for the D12S364 polymorphism. Sequencing of the gene showed that exon 3 was directly flanked by exon 6, indicating an absence of exons 4 and 5. This deletion resulted in mature IGF-I peptide truncation from 70 to 25 amino acids, followed by an out-of-frame nonsense sequence of eight residues and a premature stop codon.

The patient’s symptoms revealed much regarding the role of IGF-I in various systems. His serum IGFBP-3 and ALS concentrations were within average range, indicating that these peptides act independently of IGF-I. In addition, the bone age of the patient had been only minimally delayed, suggesting that GH directly regulates bone growth. Moreover, the severe intrauterine growth retardation displayed by the patient provides evidence that IGF-I plays a critical role in both prenatal and postnatal growth.

Laron syndrome

The first cases of Laron syndrome were identified in 1966 by Dr. Zvi Laron.⁴ Laron syndrome is an autosomal disorder characterized by elevated levels of GH and low levels of circulating IGF-I.⁵ This condition is caused by a defect in the extracellular domain; a mutation or deletion in exon 2, 3, 4, 5, 6 or 7 will result in the inability of the GH receptor to bind GH. A patient with Laron syndrome will appear relatively normal at birth because IGF-I production in utero is not dependent on GH. However, because the patient has a defect at the GH receptor level in a postnatal state, he or she cannot respond to GH intrinsically or extrinsically and therefore fails to respond to GH therapy.

Until the late 1980s, approximately 50 cases of this condition were reported worldwide, the majority of which were identified in the Mediterranean region.⁶ However, in 1988, a large cohort of individuals with apparent Laron syndrome were identified in two southern provinces in southern Ecuador, increasing the number of reported cases to approximately 100.⁷ The source of growth inhibition in the Ecuadorian patients was found to be in the proximal short arm of chromosome 5. An adenine-to-guanine switch was identified in position 180 of the amino acid sequence, resulting in an E180 splice mutation that negatively affected the ability of the GH receptor to bind GH.

The adult stature of this cohort varied from -5.3 SD to -12 SD (based on US standards). These patients exhibited some craniofacial malformations, musculoskeletal growth delay and varied intellectual functioning. All patients (as children) had elevated random GH levels, some as high as 200 µg/L, and profoundly reduced serum IGF-I concentrations (<10 ng/mL in children <100 ng/mL in adults). Furthermore, IGFBP-3 concentrations were found to be less than 1 µg/mL in children and adults.

Treatment with GH was ineffective in this cohort, as the GH receptor was unable to bind GH. IGF-I treatment (80 µg/kg and 120 µg/kg) was observed over a two-year period and compared to the effects of GH treatment. Patients in the IGF-I treatment group were found to have a greater change in SDs for height and height age, but no change was observed in the height age-bone age ratio. A greater change was observed in the mean percent body weight for height in the IGF-I treatment group compared to the GH treatment group. The difference in growth response between the two cohorts supported the hypothesis that at least 20% of GH-influenced growth is due to the effects of GH on bone.

The JAK-2/STAT-5b pathway

Intracellular domain mutations also play a role in GH-related and IGF-I-related disorders. Mutations in the intracellular domain affect the JAK-2/STAT-5b signal pathway directly or indirectly. Milward and colleagues ⁸ documented a mutant receptor incapable of activating STAT-5b.⁸ The investigation highlights a 53-year-old woman (height -8.7 SD) and her 57-year-old brother (height -6.0 SD). Her peak GH level was 45 µg/L ( >10 µg/L) and her circulating IGF-I level was 8 µg/L (normal range = 54 µg/L to 389 µg/L). Her IGFBP-3 was charted at 16 nmol/L (normal range=61 nmol/L to 254 nmol/) and GHBP was 6.8% (normal range = <10%). Respective levels in the male sibling were IGF-I 38.8 µg/L and IGFBP3 30 nmol/L.

Intracellular domain mutations also play a role in GH-related and IGF-I-related disorders. — Ron G. Rosenfeld, MD, FACE

Sequence analysis of the nine GH receptor coding exons (exons 2-10) in these patients indicated a homozygous 22-bp deletion in exon 10. The consequences of this deletion was a frameshift resulting in novel codons from position 424 to 449 and a premature stop codon at position 450; the resulting protein was truncated at amino acid 449 (GHR1-449). The consequences of this mutation—a GH receptor that could accept GH but lacked the ability to activate STAT-5b and thus could not initiate the release of IGF-I—were evident in both patients.

Mutations within the STAT-5b gene will result in a similar outcome. A study appearing in the February 2005 issue of Pediatric Nephrology described a 16-year-old girl from Argentina with severe postnatal growth failure (-7.5 SD) and a history of respiratory illness.⁹ The immunodeficiency is of interest in this case because STAT-5b is a member of the cytokine receptor family and therefore plays a role in immunity. Additionally, the parents of this patient were first cousins, further supporting the theory of a homozygous genetic mutation.

Biochemical studies identified GH sufficiency and IGF-I deficiency (serum IGF-I 36 ng/mL, N = 224 to 744 ng/mL). Levels of IGFBP-3 and ALS were also deficient (874 ng/mL [N = 2500-4800 ng/mL] and 2.9 µg/mL [normal range = 5.6-16 µg/mL], respectively), and responded poorly to exogenous GH. Genomic DNA sequencing revealed no mutation of the GH receptor gene. Immunoblotting for total STAT-5 indicated normal functioning, but immunoblotting of the STAT-5b-specific C-terminal domain revealed no phosphorylated protein. Reverse transcriptase polymerase chain sequencing (RT-PCS) of the 2.4 kb coding region of the STAT-5b gene exposed a missense mutation in codon 630 and resultant substitution of proline for wild-type alanine. Therefore, the patient could not phosphorylate STAT-5b and IGF-I signaling did not occur.

This patient was the first to be identified with a STAT-5b mutation.⁹ However, since discovering this patient, additional cases of STAT-5b mutations have been identified in Turkey and Kuwait.^10,11 The majority of these patients demonstrated some evidence of immune dysfunction, indicating that mutations within the STAT-5b gene impact immunity as well as growth.

ALS mutation

IGF-I is part of a ternary complex that is composed of IGF-I, IGFBP-3 and ALS. IGF-I circulation is mediated by both IGFBP-3 and ALS. In addition, ALS is believed to extend the half-lives of IGF-I and IGFBP-3 by forming stable ternary complexes. Also, mutations in this protein can result in impaired growth. ¹¹ A case of ALS mutation was documented by Hwa and colleagues in 2005.¹² A patient presented at age 12.5 with moderate growth retardation (-2.9 SD, Turkish standards). The patient had a normal fasting GH level of 3.7 µg/mL but IGF-I and IGFBP-3 were significantly decreased (38 ng/mL and 449 ng/mL, respectively). Height varied between -2.0 SD and -3.0 SD, and IGF-I/IGF-BP3 levels remained consistently low throughout puberty.

DNA sequencing revealed no mutations in the GH, GH receptor, STAT-5b or STAT-5b receptor genes, excluding a diagnosis of GH resistance. However, additional serum analysis revealed undetectable levels of ALS. RT-PCS analysis exposed a homozygous substitution at nucleotide 1318, altering the codon encoding aspartic acid to asparagine at residue 440. This D440N substitution resulted in the inability of ALS to bind IGF-I.

It is important to note that the extremely low levels of serum IGF-I and IGF-BP3 detected in this case are typically associated with clinical phenotypes of severe growth failure due to GH disorders. The authors of the study hypothesized that the absence of normal levels of stable ternary complexes may have failed to impact free IGF-I, which would have contributed to the growth of the patient. Additionally, because GH production may be increased in ALS deficiency, local production of IGF-I may have been elevated to compensate for deficiency in circulating IGF-I.

Figure 2: Reported defects in the GH/IGF-I Axis Figure 2. Reported cases of molecular defects that result in GH/IGF-I deficiency. Source: Ron G. Rosenfeld, MD, FACE

Bioinactive IGF-I

In utero growth and development is mediated by the GH-independent action of IGF-I.¹³ To date, one reported case of complete in utero IGF-I bioinactivity has resulted in severely compromised intrauterine growth.¹⁴ The study involves a 55-year-old man with exaggerated prenatal and postnatal growth retardation, microcephaly, deaf-mutism and mental retardation. The patient displayed markedly elevated levels of serum IGF-I (606 ng/mL) and ALS (28.9 µg/mL), and average IGFBP-3 (1.90 µg/mL).

RT-PCS revealed a homozygous guanine-to-adenine substitution at position 274 of the IGF-I gene, changing valine to methionine at position 44 of the IGF-I protein. The mutant IGF-I (V44M) was found to have an approximate 90-fold lower affinity for the normal IGF-I receptor. The result of this mutation was the inability of IGF-I to interact with the receptor. These findings support the evidence that, in utero, biologically active IGF-I is critical for growth and brain development. Furthermore, the absence of intrauterine and mental growth retardation in cases of GH disorders implies that IGF-I secretion works independently of GH in utero.

Along the GH/IGF-I axis, the majority of mutations currently identified are located in the extracellular domain of GH, resulting in the inability of GH to bind the GH receptor (Figure 2). This mutation is observed in classical cases of Laron syndrome, and treatment with IGF-I in these patients is found to be moderately effective. More rare are cases of mutations in the transmembrane and intracellular domain of GH; incidences of STAT-5b and ALS mutations fall under this category. Finally, in utero bioinactive IGF-I holds potential for severe inhibition of physical and mental development.

As our body of knowledge on this subject grows, it is inevitable that cases of “milder” short stature will be found to reflect more subtle defects along the GH/IGF-I axis. This phenomenon has been observed in other endocrine disorders, and it is possible that these understated defects will reflect a combination of heterozygous states and seemingly benign single nucleotide polymorphisms or inadequate compensatory mechanisms. These discoveries will come into play as we learn more about the molecular features of human growth.

References

1. Sherlock M, TooGood AA. Aging and the growth hormone/insulin like growth factor-I axis. Pituitary. 2007; 10:189-203.
2. Ono M, Chia DJ, Merino-Martinez R, Flores-Morales A, Unterman TG, Rotwein P. Signal transducer and activator of transcription (Stat) 5b-mediated inhibition of insulin-like growth factor binding protein-1 gene transcription: A mechanism for repression of gene expression by growth hormone. Mol Endocrinol. 2007; 21:1443-1457.
3. Woods KA, Camancho-Hübner C, Savage MO, Clark AJL. Intrauterine growth retardation and postnatal growth failure associated with deletion of the insulin-like growth factor I gene. N Engl J Med. 1996; 335:1363-1367.
4. Laron Z, Pertzelan A, Mannheimer S. Genetic pituitary dwarfism with high serum concentration of growth hormone—a new inborn error of metabolism? Isr J Med Sci. 1966; 2:152-155.
5. Godowski PJ, Leung DW, Ameacham LR, et al. Characterization of the human growth hormone receptor gene and demonstration of a partial gene deletion in two patients with Laron-type dwarfism. Proc Natl Acad Sci USA. 1989; 86:8083-8087.
6. Laron Z. Laron syndrome (Primary growth hormone resistance or insensitivity): The personal experience 1958-2003. J Clin Endocrinol Metab. 2004; 89:1031-1044.
7. Rosenbloom AL, Guevara-Aguirre, Rosenfeld RG, Francke U. Growth hormone receptor deficiency in Ecuador. J Clin Endocrinol Metab. 1999; 84:4436-4443.
8. Milward A, Metherell L, Maamra M, et al. Growth hormone (GH) insensitivity syndrome due to a GH receptor truncated after Box1, resulting in isolated failure of STAT 5 signal transduction. J Clin Endocrinol Metab. 2004; 89:1259-1266.
9. Rosenfeld RG, Kofoed E, Buckway C, et al. Identification of the first patient with a confirmed mutation of the JAK-STAT system. Pediatr Nephrol. 2005;20:303-305.
10. Hwa V, Little B, Adiyaman P, et al. Severe growth hormone insensitivity resulting from total absence of Signal Transducer and Activator of Transcription 5b. J Clin Endocrinol Metab. 2005;90:4260-4266.
11. Boisclair YR, Rhoads RP, Ueki I, Wang J, Ooi GT. The acid-labile subunit (ALS) of the 150 kDa IGF-binding protein complex: an important but forgotten component of the circulating IGF system. J Endocrinol. 2001;170:63-70.
12. Hwa V, Camacho-Hubner C, Little BM, et al. Growth hormone insensitivity and severe short stature in siblings: a novel mutation at the exon 13-intron 13 junction of the STAT5b gene. Horm Res. 2007;68:218-224.
13. Baker J, Liu JP, Robertson EJ, Efstratiadis A. Role of insulin-like growth factors in embryonic and postnatal growth. Cell. 1993; 75:73-82.
14. Walenkamp JME, Karperien M, Pereira AM, et al. Homozygous and heterozygous expression of a novel insulin-like growth factor-I mutation. J Clin Endocrinol Metab. 2005; 90:2833-2864.

Clinical and molecular aspects of IGF-I deficiency and resistance
Mitchell E. Geffner, MD, and Mark A. Sperling, MD

The growth hormone (GH)/insulin-like growth factor-I (IGF-I) signaling pathway is critical for the initiation and regulation of growth. Control of this system involves the interaction of several sites throughout the body, primarily the hypothalamus, pituitary gland and liver. The principal source of circulating GH is the somatotroph cells of the anterior pituitary gland. These cells release GH in a pulsatile manner that is regulated by two hypothalamic hormones: growth hormone-releasing hormone and somatostatin.¹

GH action to promote growth is mediated in large part by IGF-I, a GH-dependent growth factor produced predominantly in the liver for circulation and at local tissue sites for paracrine and autocrine effects. IGF-I secretion occurs as the result of GH receptor activation and a series of post-receptor signaling events. IGF-I action is responsible for the stimulation of growth and increase in bone and muscle mass. ¹ IGF-I is transported in the circulation by a number of IGF binding proteins, including IGFBP-3, the primary regulator of IGF-I bioactivity. Levels of IGF-I are also regulated by sex steroids, thyroxine and glucocorticoids.

An additional regulator of the GH/IGF-I family is insulin. GH, by antagonizing the effects of insulin on carbohydrate metabolism, increases insulin secretion by pancreatic ß-cells. This event causes synergy between insulin and GH, resulting in augmented synthesis of amino acids into protein (Figure 1). Insulin also promotes the expression of GH receptors in the liver and, hence, the subsequent generation of IGF-I. Although IGF-I inhibits insulin secretion, circulating glucose levels may fall due to the insulin-like action of IGF-I. In addition, IGF-I has been linked to increased insulin sensitivity. The insulin-like effects of IGF-I and its possible enhancement of insulin action have prompted researchers currently investigating the use of IGF-I treatment modalities in patients with insulin resistance and diabetes.^2,3

A number of genetic and nongenetic conditions are associated with defects and mutations in the GH/IGF-I axis. Genetic testing has identified numerous variations in GH and IGF-I receptor protein functioning, as well as deletions and mutations in specific portions of the genes that encode these proteins, leading to short stature. Presumed environmentally triggered differences in IGF-I receptor function, such as those observed in African Efe Pygmies, have also been identified. Additionally, conditions such as malnutrition, diabetes and HIV have been linked to GH and IGF-I pathology. Identification and diagnosis of these abnormalities are discussed in this article.

Figure 1: Role of IGF-I in normal growth and metabolism Figure 1. Insulin is a regulator of the GH/IGF-I pathway. Growth hormone and insulin act synergistically to synthesize protein from amino acids, whereas IGF-I inhibits insulin secretion. Source: Mark A. Sperling, MD

Pygmies

Pygmies are short-statured people who reside predominantly in equatorial Africa. Their consistent short stature is believed to be the result of adaptive processes to ease locomotion through the dense forest undergrowth, minimize food requirements during periods of undernutrition and improve thermoregulation by means of a smaller surface area-to-body mass ratio.⁴ Experts previously believed that the cause of the Pygmies’ short stature was related to GH resistance coupled with an absent adolescent growth spurt.⁵ However, more recent research has suggested that unresponsiveness to IGF-I may be the primary cause for the blunted height of this population.⁶ Investigators evaluated the cells from African Efe Pygmies, their Lese neighbors, a farming tribe living adjacent to them, whose men frequently reproduce with the Efe Pygmy women, and American controls. Both GH and IGF-I responsiveness of Pygmy cells were inhibited compared to the Lese and control groups. Furthermore, the responsiveness of the Lese cells was intermediate between those of the Pygmies and controls, similar to their intermediate stature. Additional studies suggest that IGF-I resistance may be the primary cause of short stature, whereas apparent GH resistance may be secondary to the IGF-I resistance.⁷ Molecular analysis of the IGF-I receptor and its post-receptor signaling in Pygmy cells revealed decreased cell surface expression of IGF-I receptors, a lack of signal transmission in response to physiological concentrations of IGF-I and a decreased level of IGF-I receptor messenger RNA. These findings suggest that African Efe Pygmy stature may be genetically controlled by modified expression and function of IGF-I receptors.⁷

IGF-I receptor mutations

Another type of disorder associated with IGF-I resistance is related to mutations involving the IGF-I receptor gene on chromosome 15. To date, 12 cases in five families have been reported.⁷ All patients presented with prenatal and postnatal growth retardation and demonstrated global postnatal growth failure, along with varying degrees of mental retardation and various dysmorphic features.

One particular family found to have this mutation was recently studied by Inagaki and colleagues.⁸ The patient was identified at age 13 when her height was noted to be -6.1 SD. Her condition was thought to be hereditary; her mother was -5.7 SD and her sister was -5.0 SD. Her IGF-I level was slightly elevated at 400 ng/mL (normal range 165-300 ng/mL) and her peak stimulated GH level was 10.6 ng/mL (normal <10 ng/mL). An IGF-I generation test following four days of GH treatment revealed a rise in serum IGF-I, pointing to an unidentified form of IGF-I resistance. Direct reverse transcriptase polymerase chain reaction sequencing of the IGF-I receptor revealed a heterozygous glutamine-to-arginine switch at position 1577, leading to an arginine-to-glutamine codon substitution at residue 481. This, in turn, caused a reduction in IGF-I receptor ß-subunit phosphorylation and decreased cell proliferation (Figure 2).

Figure 2: IGF-I stimulated cell proliferation Figure 2. In the absence of IGF-I, proliferation of the patient’s mutant fibroblast cells (NIH3T3 IGF-1R MT) decreased (right) while proliferation of wild-type fibroblast cells (NIH3T3 IGF-1R WT) increased (center) compared to that of non-transfected NIH3T3 cells (left). Mutant receptors responded poorly to the addition of IGF-I (right). Source: Mitchell E. Geffner, MD

Primary IGF-I deficiency

Researchers have questioned the possibility of primary IGF-I deficiency (the presence of IGF-I deficiency despite normal GH secretion and receptor activity).⁹ Several cases have suggested it does. One case currently being investigated by Rosenbloom and colleagues involves a young boy with above-average-sized parents and siblings.⁹ At 6 months of age, his IGF-I was less than 10 ng/mL, yet GH responses to secretagogues were normal, at 14.6 ng/mL and 11.2 ng/mL. Follow-up revealed increasingly significant short stature; at 40 months of age, the patient’s height was -3.5 SD. His IGF-I was still below 10 ng/mL and his peak GH level was 21.8 ng/mL. GH treatment, initiated at 51 months, resulted in a profound improvement in growth, despite his current IGF-I level of 50 ng/mL, suggesting that he has primary IGF-I deficiency.

Other cases of rare IGF-I mutations and deficiency have been reported. In one study, investigators reported a patient with short stature, sensorineural deafness, delayed psychomotor development and IGF-I deficiency.¹⁰ Direct sequencing analysis revealed a novel transversion of thiamine and adenine in the untranslated region of exon 6 of the IGF-I gene. This disruption of the normal amino acid sequence appeared to account for low circulating levels of IGF-I in this patient.

A case investigated by Walenkamp and colleagues highlighted a familial mutation of the IGF-I gene that resulted in severe intrauterine and postnatal growth retardation, microcephaly and sensorineural deafness in a 55-year-old man.¹¹ The investigators identified a transversion of guanine and adenine within the IGF-I gene, changing valine 44 into methionine. The inactivating nature of the mutation caused a 90-fold reduced affinity of IGF-I for the IGF-I receptor and consequent IGF-I deficiency. The researchers concluded that the similarity of the postnatal growth pattern to that of untreated GH deficiency suggests that IGF-I haploinsufficiency results in inhibition of both prenatal and postnatal growth.

IGF-I resistance/deficiency and malnutrition

IGF-I deficiency and resistance can also be observed when no molecular abnormality can be detected. One such setting is malnutrition. Studies in rats have demonstrated that a protein-restricted diet results in symptoms of IGF-I deficiency/resistance independent of the effects of insulin. In a study by Maiter and colleagues, seven days of protein restriction resulted in a 28% reduction in serum IGF-I concentrations (P>.001).¹² The effects of malnutrition on IGF-I and subsequent growth have been documented in animals.¹³ Four-week-old rats were separated into four groups: protein-restricted rats (P5), P5 rats treated with GH, P5 rats treated with IGF-I and normal protein-fed rats (P15). Body weight, tail length and tibial epiphyseal width were monitored over the course of several days. For each end point, the P15 rats demonstrated significantly more growth than the P5 rats, regardless of GH or IGF-I treatment. The investigators concluded that the absence of growth despite the administration of IGF-I therapy indicated that the growth arrest resulting from protein restriction was mediated in part by IGF-I resistance.

Poor growth and HIV infection

Poor growth is commonly observed in symptomatic children infected with HIV despite the fact that serum hormone levels usually fall within the normal range.^14,15 To further investigate the roles of GH and IGF-I deficiency and resistance, investigators evaluated six asymptomatic short (mean height SD = ±0.01±1.0) children with HIV (group P1), 10 symptomatic short (mean SD = -2.0±1.0) children with HIV (group P2) and six short (mean SD = -2.4±1.2) healthy controls. The patients in the P2 group demonstrated decreased IGF-I levels and decreased in vitro GH and IGF-I responsiveness compared to the P1 and control groups. Therefore, patients with HIV infection demonstrated symptoms of IGF-I deficiency/resistance without a discernible molecular defect known to explain the underlying mechanisms. ¹⁵

Insulin/IGF-I resistance and longevity

Insulin plays a critical role in the GH/IGF-I signal pathway. Expression of GH receptors is dependent upon the action of insulin and patients with type 1 diabetes typically demonstrate high levels of GH with equally low circulating levels of IGF-I. Such patients may present with poor growth; lack of metabolic control is related to low IGF-I levels despite no demonstrable genetic defect. Treating patients with type 1 diabetes with IGF-I has been shown to reduce GH levels, stimulate glucose uptake in muscle, enhance insulin action and block ß-cell apoptosis. ¹⁶

Although the dual resistance to insulin and IGF-I found in some states of type 1 diabetes, such as diabetic ketoacidosis, has a detrimental clinical effect, this dual resistance may lend beneficial properties as shown in several mouse models. This is at least in part due to overexpression of Klotho, a protein named after the character in Greek mythology who was responsible for determining the length of life of mortals. Mice that overexpress Klotho are hyperinsulinemic and normoglycemic, implying a state of insulin resistance, with male Klotho transgenic mice being resistant to the hypoglycemic action of administered IGF-I. ¹⁷ Researchers have found that this combined insulin/IGF-I resistance appears to be associated with longevity in Klotho mice (Figure 3), despite previous findings that enhanced, as opposed to reduced, insulin sensitivity extended life in calorie-restricted mice.^18,19 Further investigation regarding the effect of insulin and IGF-I signal suppression in Ames and Snell dwarf mice (in which GH deficiency is present) and GH receptor knock-out models (in which the GH receptor is absent) suggested a common physiological pathway between these conditions and delayed aging. ¹⁹

Figure 3: Convergence of suspected mechanisms of expected longevity Figure 3. Combined insulin and IGF-I resistance converge to provide extended longevity in trangenic Klotho mice. Source: Mitchell E. Geffner, MD

Distinguishing between GH and IGF-I malfunction

Patients with GH/IGF-I mutations and deletions share the common physiological characteristic of short stature. However, patients with an IGF-I mutation present with reduced birth weight, length and head circumference, mental retardation, facial abnormalities and malformations of the extremities. Distinguishing between GH and IGF-I malfunction can be aided by the appearance of the patient. The presence of excessive body fat suggests GH deficiency/resistance because of the lack of a direct lipolytic effect of GH in fat cells, along with lack of muscle development because of the absence of IGF-I, which normally stimulates muscle anabolism. In IGF-I resistance, the patient is not fat because the direct effect of GH on fat metabolism and lipolysis is retained and some muscle anabolism also occurs due to a still-functioning GH receptor conveying the direct (IGF-I-independent) effects of GH.

Two strategies to further distinguish between GH and IGF-I signal malfunction have been used.²⁰ Forty-two patients with unexplained intrauterine growth retardation (IUGR) were identified using single-strand conformational polymorphism sequencing followed by direct DNA sequencing of identified abnormalities. This approach resulted in a one in 42 identification rate (2.3%) of IGF-I receptor gene mutations. The second strategy consisted of studies in 50 patients with short stature and elevated IGF-I concentrations, nine of whom underwent direct DNA sequencing. In this group, one boy with a nonsense mutation in the IGF-I receptor gene was identified. This mutation reduced the number of IGF-I receptors on fibroblasts. This boy identified with the first approach and had IUGR and poor postnatal growth.²⁰

These data have contributed to an emerging model to aid in the diagnosis of GH and IGF-I pathology (Figure 4). Patients with GH deficiency typically have low levels of GH and IGF-I. They will most likely appear to be normal at birth, and their growth velocity typically begins to slow between the ages of 3 and 6 months when GH receptor action becomes evident under normal circumstances. If a patient presents with increased GH and decreased IGF-I levels, he or she may have classical GH resistance or Laron syndrome with a lack of IUGR. Suspicion for this genetic abnormality can be aided by the patient’s fatty appearance. Furthermore, recurrent respiratory infections in a severely short child may point to a defect in signal transducer and activator of transcription STAT-5b, a common mediator of the function of GH and cytokine receptors, with the latter regulating immunity. Patients who present with elevated serum GH and decreased IGF-I concentrations who demonstrate IUGR and who continue to grow poorly postnatally may have a primary IGF-I gene defect. However, patients with increased levels of GH and IGF-I, and concurrent IUGR may have IGF-I resistance and, hence, a primary or secondary defect in the IGF-I receptor.

A closer look at the molecular and genetic mechanisms of GH and IGF-I action reveals numerous specific malfunctions, deletions and mutations that are associated with stunted growth. The development of IGF-I immunoassays has been helpful in screening for GH deficiency and monitoring GH therapy. However, improved assays and adequate reference values are needed to properly interpret the data.²¹ Thus, it is likely that future research will contribute to the growing list of GH-IGF-I signal pathway disruptions, lending a deeper understanding of the pathology of short stature.

Figure 4 Figure 4. Emerging model to aid in the diagnosis of GH and IGF-I pathology. Source: Mark A. Sperling, MD

References

1. Sherlock M, Toogood AA. Aging and the growth hormone/insulin like growth factor-I axis. Pituitary. 2007; 10:189-203.
2. Yuen KC, Dunger DB. Therapeutic aspects of growth hormone and insulin-like growth factor-I treatment on visceral fat and insulin sensitivity in adults. Diabetes Obes Metab. 2007; 9:11-22.
3. Clemmons DR, Sleevi M, Allan G, Sommer A. Effects of combined recombinant insulin-like growth factor (IGF)-I and IGF binding protein-3 in type 2 diabetic patients on glycemic control and distribution of IGF-I and IGF-II among serum binding protein complexes. J Clin Endocrinol Metab. 2007; 92:2652-2658.
4. Jain S, Golde DW, Bailey R, Geffner ME. Insulin-like growth factor-1 resistance. Endocr Rev. 1998; 19:625-646.
5. Geffner MD, Bailey RC, Bersch N, Vera JC, Golde DW. Insulin-like growth factor-I unresponsiveness in an Efe Pygmy. Biochem Biophys Res Commun. 1993; 193:1216-1223.
6. Geffner ME, Bersch N, Bailey RC, Golde DW. Decreased insulin-like growth factor I receptor expression and function in immortalized African Pygmy T cells. J Clin Endocrinol Metab. 1995; 80:3732-3738.
7. Hattori Y, Vera JC, Rivas CI, et al. Insulin-like growth factor I resistance in immortalized T cell lines from African Efe Pygmies. J Clin Endocrinol Metab. 1996; 81:2257-2263.
8. Inagaki K, Tiulpakov A, Rubtsov P, et al. A familial insulin-like growth factor-I receptor mutant leads to short stature: Clinical and biochemical characterization. J Clin Endocrinol Metab. 2007; 92:1542-1548.
9. Rosenbloom AL.The role of recombinant insulin-like growth factor 1 in the treatment of the short child. Curr Opin Pediatr. 2007; 19:458-464
10. Bonapace G, Concolino D, Formicola S, Strisciuglio P. A novel mutation in a patient with IGF-I deficiency. J Med Genet. 2003; 40:913-917.
11. Walenkamp MJ, Karperien M, Pereira AM, et al. Homozygous and heterozygous expression of a novel insulin-like growth factor-I mutation. J Clin Endocrinol Metab. 2005; 90:2855-2864.
12. Maiter D, Fliesen T, Underwood LE, et al. Dietary protein restriction decreases insulin-like growth factor I independent of insulin and liver growth hormone binding. Endocrinology. 1989; 124:2604-2611.
13. Thissen JP, Underwood LE, Maiter D, Maes M, Clemmons DR, Ketelslegers JM. Failure of insulin-like growth factor-I (IGF-I) infusion to promote growth in protein-restricted rats despite normalization of serum IGF-I concentrations. Endocrinology. 1991; 128:885-890.
14. Geffner ME, Yeh DY, Landaw EM, et al. In vitro insulin-like growth factor-I, growth hormone, and insulin resistance occurs in symptomatic human immunodeficiency virus-1-infected children. Pediatr Res. 1993; 34:66-72.
15. Haugaard SB, Anderson O, Hansen BR, et al. Insulin-like growth factors, insulin-like growth factor-binding proteins, insulin-like growth factor-binding protein-3 protease, and growth hormone-binding protein in lipodystrophic human immunodeficiency virus-infected patients. Metabolism. 2004;53:1565-1573.
16. Simpson HL, Jackson NC, Shojaee-Moradie F, et al. Insulin-like growth factor 1 has a direct effect on glucose and protein metabolism, but no effect on lipid metabolism in type 1 diabetes. J Clin Endocrinol Metab. 2004; 89:425-432.
17. Kurosu H, Yamamoto M, Clark JD, et al. Suppression of aging in mice by the hormone Klotho. Science. 2005; 30:1829-1833.
18. Bartke A. long-lived Klotho mice: New insights into the roles of IGF-I and insulin in aging. Trends Endocrinol Metab. 2006; 17:33-35.
19. Bartke A. New findings in transgenic, gene knockout and mutant mice. Exp Gerontol. 2006; 41:1217-1219.
20. Abuzzahab MJ, Schneider A, Goddard A, et al. IGF-1 receptor mutations esulting in intrauterine and postnatal growth retardation. N Engl J Med. 2003; 349:2211-2222.
21. Anckaert E, Schiettecatte J, Vanbesien J, Smitz J,Velkeniers B, De Schepper J. Variability among five different commercial IGF-I immunoassays in conditions of childhood-onset GH deficiency and GH therapy. Acta Clin Belg. 2006; 61:335-339.

Genomic regulation in the induction of puberty
David R. Brown, MD, FACE

Puberty plays a significant role in the modulation and completion of growth. The pubertal process is mediated by coordinated and integrated regulation of the pulsatile secretion of the gonadotropin releasing hormone (GnRH) from the hypothalamus and represents the reactivation of gonadotropins after a prolonged period of childhood quiescence.

Puberty plays a significant role in the modulation and completion of growth . . . Kisspeptin neurons are direct targets for gonadal steroids and are involved in both negative and positive feedback regulation of gonadotropin secretion. — David R. Brown, MD, FACE

GnRH pulsatility is first active in utero, where gonadotropins are secreted at high levels beginning toward the end of the first trimester and continuing throughout neonatal life up to about six months in males and two years in females. GnRH pulsatility is then dampened and remains inactive until puberty, at which point the hypothalamic-pituitary axis is reactivated and gonadotropins are secreted. Previously, traditional neurophysiologic mechanisms were thought to facilitate a GnRH pulse generator; however, recent clinical and laboratory investigations reveal a genomic-based regulatory system.

Gonadotropin deficiency

Gonadotropin deficiency occurs when the release of gonadotropins is interrupted. Two conditions are related to gonadotropin deficiency, isolated hypogonadotrophic hypogonadism (IHH) and Kallmann syndrome (KS). IHH is characterized by a reduction in plasma luteinizing hormone (LH) and follicle stimulating hormone (FSH) levels and depressed gonadal hormone secretion.¹ KS constitutes IHH plus manifestations of anosmia. The genes involved in KS and IHH are active in different areas of the gonadotropic gonadal axis. The KAL-1 and the fibroblast growth factor receptor-1 (FGFR-1) genes are associated with KS and are active primarily within the hypothalamus.² The GnRH receptor (GnRHR) gene is located in the anterior pituitary, whereas GPR-54 is an intermediary between the hypothalamic and pituitary components of the gonadotropic axis.

Kallmann syndrome genomics

The primary genes involved in KS are KAL-1 and FGFR-1. KAL-1 is located on the short arm of the X chromosome and encodes for the fibronectin-containing protein anosmin. The key role of KAL-1 is to facilitate gonadotropin neuron migration from the olfactory placode to the GnRH neurons of the hypothalamus. ^3-5 KAL-1 also facilitates synapse formation in the olfactory bulb neurons. This gene is composed of 14 discrete exons.⁶ Multiple mutations (slice, frameshift, nonsense, missense) in addition to short and long sequence deletions have been identified on KAL-1, which involve almost every exon. KAL-1 is expressed in higher CNS centers, and patients with mutations or deletions in this gene may lack their olfactory bulbs and tracts, resulting in anosmia. This neuronal migration defect will also result in GnRH deficiency and hypogonadism. Patients with KAL-1 defects often present with synkinesis and unilateral renal agenesis.^7,8

FGFR-1 is an autosomal gene with significant genetic heterogeneity and appears to act through the activation of tyrosine kinase receptors.⁹ Like KAL-1, it is involved in the embryonic development of the olfactory bulbs but is also expressed in the second brachial arch and lower face. Patients with deletions or loss of function mutations in this gene will present with mandibular hypoplasia, midline defects and dental anomalies such as a solitary central maxillary incisor.^10,11 ProKR₂ and ProK₂ are olfactory bulb genes active in the fetus, which are synergistic with FGFR-1. FGF-8 and NELF are also ligands for FGFR-1 in GnRH neuron ontogeny.

Isolated hypogonadotropic hypogonadism genomics

Two genes are associated with IHH, GnRHR and GPR-54. Defects in the GnRHR gene are autosomal recessive or loss-of-function mutations. GnRH-related IHH is not observed as frequently as GPR-54-related IHH. In fact, fewer than 30% of clinical conditions associated with IHH are caused by GnRHR mutations. Therefore, GPR-54 deletions or mutations are responsible for the majority of IHH cases.¹² GPR-54 is a 398-amino acid G-coupled protein receptor whose primary effect is phosphorylation activation. It has nine classic domains: an extracellular domain, seven looped transmembrane domains and an intracellular domain. It is part of the family of rhodopsin-like receptors that include galanin and somatostatin receptors. This gene is expressed primarily in the hypothalamus but demonstrates some expression within the greater CNS and the anterior pituitary. GPR-54 plays a key role in the reactivation of the GnRH axis and regulation of GnRH pulsatility. Patients with GPR-54 defects demonstrate partial gonadotropin deficiency with normal or partial responses to exogenous GnRH.

GnRH knock-out studies

GnRH and GPR-54 are involved in gonadotropin (LH) release. In healthy males, LH is released in four-hour pulses of similar amplitude (Figure 1). However, when the GnRH receptor is inactive in murine knock-out studies, a complete absence of pulsatility of the gonadotropins is observed. This leads to secondary diminished gonadal hormones.

Figure 1: GnRH-induced LH secretion in men with GnRH deficiency Figure 1. These graphs illustrate the spectrum of GnRH-induced LH secretion in normal men, men with GnRH deficiency and men with IHH caused by +GPR-54 mutations. Source: Copyright 1998, The Endocrine Society

GPR-54 knock-out studies

Unlike those with GnRH mutations, patients with defects in the GPR-54 system do not demonstrate a complete loss of LH pulsatility. Studies have shown that ineffective GPR-54 signaling leads to a normal frequency of LH pulses but of significantly lower amplitude to that observed in unaffected males. This diminished LH pulsatility has multiple effects. Knock-out models of GPR-54 in male mice demonstrate decreased gonadotropin secretion, diminished androgenital length, small phallus and decreased gonadal size compared to wild-type, as well as infertility.¹ Female mice lacking functioning GPR-54 demonstrate decreased ovarian size, smaller fallopian tubes, infantile uterus and corresponding histologic demonstration of diminished gonadal maturation.

Kisspeptin-54

Kisspeptin-54, also known as metastin, is the endogenous ligand of GPR-54 and plays a critical role in GPR-54 signaling. Kisspeptin-54 is a 54 amino acid post-translational product of the primary KISS-1 protein, a peptide of 145 amino acids that is expressed in the arcuate and anterioventral periventricular (AVPV) nuclei of the forebrain. KISS-1 produces Kisspeptin-54 through dibasic cleavages between amino acid positions 67/68 and 121/122.

KISS-1 and GPR-54 play a role in timing the onset of puberty. KISS-1 and GPR-54 mRNA levels increase within the arcuate nucleus as a function of sexual development in monkeys.¹³ GPR-54 is the only receptor for Kisspeptin-54 on GnRH neurons, suggesting that Kisspeptin-54 GPR-54 signaling is a prerequisite for gonadotropin secretion. ¹⁴

The administration of Kisspeptin-54 (metastin) to juvenile orchidectomized monkeys results in a rapid, quantitative response in the release of gonadotropin (LH) from the hypothalamus.¹⁵ This suggests that hypothalamic neurons have GPR-54 receptors on their surfaces that bind Kisspeptin-54. When Kisspeptin-54 binds to these sites, the pulsatile release of GnRH occurs, which initiates LH and FSH leading to gonadal steroid secretion. Interruption in this signal pathway results in the continued quiescence of GnRH pulsatility and IHH.

Gain of function mutations has been described in GPR-54 and KISS-1 genes, resulting in gonadotropin dependent precocious puberty. These result in a prolonged activation of intracellular GPR-54 signaling in response to kisspeptin, decreasing GPR-54 sensitization, and increased GPR-54 leading to enhanced GnRH secretion and increased gonadotropins resulting in central precocious puberty.

Steroidal feedback

Estradiol and testosterone regulate KISS-1 gene expression and are involved with up-regulation and down-regulation of the GnRH signal pathway (Figure 2).¹⁶ When the GnRH-releasing neuron receives a positive signal, it secretes GnRH, stimulating the secretion of LH and FSH. These gonadotropins stimulate the gonads, which in turn initiate the secretion of estradiol or testosterone. If the sex steroid signal is received in the arcuate nucleus, a negative feedback message to the KISS-1 neuron down-regulates activity and inhibits the GnRH activation sequence. However, if the message is received at the AVPV nucleus, positive stimulation of the KISS-1 neuron up-regulates GPR-54, activating the GnRH signaling cascade. The AVPV- mediated positive feedback also facilitates the post-ovulatory LH surge in the female, facilitating gender dimorphism and sexual differentiation in the human brain. Kisspeptins also induce Fos expression in GnRH neurons, enabling DNA transcription. Consequently, KISS-1 m-RNA is differentially regulated by estradiol in the female AVPV. The ER α gene is a moderator for differential expression. The Kisspeptin system via GPR-54 provides a genomic-based mechanism for the feedback regulation of GnRH secretion.

Although the Kisspeptin-54/GPR-54 signal pathway is only one of many factors involved in the transition into puberty, it is the critical regulator in the release of gonadotropins. Kisspeptin-54 signaling is a central component of the neuroendocrine reproductive axis, and hypothalamic GPR-54 receptors initiate as well as coordinate gonadotropin-mediated reproductive functions at the time of puberty. Furthermore, Kisspeptin neurons are direct targets for gonadal steroids and are involved in negative and positive feedback regulation of gonadotropin secretion. Truly, puberty begins with a KISS.

Figure 2: Kisspeptin: Neuroendocrine axis Figure 2. Kisspeptin-54 signaling is a central component of the neuroendocrine reproductive axis. Source: Dungan HM, Clifton DK, Steiner RA. Minireview: Kisspeptin neurons as central processors in the regulation of gonadotropin-releasing hormone secretion. Endocrinology. 2006;147:1154-1158.

References

1. Seminara SB, Hayes FJ, Crowley Jr WF. Gonadotropin-releasing hormone deficiency in the human (idiopathic hypogonadotropic hypogonadism and Kallmann’s syndrome): Pathophysiology and genetic considerations. Endocr Rev. 1998; 19:521-539.
2. Karges B, de Roux N. Molecular genetics of isolated hypogonadotropic hypogonadism and Kallmann syndrome. Endocr Dev. 2005; 8:67-80.
3. Soussi-Yanicostas N, Faivre-Sarrailh C, Hardelin JP, et al. Anosmin-1 underlying the X chromosome-linked Kallmann syndrome is an adhesion molecule that can modulate neurite growth in a cell-type specific manner. J Cell Sci. 1998; 111:2953-2965.
4. Soussi-Yanicostas N, de Castro F, Julliard AK, Perfettini I, Chedotal A, Petit C. Anosmin-1, defective in the X-linked Kallmann syndrome, promotes axonal branch formation from olfactory bulb output neurons. Cell. 2002; 109:217-228.
5. Cariboni A, Pimpinelli F, Colamarino S, et al. The product of X-linked Kallmann’s syndrome gene (KAL1) affects the migratory activity of gonadotropin-releasing hormone (GnRH)-producing neurons. Hum Mol Genet. 2004; 13:2781-2791.
6. Legouis R, Hardelin JP, Levilliers J, et al. The candidate gene for the X-linked Kallmann syndrome encodes a protein related to adhesion molecules. Cell. 1991; 67:423-435.
7. Quinton R, Duke VM, de Zoysa PA, et al. The neuroradiology of Kallmann’s syndrome: a genotypic and phenotypic analysis. J Clin Endocrinol Metab. 1996; 81:3010-3017.
8. Soderlund D, Canto P, Mendez JP. Identification of three novel mutations in the KAL1 gene in patients with Kallmann syndrome. J Clin Endocrinol Metab. 2002;87;2589-2592.
9. Ruta M, Burgess W, Givol D, et al. Receptor for acidic fibroblast growth factor is related to the tyrosine kinase encoded by the fms-like gene (FLG). Proc Natl Acad Sci USA. 1989; 86:8722-8726.
10. Dode C, Levilliers J, Dupont JM, et al. Loss-of-function mutations in FGFR1 cause autosomal dominant Kallmann syndrome. Nat Genet. 2003; 33:463-465.
11. Sato N, Katsumata N, Kagami M, Kagami M. Clinical assessment and mutation analysis of Kallmann syndrome (KAL1) and fibroblast growth factor receptor 1 (FGFR1, or KAL2) in five families and 18 sporadic patients. J Clin Endocrinol Metab. 2004; 89:1079-1088.
12. Seminara SB, Messager S, Chatzidaki EE, et al. The GPR54 gene as a regulator of puberty. N Engl J Med. 2003; 349:1614-1627.
13. Sahab M, Mastronardi C, Seminara SB, Crowley WF, Ojeda SR, Plant TM. Increased hypothalamic GPR54 signaling: A potential mechanism for initiation of puberty in primates. Proc Natl Acad Sci USA. 2005; 102:2129-2134.
14. Gottsch ML, Clifton DK, Steiner RA. Mol Cell Endocrinol. Kisspepeptin-GPR54 signaling in the neuroendocrine reproductive axis. 2006; 254:91-96.
15. Seminara SB, Dipietro MD, Ramaswamy S, Cowley WF, Plant TM. Continuous human metastin 45-54 infusion desensitizes G protein-coupled receptor 54-induced gonadotropin-releasing hormone release monitored indirectly in the juvenile male Rhesus monkey (Macaca mulatta): a finding with therapeutic implications. Endocrinology. 2006; 147:2122-2126.
16. Dungan HM, Clifton DK, Steiner RA. Minireview: Kisspeptin neurons as central processors in the regulation of gonadotropin-releasing hormone secretion. Endocrinology. 2006;147-1154-1158.

Growth hormone treatment information for nurses
Gaye Madigan, APRN, BC, and Jan Penn, BSN, RN

Large steps have been taken toward understanding of growth hormone (GH) disorders. They have significant implications for pediatric endocrine nurses who treat these conditions. Novel genomic diagnostic modalities may replace provocative testing, and identification of additional defects in the GH and insulin-like growth factor-I (IGF-I) will enhance the current understanding of primary and secondary IGF-I deficiency.

Nurses will be on the forefront of this new frontier, working closely with clinicians and patients to facilitate and supervise treatment. It will be the responsibility of pediatric endocrine nurses to increase the understanding of GH disorders as the body of knowledge on this subject continues to expand (Figure). Testing will be modified to isolate pre- and post-receptor signal interruptions by means of genomic analysis; nurses will need to stay abreast of recent advances in GH genomics. New therapies are likely to evolve that will target subtle defects within the GH/IGF-I pathway, and nurses must understand each aspect of this pathway to apply treatment effectively.

Figure: Preparing for New Challenges: The Role of Pediatric Nurses in Treating GH and IGF-I Disorders

Patient care will also change dramatically in response to increased genomic understanding, and nurses will be leading the way toward more effective care. Therapeutic compliance is comprised of three aspects—dosage, frequency and duration. To manage each patient effectively, pediatric endocrine nurses must not only distinguish between GH, IGF-I-based, or combined therapy, but match each patient to the therapeutic delivery system that best suits his or her needs. Currently, three delivery systems are available: needle and syringe, pen devices and needle-free devices. Potential delivery systems for future administration may include depot formulations, pulmonary and transdermal delivery methods. It will be the nurses’ responsibility to manage treatments utilizing these systems. They must continually be aware of new technologies and biopharmaceutical developments.

Pediatric endocrine nurses also oversee other aspects of GH disorders: bone mineralization, body composition, lipid management and overall quality of life. Their understanding of GH disorders must extend beyond molecular and genomic mechanisms of GH to broader patient care. Moreover, as nurses are often the conduit between patients and their clinicians, they will be charged with continual education and support of not only the patient but the family. They are key in facilitating the transition from childhood to adult GH and IGF therapy. Pediatric endocrine nurses have a unique and vital role to play in the treatment of patients with GH disorders; to do this effectively, they must be armed with knowledge and understanding of each and every aspect of these conditions and their underlying molecular diagnoses and appropriate therapeutic options.