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The Growth Response to Growth Hormone (GH) Treatment in Children with Isolated GH Deficiency Is Independent of the Presence of the Exon 3-Minus Isoform of the GH Receptor

Eli Lilly & Co. (W.F.B.), D-61350 Bad Homburg, Germany; University Hospital for Children and Adolescents (W.F.B., A.K., H.S., R.W.P.), D-04317 Leipzig, Germany; Institut National de la Santé et de la Recherche Médicale Unit 654 (K.M., S.A.), 94010 Créteil, France; and Pharma Support Inc. (E.P.S.), 191119 St. Petersburg, Russia

Address all correspondence and requests for reprints to: Werner F. Blum, Eli Lilly & Co., Saalburgstrasse 153, D-61350 Bad Homburg, Germany. E-mail: blum_werner{at}lilly.com.

	Abstract

Top Abstract Introduction Patients and Methods Results Discussion References

 
Context: A variant of the human GH receptor (GHR) lacks a 22-amino-acid sequence derived from exon 3 (d3-GHR). It was reported that pediatric patients, born small for gestational age or with idiopathic short stature who were homozygous or heterozygous for this variant responded better to GH treatment than those homozygous for the full-length allele (fl-GHR).

Objective: The objective was to study the impact of the GHR genotype on the phenotype and growth response in patients with isolated GH deficiency (IGHD) treated with GH.

Design: This was a retrospective, multinational, multicenter observational study.

Patients: Patients with IGHD (n = 107) were recruited.

Interventions: All patients received GH treatment at replacement doses. The GHR genotype (fl-GHR/fl-GHR, fl-GHR/d3-GHR, or d3-GHR/d3-GHR) was determined by PCR amplification.

Main Outcome Measures: Measures included height SD score, height velocity, height velocity SD score at baseline and 1 yr of GH treatment, and their changes.

Results: There was no statistically significant difference of the main outcome measures between patients with the d3-GHR allele (n = 48) and patients who were homozygous for the fl-GHR allele (n = 59). Moreover, the genotype group did not contribute significantly to the growth prediction in multiple linear regression models.

Conclusions: Our results indicate that the d3-GHR allele does not affect response to GH treatment or contribute to growth predictions in patients with IGHD who received replacement doses of GH aiming to restore a normal GH status. We did not confirm the previously reported data obtained in patients small for gestational age or with idiopathic short stature who received supraphysiological GH doses.

	Introduction

Top Abstract Introduction Patients and Methods Results Discussion References

 
THE HUMAN GH receptor (GHR) gene is a single copy gene that spans 90 kb of the 5p13-p12 chromosomal region (1, 2). It contains nine coding exons (exons 2–10) and several untranslated exons: exon 2 codes for the signal peptide, exons 3–7 encode the extracellular domain, exon 8 encodes the transmembrane domain, and exons 9 and 10 encode the cytoplasmic domain (1). One of several variants of the GHR lacks a 22-amino-acid sequence derived from exon 3. It was believed for many years that the full-length GHR (fl-GHR) and the exon 3-deleted GHR transcripts (d3-GHR) derive from the same primary GHR transcripts through alternative splicing. However, a more recent study demonstrated that skipping of exon 3 results from an ancestral recombination event that occurred at the DNA level during primate evolution between two homologous retroviral sequences that flank this exon (3). This mechanism, which mimics alternative splicing, is unique to humans, but its importance for the function of the GHR is unknown.

The response of short children to GH treatment is highly variable. Therefore, efforts have been made to identify indicators of good or poor response, and mathematical models have been developed to predict response (4, 5). Indicators for a good response to GH treatment include young age, low baseline height, low baseline IGF-I, and high GH dose. Genetic factors influencing the GH response remain poorly defined; however, Dos Santos et al. (6) recently reported that pediatric patients, born small for gestational age (SGA) or with idiopathic short stature (ISS) who were homozygous or heterozygous for the d3-GHR variant responded better to GH treatment than those who were homozygous for the fl-GHR allele. Moreover, these authors demonstrated that HEK fibroblasts transfected with a plasmid encoding the d3-GHR isoform showed a significantly greater dose-dependent response to GH than those expressing the fl-GHR isoform only.

We set out to study the impact of the genotype comprising at least one d3-GHR allele, compared with the homozygous full-length genotype (fl-GHR/fl-GHR), on the phenotype and response to recombinant GH treatment in pediatric patients with isolated GH deficiency (IGHD). Furthermore, we asked whether the presence or absence of the d3-GHR allele significantly adds to the accuracy of growth prediction models in these patients.

	Patients and Methods

Top Abstract Introduction Patients and Methods Results Discussion References

 
We studied 107 pediatric patients with IGHD, naïve to GH treatment at baseline, who underwent analysis of genomic DNA to determine the presence or absence of the d3-GHR genotype. The patients were recruited from the Genetics and Neuroendocrinology of Short Stature International Study, the Eli Lilly & Co. postmarketing research program. The diagnosis of IGHD was based on a GH peak value of less than 10 µg/liter in at least two GH stimulation tests, as well as the absence of any additional pituitary hormone deficiencies. In addition, investigators were requested to apply a stringent score system to focus on patients with severe GH deficiency. The score included the following items (score points): height SD score (SDS) less than –3 (2), height velocity less than 25th percentile (1), neonatal signs of hypopituitarism (1), GH peak response to GHRH less than 10 µg/liter (1), IGF-I SDS less than –3 or IGF binding protein (IGFBP)-3 SDS less than –1.6 (1), morphologic anomalies of the hypothalamic-pituitary region (1), and GH deficiency in first-degree relatives (2). Patients were eligible if they achieved five of nine score points (7). All patients received recombinant human GH treatment at a replacement dose. Growth parameters were studied at baseline and after 1 yr of GH treatment. Patients included in this study were from the following countries: Australia (n = 3), Belgium (n = 2), Czech Republic (n = 3), Germany (n = 46), India (n = 2), Italy (n = 18), Lithuania (n = 1), Russia (n = 14), Singapore (n = 2), Spain (n = 3), Taiwan (n = 8), Thailand (n = 3), and the United States (n = 2). To exclude a possible impact of ethnic differences, the described analyses were also performed excluding patients from India, Singapore, Taiwan, and Thailand (n = 15). Because similar results were obtained in both cases, only those from the entire group are presented. Legal guardians of all patients gave written informed consent, and the performance of DNA analyses was approved by local ethics review committees.

To test for the presence of the fl-GHR/fl-GHR, fl-GHR/d3-GHR, or d3-GHR/d3-GHR genotype, we performed a multiplex PCR amplification as described previously (3). Briefly, this assay, which is based on the use of three primers (two primers bracketing exon 3 and the third located within this exon), allows for discrimination between fl-GHR and d3-GHR alleles that are amplified as two products of different size (935 and 532 bp, respectively).

The SDS for an individual patient for some parameters was calculated by reference to healthy populations as: (patient value – mean value according to age and sex)/SD according to age and sex.

Bone age SDS was calculated using the tables of Greulich and Pyle (8). Height SDS was calculated using the growth charts of Kuczmarski et al. (9). Height velocity SDS was calculated using the results of Preece (10). The majority of IGF-I and IGFBP-3 concentrations were measured in a central laboratory as described by Blum and Breier (11). Some values, which were determined locally, were converted to central laboratory values after cross-calibration of the assays used. SDS values were calculated using the method and reference values described by Blum and Schweizer (12). P values for categorical variables were calculated by Fisher’s exact test and continuous variables by two-sided t test. Statistical significance of differences was assumed if P < 0.05. Multiple linear regression analysis was performed with growth response parameters as the dependent variable and various combinations of potentially influencing parameters available at baseline, including the GHR genotype group in all models.

	Results

Top Abstract Introduction Patients and Methods Results Discussion References

 
Fifty-nine of 107 patients (55%) were homozygous for the fl-GHR allele, three (3%) were homozygous for the d3-GHR allele, and 45 (42%) were heterozygous; these percentages comply with the Hardy-Weinberg equilibrium. Because the previous study (6) showed no difference between patients who were homozygous or heterozygous for the d3-GHR allele, patients with the d3/d3 or d3/fl genotype were combined for further analyses and are designated d3/fl(d3).

Table 1 presents demographic information and baseline characteristics of the study population according to GHR genotype. As expected on the basis of the higher frequency of short stature referrals for male children, there were significantly more males, especially in the fl/fl group. Most patients were prepubertal (93%); a few patients were at Tanner stage 2 or 3. There were no statistically significant differences in baseline characteristics between the two genotype groups, with the exceptions of sex and serum IGF-I concentration, which were lower in the fl/fl group.

View larger version (18K): [in this window] [in a new window]   FIG. 1. Height SDS (A), height velocity (B), and IGF-I SDS (C) in pediatric patients with IGHD. Height SDS, height velocity at baseline and 1 yr of GH treatment, and first-year change in height SDS are based on all 107 patients; change in height velocity is based on 64 patients in whom pretreatment height velocities were available. Mean and SE are shown by genotype group for baseline, after 1 yr of GH treatment, and for change from baseline to 1 yr.

	Discussion

Top Abstract Introduction Patients and Methods Results Discussion References

 
The conclusions from these findings are 3-fold: first, the presence or absence of the d3-GHR allele has no impact on the baseline phenotype in patients with IGHD, with the possible exception of a lower mean serum IGF-I concentration in the fl/fl group. In patients with GHD, an inverse association between baseline IGF-I levels and growth response to GH treatment has been reported (5) and has also been observed in this study applying multiple regression models. Therefore, one may speculate that the fl/fl group responded better to GH treatment due to the lower IGF-I levels, which may have attenuated a possible difference in GH responsiveness between groups. However, the positive effect of low serum IGF-I at baseline on the growth response is small (5). Moreover, when serum IGF-I was included in multiple regression models to adjust for the imbalance between the genotype groups, the variable genotype group was always far from reaching statistical significance regarding prediction of the growth response. Therefore, the statistically significant difference in IGF-I between the two groups should be interpreted with caution because no adjustment for multiple testing was made, and a statistical type 1 error cannot be excluded. In addition, this finding should be interpreted in the context of the lack of difference in IGFBP-3 values between groups. Further studies are needed to evaluate the effect of GHR genotype on IGF-I secretion. Second, the presence or absence of the d3-GHR allele has no impact on the response to GH treatment in patients with IGHD using replacement doses. Third, the presence or absence of the d3-GHR allele does not significantly contribute to growth prediction in multiple regression models in GH-treated patients with IGHD. This finding is notable because the impact of potential imbalances between the two genotype groups has been adjusted for by the multiple regression approach.

When comparing the findings of this study to the previous reference study (6), the following caveats need to be considered: at first glance, our study does not confirm the findings of the study by Dos Santos et al. (6) regarding the influence of the d3-GHR allele on the growth response to GH treatment. However, Dos Santos et al. studied patients with SGA or ISS, with normal GH secretion, and therefore treated with supraphysiological doses of GH, whereas our patients had IGHD and received low replacement doses of GH. It is therefore tempting to speculate that the impact of the presence or absence of the d3-GHR allele on linear growth would be detectable with only supraphysiological doses of GH. A partial answer to this open question has been provided by studies published while our manuscript was under review. Binder et al. (13) showed an increased responsiveness to high doses of GH in patients with SGA or Turner syndrome carrying a d3-GHR allele. By contrast and in keeping with our data, Pilotta et al. (14), who investigated GH-deficient patients (all but one had IGHD) receiving low replacement doses of GH, did not find such an association of GH responsiveness with the GHR genotype. A similar observation was made in a Japanese population of prepubertal patients with partial IGHD who were also treated with low doses of GH (15). Taken together, these studies suggest that the possible impact of the GHR genotype on GH responsiveness is a dose-dependent phenomenon. However, one group who investigated a population of patients with profound GHD, the majority displaying combined pituitary hormone deficiency, observed that patients with at least one d3-GHR allele had a better response to low doses of GH than those homozygous for the fl-GHR allele (16). Although the reason for this discrepancy is so far unknown, a reconciling hypothesis is that the severity of GH deficiency may represent another factor that interferes with responsiveness to GH.

In summary, our study and two other studies in patients with IGHD who received replacement doses of GH did not show a modulating effect of the presence or absence of the d3-GHR allele on GH responsiveness. In contrast, such an impact has been reported in a mixed population of patients with isolated GHD or combined pituitary hormone deficiency. In two studies on patients with SGA, ISS, or Turner syndrome, who received supraphysiological GH doses, a better growth response was observed if at least one d3-GHR allele was present. Considering these studies together, it appears that the impact of the d3-GHR allele on GH responsiveness is controversial and may become detectable primarily with supraphysiological doses of GH.

	Acknowledgments

 
We thank the patients, their families, and the attending physicians for participating in this study.

	Footnotes

 
This work was supported by Eli Lilly & Co.

First Published Online July 25, 2006

Abbreviations: d3-GHR, GHR missing the amino-acid sequences derived from exon 3; fl-GHR, full-length allele of GHR; GHR, GH receptor; IGFBP, IGF binding protein; IGHD, isolated GH deficiency; ISS, idiopathic short stature; SDS, SD score; SGA, small for gestational age.

Received January 11, 2006.

Accepted July 7, 2006.

	References

Top Abstract Introduction Patients and Methods Results Discussion References

 

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