Endocrinol Metab > Volume 30(3); 2015 > Article
Park, Hwang, Seo, Kim, Seo, Lee, and Kim: Association between the Growth Hormone Receptor Exon 3 Polymorphism and Metabolic Factors in Korean Patients with Acromegaly

Abstract

Background

This study investigated the association between the frequency of growth hormone receptor (GHR) exon 3 polymorphism (exon 3 deletion; d3-GHR) and metabolic factors in patients with acromegaly in Korea.

Methods

DNA was extracted from the peripheral blood of 30 unrelated patients with acromegaly. GHR genotypes were evaluated by polymerase chain reaction and correlated with demographic data and laboratory parameters.

Results

No patient had the d3/d3 genotype, while four (13.3%) had the d3/fl genotype, and 26 (86.7%) had the fl/fl genotype. Body mass index (BMI) in patients with the d3/fl genotype was significantly higher than in those with the fl/fl genotype (P=0.001). Age, gender, blood pressure, insulin-like growth factor-1, growth hormone, fasting plasma glucose, triglycerides, high density lipoprotein cholesterol, and low density lipoprotein cholesterol levels showed no significant differences between the two genotypes.

Conclusion

The d3-GHR polymorphism may be associated with high BMI but not with other demographic characteristics or laboratory parameters.

INTRODUCTION

Acromegaly is a rare chronic pathology that results from excessive secretion of growth hormone (GH) [1]. The most common cause of acromegaly is a somatotroph (growth hormone-secreting) adenoma of the anterior pituitary that accounts for about one-third of all hormone-secreting pituitary adenomas [2]. The clinical features of acromegaly are attributable to high serum concentrations of both pituitary-derived GH and liver-derived insulin-like growth factor-1 (IGF-1), which are GH dependent. The clinical spectrum of acromegaly ranges from a relatively asymptomatic condition with slowly evolving acral changes to a severe incapacitating disorder that includes headaches, arthropathy, nerve entrapment syndrome, diabetes, hypertension, and cardiomyopathy [3].
Several recent studies have investigated between clinicopathological characteristics in patients with acromegaly and polymorphisms in a number of genes, including the growth hormone receptor (GHR) gene. The GHR gene is located on the short arm of chromosome 5 (p13.1-p12) and contains nine coding exons [4,5]. The GHR protein is a single polypeptide consisting of an intracellular domain, transmembrane portion and an extracellular, ligand-binding domain encoded by exons 3 to 7 [4,5]. Several GHR isoforms have been identified; one of these lacks a 22-amino-acid sequence encoded by exon 3 to create two isoforms: one containing exon 3, or a full-length GHR (fl-GHR), and one missing exon 3 (d3-GHR) [6,7]. Pantel and colleagues [8] recently hypothesized that these two isoforms are the consequence of an in-frame deletion derived from an intrachromosomal recombination event between two similar primate-specific retro-elements (DNA sequences derived from a retrovirus) that flank exon 3, which occurred late during primate evolution. During cell division, these flanking sequences may undergo recombination, which results in a 2.7-kb genomic deletion that spans exon 3 [8]. This polymorphism enhances signal transduction by GH although its binding to the GHR is not altered [9,10,11]. In most studies investigating the frequency of these GHR isoforms in healthy control subjects, approximately half are homozygous for the fl/fl-GHR genotype, while the fl/d3-GHR genotype is present in 30% to 40% of individuals; the d3/d3 genotype is present in 10% to 20% of subjects [8,11,12]. About one-half of patients with acromegaly have at least one d3 allele [13]. In addition, patients with acromegaly carrying a d3-GHR allele may be at increased risk of more severe complications and may achieve biochemical control with great difficulty [7].
Acromegaly is potentially a very severe condition because chronic GH hypersecretion lead to cause cardiometabolic complications that reduce life expectancy and increase morbidity. While several studies have attempt to correlate cardiometabolic complications with the presence of the d3-GHR polymorphism, the role of two isoforms remains controversial. While the presence of the d3-GHR allele has been related to clinical manifestation of acromegaly in some studies, these results are controversial [7,13,14].
The present study investigated the association between the d3-GHR polymorphism and demographic and laboratory parameters in Korean patients with acromegaly.

METHODS

Study cohort

Thirty unrelated patients with acromegaly were recruited for the present cross-sectional study among those attending Kyungpook National University Hospital, Daegu, South Korea, from January 2008 to June 2012. The inclusion criteria for this study was described as following; first, a diagnosis of acromegaly was established by documenting an elevated IGF-1 level in combination with failure of GH to suppress after oral glucose to below 0.3 µg/L [15] and second, patients was confirmed by pathological examination of surgically resected tissues. We excluded patients with any medication which can affect their obese state (e.g., oral pills, herbal medication, lipid lowering agents). All patients agreed to participate in the Korean Genomic Cohort Study and provided written informed consent for the investigation. The study protocol was approved by the Institutional Review Board of Kyungpook National University Hospital.

Demographical and laboratory assessments

Demographic data from 30 patients with acromegaly were reviewed. Demographic parameters (age, gender, height, weight, and systolic blood pressure [SBP] and diastolic blood pressure [DBP]) were collected from the medical charts when they visited our hospital at first visit. Body mass index (BMI, kg/m2) was also calculated based on their height and weight.
Blood samples were collected in the morning after overnight fasting. Fasting plasma glucose (FPG) was measured by the hexokinase method (Roche Diagnostics, Basel, Switzerland). Low density lipoprotein cholesterol (LDL-C) and high density lipoprotein cholesterol (HDL-C) were measured using a selective enzymatic protection assay. Triglycerides were measured by enzymatic assay without a glycerol blank using a Hitachi Molecular Analytics D2400 apparatus (Roche, Tokyo, Japan). Serum GH and IGF-1 were measured by radioimmunoassay using SR-300 (STRATEC, Birkenfeld, Germany).

Genetic analysis

DNA was extracted from the leukocyte of peripheral blood by using the Puregene DNA isolation kit was used according to the manufacturer's instructions. Genotyping for the d3-GHR gene was performed using polymerase chain reaction (PCR) with the following primers: G1 5'-TGT GCT GGT CTG TTG GTC TG-3', G2 5'-AGT CGT TCC TGG GAC AGA GA-3', and G3 5'-CCT GGA TTA ACA CTT TGC AGA CTC-3' [14]. Amplification was performed for 40 cycles of 94℃ for 30 seconds, 57℃ for 30 seconds, and 68℃ for 30 seconds, followed by a final extension time at 68℃ for 10 minutes. The polymorphism detected by PCR was evident as a 592 bp product in the presence of the deletion (d3/d3) and a 935 bp fragment in the presence of the full length fragment (fl/fl). Each sample was geno-typed as d3/d3, d3/fl, or fl/fl.

Statistical analysis

Continuous variables were expressed as mean±standard deviation (SD). Independent sample t test and Mann-Whitney U test were used to analyze differences in continuous variables between the different genotypes. A chi-square test was used for comparison of nominal variables between groups. P values <0.05 were considered statistically significant. Statistical significance of genotypic frequencies was evaluated according to the Hardy-Weinberg rule considering the expected genotypic frequencies and evaluated by the chi-square test. All analyses were carried out using SPSS version 18.0 (SPSS Inc., Chicago, IL, USA).

RESULTS

Thirty patients with acromegaly met the inclusion criteria. Selected demographic characteristics are shown in Table 1. The d3-GHR genotypes were distributed as follows: four patients (13.3%) had the d3/fl genotype and 26 (86.7%) had the fl/fl genotype while none had the d3/d3 genotype. There frequencies are different from previous reports in other population. For example, Montefusco et al. [14] reported a distribution of 9.2% for d3/d3 and 31.6% for d3/fl in 76 patients with acromegaly in Italy, while Mercado et al. [7] documented 32.4% of d3/d3 and 21.6% of d3/fl in 148 patients with acromegaly in Mexico.
Demographic and laboratory parameters were compared between the different genotypes. No significant differences were seen between age, gender, SBP, DBP, IGF-1, GH, FPG, triglycerides, and HDL-C and LDL-C between the d3/fl and fl/fl genotypes (Mann-Whitney U test) (Table 2). BMI was significantly higher in patients with the d3/fl genotype compared to fl/fl (P=0.001).

DISCUSSION

In this study of Korean patients of acromegaly, the distribution of the d3-GHR genotype was somewhat dissimilar to that reported in previous studies shown in Table 3. In particular, in patients with acromegaly from Italy, France, and Mexico, the d3/d3 genotype was present 14.3% to 32.4% of cases, compared to no cases herein. On the other hand, comparing with healthy controls in same ethnic groups, especially Korea and China, the d3/d3 genotype was present lower (3.2% to 7.2% of cases) than that of other ethnic groups spoken above. In distribution of genotype with d3 allele (d3/d3+d3/fl), patients with acromegaly from Italy, France, Mexico were higher as 45.7% to 54% than the present study (13.3%) and the study with healthy controls in Korea (27.4%) and China (25.4%) [7,14,16,17,18,19]. While this may suggest the existence of ethnic differences in the frequency of d3-GHR genotypes, some limitations to the present study must be considered. First, our study was conducted a local area of South Korea, and thus reflects only this ethnic group. Second, this study enrolled a small number of patients, and no patient was homozygous for d3-GHR. Obviously, the heterozygote d3-GHR cannot represent all d3 GHR allele carriers.
None of the parameters assessed showed any significant differences between d3-GHR genotypes with the exception of BMI, in agreement with previous studies [7,14,16,17]. Cinar et al. [20] showed that the d3-GHR variant genotype did not have an effect on clinical features or comorbidity in patients with acromegaly, but it might play a role in GH/IGF-1 level discordance in acromegaly. There are also some previous studies reported that basal levels of GH were lower in patients bearing the fl/fl genotype [7,14,16,17], while no significant differences between genotypes were seen in the present analysis. Likewise, IGF levels are reported to be higher in patients with the d3/fl genotype, but no significant difference was seen in this cohort of Korean patients. SBP was somewhat higher in d3/fl heterozygotes, but these levels were under 140 mm Hg in both genotypes, no significant difference was observed between genotypes. The same is true of DBP, which was under 85 mm Hg in both groups.
Considering the lipid profile, triglycerides and LDL-C were lower in the d3/fl genotype, but with no significant differences between groups. One limitation in interpreting these results that data on lipids was not available for all patients, further decreasing the sample size, because we only could collect laboratory results of lipid profile depending their medical records and some were missed from their initial laboratory test when patients diagnosed acromegaly.
BMI was the only parameter showing significant differences between groups, and was higher in patients with the d3/fl genotype (P=0.001) as shown in Table 2, Fig. 1. Interestingly, most previous studies reported no significant difference between GHR genotypes and BMI [13,16,17], although one found a significant relationship between lower BMI and patients bearing the d3 GHR allele compared to fl/fl carriers [14]. Turgut et al. [21] reported that BMI was significantly higher in individuals with the d3/fl genotype, similar to the present study.
Some previous reports have documented slightly higher prevalence of type 2 diabetes in patients with acromegaly bearing the d3-GHR genotype, along with possible differences in glucose metabolism in individuals with the d3/d3 [7,21]. On the basis of these studies, we presumed the higher levels of FPG may be associated with the higher BMI in patients with the d3-GHR genotype, at least to some degree. Nonetheless, FPG seen herein did not reach statistical significance (P=0.526). Thus, the d3-GHR genotype is unlikely to affect glucose metabolism in this study.
In conclusion, the d3-GHR polymorphism may be associated with higher BMI. Although this study has some inherent limitation, it is our belief that further investigations are warranted on large cohorts of patients with acromegaly to validate these findings.

ACKNOWLEDGMENTS

This study was sponsored by Yuhan pharmaceuticals (2012).

NOTES

CONFLICTS OF INTEREST: No potential conflict of interest relevant to this article was reported.

REFERENCES

1. Melmed S. Acromegaly. N Engl J Med 1990;322:966-977.
[CROSSREF]  [PUBMED] 
2. Zhang X, Horwitz GA, Heaney AP, Nakashima M, Prezant TR, Bronstein MD, et al. Pituitary tumor transforming gene (PTTG) expression in pituitary adenomas. J Clin Endocrinol Metab 1999;84:761-767.
[CROSSREF]  [PUBMED] 
3. Ben-Shlomo A, Melmed S. Acromegaly. Endocrinol Metab Clin North Am 2008;37:101-122.
[CROSSREF]  [PUBMED]  [PMC] 
4. Leung DW, Spencer SA, Cachianes G, Hammonds RG, Collins C, Henzel WJ, et al. Growth hormone receptor and serum binding protein: purification, cloning and expression. Nature 1987;330:537-543.
[CROSSREF]  [PUBMED]  [PDF]
5. Godowski PJ, Leung DW, Meacham LR, Galgani JP, Hellmiss R, Keret R, et al. Characterization of the human growth hormone receptor gene and demonstration of a partial gene deletion in two patients with Laron-type dwarfism. Proc Natl Acad Sci U S A 1989;86:8083-8087.
[CROSSREF] 
6. Urbanek M, MacLeod JN, Cooke NE, Liebhaber SA. Expression of a human growth hormone (hGH) receptor isoform is predicted by tissue-specific alternative splicing of exon 3 of the hGH receptor gene transcript. Mol Endocrinol 1992;6:279-287.
[PUBMED] 
7. Mercado M, DaVila N, McLeod JF, Baumann G. Distribution of growth hormone receptor messenger ribonucleic acid containing and lacking exon 3 in human tissues. J Clin Endocrinol Metab 1994;78:731-735.
[PUBMED] 
8. Pantel J, Machinis K, Sobrier ML, Duquesnoy P, Goossens M, Amselem S. Species-specific alternative splice mimicry at the growth hormone receptor locus revealed by the lineage of retroelements during primate evolution. J Biol Chem 2000;275:18664-18669.
[CROSSREF]  [PUBMED] 
9. Dos Santos C, Essioux L, Teinturier C, Tauber M, Goffin V, Bougneres P. A common polymorphism of the growth hormone receptor is associated with increased responsiveness to growth hormone. Nat Genet 2004;36:720-724.
[CROSSREF]  [PUBMED]  [PDF]
10. Audi L, Esteban C, Carrascosa A, Espadero R, Perez-Arroyo A, Arjona R, et al. Exon 3-deleted/full-length growth hormone receptor polymorphism genotype frequencies in Spanish short small-for-gestational-age (SGA) children and adolescents (n=247) and in an adult control population (n=289) show increased fl/fl in short SGA. J Clin Endocrinol Metab 2006;91:5038-5043.
[CROSSREF]  [PUBMED]  [PDF]
11. Jorge AA, Marchisotti FG, Montenegro LR, Carvalho LR, Mendonca BB, Arnhold IJ. Growth hormone (GH) pharmacogenetics: influence of GH receptor exon 3 retention or deletion on first-year growth response and final height in patients with severe GH deficiency. J Clin Endocrinol Metab 2006;91:1076-1080.
[CROSSREF]  [PUBMED] 
12. Sobrier ML, Duquesnoy P, Duriez B, Amselem S, Goossens M. Expression and binding properties of two isoforms of the human growth hormone receptor. FEBS Lett 1993;319:16-20.
[CROSSREF]  [PUBMED] 
13. Schmid C, Krayenbuehl PA, Bernays RL, Zwimpfer C, Maly FE, Wiesli P. Growth hormone (GH) receptor isoform in acromegaly: lower concentrations of GH but not insulin-like growth factor-1 in patients with a genomic deletion of exon 3 in the GH receptor gene. Clin Chem 2007;53:1484-1488.
[CROSSREF]  [PUBMED]  [PDF]
14. Montefusco L, Filopanti M, Ronchi CL, Olgiati L, La-Porta C, Losa M, et al. d3-Growth hormone receptor polymorphism in acromegaly: effects on metabolic phenotype. Clin Endocrinol (Oxf) 2010;72:661-667.
[CROSSREF]  [PUBMED] 
15. Freda PU. Current concepts in the biochemical assessment of the patient with acromegaly. Growth Horm IGF Res 2003;13:171-184.
[CROSSREF]  [PUBMED] 
16. Bianchi A, Giustina A, Cimino V, Pola R, Angelini F, Pontecorvi A, et al. Influence of growth hormone receptor d3 and full-length isoforms on biochemical treatment outcomes in acromegaly. J Clin Endocrinol Metab 2009;94:2015-2022.
[CROSSREF]  [PUBMED]  [PDF]
17. Kamenicky P, Dos Santos C, Espinosa C, Salenave S, Galland F, Le Bouc Y, et al. D3 GH receptor polymorphism is not associated with IGF1 levels in untreated acromegaly. Eur J Endocrinol 2009;161:231-235.
[CROSSREF]  [PUBMED] 
18. Kang EH, Yamaguchi T, Tajima A, Nakajima T, Tomoyasu Y, Watanabe M, et al. Association of the growth hormone receptor gene polymorphisms with mandibular height in a Korean population. Arch Oral Biol 2009;54:556-562.
[CROSSREF]  [PUBMED] 
19. Shen Y, Wang L, Zhao Y, You L, Geng L, Gu HF, et al. Evaluation of the association between GHR exon 3 polymorphism and polycystic ovary syndrome among Han Chinese women. Growth Horm IGF Res 2011;21:248-251.
[CROSSREF]  [PUBMED] 
20. Cinar N, Dagdelen S, Yorgun H, Canpolat U, Kabakci G, Erbas T. The clinical and cardiometabolic effects of d3-growth hormone receptor polymorphism in acromegaly. Pituitary 2015;18:116-125.
[CROSSREF]  [PUBMED]  [PDF]
21. Turgut S, Akin F, Ayada C, Topsakal S, Yerlikaya E, Turgut G. The growth hormone receptor polymorphism in patients with acromegaly: relationship to BMI and glucose metabolism. Pituitary 2012;15:374-379.
[CROSSREF]  [PUBMED]  [PDF]
Fig. 1

Scatter plot of growth hormone receptor exon 3 deletion gene polymorphism and body mass index (BMI). aBMI was significantly higher in patients with the d3/fl genotype compred to fl/fl.

enm-30-312-g001.jpg
Table 1

Demographic and Laboratory Parameters of Korean Patients with Acromegaly (n=30)

Values are expressed as mean±SD.

BMI, body mass index; SBP, systolic blood pressure; DBP, diastolic blood pressure; FPG, fasting plasma glucose; LDL-C, low density lipoprotein cholesterol; HDL-C, high density lipoprotein cholesterol; GH, growth hormone; IGF-1, insulin like growth factor-1.

Parameter Value
Age, yr 47.5±9.32
Sex, male:female 12:18
BMI, kg/m2 25.3±2.94
SBP, mm Hg 130.2±12.65
DBP, mm Hg 78.8±12.35
FPG, mg/dL 133.2±71.70
Triglycerides, mg/dL 144.8±56.84
LDL-C, mg/dL 126.1±28.19
HDL-C, mg/dL 44.9±12.92
GH, ng/mL 33.9±35.63
IGF-1, ng/mL 888.3±413.71
Table 2

Comparison of d3-GHR Genotypes with Demographic Characteristics and Laboratory Parameters in Korean Patients with Acromegaly

Values are expressed as number (%) or mean±SD.

d3-GHR, growth hormone receptor exon 3 deletion; fl, full length; DM, diabetes mellitus; BMI, body mass index; SBP, systolic blood pressure; DBP, diastolic blood pressure; GH, growth hormone; IGF-1, insulin like growth factor-1; FPG, fasting plasma glucose; LDL-C, low density lipoprotein cholesterol; HDL-C, high density lipoprotein cholesterol.

aChi-square test; bIndependent t test; cMann-Whitney U test.

d3/d3 d3/fl fl/fl P<0.05
All patients (n=30) 0 4 (13.3) 26 (86.7)
Age, yr 41.25±7.805 48.50±9.283 NS
Sex
 Male 3 9 NSa
 Female 1 17 NSa
Non-DM 3 22 NSa
DM 1 4 NSa
BMI, kg/m2 28.5±0.80 24.7±2.87 0.001b, 0.045c
SBP, mm Hg 137.3±6.43 129.3±13.10 NS
DBP, mm Hg 83.0±5.196 78.24±13.034 NS
GH, ng/mL 22.8±20.01 34.9±36.85 NS
IGF-1, ng/mL 1017.9±192.64 869.8±436.16 NS
FPG, mg/dL 196.7±165.71 123.7±48.27 NS
Triglycerides, mg/dL 88 147.7±56.76 NS
LDL-C, mg/dL 99 127.5±28.20 NS
HDL-C, mg/dL 48 44.7±13.26 NS
Table 3

d3-GHR Gene Polymorphism Genotype of Variable Populations

d3-GHR, growth hormone receptor exon 3 deletion; fl, full length.

Study Population State of disease Number Genotype, %
fl/fl d3/fl d3/d3 d3/fl+d3/d3
This study Korean Acromegaly 30 86.7 13.3 0 13.3
Mercado et al. [7] Mexican Acromegaly 148 45.9 21.6 32.4 54.0
Kamenicky et al. [17] French Acromegaly 105 51.4 29.5 19.0 48.5
Bianchi et al. [16] Italy Acromegaly 84 52.4 29.8 17.9 47.7
Montefusco et al. [14] Italy Acromegaly 76 59.2 31.4 14.3 45.7
Kang et al. [18] Korean Healthy control 125 73.6 19.2 7.2 27.4
Shen et al. [19] Chinese Healthy control 441 74.6 22.2 3.2 25.4


Editorial Office
101-2503, Lotte Castle President, 109 Mapo-daero, Mapo-gu, Seoul 04146, Korea​
Tel: +82-2-716-2428    Fax: +82-2-714-5103    E-mail: journal@endocrinology.or.kr                

Copyright © 2020 by Korean Endocrine Society. All rights reserved.

Developed in M2community

Close layer
prev next