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Section of Pediatric Endocrinology and Growth Research Center, University-Children’s Hospital, 72076 Tübingen, Germany
Address all correspondence and requests for reprints to: PD Dr. Gerhard Binder, Pediatric Endocrinology Section, University-Children’s Hospital, Hoppe-Seyler-Strasse 1, 72076 Tübingen, Germany. E-mail: gerhard.binder{at}med.uni-tuebingen.de.
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Abstract |
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Objective: This study tests the novel association in two distinct groups of rhGH-treated patients, short girls with Turner syndrome and short children born small for gestational age (SGA).
Design: The retrospective study included all children who were treated with rhGH during the last 18 yr at our hospital.
Patients: Patients with Turner syndrome were defined by the specific karyotype (n = 53), short children born SGA were determined by birth length and/or weight less than –2.0 SD score and a height at start of rhGH therapy less than –2.0 SD score (n = 60). Exclusion criteria were puberty, an age less than 3.5 or more than 14 yr, and GH deficiency.
Materials and Methods: Growth prediction for the first year of therapy was calculated on the basis of rhGH dose, age, weight, height, and gender-adjusted midparental height according to the prediction models by Ranke et al. The GHR-exon 3 locus was genotyped using a PCR multiplex assay. GH, IGF-I, and IGF binding protein 3 (IGFBP-3) were measured by RIA.
Intervention: For growth promotion, a mean rhGH dose of 38 µg/kg·d (SD, ±8) was administered in Turner syndrome patients and 56 µg/kg·d (SD, ±11) in short children born SGA.
Results: No significant difference in height, spontaneous height velocity, IGF-I, and IGFBP-3 levels was found at the start of rhGH therapy in the three GHR genotype groups studied. At the first year of treatment, girls with Turner syndrome carrying one or two d3-GHR alleles showed a significantly higher increment in height velocity (P = 0.019) and exceeded their growth prediction significantly (P = 0.007), whereas their increments of IGF-I and IGFBP-3, weight, and height were not significantly different. Carriers of d3-GHR in the group of short children born SGA grew significantly faster than predicted (P = 0.023). However, in comparison to the carriers of full-length GHR, gain of height velocity was not significantly higher (P = 0.067). The mean gain of height associated with d3-GHR accounted for approximately 0.75 cm in SGA and 1.5 cm in Turner syndrome during the first year of rhGH therapy.
Conclusions: Our data support the theory that there is increased responsiveness to high-dose rhGH in association with the d3-GHR genotype. The magnitude of this effect may depend on the primary origin of the short stature.
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The GH receptor (GHR) is the gateway to the GH signaling cascade. This receptor contains a very unusual genetic polymorphism caused by a genomic deletion of exon 3 (d3-GHR), which mimics alternative splicing, with high frequency (5). This protein polymorphism results in the loss of amino acid (aa) residues 7–28 and the aa substitution A6D at the N-terminal part of the extracellular receptor domain (5), which is conserved in mammalian species (6). The prevalence of the d3-GHR allele in humans is around 25–32% with a homozygosity frequency of 9–14% (5, 7). The aa residues 6–28 of the GHR could not be modeled by any crystallography so far and do not participate in GH binding directly (7). Consequently, binding affinity to GH was not affected by this receptor protein polymorphism in vitro (8, 9). It has been speculated that this supposedly very flexible region may play a role in the conformational changes during transactivation of the GHR dimer by GH (7). However, other effects on gene transcription, RNA splicing, protein stability, and glycosylation as well as transport to the cell membrane are possible as well. The presence of a single copy of the d3-GHR seems to be sufficient for normal human growth (10).
Dos Santos et al. (7) have recently reported an association of the d3-GHR genotype with increased responsiveness to high-dose rhGH therapy in short children without GH deficiency. The children genotyped consisted of short children born small for gestational age (SGA) and those with idiopathic short stature. The responsiveness to GH was measured in increments in height velocity during the first and second year of therapy. Children having one or two d3-GHR alleles showed a 75% higher increment in height velocity in comparison to those with full-length GHR receptors (7). The same research group performed in vitro studies using human embryonic kidney fibroblasts cotransfected with a lactogenic response element containing luciferase as reporter (11) and full-length GHR as well as d3-GHR. The observed luciferase activity stimulated by GH suggested a reduced in vitro bioactivity of the full-length GHR in comparison to the d3-GHR (7).
Because these pharmacogenetic findings on the GH axis have the potential to promote a basis for a rational individualization of rhGH therapy (12), we were interested to test these novel mechanisms in two distinctly defined groups of patients treated with a high-dose GH: a group consisting solely of short children born SGA and a group of short girls with Turner syndrome. Our data confirm the association of the d3-GHR genotype with increased responsiveness to high-dose rhGH therapy.
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Subjects and Methods |
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The clinical characteristics of Turner girls at the start of therapy are summarized in the first column of Table 1
. SGA short stature was defined by birth length and/or weight of two or more SD below the mean according to Niklasson et al. (13) and a height at start of therapy below –2.0 SD score (SDS) according to Prader et al. (14). Mean birth length was –2.96 SDS (±1.27) and mean birth weight was –2.91 SDS (±1.25). The first column of Table 2 shows the clinical characteristics of the group of SGA children treated.
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The clinical data were collected from the patients’ records by F.B., who was blinded to the results of the genetic analysis. The spontaneous growth immediately before therapy was documented for at least 9 months in each child. The parents’ heights were measured in all but three patients, who were adopted. Height, weight, and gender-adjusted midparental height (MPH) were transformed in SDS according to Prader et al. (14); height velocities are given in SDS according to Tanner et al. (15, 16). For weight, the reference values reported by Freeman et al. (17) were used. Height gain (
height = height at start minus height after 12 months of therapy), increment in height velocity ( height velocity = height velocity at start minus height velocity during the first 12 months of therapy), and weight gain ( weight) were calculated. rhGH therapy was performed using a mean dose of 38 µg/kg·d (±8) in Turner syndrome and of 56 µg/kg·d (±11) in short children born SGA. There was no significant difference in the GH dose between subgroups according to the genotype (Tables 3 and 4).
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Growth prediction
For each child, a growth prediction for the first year of therapy was calculated according to Ranke et al. (2, 3). Their prediction model incorporates rhGH dose, age, and weight at start of therapy [given in SDS according to Freeman et al. (17)] as well as gender-adjusted MPH (in SGA) or the distance to MPH [given in height SDS according to Tanner et al. (15, 16)] as the main prognostic factors.
The equation for prediction in Turner syndrome is: centimeters of growth during the first year = 8.1 + [2.2 x rhGH dose (ln; IU/kg·wk)] + [–0.3 x age at onset (yr)] + [0.4 x body weight SDS] + [–0.2 x (height SDS – MPH SDS)] + [0.4 x number of injections per week] + [1.6 x (oxandrolone = 1; no oxandrolone = 0)].
The equation for prediction in short SGA children is: centimeters of growth during the first year = 9.4 + [56.51 x rhGH dose (mg/kg·d)] + [–0.31 x age at onset (yr)] + [0.30 x body weight SDS at start] + [0.11 x MPH SDS].
The individual deviations from the prediction were divided by the SE of the prediction, which is 1.26 cm in Turner syndrome and 1.30 cm in SGA children (2, 3), resulting in the Studentized residuals. A Studentized residual of 0 means that the prediction was completely fulfilled; positive values indicate growth exceeding the prediction, negative values are the expression of growth slower than predicted.
Genotyping
Genomic DNA was extracted from blood lymphocytes using NucleoSpin Blood XL (Macherey-Nagel, Düren, Germany). For genotyping of the GHR-exon 3 locus, the PCR multiplex assay invented by Pantel et al. (5) was used. Products were analyzed on a 1% agarose gel stained with ethidium bromide. Sixty-two control individuals were genotyped as well.
Hormone measurements
hGH levels in serum were measured by a polyclonal in-house RIA and were calibrated against the World Health Organization International Reference Preparation 88/624 (1 mg = 3 IU). The lower detection limit was 0.1 µg/liter. The mean intraassay coefficient of variation was 6.9%, the mean interassay coefficient was 9.5% (18). Serum levels of IGF-I and IGF binding protein 3 (IGFBP-3) were both measured by RIA as described by Blum et al. (19, 20). The mean intra and interassay coefficients of the IGF-I assay were less than 10%. For the IGFBP-3 assay, the intraassay coefficient of variation was 4.1%, the interassay coefficient of variation was 9.7%. The age and gender-dependent IGF-I and IGFBP-3 serum levels were expressed in SDS. The of the serum levels after 12 months of therapy and those at start were calculated.
Statistical analysis
Statistical analysis was performed using the two-tailed Student’s t test for the continuous characteristics. P values <0.05 were considered to indicate significance.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The multiplex assay of the GHR exon 3 locus yielded the expected fragments of 935 bp (indicating the presence of full-length GHR) and 532 bp (d3-GHR), which accurately discriminated between the three possible genotypes fl/fl, d3/fl, and d3/d3. The distribution of the genotypes in the 175 children tested was not significantly different between controls, short children born SGA, and Turner syndrome girls (Table 5). The calculated allele frequencies for full-length GHR and d3-GHR were 66 and 34%, respectively.
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Tables 1
and 2 show the means and SDs of the growth parameters at start of rhGH therapy. There was no significant difference in height and spontaneous height velocity between the three different genotype groups, within Turner girls, and short children born SGA. The same was true for the IGF-I and IGFBP-3 levels. In the Turner girls, the gender-adjusted MPH of the d3/d3 group (mean, –0.16) was significantly lower than of the fl/fl group (mean, +0.72) (P = 0.037).
GHR exon 3 genotypes and response to rhGH in Turner syndrome
Table 3 and Figs. 1 and 2 show the clinical and biochemical changes under rhGH therapy in girls with Turner syndrome. The increment in height velocity was significantly higher in the d3/d3 group in comparison to the other two groups tested (P = 0.0031; d3/d3 vs. fl/fl; P = 0.048; d3/d3 vs. d3/fl). This difference in height gain was also significant when d3/d3 and d3/fl girls with Turner syndrome were pooled and compared with the fl/fl group (P = 0.019) (Fig. 1).
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). Consequently, the majority of the girls with Turner syndrome carrying the fl/fl genotype did not meet their prediction (Fig. 2). The higher mean MPH of this group in comparison to the d3/fl group and the d3/d3 group contributed to this fact. The increments of IGF-I and IGFBP-3 levels as well as the weight and height gain during therapy expressed in SDS were not significantly different between the three genotype groups.
GHR exon 3 genotypes and response to rhGH in short SGA children
The same parameters of the SGA children grouped into the three different genotypes of the GHR are shown in Table 4 and Figs. 1 and 2. The effects associated with the d3-GHR allele that were observed were milder in this group of patients; mean height velocity was modestly higher in the d3/fl and d3/d3 genotyped groups, but this difference did not reach statistical significance (P = 0.067) (Fig. 1). The combined group of d3/d3 and d3/fl children showed significantly higher Studentized residuals (+0.27) than the fl/fl children (–0.36) (P = 0.023), the same was true for the d3/fl children in comparison to the fl/fl children (P = 0.047) (Fig. 2). No level of significance was reached for the small group of d3/d3 children (n = 8) in comparison to the fl/fl children. As in Turner syndrome, the increase of IGF-I and IGFBP-3 levels in serum or changes in weight and height during therapy were not significantly different between the three genotype groups.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The girls with Turner syndrome are a specific female subgroup of short children with a basically skeletal short stature in combination with hypogonadism (23). The girls with Turner syndrome of our study who were homozygote for the d3-GHR variant showed the highest increment in height velocity and exceeded their growth prediction by far. This prediction was based on rhGH dose, age, height, weight, and gender-adjusted MPH (2), factors that showed no correlation to the GHR genotype studied. Accordingly, the majority of Turner girls with the full-length GHR failed to reach their height prediction and the heterozygotes (d3/fl) showed intermediate responsiveness to GH. These data collected in a group of 53 Turner girls may suggest that the responsiveness to GH is correlated to the gene dosage of the d3-GHR allele resembling the in vitro findings in human embryonic kidney fibroblasts where transactivation of the lactogenic response element was correlated to the amount of d3-GHR transfected (7). The overall effect associated with d3-GHR observed seems to account for 1.5 cm of height gain during the first year of rhGH therapy.
The second group tested, the short children born SGA, demonstrated only a mildly increased response to high-dose rhGH in the presence of the d3-GHR variant when estimated by the prediction model. The Studentized residuals indicated a significantly higher responsiveness in carriers of one or two d3-GHR alleles, but the difference was smaller, around half of that observed in Turner syndrome. Consequently, the mean height velocity increment of the carriers of d3-GHR was not found to be significantly higher than in children of the fl/fl group.
In contrast to girls with Turner syndrome, short children born SGA are a quite heterogeneous group regarding the origin of short stature (24, 25). Height gain during high-dose rhGH therapy associated with the presence of the d3-GHR allele may depend on the specific mechanisms that are involved in short stature. This gain in height may be small or absent in primary IGF-I deficiency, but substantial in IGF-I insensitivity like in defects at the epiphyseal growth plate. Interestingly, the significant differences of height increment associated with the GHR genotypes were not reflected by different increases in IGF-I levels in this study. Therefore, one may speculate that IGF-I-independent effects of high-dose rhGH at the epiphyseal growth plate could also play a role (26). Alternatively, IGF-I serum levels may not display the total IGF-I mediated effect at the epiphyseal growth plate, as autocrine and paracrine effects of locally secreted IGF-I—which are also influenced by GH therapy—may not be reflected by the circulating IGF-I concentration (26).
The short children genotyped at the GHR locus by Dos Santos et al. (7) were treated with a similar rhGH dose (in cohort 1) as our SGA group, but were, in the mean, 6 months younger and almost 1 SD taller. Most importantly, only 37% of these short children were born SGA, the majority having idiopathic short stature. Their observed increment in height velocity of 3.64 cm/yr during the first year reported for the fl/fl children of the mixed cohort 1 is almost identical to our findings for the total group independent of the GHR genotype (3.84 cm/yr). In contrast, increments in height velocity of 6.30 cm/yr or higher that were reported as mean increments in the d3/fl and d3/d3 groups by Dos Santos et al. (7) could not be confirmed by us. The difference in height velocity found here is likely to account for less than 1 cm in the first year of therapy, which is significantly less than 2 or 3 cm/yr as suggested previously (7).
It has been shown that association studies like this can be biased by the accidental collection of a subgroup of individuals that ultimately causes spurious results, the so called population stratification (27). We cannot completely exclude effects of population stratification, which may mask effects of the d3-GHR variant in our relatively small group studied. However, our group was quite homogeneous, which substantially decrease the chance that a relevant population substructure might have been present. The mechanism at the cellular level that contributes to the higher GH sensitivity associated with the d3-GHR allele may involve altered transactivation, production, stability, glycosylation, or transport of GHR. Uncovering this mechanism will promote our understanding of the GH-IGF-axis. In addition, prospective studies are needed for corroboration of the retrospective data presented in this study.
This study indicates that genotyping of the d3-GHR protein polymorphism may provide a tool for a more precise understanding of rhGH effects on growth and for the individualization of rhGH dosing in growth promoting therapy when GH deficiency is absent.
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Acknowledgments |
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Footnotes |
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First Published Online November 15, 2005
Abbreviations: aa, Amino acid; GHR, GH receptor; IGFBP-3, IGF binding protein 3; MPH, midparental height; rhGH, recombinant human GH; SDS, SD score; SGA, small for gestational age.
Received July 15, 2005.
Accepted November 9, 2005.
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