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Relationship of the Human Growth Hormone Receptor Exon 3 Genotype with Final Adult Height and Bone Mineral Density

Endocrine Research Laboratory (G.K., Z.S., C.G.G.), McGill University Health Centre-Montreal Children’s Hospital Research Institute, Montreal, Quebec, Canada H3Z 2Z3; Departments of Laboratory Medicine and Pathobiology and Pediatrics (D.E.C.C.), University of Toronto, Toronto, Ontario, Canada M5G 1L5; and Division of Experimental Medicine (G.K., C.G.G.), and Department of Pediatrics (C.G.G.), McGill University, Montreal, Quebec, Canada H3Z 2Z3

Address all correspondence and requests for reprints to: Dr. Cynthia Gates Goodyer, Endocrine Research Laboratory, Room 415/1, McGill University Health Centre-Montreal Children’s Hospital Research Institute, 4060 Ste Catherine Street West, Montreal, Quebec, Canada H3Z 2Z3. E-mail: cindy.goodyer{at}muhc.mcgill.ca.

	Abstract

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Context: Three recent clinical studies have reported that two of the most common isoforms of the human GH (hGH) receptor (hGHR), exon 3 full-length (3+) and exon 3 deleted (3–), may have differential effects on the growth response of children receiving hGH therapy, whereas others refute this. However, none of the investigations has explored the relationship between these hGHR isoforms and final adult height (FAH) or measures of bone mineral density (BMD) within a healthy adult population.

Objective: The aim of this study was to investigate the possible influences of hGHR exon 3 isoforms on FAH and BMD measures of a normal population.

Design: The study was designed to correlate the hGHR exon 3 genotype of a cohort of healthy adults with FAH, BMDs [spine (L2–L4) and hip (femoral neck)], and quantitative ultrasound (QUS) of the heel.

Patients: Participants were 368 unrelated healthy adult white women, aged 18–35 yr.

Main Outcome Measures: We analyzed association of hGHR exon 3 genotypes with FAH, BMD, and QUS. Heights were measured using a stadiometer, BMDs using dual-energy x-ray absorptiometry, and QUS by standard technique. Detailed medical histories, including lifestyle factors, were obtained using a standardized interview.

Results: The distribution of hGHR genotypes in the 368 samples was 53.3% for 3+/3+, 35.6% for 3+/3–, and 11.1% for 3–/3–. There was no correlation between the hGHR exon 3 genotypes and FAH, BMD, or QUS in this cohort.

Conclusion: The hGHR 3+ and 3– isoforms appear not to have differential effects on two major growth outcomes of hGH action, FAH, and BMD in a population of healthy adult women.

	Introduction

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HUMAN GH PLAYS a major role in regulating growth and metabolic activity during childhood and adolescence (1). The biological effects of this hormone are initiated after binding to a dimer of its cell surface receptor [human GH (hGH) receptor (hGHR)], a 620-amino-acid single-transmembrane protein (2, 3).

Pantel et al. (4) have shown that, because of a retroviral-mediated mechanism at the genomic level, both exon 3+ and exon 3– hGHR isoforms exist in the normal population. The lack of exon 3 results in a 22-amino-acid truncation of the extracellular domain, N terminal to the hGH binding domain, and loss of one glycosylation site (4). There is a continuing controversy as to whether the exon 3– isoform modifies hGH effects on human growth or metabolism. It appears not to alter binding of hGH (5, 6), but the crystal structure of this region has not been modeled to date.

Recent clinical studies characterizing the relationship between the hGHR exon 3 genotype and the growth response in patients with short stature undergoing hGH therapy are summarized in Table 1. Three groups have reported significant differences in height velocity between those with the 3+/3+ genotype vs. those with at least one 3– allele, including patients who have been diagnosed as small for gestational age (SGA), hGH-deficient, idiopathic short stature (ISS), or Turner syndrome (7, 8, 9). Dos Santos et al. (7) also reported a small significant increase in the ability of HEK293 cells expressing 3+/3– or 3–/3– hGHR to respond to hGH with stimulation of a signal transducer and activator of transcription 5 (STAT5) reporter vector compared with 3+/3+-expressing cells. In contrast, other groups report no significant association between the genotypes and response to hGH therapy in their SGA and hGH-deficient cohorts (9, 10, 11, 12).

	Subjects and Methods

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Experimental subjects

This study analyzed 368 individuals from a cohort of 678 unrelated healthy white young women, aged 18–35 yr, and recruited in the City of Toronto (Ontario, Canada) for large-scale investigations of peak BMD determinants (14). Subjects recruited by newspaper advertisements and posted flyers were screened by a telephone interview and excluded if they: 1) were not 18–35 yr old; 2) had a history of a comorbid condition; or 3) were taking medications known to affect bone. Approval was obtained from the local Research Ethics Board and written informed consent from each subject. Height was measured using a stadiometer, BMD of the lumbar spine ([L2–L4 (LS)] and hip [femoral neck (FN)] using dual-energy x-ray absorptiometry, and bone volume quality by a stiffness index (SI) by quantitative ultrasound (QUS) of the calcaneus (14, 15). A standardized interview was conducted to evaluate medical history and lifestyle factors. The database of physical, medical and lifestyle information was coded to preserve anonymity.

hGHR genotyping

Genomic DNA was isolated from peripheral blood lymphocytes by standard procedures. A 38-cycle amplification of the region around exon 3 of the hGHR gene was performed on 100-ng samples using specific primers (GenBank accession no. AF155912) (4) that amplify a fragment of 935 bp for the 3+ allele and 532 bp for the 3– allele. For each set of unknown samples, 3+/3+ and 3+/3– controls were amplified and run on the same gel.

Statistical methods

Genotypic and relevant phenotypic information was coded and analyzed for association with FAH, BMD, and QUS-SI. The significance of associations among the three hGHR genotypes and outcome variables was determined using SPSS (version 12.0.1). Spearman rho coefficients were tested for significance and ANOVA with Tukey post-hoc testing was used to complete the assessment of univariate associations with genotype. For FAH, a multiple regression analysis included age at menses and calcium intake as covariates, with and without the PTH receptor 1 (PTHR1) genotype, a known genetic determinant in this cohort (16). For QUS-SI, general linear modeling was used to incorporate the hGHR genotype information into a univariate model, with original significant covariates, including the estrogen receptor 1 (ER1) XbaI and PvuII single nucleotide polymorphisms (SNPs), and the amplified in breast cancer 1 (AIB1) genotype and their interaction, as described before (15). For BMD at FN and LS, the same approach was used but only the TaqI genotype for vitamin D receptor (VDR) was included, because the linkage disequilibrium with the other significant VDR genotype covariate, the BsmI SNP, is virtually complete in this cohort (14). P < 0.05 was considered significant.

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Genotyping and distribution of hGHR exon 3

Amplified fragments of the hGHR exon 3 locus were as expected: 935 bp for the full-length (3+) allele and 532 bp for the exon 3– allele. The distribution of hGHR genotypes in the 368 samples was 53.3% (3+/3+), 35.6% (3+/3– and 11.1% for 3–/3– and did not deviate significantly from Hardy-Weinberg equilibrium. The allele frequencies for 3+ and 3– alleles were 71.4% and 28.6%, respectively, similar to what has been reported by Pantel et al. (4), as well as all six of the clinical studies in Table 1: approximately half of Europeans and North and South Americans are 3+/3+ and the other half have at least one 3– allele.

Physical characteristics of the cohort and genetic associations

No significant differences in age, FAH, weight, BMI, age at menses, or BMD (LS, FN) were noted among the three subgroups (Table 2).

Because complete data were available on only a subset of the original cohort (14), previously reported associations were reexamined to rule out bias of attrition. Overall, means and SD values for the variables of interest (FAH, LS-BMD, FN-BMD, QUS-SI) in the subset (see Table 2) were essentially the same as those published for the larger cohort (14, 15). The Spearman rank coefficient between hGHR genotype and FAH was not significant, nor was ANOVA with genotype as factor (data not shown). Multivariate regression modeling of FAH, based on 358 cases with no missing values, showed that age at menarche, calcium intake, and PTHR1-(AAAG)₆ genotype remained significant independent predictors (P < 0.05, P < 0.02, and P < 0.01, respectively), as reported before for a larger cohort (n = 569) (16). In contrast, hGHR genotype was not significant (P > 0.65) as a predictor, whether or not the PTHR1 genotype was included in the model.

In a reexamination of QUS-SI, the original model (model 2 for a cohort of n = 663) (15) was readily validated by general linear modeling. In the smaller cohort, the following dependent variables were still significant predictors of QUS-SI: BMI (P = 0.006), cups of coffee per day (P = 0.034), and current exercise (P = 0.010). Addition of hGHR genotype to the model suggested a marginally significant (P = 0.046) predictive effect, although it did not persist when ER1 and AIB1 genotypes were also inserted.

Similar reexamination of BMD at LS and FN by general linear modeling confirmed that the TaqI VDR SNP is a significant predictor for FN but not LS in the original cohort (P = 0.031 and P = 0.217, respectively) (14), as well as the present subset (P = 0.021 and P = 0.217). Whether the hGHR genotype was used to replace the VDR SNP predictor or both were coinserted, hGHR was not a significant predictor for BMD at either FN (P = 0.852 and P = 0.936, respectively) or LS (P = 0.640 and P = 0.212).

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Although nutritional, steroidal, growth factor, and environmental factors are also important (1), it is well known that hGH plays a major role in regulating postnatal growth and BMD in both children and adults, and that its specific receptor, hGHR, is essential for these effects (1, 2, 13, 17, 18). What is not clear is whether the exon 3– deleted form of the receptor has a special physiological role in regulating responsiveness to hGH, as suggested by three recently published clinical studies but refuted by others (7, 8, 9, 10, 11, 12) (see details in Table 1). The loss of 22 amino acids at the N terminus of the extracellular domain, external to the ligand binding domain, has not been shown to alter hGH binding (5, 6). Dos Santos et al. (7) showed a small positive effect of the 3– allele on the responsiveness of transiently transfected cells to hGH in terms of STAT5 signaling, but otherwise no investigations of intracellular signaling differences have been reported.

Although it was initially thought that the 3– hGHR isoform was caused by individual-specific alternative splicing mechanisms (19, 20), Pantel et al. (4) showed that it is more likely caused by a homologous recombination event occurring between retroviral elements that surround exon 3 in the hGHR gene. The production of both full-length and 3– isoforms is a species-specific event: other mammalian species (e.g. rabbits, rodents) express only the full-length GHR, whereas birds, bony fish, marsupials, and amphibians express only an exon 3– GHR (National Center for Biotechnology Information, NCBI). Interestingly, the hPRLR, closely related to the hGHR, always lacks an exon 3 equivalent (NCBI).

Our study demonstrates a lack of association of FAH and BMD with hGHR exon 3 genotypes in a healthy adult cohort. It is possible that there is a weak association between genotype and bone quality measured by ultrasound, but a more likely explanation of our marginal results is chance stratification, particularly because it is the heterozygous group that differs from both homozygous ones. Overall, our results support the findings of Pilotta et al. (10), Carrascosa et al. (11), and Blum et al. (12) that suggest no effect of the 3– allele on hGH responsiveness in children with short stature, although our biological endpoints and the hGH "physiologies" are different. Although it is impossible to compare our genotyping study directly with therapeutic trials of supraphysiological levels of hGH (7, 9, 11), three of the investigations used "replacement doses" of hGH to treat hGH-deficient children (8, 10, 12). Yet even among these studies, there is no consensus: Jorge et al. (8) found a significant effect of at least one 3– allele on growth velocity, whereas Pilotta et al. (10) and Blum et al. (12) found no association. In addition, hGH dose does not appear to be a predisposing factor: higher dose treatments in SGA cohorts have had both positive (7) and not significant (9, 11) results.

Thus, although we cannot clarify the present controversy, we have shown that the hGHR 3+ and 3– isoforms do not differentially affect two major outcomes of hGH action, FAH and BMD, within a cohort of healthy adult Caucasian females.

	Acknowledgments

 
We thank Dr. Laurent Legault for his helpful review of the manuscript and Dr. Geoffrey N. Hendy for facilitating this study.

	Footnotes

 
Funding for this study came from the Canadian Institutes of Health Research (CIHR) (to C.G.G.) and an Industrially Oriented Research (IOR) grant from NSERC-Dairy Farmers of Canada (to D.E.C.C.). G.K. was supported by a studentship from the McGill University Health Centre-Montreal Children’s Hospital Research Institute, and Z.S. by a bursary from the CIHR Training Program in Skeletal Health Research.

First Published Online November 7, 2006

G.K. and Z.S. contributed equally to this work.

D.E.C.C. and C.G.G. contributed equally to this work.

Abbreviations: AIB1, Amplified in breast cancer 1; BMD, bone mineral density; ER1, estrogen receptor 1; FAH, final adult height; FN, femoral neck; hGH, human GH; hGHR, hGH receptor; ISS, idiopathic short stature; LS, lumbar spine; PTHR1, PTH receptor 1; QUS, quantitative ultrasound; SGA, small for gestational age; SI, stiffness index; SNP, single nucleotide polymorphism; STAT5, signal transducer and activator of transcription 5.

Received August 8, 2006.

Accepted October 30, 2006.

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This Article Abstract Full Text (PDF) All Versions of this Article: 92/2/725    most recent Author Manuscript (PDF) Submit a related Letter to the Editor Purchase Article View Shopping Cart Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kenth, G. Articles by Goodyer, C. G. Search for Related Content PubMed PubMed Citation Articles by Kenth, G. Articles by Goodyer, C. G. Related Collections Calcium and Bone Metabolism Female Endocrinology Neuroendocrinology and Pituitary Pediatric Endocrinology