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Department of Pediatrics, Federico II University (M.S., B.B., E.M., A.O., S.D.M., C.P.), 80131 Naples, Italy; Department of Pediatrics, Oregon Health Sciences University (R.G.R.), Portland, Oregon 97201-3098; and International Institute of Genetics and Biophysics (G.F., M.V.U.), Consiglio Nazionale delle Richerche, 80131 Naples, Italy
Address all correspondence and requests for reprints to: Claudio Pignata, M.D., Ph.D., Department of Pediatrics, Unit of Immunology, Federico II University, Via S. Pansini 5, 80131 Naples, Italy. E-mail: pignata{at}unina.it
Abstract
Peripheral GH insensitivity may underlie idiopathic short stature in children. As the clinical and biochemical hallmarks of partial GH insensitivity have not yet been clearly elucidated, the identification of such patients is still difficult. We integrated functional, biochemical, and molecular studies to define the more reliable marker(s) of GH insensitivity. In particular, we measured GH receptor transducing properties through GH-induced protein tyrosine phosphorylation in patients’ peripheral blood mononuclear cells and performed direct sequencing analysis of GH receptor-coding exons. Five of 14 idiopathic short stature patients with low basal IGF-I levels showed low or absent IGF-I increment after 4 d of GH administration. However, a prolonged GH stimulation induced in 3 of them an increase in IGF-I 40% above the baseline value. The IGF-binding protein-3 behavior paralleled that of IGF-I. The 2 GH-unresponsive subjects showed an abnormal tyrosine phosphorylation pattern after GH challenge. Sequence analysis of the GH receptor gene revealed a heterozygous mutation resulting in an Arg to Cys change (R161C) in exon 6 in only 1 patient, who had normal GH receptor responsiveness. Our findings indicate that abnormal GH receptor signaling may underlie idiopathic short stature even in the absence of GH receptor mutations. Thus, combining the 4-d IGF-I generation test and the analysis of GH-induced protein tyrosine phosphorylation is a useful tool to help identify idiopathic short stature patients with partial GH insensitivity.
THE MANAGEMENT of patients with short stature and low growth velocity who do not meet accepted criteria for GH deficiency remains controversial. In the absence of systemic diseases or hormone deficiencies, these patients are often classified as having idiopathic short stature (ISS), which implies that the intimate pathogenic mechanism has yet to be defined (1). It has been recognized that some children with ISS may have peripheral insensitivity to GH at different extents of severity (2). Overall, studies in the last few years have elucidated that receptor defects or molecular alterations of downstream elements in the signaling cascade may lead to a considerable number of diseases characterized by total or partial unresponsiveness of the target organ to the appropriate stimuli. GH insensitivity (GHI) is characterized by the peripheral resistance to the physiological action of GH (3). Clinical features include short stature with high circulating levels of GH and low levels of IGF-I and IGF-binding protein-3 (IGFBP-3). Children with GHI exhibit significant heterogeneity in both phenotype and genotype, resulting in a wide spectrum of severity (4, 5). In classic Laron syndrome, mutations in the GH receptor (GH-R) gene have been extensively documented in the last 10 yr. Along with exon deletions, nearly 40 distinct mutations of the GH-R, including nonsense, missense, splice, or frameshift mutations, have been identified (6). Mostly, these mutations have been found in regions coding for the extracellular domain of the GH-R, consisting of the exons 2–7 (3, 7). Only a few mutations have been documented in regions coding for intracellular domains, such as exons 9 and 10, implicated in the intracytoplasmic intermolecular connections (8, 9, 10, 11). The possibility that GHI might also underlie ISS in those children who do not have features of Laron syndrome led to a search for alterations in the GH-R. However, mutations in the GH-R gene have been found in fewer than 5% of these patients (12, 13), thus suggesting that other molecules involved in GH-R signaling might be altered and cause short stature in those patients with ISS due to GHI (14).
As in the case of other receptors, the signal transduction induced by GH-R triggering is a complex array of biochemical events acting in a coordinated fashion and involving a large number of distinct molecules (15, 16). Protein tyrosine phosphorylation (P-Tyr) of several proteins represents a key event, ultimately resulting in nuclear factor activation and gene transcription (17).
As GHI encompasses a wide spectrum of clinical phenotypes, ranging from classical complete forms and partial deficiencies, the identification of these patients is often difficult, and the prevalence of such cases may be underestimated (18). Although diagnostic criteria to define patients with severe GHI are well established (5), the identification of patients with milder forms is still debated (19).
In this study we ascertain whether integrating functional provocative assays, analysis of receptor-induced biochemical alterations and molecular studies may help unravel the complexity of ISS and lead to identification of more reliable marker(s) of GHI. These patients might also be good candidates for studies aimed at identifying downstream targets in GH-R signaling.
Subjects and Methods
Study subjects
The study population consisted of 14 children (2 girls and 12 boys), aged 1.5–14.1 yr (mean age ± SD, 8.8 ± 4.3 yr) with ISS who met the following inclusion criteria: 1) height below 2 SD for chronological age, 2) bone age delay more than 2 SD for chronological age, 3) growth velocity below the 25th percentile for chronological age, 4) normal birth weight, 5) absence of known endocrine disease or skeletal dysplasia, and 6) no other reason for short stature. Data on height, growth velocity, and sex-corrected midparental height (target height) were transformed into SD scores according to the standards of Tanner (20). Growth velocity was evaluated at 6- to 12-month intervals. Bone age was assessed according to the method of Greulich and Pyle (21). GH secretion was evaluated after clonidine and arginine provocative tests by standard procedures.
Informed consent was obtained from the parents of the patients enrolled
in the study. The clinical features of the 14 children with ISS are
shown in Table 1
. Patient 3 was initially
considered to have classical GH deficiency based on low GH values after
two stimulation tests performed in another hospital. However, after 1
yr of GH therapy (0.1 U/kg daily) growth velocity remained abnormal
(-2.6 SD score); therefore, therapy was discontinued, and
the patient was enrolled in the study.
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To assess the ability to respond to exogenous GH, each patient underwent an IGF-I generation test. This involved daily sc injections of recombinant GH (0.1 IU/kg) for 14–21 consecutive d. Blood samples were taken before the first injection and on d 5, 8, 15, and 22 for measurements of fasting IGF-I and IGFBP-3 levels. IGF-I and IGFBP-3 were measured using a two-site immunoradiometric assay kit (Diagnostics Systems Laboratories, Inc., Webster, TX). Values were expressed as absolute values (micrograms per liter) or were normalized for age and sex with the normative data provided by the manufacturer and expressed as the SD score. The increase in IGF-I and IGFBP-3 values during the generation test were also expressed as the percent increase. The IGF-I intra- and interassay coefficients of variation were, respectively, 3.4% and 8.2%, whereas the IGFBP-3 intra- and interassay coefficients of variation were 1.8% and 1.9%.
GH-induced P-Tyr
The transducing properties of the GH-R were studied through the analysis of GH-induced protein tyrosine phosphorylation on peripheral blood mononuclear cells (PBMC). PBMC were isolated by Ficoll-Hypaque (Biochrom, Berlin, Germany) density gradient centrifugation using standard procedure. GH stimulation (500 ng/ml) was performed with 5- to 15-min stimulation. The pattern of P-Tyr proteins was analyzed on whole cell lysates. Cells (3 x 106) were lysed in a buffer containing 20 mM Tris (pH 8), 10% glycerol, 137 mM NaCl, 1% Nonidet P-40, 10 mmol EDTA, 1 mM phenylmethylsulfonylfluoride, 1 mM sodium orthovanadatum (Na3VO4), 5 µg/ml leupeptin, and 5 µg/ml aprotinin. Proteins were resolved by 10% SDS-PAGE and then blocked with 3% BSA. Immunoblotting was performed by a 1- to 4-h incubation with anti-P-Tyr. In a few experiments membranes were stripped with a buffer consisting of 2% SDS, 6.25 mM Tris-HCl, and 100 mM ß-mercaptoethanol and were reprobed with an anti-Janus kinase 2 (JAK2; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or antisignal transducers and activators of transcription 5 (anti-STAT5) monoclonal (Santa Cruz Biotechnology, Inc.) antibody. In immunoprecipitation experiments, JAK2 was precipitated from 7–10 x 106 PBMC, resting or stimulated for 5 min with GH, and lysed in glycerol-free buffer by a 2-h incubation with protein A-Sepharose precoated with 5 µl anti-JAK2 (Santa Cruz Biotechnology, Inc.). The enhanced chemiluminescence detection system (Amersham Pharmacia Biotech, Little Chalfont, UK) was used after the secondary appropriate Ab incubation. Densitometric analysis of the signals was performed.
GH-R mutational analysis
Automated sequence analysis of exons 2–10 of GH-R was performed in those patients who failed to show an IGF-I increase after the standard 4-d IGF-I generation test and in three of the remaining patients with a borderline IGF-I response. Genomic DNA was isolated from peripheral blood leukocytes. Each exon and corresponding donor and acceptor splice sites were individually amplified by PCR. The coding portion of exon 10 was amplified in three overlapping fragments. Primers were derived from published sequence (22), and amplification was performed using standard PCR conditions (35 cycles of 95 C for 30 sec, 52 C for 45 sec, and 72 C for 1 min). PCR products were recovered from 1.5% agarose gel and sequenced (both coding and noncoding strands) with an ABI Prism dye terminator cycle sequencing kit using the PCR primers, and an ABI Prism 377 Automated DNA Sequencer (PE Applied Biosystems, Foster City, CA). The mutation detected in exon 6 and the polymorphism detected in exon 10 were confirmed by NlaIII or FokI restriction enzyme (New England Biolabs, Inc., Beverly, MA) digestion, respectively.
Results
IGF-I and IGFBP-3 levels during the generation test
As illustrated in Table 2, the IGF-I
generation test, performed over a 4-d standard schedule, revealed an
increase of 40% above the baseline value in 8 (patients 1, 2, 4, 5, 6,
7, 10, and 11) of 13 patients (patient 14 was lost to follow-up at the
time of the generation test). However, in 2 subjects (patients 2 and 5)
the increase in IGF-I expressed as the SD score, remained
below the basal IGF-I mean value for age.
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. In particular, all patients with a
small IGF-I increase also had low basal IGF-I SD
scores. Similarly, the stimulated IGFBP-3 level during the generation
test correlated to the basal IGFBP-3 value (r = 0.74;
P < 0.004; data not shown). As expected, the increase
in IGF-I during the generation test paralleled the increase in IGFBP-3
(r = 0.80; P = 0.001), as depicted in Fig. 1B.
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depicts the behavior
of IGF-I in the five patients who failed to respond with an IGF-I
increase in the 4-d test. Interestingly, in three of these five
patients a late response was observed with increases in IGF-I values at
14 d of 192% and 82% in patients 9 and 13, and an increase of
137% at 21 d in patient 8, respectively. In contrast, in patients
3 and 12 no response was observed. Paternal heights in patients 3 and
12 were 158 and 168 cm, whereas maternal heights were 147 and 158 cm,
respectively.
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In control PBMC, short-term GH stimulation induced P-Tyr. In
particular, two major proteins of 119 and 105 kDa were promptly
phosphorylated; signals reached maximal intensity at 5 min and remained
appreciable 15 min after the stimulation, as shown in Fig. 3A. As illustrated in Fig. 3, B and C,
these proteins should correspond to JAK2 and STAT5, important signaling
molecules in GH-R signaling. In the patient group GH-induced P-Tyr was
normal in all subjects who showed a good response to IGF-I generation
test, including those with a delayed response at 14 or 21 d of
in vivo stimulation. In two patients (no. 3 and 12) who
failed to respond to the sustained IGF-I generation test, in
vitro GH stimulation of the receptor on PBMC induced abnormal
P-Tyr events. Although in patient 3 no tyrosine phosphorylation was
observed, in patient 12 the abnormality was selective, in that it
concerned exclusively the protein migrating in the 119 kDa area, whose
size corresponds to that of JAK2. Figure 3A shows the abnormal P-Tyr in
this patient. This abnormality was not due to an overall low amount of
the protein, in that, as depicted in Fig. 3, B and C, showing reprobing
of the membranes with anti-JAK2 or anti-STAT5, both JAK2 and STAT5
molecules were expressed to an extent comparable to that in the
controls. Moreover, in three experiments immunoprecipitated JAK2 from
unstimulated or GH-stimulated PBMC was properly phosphorylated (data
not shown). In all other patients, the number of P-Tyr proteins and the
timing of the phosphorylation events were m comparable to those in the
controls, as indicated by the densitometric analysis of the behavior of
105- and 119-kDa protein phosphorylation shown in Fig. 4, A and B.
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To rule out a genetic alteration of the GH-R gene in patients who
failed to show an IGF-I increase after GH stimulation (patients 3, 8,
9, 12, and 13) and in three of the remaining patients, chosen on the
basis of a borderline IGF-I response (patients 2, 5, and 10), GH-R
sequence analysis of the exons 2–10 was performed. In patient 5, a
heterozygous missense mutation resulting in an Arg to Cys change
(R161C) in exon 6 was found. As previously shown, this patient had a
low basal value of IGF-I, which normally increased by 175% after
4 d of GH stimulation; however, the stimulated IGF-I value,
expressed as the SD score, remained below the basal mean
value for age. Basal and stimulated IGFBP-3 values were normal. GH
stimulation induced a normal pattern of P-Tyr in PBMC. Genetic analysis
of the patient’s family members revealed that the mother and three
older sisters of the index case were heterozygous for the same
mutation, as depicted in Fig. 5, and none
of them had short stature or mild features of Laron syndrome. In one of
the three sisters a delay in growth and puberty (menarche at age 16 yr)
was reported.
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Discussion
To ascertain whether GH-R hyporesponsiveness might be implicated in the pathogenesis of ISS, in this study a combination of several functional and molecular assays was used to evaluate a number of events induced by GH-R in vivo or in vitro signal transduction. This approach led to the identification of 2 of 14 ISS patients exhibiting partial GHI, thus indicating that GH-R hypo/unresponsiveness may underlie ISS in childhood.
Mutations of the GH-R gene itself in the heterozygous state have been implicated in the pathogenesis of ISS (2, 12, 13). In particular, in a large cohort of patients, 8 of 100 children affected with ISS carried mutations in the GH-R locus (12). Nevertheless, the role of heterozygous mutations in the pathogenesis of short stature has yet to be defined, as these studies did not provide information on IGF generation or GH intracellular signaling. In our patients, heterozygosity for an Arg161Cys mutation of the GH-R gene was found in only 1 patient affected with ISS, who had, however, a normal incremental response to GH. Interestingly, 3 sisters and the mother of the index case, none of whom had short stature, also carried the same mutation. In 1 sister, a delay in growth and puberty, eventually resulting in regular menses, was reported, similar to what is frequently observed in patients with complete GHI (6). The Arg161Cys mutation, which is responsible in the homozygous state for complete GHI, has been previously described in the heterozygous state in 2 ISS patients originating from different geographic areas (12). However, the family data associated with the nucleotide variant reported here would argue against a pathological role of this mutation. Taken together, the low prevalence of GH-R mutations in large cohorts of ISS patients and the lack of a clear phenotype/genotype relationship indicate that a genetic alteration of the receptor itself, although possible, is a relatively rare mechanism for ISS in childhood.
In our patients several polymorphisms of the GH-R gene have been found. In particular, the guanine to adenine polymorphism at position 3 of codon 168 (G168) was found in three patients. The I526L polymorphism in exon 10 was found at the same frequency in patients and controls originating from the same geographic area. A higher frequency of G168 polymorphism has been described in subjects with ISS compared with normal controls (12). However, the significance of this genetic alteration in influencing linear growth as well as the overall clinical phenotype remains an open and intriguing issue.
The GH-R, like all other receptors, exerts its function by delivering intracellular signals through biochemical alterations of functionally related signaling molecules (23). This complex array of biochemical events is redundant in that several genes are involved in amplifying signal transduction in the individual pathway (24). A proper functional interaction in a well coordinated fashion of several gene products is, therefore, required for fully functioning receptor signaling. Thus, a possible mechanism to explain the wide phenotypic variability of heterozygous mutations of GH-R is that there are other genes that may interact with the signaling pathway, thus modifying the clinical phenotype.
In the two patients with peripheral hyporesponsivity herein reported, an abnormal P-Tyr after in vitro stimulation with GH of the GH-R expressed on the surface of PBMC (25), was documented. In one of these two patients, the effect selectively involved a 119-kDa protein, a size corresponding to the JAK2 molecule, which plays a major role in GH-R signaling. However, the absence of tyrosine phosphorylation of the protein was not due to a reduced amount of the kinase itself, thus suggesting a possible abnormal posttranslational regulation of the molecule. Moreover, through immunoprecipitation experiments showing a normal JAK2 phosphorylation this hypothesis was ruled out, thus suggesting that a different molecule migrating in the same area of JAK2 and yet to be defined was involved in the process. Dimerization of the receptor that follows GH binding represents the first key event in the activation of target cells (24). Subsequently, tyrosine phosphorylation of JAK2 and STAT5 proteins plays a crucial role in the activation process, which ultimately results in gene transcription (16, 17). In the context of the overwhelming number of distinct molecules implicated in the process, the pivotal role of STAT5 in the signaling pathway related to GH-R, and in particular of the STAT5b protein, is also supported by the experimental evidence of reduced growth in male mice with STAT5b gene disruption (26, 27). Furthermore, the importance of the JAK-STAT pathway is also underlined by the evidence of defective GH-induced tyrosine phosphorylation and activation of STAT5 in patients affected with short stature carrying GH-R gene mutations and in patients with Laron syndrome (28, 29, 30). To our knowledge, the two cases reported here are the first patients with ISS in whom an abnormal tyrosine phosphorylation induced by GH-R signaling in PBMC has been documented in the absence of any genetic alteration of the receptor itself or classical features of Laron syndrome.
Overall, as the clinical and biochemical hallmarks of partial GHI have not yet been clearly elucidated, the identification of such patients is still difficult. Our approach was to integrate functional, biochemical, and molecular studies in an effort to identify the most reliable markers of GHI. Our findings indicate that none of the individual functional parameters, including basal and stimulated IGF-I and IGFBP-3 values, led to unequivocal identification of such patients according to previously reported observations (31). It should be noted that four patients (patients 1, 4, 7, and 10) with an ISS clinical phenotype indistinguishable from that of the two patients with the GH-R unresponsiveness had normal or close to normal basal IGF-I values, which further increased in response to GH stimulation. These patients deserve further attention, because they could represent a distinct clinical entity, such as peripheral resistance to IGF-I. The frequently used 4-d IGF-I generation test led to an overestimate of unresponsive patients. Indeed, a sustained GH stimulation induced, in thre of five unresponsive patients, a significant IGF-I increase. In three subjects (patients 5–7) a discrepancy between the increases in IGF-I and IGFBP-3 after GH administration was observed. In all of these cases only an increase in IGF-I was observed, suggesting a possible selective abnormality of the binding protein. However, arguing against this hypothesis, the amount of the binding protein was normal in two of them, and in the third patient it significantly increased after a prolonged GH stimulation. Of note, the two patients who failed to respond to the long-term IGF-I generation test also showed an abnormal pattern of P-Tyr after in vitro GH stimulation of PBMC, thus suggesting that different cell targets, such as hepatocytes and PBMC, share the same transducing elements. As the 21-d IGF-I generation test cannot be proposed as a diagnostic tool on a routine basis, due to the high cost and the uncomfortable nature of the test, our findings also suggest that combining the 4-d IGF-I generation test with analysis of GH-induced P-Tyr on PBMC may help identify ISS patients with partial GHI.
Footnotes
This work was supported by MURST-99-PRIN (Progetto di Rilevante Interesse Nazionale; to C.P.).
Abbreviations: GHI, GH insensitivity; GH-R, GH receptor; IGFBP, IGF-binding protein; ISS, idiopathic short stature; JAK2, Janus kinase 2; PBMC, peripheral blood mononuclear cells; P-Tyr, protein tyrosine phosphorylation; STAT5, signal transducers and activators of transcription 5.
Received August 31, 2000.
Accepted April 20, 2001.
References
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