Review cuprinzător privind mutațiile și variantelor secvențiale ale hormonului de creștere (GH) la copiii cu deficit de hormon de creștere

Background

Growth hormone (GH), which is responsible for growth and the integral feature of living being, is a peptide hormone of 190 amino acids, secreted by the somatotroph cells in the anterior pituitary gland⁽⁴⁹⁾. The hormone is encoded by the GH1 gene (2 kb), which consist of five exons and four introns, being a member of the five genes cluster (50 kb) on the chromosome 17q 22-24 region⁽⁴⁷⁾. Growth hormone is associated with many physiological functions and is very important in the determination of the stature and in the reproduction function.

There are mainly two types of genes involved in determining the stature and the growth, known as the genes involved in the hypothalamic-pituitary-GH-IGF-1 axis and the genes involved in the growth of skeletal growth plate⁽⁵³⁾. Growth hormone deficiency is caused by various defects in the GHRH-GH axis, being creditworthy for dwarfism⁽²⁰⁾. Growth hormone is stimulated by growth hormone releasing hormone and inhibited by growth hormone inhibiting factor, as the pituitary cells contain receptors for both enzymes. Growth hormone is secreted in a pulsatile manner and the highest level of GH is obtained at night. The GH binding receptors are present in liver and in some other target organs⁽¹⁷⁾. Due to the binding of growth hormone, IGF-1 is released and binds with the IGF-1 receptors in the target tissues. Apart from these, the GH-IGF axis is regulated by several other genes and pituitary transcription factors (Figure 1). The mutations in genes in the GH-IGF1 axis cause GH insensitivity, and mutations in the GH receptor, IGF-1 gene, IGF-1 receptor, and mutation in signal transducers and activator of transcription (STAT 5b) also cause growth hormone insensitivity and low levels of IGF-1⁽³⁶⁾.

The growth hormone deficiency (GHD) is the most common pituitary hormone deficiency, being possible in two ways, such as isolated growth hormone defici­ency (IGHD; i.e., GH is deficient) and combined growth hormone deficiency, where several other pituitary hormones are deficient⁽⁴⁴⁾. Currently, many mutations associated with isolated GHD have been identified and have been categorized into several types, depending on the mutation and the pattern of inheritance. Growth hormone deficiency is primarily identified due to the reduced growth rate after the birth, and it is confirmed using biochemical assay methods. Many mutations associated with the GH gene and the GHRHR gene have been identified. Mainly, the complete GH1 gene deletion, which differs in size as 6.7kb, 7.1 kb and 7.6kb as a result of unequal recombination and crossing over during meiosis^(13,50), and additionally several small deletions, frame shift and nonsense mutations are categorised as type I IGHD, and several other mutations in the intronic and exonic regions have been identified so far and categorized as type II, where all differs upon ethnicity⁽²¹⁾. The type III or the X-linked inheritance is a rare form. The biochemically defined growth hormone deficiency children are treated with the exogenous growth hormone, and the best results can be obtained when the GHD is identified in early ages.

Growth hormone

The human growth hormone is categorized with prolactin and human chorionic somatomammotropin under the same protein family. Among these, the growth hormone and the somatomammotropin are closely related, with 85% homology, being present as a cluster on chromosome 17 q 22-24 (22-24 band in small arm of the 17^th chromosome), where prolactin shows only 35% homology and is present on the 6^th chromosome. The GH expression is tissue-specific and limited only to the pituitary⁽¹¹⁾.

Growth hormone gene

The human growth hormone gene is arranged on a gene cluster as a five-gene family of two HGH genes and three HCS genes as 5’ HGH- N, HCS- L, HCS- A, HGH- V, HCS-B 3’ and spanning about 50 kb⁽⁴⁵⁾ where HGH-N and HCS-B are separated by 45 kb.

HGH gene is termed as the normal form, and the HGH-V gene is known as variant form. HGH-N and the HGH-V genes are located on 2.6 kb ECoR1 fragment and can be readily discriminated by the presence of BamH1 site present in the fourth intron of the HGH-V gene. The HCS-A and HCS-B are also present on 2.9 kb EcoR1 fragments, where HCS-A contains two PvuII sites, but HCS-B contains only one such site (Figure 2). The 2.9 kb genomic DNA fragment corresponds to the HCS-B gene, while the cloned cDNA sequence represents the HCS-A gene; although these genes generate distinct mRNA transcripts, they encode identical polypeptides. The HCS-L gene, characterized by a 9.5 kb fragment, is closely related to both HCS-A and HCS-B⁽¹⁶⁾. The most remarkable feature of the GH gene cluster is the existence of high-density repetitive sequence. Many Alu sequence repeats with complete and partial sequences are present on the 3’ site of HCS and HGH genes correspondingly, and there are about 20 Alu repeats in the cluster⁽³¹⁾.

The DNA analysis of the five genes in the cluster revealed that five genes are highly conserved within 500 nucleotides 5’ to the transcription initiation site. The 3’ site is highly conserved within 700 nucleotides in the HCS gene and 400 nucleotides within the HGH gene. There is an inverted Alu repeat in about 100 nucleotides downstream of polyadenylation sites in HGH-N gene which distinguish HGH gene from the HCS gene. This Alu sequence lasts about 100 bases of HGH-N gene and stops 25 bases of HGH-A, HGH-B and HGH-L gene⁽⁴⁰⁾.

Furthermore, the human growth hormone (HGH) gene cluster comprises three types of duplicated units: the coding regions of the genes, the 5’ flanking sequen­ces, and the 3’ flanking regions. The 3’ flanking regions of the HCS genes, in particular, exhibit characteristics of ancestral duplication events. These findings support the hypothesis that the five genes within the cluster arose through duplication and homologous but unequal recombination of the HGH and HCS genes. The ancestral form can be denoted as 5’ HGH:HCS 3’ as the gene cluster contains 5’ HGH-N to HCS-B 3’. The duplication events occur between homologous Alu repeat elements, highlighting the pivotal role of Alu sequences in genomic evolution and their function as recombination hotspots. The five genes give rise to different proteins, as the HCS-A and HCS-B genes give rise to identical proteins and the chorionic somatomammotropin acts during the pregnancy and improve the fatal growth during starvation period and have mammotrophic and lactogenic effects⁽³⁾. The structural difference in the genes determines the site of expression as the genes in the left-hand domain of the cluster, that is the HGH-N gene, are expressed in the pituitary, and the genes in the right-hand domain of the cluster, HCS-A and HCS-B, tend to express in the placenta^(25,35).

The HGH-N gene

The HGH-N gene is present in the 5’ of the gene locus and includes five exons (Figure 3) and four introns⁽²⁸⁾.  The expression of the gene is controlled by the cis acting elements and trans acting elements. The most well-defined cis sequences are the sequences present near the promoter sequence as CAT sequence present at 85 base pairs and TATA sequence located at 30 base pairs and pit-1 transacting factor binding sequence⁽³³⁾. Binding sites for specific factors, like NF1, AP2, USF and Sp1, are present upstream of the gene. The binding sites for glucocorticoid receptor, thyroid hormone receptor and retiboicacir receptor are also present in the gene^(24,34).

Growth hormone protein

The growth hormone is a monomeric protein of 191 amino acids, and the 26 amino acid leader sequence is enzymatically cleaved in order to entry into the endoplasmic reticulum and the resulting mature growth hormone (Figure 4); a four-helical bundle with an unusual structure of antiparallel 4 a helix bundle with intramolecular disulphide bridges, of 22 kDa which act as a major circulating GH (approximately 75%)⁽¹²⁾.

Helix one and four (26 and 30 amino acid residues, respectively), which contain the NH₂ and COOH termini, are longer than the helix two and three (21 and 23 amino acid residues). The NH₂ termini is resides away from the molecule, while the COOH termini remains linked with the helix 4 through a disulphide bond between Cys¹⁸² and Cys¹⁸⁹. Helix 2 is linked via a Pro⁸⁹, helixes 1 and 2 are cross connected by 35-71 residues, and helixes 3 and 4 are connected through 129-154 residues. A disulphide bond is found between Cys⁵³ and Cys¹⁶⁵ which connects the helix 4. Helix 2 and 3 are connected by a 93-105 residue. Apart from that, small helixes are present in each beginning and end of the helices in the connecting loops, as helix 1 and 2, and one between helix 2 and 3 (38-47 and 64-70, and 94-100, respectively). The core of the molecule is hydrophobic, as many of the residues are hydrophobic amino acids⁽¹⁹⁾.

The growth hormone contains two sites to bind with the receptor molecule as site I formed by residues on exposed sites of helix 1 and 4 and the connecting loops between helix 1 and 2 (Figure 5). The site II is composed of residues of helix 1 and 3⁽²²⁾. A minor amount of circulating GH is a 20 kDa product, an alternating splicing product of GH1 gene because of deleting amino acid 32-46 due to the joining of exon 2 with splice acceptor site present internal to the exon 3⁽⁵⁴⁾.

Growth hormone action

Growth hormone release is stimulated by the GHRH and correspondingly by somatotrophs, only when the GH releasing peptide receptors are stimulated; besides, the GH is inhibited by the action of somatostatin⁽⁵⁾ and the production of GH is controlled by hypothalamic GH releasing inhibiting factor and negative feedback regulation of GHRH and positive feedback regulation of somatostatin production. The pituitary specific transcription factor POU1F1 regulates the GH expression. The mutations in the pit1/GHF 1 which is homologous to POU1F1 do not express the GH and cause hypoplasia in cells (Figure 6). The Pit1/GHF1 action is autoregulated and can be enhanced by the action of growth hormone releasing hormone when bound with the receptor.

Growth hormone has a wide range of activities due to the presence of GH receptors on many target tissues, and it is mediated by insulin-like growth factor 1, which release in response to GH, act as an autocrine and paracrine action in neighboring tissues and act by means of a hormone in distant tissues. The growth and proliferation of tissues are directly (via GH) and indirectly (IGF1) stimulated by the growth hormone⁽²⁶⁾. The responsiveness to growth hormone develops progressively with the age due to the increased number of GH receptors, and the binding sites are fewer in fetal and neonatal period, but begin to increase later, and the higher the number of receptors, the greater the GH dependence responsiveness during the growth process⁽²⁷⁾. The growth hormone binds with two growth hormone receptor molecules in a sequential manner. First, the growth hormone binds with one receptor molecule in a high affinity and then the alternative site of the hormone binds with one more receptor and stabilizes the receptor dimer⁽¹⁷⁾.

The receptor contains two domains, consisting of 7 b strands, arranged in two antiparallel b sheets that represent one with four strands and one with three strands, as shown, and the two domains (1-123 and 128-238 residues, respectively) are linked by a single four-residue segment. The NH2 terminal domain contains three disulphide bridges, and the two domains are linked by short four-residue segment⁽¹⁸⁾.

The COOH termini domain of the growth hormone bound receptor complex (hGH.(hGHbp)₂ present as closely parallel, and the termini is present away from the hormone (Figure 7). The eight-residue segment present between the COOH termini, and the membrane spanning region helps to retain the flexibility and provides the freedom to bring the extracellular domains together⁽⁴⁾.

The growth hormone, after binding with the receptor dimer, two non-receptor tyrosine kinase (JAK2) links with the receptor with high affinity and phosphorylates the tyrosine kinase in the other JAK2 molecule and make it activated (Figure 8). The tyrosine residues in the domains of the GH receptor and the JAK2 itself is phosphorylated by the activated JAK2. The phosphorylated tyrosine residues have high affinity for other signaling protein molecules like SH2⁽¹⁰⁾.

The role of growth hormone

Growth hormone reflects an endocrine, paracrine and autocrine action. Nonetheless, the growth hormone is not a foremost player throughout reproduction, rather it acts as a tuner and it regulates the growth in a complex process by combining the mitogenic and metabolic actions through IGF-I dependent or independent manner in the liver cells where IGF-I and other sex steroids produces henceforth the main target organ of the GH⁽⁵¹⁾. The growth hormone brings transcription of many genes as IGF-1, transcription factors and other metabolic enzymes, the JAK2 phosphorylate STAT molecules and make them activated by dimerization, nuclear localization DNA binding and in turn activating the transcription⁽²³⁾. GH is also important in activating the Ras-MAP kinase pathway by phosphorylating the SHC protein (Figure 9). The growth hormone increases the lipogenesis, protein synthesis and also helps to transport glucose and amino acids. The GH stimulates the insulin receptor substrates (IRS) 1, 2 and 3, which then provides a binding site for SH2 domain of phosphotidylinositol 3’ kinase, an important protein for insulin-like GH action⁽²³⁾.

The action of growth hormone is regulated by the estrogen

Growth hormone is important in testicular development and function, gametogenesis and steroidogenesis. The sperm fertility, morphology and concentration are retained by the growth hormone; GH deficiency is associated with reduced sperm fertility. The growth hormone plays an important role in the progenitor Leydig cells, the cells that synthesize steroidogenic enzyme during the maturation into mature Leydig cells, by increasing the synthesis of testosterone. The growth hormone also prompts the expression of genes that code for steroidogenic enzymes in the differentiated and immature Leydig cells. The conversion of pregnenolone to progesterone is stimulated by GH by increasing the activity of b-hydroxysteroid dehydrogenase by a mechanism dependent on de novo protein synthesis and tyrosine kinase (Figure 10). The growth hormone also stimulates the production of StAR (steroidogenic acute regulatory protein), which mediates the translocation of cholesterol into mitochondria by a process independent of de novo protein synthesis and tyrosine kinase, which is then convert to pregnenolone⁽¹⁵⁾.

The growth hormone promotes fertility in women, and it has been demonstrated in many mammals that ovarian steroid production has been increased after GH administration. The growth hormone stimulates the steroidogenesis directly or by inducing the gonadotropin action (Figure 11). The growth hormone stimulates the LH receptors and induces the luteinization, achieving the progesterone synthetic ability. Furthermore, the GH may induce steroidogenic enzyme production through direct, IGF mediated mechanism, cAMP dependent pathway involving de novo protein synthesis, and the rest by cAMP independent pathway independent of protein synthesis⁽⁹⁾.

The growth hormone also induces gonadotropin-induced folliculogenesis, and there were observed a reduced small follicular development and follicular atresia in hypophysectomised sheep. The GH further induces the luteinized granulosa cell development in an IGF-I and FSH independent manner (Table 1). In turn, GH increase the activin synthesis, thereby stimulating early follicular development. On the other hand, GH stimulate nuclear maturation by an IGF-I independent manner via cAMP in cumulus cells⁽³⁸⁾.

The role of growth hormone in stature

The genetic factors, local hormones and nutritional factors determine the growth of the skeletal system and the height of an individual, but the genetic factors like GH-IGF1 axis play a predominant effect on stature throughout the postnatal period⁽³⁹⁾. The multiplication of cells and the formation of multinucleated myotubes are the two physiognomies of the growth where GH directly or indirectly participates. The number of nuclei in the skeletal muscle increases with the age, from the neonatal period to the period of sexual maturation, henceforth the DNA content is low in GH-deficient people⁽⁴⁶⁾. According to the somatomedin hypothesis, growth hormone (GH) stimulates the production of somatomedins, primarily insulin-like growth factors (IGFs), in liver cells, which in turn mediate skeletal growth⁽⁴⁶⁾.

The longitudinal bone growth is occurring as the cells of the chondrocytes in the epiphyseal growth plate begin to proliferate. As the exogenous GH is administered to the epiphyseal growth plate, there have been observed that the longitudinal bone grew, as well as the width of the bone grew, signifying the stimulated chondrogenesis by the growth hormone. The growth plate is arranged in a typical manner, with the top layer of the resting or the germinal cells or the stem cells, and these cells are able to differentiate and enter into the proliferative zone. After undergoing maturation, these enter the hypertrophic cell zone. The cells are then calcified and enter metaphyseal bone. The chondrocytes are arranged in a fascicular pattern in the direction of growth. The primary target of the GH is the germinal cells of the epiphyseal growth plate⁽⁶⁾.

Growth hormone deficiency

The growth hormone deficiency has two forms: isolated growth hormone deficiency (IGHD), where only the growth hormone is deficient, and combined pituitary hormone deficiency, where the growth hormone along with several other hormones resembling ACTH, TSH and gonadotropin are being deficient⁽⁴²⁾. Thirty percent of the cases in GHD are owing to the genetic etiology – in consanguineous families. It has been estimated that the IGHD stands to occur 1 in 4000-10,000 births⁽⁴²⁾. Four familial isolated GH deficient types have been found so far: IGHD type I A, autosomal recessive and due to the absence of the GH 1 gene, IGHD I B, autosomal recessive and reduced GH, IGHD II, autosomal dominant and with diminished GH, IGHD III, X-linked and diminished GH (Table 2).

IGHD-I is present in two ways, as GH-1 gene defects and GHRHR gene defects (Hayashi et al, 2002). The former one is associated in two ways, as the complete deletion of GH-1 (both the alleles) accompanied by the lack of GH in the circulation, and this category of mutation is known as IGHD type IA, and the affected patients are assumed as homozygous or compound heterozygous for the mutations in the GH 1 gene. IGHD type IA, characterized by growth retardations, characteristic facial appearance and hypoglycemia in infancy, is the most severe form of IGHD. The size of the gene deletion differs in individuals, depending upon ethnicity. Deletions of 6.7 kb, 7.0 kb, 7.1 kb, 7.6 kb and 45 kb are observed in different population studies⁽⁴³⁾ (Figure 12).

The deletion of GH1 gene is due to the unequal recombination events taking place between the homologous regions flanking the GH1 gene at meiosis⁽⁵²⁾.

This was first demonstrated in 1981 by Southern blot analysis and currently by PCR amplification of two DNA sequences of 1.9 and 1.919 kb flanking the GH1 gene at 5’ side 2.6 kb and 3’ side 1.5 kb site followed by restriction digestion method (Figures 13-15).

The individuals with the complete lack of the GH1 respond well to exogenous human growth hormone (hGH) replacement initially, then become resistant to the treatment because of the development of anti-hGH antibodies. Insulin-like growth factor-1 (IGF-I) therapy for these patients was introduced recently⁽³²⁾. IGHD patients, heterozygous for deletions and frameshift mutations of the GH1 gene, fall into the second form of the IGHD type IA. The sequence analysis detected a deletion in cytosine at 371 positions – i.e., in the signal peptide of the coding region –, creating a frameshift mutation which prevents the mature GH synthesis.

A nonsense mutation caused by a G-to-A transition in the 20^th codon of the GH1 signal peptide converts a tryptophan codon (TGG) into a premature stop codon (TAG), resulting in translation termination after 19 residues; this mutation is classified under isolated growth hormone deficiency type IA (IGHD IA). The homozygous patients for this mutation also develop the anti-GH antibodies. This can be screened by PCR amplification followed by restriction digestion using AluI restriction enzyme, as it creates a new Alu site. The IGHD type IB is due to point mutation, deletion or nonsense mutation in the GH1 gene, and is milder than that of type IA. The patients do not develop antibodies against exogenous GH therapy. Splice site mutations, deletions and frameshift mutations can be detected in this type of IGHD IB.

Some mutations of this type occur in the intron 3 (IVS3) and intron 4 (IVS4). The IVS4+1G>C mutation in intron 4 disturbs the splicing and give rise to an altered protein product. The 20^th codon of GH1 contains a homozygous G>A transition which results in a premature stop codon in the exon 2. Furthermore, an IVS4 +1G>T transition is present in IGHD IB patients⁽²⁸⁾.

The latter one explains the growth hormone releasing hormone receptor (GHRHR) gene mutations (Figure 16). GHRH is one of the regulators of GH which stimulate the GH release. A nonsense mutation at codon 72 of the GHRH gene introduces a premature stop codon, resulting in truncated and nonfunctional growth hormone-releasing hormone (GHRH). Several other mutations associated with GHRHR have been identified. In IGHD type II, several mutations have been observed in different populations; the patients are heterozygous for the deletions, hence diminished levels of GH can be detected, and patients under exogenous GH therapy do not produce antibodies. The mutation in intron 3 is the most commonly occurring in populations.

Five splicing mutations categorized under the dominant inheritance

IVS3+6T>C transition mutation inactivates the donor splice site and, in turn, the synthesis of a protein product with skipping of exon 3 (List et al., 2024). IVS3+1G>A transition in the invariant CpG dinucleotide of exon 3/IVS3 boundary region results in skipped exon 3, with a loss of amino acids 32-71 resulting in a 151 amino acid shortened protein product⁽³⁰⁾. The resulting isoform is a 17.5 kDa mutant form which lacks the connecting loop between helix 1 and 2 with Cys53 of internal disulphide bridge⁽¹⁾.

Apart from these, the heterozygous mutation in IVS3+1G>A, IVS3+1G>C, IVS3+28G>A, IVS3+5G>A, IVS3+2T>C, IVS3+6T>C mutation is commonly seen in this type. Furthermore, the dominant-negative novel mutation IVS2–2A>T disrupts the highly conserved tagGAA sequence at the 3’ splice acceptor site, abolishing the essential AG dinucleotide required for accurate mRNA splicing. IVS3+2T>C mutation in intron 3 in TCCgtg sequence of donor spice site disturbs the splicing pattern. Moreover, IVS-1G>A in the intron 2 acceptor splice site mutation is detected among IGHD type II. Among these, the IVS3+1G>A transition mutation is the most common mutation among different ethnic groups. Other two dominant negative mutations in the intron 4 are IVS4+1G>C and IVS4+1G>T transitions. Some of the cases show a mutation in 456 positions of GGGgtg sequence, the last base of the 3’ acceptor splice site in exon 4 results in G to A transition, resulting the skipping of exon 4⁽³⁰⁾.

The exon splice enhancer mutations (ESE) disturb the splicing and lead to a weak recognition of exon 3, ESE3+1G>T, ESE3+5A>G mutations observed⁽⁴¹⁾. Isolated growth hormone deficiency type III (IGHD III) is a rare X-linked form of the disorder and is associated with X-linked agammaglobulinemia (XLA)⁽⁷⁾.

Diagnosis of growth hormone deficiency

The most common feature of GHD is the short stature associated with low growth velocity for age and puberty, and these are correlated with the severity of the GHD. Complete absence of circulating growth hormone (GH), resulting from a deletion of the GH1 gene, is characterized by a height standard deviation score (SDS) below –3 and a growth velocity below the 3^rd percentile.

Growth hormone therapy

Earlier, the replacement therapy was done using the pituitary-extract GH obtained from human cadavers, hence the amount of the GH was limited, therefore the severe GHD was detected initially. Subsequently, after 1985, the recombinant DNA-derived GH became available, and the supply was unlimited. Unfortunately, growth hormone replacement is an expensive method when compared with others. Currently, in certain countries, children with all forms of GH insufficiency, from severe to mild, are treated with exogenous GH, while in developing countries or in poor ones, due to economic conditions, only those with severe GHD are treated.

Growth hormone is given by subcutaneous injections; for better results, the children should be treated as soon as they are diagnosed. The dosage of GH is decided according to body weight, and the amount and the frequency of administration vary upon the protocol. The amount increases with the increased body weight, and it is maximum in the puberty and becomes discontinuous at 17 years of age. The responsiveness to GH depends on: 1) the severity of the disease and the age of the patient; 2) the associated diseases, like thyroid deficiency; 3) the development of anti-GH antibodies. Type IA IGHD pa­tients respond well to the GH therapy initially, but they become resistant in time, due to the formation of anti-GH antibodies. Type IB IGHD patients respond well at the administration of growth hormone.

Autor corespondent: F.M.M.T. Marikar E-mail: faiz@kdu.ac.lk

CONFLICT OF INTEREST: none declared.

FINANCIAL SUPPORT: none declared.

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