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Short Stature Caused by a Biologically Inactive Mutant Growth Hormone (GH-C53S)

University Children’s Hospital (A.B., S.S., J.D., J.-M.V., A.E., C.F., P.E.M.), Pediatric Endocrinology and Metabolism, Inselspital, and Department of Pharmacology (S.B., U.H.), University of Bern, CH-3010 Bern, Switzerland; and Medizinische Klinik-Innenstadt (M.B.), Munich University, D-80336 Munich, Germany

Address all correspondence and requests for reprints to: Professor Dr. Primus E. Mullis, University Children’s Hospital, Pediatric Endocrinology, Inselspital, CH-3010 Bern, Switzerland. E-mail: primus.mullis{at}insel.ch.

	Abstract

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Human GH has two disulfide bridges linking Cys-53 to Cys-165 and Cys-182 to Cys-189. Although absence of the first disulfide bridge has been shown to affect the bioactivity of GH in transgenic mice, little is known of the importance of this bridge in mediating the GH/GH-receptor (GHR) interaction in humans. However, we have identified a missense mutation (G705C) in the GH1 gene of a Serbian patient. This mutation was found in the homozygous state and leads to the absence of the disulfide bridge Cys-53 to Cys-165. To study the impact of this mutation in vitro, GHR binding and Janus kinase (Jak)2/signal transducer and activator of transcription (Stat)5 activation experiments were performed, in which it was observed that at physiological concentrations (3–50 ng/ml) both GHR binding and Jak2/Stat5 signaling pathway activation were significantly reduced in the mutant GH-C53S, compared with wild-type (wt)-GH. Higher concentrations (400 ng/ml) were required for this mutant to elicit responses similar to wt-GH. These results demonstrate that the absence of the disulfide bridge Cys-53 to Cys-165 affects the binding affinity of GH for the GHR and subsequently the potency of GH to activate the Jak2/Stat5 signaling pathway. In conclusion, we have demonstrated that GH-C53S is a bioinactive GH at the physiological range and that the disulfide bridge Cys-53 to Cys-163 is required for mediating the biological effects of GH.

	Introduction

Top Abstract Introduction Subjects and Methods Results Discussion References

 
SHORT STATURE ASSOCIATED with bioinactive GH was first suggested and described by Kowarski et al. in 1978 (1). It is clinically characterized by normal or slightly increased GH secretion, pathologically low IGF-I levels, and normal catch-up growth on GH-replacement therapy. On a clinical basis, additional cases of bioinactive GH were described in the 1980s (2, 3, 4, 5, 6). Takahashi and coworkers (7, 8) reported two missense mutations (R77C and D112G) in the GH1 gene leading to Kowarski’s syndrome in two Japanese patients. However, these mutations were both found in the heterozygous state only, and furthermore, the mutation R77C was also identified in the normal-stature father. Six GH variants were then suggested to be bioinactive by Millar and coworkers (9). Again, all these mutations were found in the heterozygous state, and no clear correlation between laboratory/clinical phenotype and patient genotype was shown.

GH plays a major role in postnatal growth. The growth-promoting effects of GH are achieved through GH’s diverse and pleiotropic effects on cellular metabolism and differentiation and are mediated through the activation of a cell surface receptor (GHR). A single GH molecule contains two GHR-binding sites, and these bind two GHR molecules sequentially, inducing receptor dimerization and hence activation (10). Interaction of the dimerized GHR with the intracellular tyrosine kinase Janus kinase (Jak)2 leads to phosphorylation of downstream signal transduction molecules, including signal transducer and activator of transcription (Stat)5 (11). Activated Stat5 is translocated in the nucleus in which it transactivates a series of GH-responsive genes (11).

Structurally human (h) GH comprises four antiparallel -helices separated by connecting loops as well as four Cys residues located at position 53, 165, 182, and 189 (12). When aligned to optimize amino acid similarity, the four Cys residues were found to be conserved among GH molecules from different vertebrates (13, 14). This conservation may indicate that these residues are important for structural integrity and biological activity of the protein (15). The four Cys residues form two disulfide bridges: one between Cys-53 and Cys-165, which results in a large loop, and the other one between Cys-182 and Cys-189, which forms a small loop. Previous studies have shown that the integrity of the small loop of hGH (Cys-182 to Cys-189) is nonessential for the secretion (16) and the biological activity of GH (17, 18). A drastic reduction of GH secretion was, however, observed when the disulfide bridge Cys-53 to Cys-165 was disrupted (16). Site-directed mutagenesis techniques have been used to perform Cys residue conversion experiments. When the disulfide bonds in bovine (b) GH were disrupted by amino acid substitution of Cys to Ser residues and assayed for their ability to enhance growth in transgenic mice, only animals that expressed bGH analogs with the large loop intact demonstrated a growth-enhanced phenotype (16). Activity of GH having lost its disulfide bond Cys-53 to Cys-165 was also studied by Uchida et al. (19) using the adipose conversion assay. The GH variant without the disulfide bridge Cys-53 to Cys-165 showed lower activity than the normal GH. Its binding capacity was also reduced (19). Furthermore, a loss of biological activity of hGH and porcine GH was observed when both disulfide bridges were disrupted (20, 21), even though these results were controversial (22, 23).

In this report, we describe the first patient with short stature carrying this GH-C53S mutation. This mutation was found in a homozygous state, which is in contrast to the patients described so far (7, 8, 9). Furthermore, the parents of this patient, presenting in a heterozygous state, were of normal height. Clinical data of the patient are presented. Furthermore, in vitro studies, and more precisely GHR binding experiments and activation of Jak2/Stat5 signaling pathway experiments, were performed to study the impact of this mutation on bioactivity.

	Subjects and Methods

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Subjects

Family. We studied a family of five members from Serbia: two parents and three offspring. The pedigree is shown in Fig. 1. The parents are first cousins in good health and with height and weight within normal limits (24). The father is 171 cm [–0.9 SD score (SDS) for age and sex (24)], and the mother is 163 cm (–0.1 SDS). The only sister tested negative for the mutation [wt (wild-type)/wt) and was 164.2 cm (0.0 SDS) at the age of 18 yr when they left Switzerland for Serbia.

View larger version (7K): [in this window] [in a new window]   FIG. 1. Pedigree of the patient’s family. The distribution of the GH-C53S mutation is shown. The open symbol represents wild-type, whereas the solid symbol represents homozygosity for the mutation. The open symbols containing smaller solid symbol represent heterozygosity.

The boy was born at term (38 wk 2 d of pregnancy) after a normal pregnancy. The delivery and post- and perinatal course were uncomplicated. The patient’s birth weight and length were 2810 g (–1 SDS) and 46 cm (–1.8 SDS), respectively (24). Growth retardation became obvious at the age of 3.5 yr, at which stage a local doctor followed the progress of the boy. The psychomotor development was reported normally. At the age of 8 yr, the patient immigrated to Switzerland with his family. Thereafter, at the age of 9 yr, he was referred to the Outpatient Clinic of the University Children’s Hospital in Bern, Switzerland. At this time he presented with short stature (–3.6 SDS for age and sex) (Fig. 2) (24). At the beginning the possibility of GH insensitivity syndrome was suggested: rather high basal GH concentration, high peak GH level after provocation test, low basal IGF-I concentration, although unexplained concentrations of IGF binding protein (IGFBP)-3 in the lower normal range (P10) and normal GH binding protein (GHBP) levels were found (Table 1). Further detailed assessment, however, presented a normal IGF-I and IGFBP-3 response after an IGF-I generation test (Table 1) (25), and together with the normal sequence of the GHR-gene, a functionally altered GH was hypothesized. In addition, the other pituitary-derived hormonal axes were studied in detail and were normal. Furthermore, renal, intestinal, or metabolic reasons for failure to thrive were excluded. A magnetic resonance imaging analysis of the head, especially the region of hypothalamus and pituitary gland, showed no abnormalities. Subsequently recombinant human (rh)GH-replacement therapy (Norditropin, at a dose of 30 µg/kg; Novo-Nordisk, Copenhagen, Denmark) was administered sc in the evening on a daily basis. The treatment resulted in growth velocity increase to 9.6 cm (+8.4 SDS) during the first year (pretreatment growth velocity, 3.9 cm; –2.9 SDS). Thereafter, the doses were continuously adapted (30–45 µg/kg·d) at a regular basis according to the effect on growth velocity and IGF-I measurements. When the boy left Switzerland with his family, he was 16 yr 4 months of age, his height was 164 cm (–1.6 SDS), his weight 50 kg (–1.5 SDS), and the pubertal stages according to Tanner were P5 and G5 with testicular volumes of 16 ml (left) and 18 ml (right) (24, 26). He was in good health and finished school with good academic results.

View larger version (122K): [in this window] [in a new window]   FIG. 2. Growth chart of the patient. Percentiles are shown on the extreme right. The solid circles indicate the height measurements, the open circles the bone ages. The arrow pointing down marks the beginning of rhGH therapy, whereas the arrow pointing up the end of the therapy (the time the patient left Switzerland). Asterisk indicates start of puberty (both testicles 4 ml).

The Ethical Committee of the University Children’s Hospital approved the experimental protocol. Informed consent was obtained from parents as well as family members involved.

Production of GH peptides

The plasmid pcDNA3.1(–) containing hGH cDNA (29) was used as a template to amplify the hGH cDNA and inserted in the pSecTag2 plasmid (Invitrogen AG, Basel, Switzerland).

Site-directed mutagenesis was performed on pSecGHwt to generate the GH mutant studied (GH-C53S) using the QuickChange site-directed mutagenesis kit (Stratagene AG, Basel, Switzerland). Mutagenesis was confirmed by sequencing (Fig. 3) (Big Dye Terminator sequencing kit; PerkinElmer, Applied Biosystems, Rotkreuz, Switzerland). Products were analyzed on an ABI 373 automated DNA sequencing system (Applied Biosystem) and sequences confirmed using GenBank accession no. NM_000515 as reference sequence.

View larger version (19K): [in this window] [in a new window]   FIG. 3. Part of the sequences showing the substitution of the cysteine at amino acid 53 with serine.

The concentrations of the GH produced by the CHO cells during 3 d in Ham’s F12 plus 2% fetal calf serum media were measured by the DSL-GH ELISA kit (Diagnostic Systems Laboratories). To confirm that the mutation C53S does not affect the affinity of the antibody used in the DSL-ELISA, two different GH assays were performed on two samples of CHO supernatant, and the results were compared.

Measurements of GH

Human GH immunochemiluminometric assay (ICMA). Serum concentrations of various forms of hGH were measured using automated Advantage chemiluminescent assay system (Nichols Diagnostics Institute, Bad Vilbel, Germany). This sandwich-type immunoassay involves a monoclonal capture and a polyclonal detection antibody. Within-assay coefficient of variance was 3.5, 2.2, and 2.9% at concentrations of 1.4, 10.5, and 28 ng/ml, respectively. Between-assay variability at the same concentrations was 7.9, 2.7, and 5.9%, respectively. The lower limit of quantification was 0.2 ng/ml and the linear working range between 0.2 and 50 ng/ml.

DSL-GH ELISA. The DSL-10–1900 active hGH ELISA is an enzymatically amplified two-step sandwich-type immunoassay (Diagnostic Systems Laboratories). Intraassay CV was 4.1, 4.0, and 3.2% at concentrations of 0.9, 3.5, and 20.3 ng/ml, respectively. Interassay CV was 6.1, 6.5, and 6.8% at the same concentrations. The lower limit of detection was 0.1 ng/ml and the linear working range between 0.1 and 36 ng/ml.

Immunofunctional assay (IFA). This assay has been previously described by Strasburger et al. (30).

Receptor binding assay

Receptor binding assays were performed using 293 human embryonic kidney cells stably expressing human GHR (293GHR) (31, 32, 33). 293GHR cells were a gift from Professor R. Ross (Northern General Hospital, Sheffield, UK) and were cultivated as described by Ross et al. (33). Twenty-four hours after plating 293GHR cells in 12-well plates (1.5 x 10⁵ cells/well), cells were serum starved for 12 h. Cells were then incubated with ¹²⁵I-hGH (PerkinElmer, Schwerzenbach, Switzerland, 200,000 cpm/well) for 3 h at room temperature in starvation media in the absence (negative control) or presence of various concentrations of unlabeled GH peptides produced by the CHO cells (wt-GH and GH-C53S) as well as rhGH (positive control). The cells were then washed with PBS buffer and solubilized in 1 ml of 1 M NaOH for counting radioactivity with a -counter (1470 Wizard, Wallac, PerkinElmer, Hünenberg, Switzerland). Experiments (n 3) were performed in triplicate wells. IC₅₀ values for the different GH peptides were determined by nonlinear regression, using a single-site competition model (version 2.0 Prism software, GraphPad Inc., San Diego, CA).

Luciferase reporter gene assay of Stat5 activation

293GHR cells were used to assay Stat5 activation as described before (32, 33). Briefly, cells were transfected with a Stat5-responsive luciferase reporter gene construct (34, 35) and treated with increasing amounts of GH (rhGH, wt-GH, and GH-C53S) for 6 h. Luciferase expression was then measured with a luminometer (Mediators PhL; Aureon Biosystems, Vienna, Austria). EC₅₀ values were obtained from sigmoidal dose-response curves generated using GraphPad Prism software.

Statistical analysis

Data were analyzed using ANOVA (one-way ANOVA) and Dunnett’s posttest.

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Clinical diagnosis and follow-up on rhGH

One patient originally from Serbia presented with severe short stature (Fig. 2). Sequencing of the GH1 gene revealed a mutation leading to the substitution of the cysteine at amino acid 53 with serine (Fig. 3). This mutation causes the disruption of the disulfide bridge linking Cys-53 to Cys-165. The distribution of this mutation in the family is shown in Fig. 1. Both parents and the brother are heterozygous for the mutation. The sister does not carry the mutation, whereas the patient is homozygous for the mutation. Clinical assessment (Table 1), a normal GHR sequence, and the fact that rhGH-replacement therapy restored normal growth rate in the patient, excluded a GHR defect or an IGF-I defect but indicated the presence of a bioinactive GH. Before treatment, the secretion of GH in the patient was normal to slightly increased. Functional significance of the lesion was then explored with in vitro studies including binding and signaling experiments.

Functional characterization of the C53S variant

To functionally characterize the GH missense variant C53S, site-directed mutagenesis was performed on the plasmid pSecGHwt, before expression in CHO-K1 cells. The concentration of GH in the supernatant of the CHO cells was measured by three different GH assays. This was done to exclude the possibility that the mutation altered antibody binding affinity and subsequent determination of GH concentration (Table 2). The DSL and the ICMA assays are both conventional ELISAs functioning with antibodies against GH. The IFA has been developed by Strasburger et al. (30) and for detection uses labeled GHBP, which binds to binding site 1 of the human GH. The ratios from both conventional ELISAs (ICMA/DSL) were the same for both wt-GH and GH-C53S. Using the IFA, GH-C53S was almost undetectable as will be discussed further down. Nevertheless, the ratios IFA to DSL and IFA to ICMA were comparable.

View larger version (14K): [in this window] [in a new window]   FIG. 4. A, Competitive displacement of 125I-GH binding to the GHR by rhGH (positive control), wt-GH, and GH-C53S. Results are normalized to the amount of 125I-GH bound in the absence of any unlabeled GH. Each point is the mean of three separate experiments performed in triplicate. Bars, ± 1 SD. B, Jak2/Stat5 signaling capacity of rhGH, wt-GH, and GH-C53S. Results are expressed as x-fold induction relative to the basic activity of unstimulated cells and represent the means ± 1 SD of three separate experiments performed in duplicate.

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

 
In this report we describe a patient suffering from bioinactive GH due to a mutation that abolishes the disulfide bridge Cys-53 to Cys-165. The patient affected by this mutation showed typical clinical characteristics of GH deficiency and further assessment revealed the presence of bioinactive GH. The patient displayed no alteration in GH secretion, having normal to slightly increased GH concentrations in circulation. Although impaired secretion has been reported in mice L-cells transfected with either bGH-C53S or bGH-C164S (bovine Cys-164 corresponding to human Cys-165) in vitro, no secretion problems were observed in vivo (16). Indeed, Chen et al. (16) established a mouse line expressing bGH-C53S and observed that this GH variant was found in the sera of these mice at levels comparable with those in transgenic mice expressing bGH-wt. The different secretion patterns of GH-C53S observed in vivo and in vitro may be explained by the fact that in vivo a low concentration of IGF-I in circulation may stimulate the production and secretion of GH, as it is also observed in Laron’s syndrome (36, 37). Eventually, GH-replacement therapy in the patient restored normal growth and height. This study examined the phenotypic significance of this new mutation through binding and functional assays.

Evolutionary conserved data (13, 38) as well as molecular modeling have confirmed the importance of both disulfide bridges for GH protein stability. However, only the disulfide bond between Cys-53 and Cys-165 is essential for the interaction of this hormone with the GHR (16, 17, 18, 19). Indeed, three discontinuous segments have been shown to be crucial for the GH/GHR interaction: the NH₂ terminus of helix I, the loop from Cys53 to the start of helix II (amino acid 54 to 74), and the COOH-terminal portion of helix IV (38). Although the disruption of the disulfide bridge between Cys-53 and Cys-165 may not induce any change in the four-helice bundle structure of hGH (Fig. 5), the mutation causes the rigid association between the loop from helix I and II and the helix IV to become loose. Uchida et al. (19) hypothesized that this may alter the spatial arrangement of the three discontinuous segments, resulting in reduced binding affinity and subsequent biological activity. Our in vitro studies confirm this hypothesis. Indeed, the GH-C53S concentration was almost undetectable with the IFA, implying that this variant did not bind to the GHBP used for detection in this assay. In this assay GHBP binds to binding site 1 of hGH. The fact that GH-C53S could not be detected in the IFA indicates that the binding site 1 of this variant is affected and does not allow this variant to have proper binding. The other assays, DSL and ICMA, showed the same ratio for both GH variants, indicating that the mutation C53S does not affect the affinity for the antibody used in these two conventional ELISAs. Thereafter, for quantification, hGH-DSL was the ELISA used for the rest of the study. In the GHR binding experiments and the physiological range, GH-C53S showed a significantly lower binding affinity for the GHR than wt-GH, whereas at high, supraphysiological concentrations (>400 ng/ml), GH-C53S reached the same binding efficacy as wt-GH. However, these high concentrations are not physiologically relevant. Being in line with the results obtained from the binding studies, GH-C53S showed a lower capacity to activate the Stat5 pathway in the physiological concentrations, but at supraphysiologically high concentrations, that variant again showed responses comparable with wt-GH.

View larger version (39K): [in this window] [in a new window]   FIG. 5. Three-dimensional model of hGH performed with the PyMOL molecular graphics system (2002; http://pymol.sourceforge.net). The four -helices are shown, numbered I-IV. The four cysteines are shown with the disulfide bonds they form.

The data also show that GH-C53S is recognized by the GHR and exhibits partial activity (supraphysiological concentrations). In this way, the disulfide bridge between Cys-53 and Cys-165 may not be essential but necessary to express full biological activity for the Jak2/Stat5 signaling pathway. Other pathways have been implicated in GH actions, but the Jak2/Stat5 pathway is currently thought to be the most important pathway attributed to the growth-promoting effects of GH (39). Lewis et al. (40) described a new mutation affecting the ERK pathway of GH but not the Jak2/Stat5 pathway. However, no connection could be presented with the phenotype of the patient described.

In conclusion, we described a homozygous missense mutation, C53S, in the GH molecule of one Serbian patient with growth retardation showing all the clinical characteristics of a bioinactive GH. The bioinactivity of this mutant has been confirmed on a molecular and cellular basis and is due to lower affinity of the C53S variant for the GHR, presumably caused by the disruption of the disulfide bond between Cys-53 and Cys-165. Therefore, this research contributes new evidence for the importance of these conserved cysteines in mediating the biological effects of this hormone.

	Acknowledgments

 
We thank Professor Richard J. M. Ross and Dr. Mabrouka Maamra (Sheffield, UK) for their help. We are also very grateful to Chris Towne (Lausanne, Switzerland) for reviewing and discussing the manuscript.

	Footnotes

 
This work was supported by Grant 3200-064623.01 from the Swiss National Science Foundation (to P.E.M.).

First Published Online February 15, 2005

Abbreviations: b, Bovine; CHO, Chinese hamster ovary; DSL, Diagnostic Systems Laboratories (assay); GHBP, GH binding protein; GHR, GH-receptor; 293GHR, 293 human embryonic kidney cells stably expressing human GHR; h, human; ICMA, immunochemiluminometric assay; IFA, immunofunctional assay; IGFBP, IGF binding protein; Jak, Janus kinase; rh, recombinant human; SDS, SD score; Stat, signal transducer and activator of transcription; wt, wild type.

Received September 16, 2004.

Accepted February 3, 2005.

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Top Abstract Introduction Subjects and Methods Results Discussion References

 

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