Response to growth hormone therapy can be considered a continuous quantitative trait that has measurable phenotypic variation resulting from both genetic and nongenetic effects. Response to growth hormone therapy is variable in short children who are not deficient in growth hormone1,2,3. Age, body mass and growth hormone dose have a role in this individual variability, but the genetic factors influencing response to growth hormone ('growth hormone pharmacogenetics') are still unknown. Candidate genes, however, can easily be identified from the molecular network by which target tissues respond to growth hormone. The first step of growth hormone action is the binding of growth hormone to the growth hormone receptor (GHR), followed by the activation of the JAK-STAT pathway and subsequent increase in expression of insulin-like growth factor 1 (IGF1) and other growth hormone–dependent genes. GHR consists of an extracellular domain of 246 amino acids, a single transmembrane domain and a cytoplasmic domain. The human gene GHR contains nine coding exons. Exons 3–7 encode the extracellular domain. There are two isoforms of GHR in humans, generated by retention or exclusion of exon 3 during splicing: a full-length isoform and an isoform that lacks exon 3 (d3-GHR). The generation of two transcripts that differ by the skipping of a coding exon results from homologous recombination, which mimics alternative splicing between the two retroviral sequences that flank the skipped exon4. The allele encoding d3-GHR is therefore specific to humans. The importance of the region encoded by exon 3 is unknown. The region is conserved in GHR proteins in mammalian species but is absent in the prolactin receptor5. This pattern of evolutionary conservation suggests that the loss or retention of exon 3 could affect receptor expression or function, specifically by affecting binding of human growth hormone, receptor processing, transport, stability, binding to other ligands, dimerization of GHR monomers or signal transduction.

The 22 residues encoded by exon 3 could not be modeled from the crystal structure of the complex between growth hormone and the extracellular domain of GHR6, but the peptide is located away from binding interfaces. The intramolecular disulfide bond pattern of the extracellular domain does not seem to be affected by the loss of exon 3, and so the global folding of the extracellular domain is supposed not to be altered. Exclusion of exon 3 results in the loss of one potential glycosylation site and the substitution A6D at the end of exon 2. This latter modification involves a highly conserved amino acid and leads to a change in charge, size and hydrophobicity of the receptor domain.

The binding of growth hormone to the two GHR isoforms has been studied in various experiments. When transiently expressed in COS-7 cells, full-length GHR and d3-GHR have comparable growth hormone–binding properties7. In another study, mRNAs were transcribed from cDNA encoding human full-length GHR and d3-GHR and microinjected into Xenopus laevis oocytes8. d3-GHR was as efficient as full-length GHR in binding and internalizing growth hormone in this study, but both isoforms also bound human placental lactogen and ovine prolactin, calling into question the specificity of the binding assay. In other experiments using soluble binding proteins, monomeric d3-GHR binding protein and full-length GHR binding protein showed no difference in binding to recombinant human growth hormone9. In addition, recent clinical data indicate that a single allele encoding d3-GHR is functional in humans10. To our knowledge, however, there has been no comparative evaluation of the efficacy of the two GHR isoforms in transducing the signals generated by growth hormone binding.

After the dimerization of two transmembrane chains to form functional GHR, homozygous individuals have either full-length GHR or d3-GHR homodimers on their cell surfaces, whereas heterozygotes have full-length GHR homodimers, d3-GHR homodimers and full-length GHR–d3-GHR heterodimers. Because of its prominent function in growth hormone signaling and the frequency of its polymorphic variation, we considered GHR to be a good candidate gene for involvement in the pharmacogenomics of growth hormone therapy.

We studied the effect of the exon 3 polymorphism in GHR on response to growth hormone therapy in a large sample of short children. We selected children born small for gestational age (SGA) and children with idiopathic short stature (ISS), who have normal birth size but grow at a decreased rate1,2,3. During the study, the children were in the phase of linear growth before the onset of puberty; this allowed us to avoid the extensive variations in growth related to individual patterns of sexual maturation. We recruited two independent cohorts of ISS and SGA children with short stature (Table 1) and genotyped them for the GHR polymorphism. The frequency of the d3-GHR variant was comparable in the short children and in 283 control adults of normal height, suggesting that this polymorphism is not primarily related to the genesis of short stature in individuals with ISS and SGA. GHR genotype had no significant effect on growth rates or hormonal parameters before therapy (Table 1). We concluded from this data that potential variations in growth hormone sensitivity due to GHR differences in short children can be compensated by endogenous pituitary growth hormone secretion, which masks the effects of the GHR polymorphism on basal growth rate. Unfortunately, growth hormone stimulation tests used in clinical endocrinology are unreliable reflections of endogenous growth hormone secretion and did not allow us to evaluate this hypothesis properly.

Table 1 Main clinical and endocrine characteristics of children with SGA and IDSS grouped by GHR genotype Full size table

Our primary question was whether response to growth hormone differed across GHR genotypes. We studied children with SGA and ISS during the first two years of growth hormone administration. The distribution of individual growth responses, indicating various degrees of sensitivity to growth hormone, closely fit that of the normal distribution, as expected for a multifactorial trait (Fig. 1). But the distribution of genotypes across the range of growth acceleration values indicated that the growth responses to growth hormone were greater in children bearing at least one allele encoding the d3-GHR isoform. The unadjusted mean growth responses stratified by GHR genotype are shown in Table 2. In the first sample of 76 short children, GHR genotype had a significant effect on response to growth hormone during the two years of growth hormone administration (P < 10−5 and P < 0.001 for the first and second years, respectively). Response to growth hormone was greater in children carrying at least one allele encoding the d3-GHR isoform. To further characterize the effect of the allele encoding the d3-GHR isoform on response to growth hormone, we built a general linear regression model taking into account age, sex and growth hormone dose (Table 3). We found a strong relationship between GHR genotype and growth acceleration (P < 10−6 and P < 0.005 for the first and second years of growth hormone administration, respectively; Table 2). The regression model also showed the expected relationship between growth hormone dose and growth response, which was attenuated during the second year of growth hormone administration because many subjects were switched from a fixed 'per protocol' growth hormone dose (in the first year) to a personalized dose regimen (in the second year).

Figure 1: Frequency histogram showing the distribution of individual growth rate increments (Δgr) during the first year of growth hormone therapy. The distribution of values was normal and had a large variance. The two cohorts of short children treated with growth hormone were pooled for analysis. Increments in growth rates were greater in children carrying alleles encoding the d3-GHR isoform. fl, allele encoding the full-length GHR isoform; d3, allele encoding the d3-GHR isoform. Full size image

Table 2 Response to growth hormone administration in children with SGA and IDSS grouped by GHR genotype Full size table

Table 3 General linear model for regression of GHR genotype, growth hormone dose and test factors on growth rate increment Full size table

We replicated these findings in a second, independent sample of 96 short children (Table 1). Response to growth hormone in this sample differed across GHR genotypes (Table 1), comparable to that in the first sample (Table 2). As expected, we observed no differences in the effects of GHR genotype between children with SGA and children with ISS.

To verify that the observed effect could be reliably attributed to GHR, we examined nine polymorphisms of high heterozygosity in genes unlinked to GHR. None showed evidence of association with treatment response or GHR genotypes in our cohorts. Because we carefully matched the children with SGA and ISS with respect to ethnic background, the observed association is probably not due to the effects of population stratification.

We then investigated in vitro whether the deletion of exon 3 affects the functional properties of GHR. We transiently cotransfected 293 HEK fibroblasts with vectors expressing full-length GHR, d3-GHR or both using the LHRE-luciferase reporter plasmid, which is activated by full-length GHR2,11. When cells were exposed to various growth hormone concentrations, d3-GHR induced a higher transcriptional activity of the reporter construct than full-length GHR (Fig. 2). The results were linear and consistent at all levels of growth hormone stimulation between 0 and 50 ng ml−1, a range of low growth hormone concentrations supporting the in vivo relevance of our findings. 293 HEK fibroblasts expressing d3-GHR had a greater response to growth hormone stimulation in all our experiments (Fig. 2). To our knowledge, this is the first report comparing the bioactivity of full-length GHR and d3-GHR in vitro. The molecular mechanisms underlying the higher bioactivity of d3-GHR are not yet elucidated, but they do not seem to involve more binding7,8,9 or less internalization8. In vivo, full-length GHR and d3-GHR isoforms might be discriminated at the level of expression in lymphocytes12. This is not the case in our assay, as both receptors are under the control of the constitutively active CMV promoter.

Figure 2: In vitro bioactivity of full-length GHR and d3-GHR. HEK 293 cells transiently expressing full-length GHR, d3-GHR or both were stimulated by increasing concentrations of growth hormone ([GH]) for 8 h. Relative induction of LHRE-luciferase reporter gene is expressed relative to unstimulated cells (value of 1, horizontal line). The number of experiments done for each condition is indicated in parentheses. *P < 0.005, **P < 0.0005 and ***P < 0.0001 for comparison of cells transfected with two alleles encoding the full-length GHR isoform versus cells transfected with at least one allele encoding the d3-GHR isoform. We pooled cells carrying one and two alleles encoding the d3-GHR isoform because their individual effects were each consistently higher than those of cells carrying two alleles encoding the full-length GHR isoform (P = 0.006–0.0005 and P = 0.004–0.0001, respectively). fl, allele encoding the full-length GHR isoform; d3, allele encoding the d3-GHR isoform. Full size image

Recent studies showed that growth hormone receptors exist at the cell membrane as inactive, preformed dimers13 whose activation is triggered by conformational changes induced by growth hormone binding, possibly involving the rotation of receptor chains (M. J. Waters et al., unpublished data). The first 32 residues (including residues 7–28 encoded by exon 3) could not be modeled in any of the available crystal structures involving GHR6,14, suggesting that the N-terminal loop has a high degree of flexibility. It is not known how the deletion of the region encoded by exon 3 could increase receptor activity, but removal of this flexible loop may propagate subtle conformational changes along the extracellular domain that facilitate hormone-triggered activation of the receptor.

In 2001 and in 2003, the US Food and Drug Administration and the European regulatory agency, respectively, allowed the treatment of short children born SGA with recombinant growth hormone. In July 2003, growth hormone was also approved by the US Food and Drug Administration for the treatment of growth failure in children with ISS. Children whose height is 2.25–3 s.d. below the mean for their ages are eligible for growth hormone prescription; this includes hundreds of thousands of children in the US and Europe. For those short children, pharmacogenomic testing could be used in the future to avoid the use of ineffective regimens of growth hormone, to improve the personalized design of therapy and to decrease its cost:benefit ratio. Our study on GHR polymorphism is a preliminary step in this direction. If it holds true, our observation will have more useful consequences for the therapy of short children than for direct diagnostic purposes, because although it is involved in the multifactorial network of growth genetics, the exon 3 variation in GHR is not expected to be associated with physiologic variations in human growth or to be a primary cause of short stature in humans. This is because individual differences in growth hormone sensitivity due to GHR structure and function are probably easily compensated for by pituitary growth hormone secretion in a secretion-sensitivity balance that allows optimal growth. When this compensation is impossible, however, as in growth hormone–deficient children, or limited by its own genetic variation, homozygosity with respect to the allele encoding full-length GHR could aggravate the growth deficit associated with low growth hormone secretion.

The main finding of our study is that, in response to a given dose of exogenous growth hormone, and regardless of the cause of shortness, individual differences in GHR genotype have medically relevant effects on growth rate. Prospective dose-response studies in all categories of children treated with growth hormone will determine the optimal growth hormone dosage for their GHR genotype. This may contribute to the progressive switch in growth hormone prescription from a 'fixed dosage' mode of therapy, which prevails now, to a more personalized adjustment of dose.