help button home button Endocrine Society JCEM
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2007-0857
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow Submit a related Letter to the Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Petkovic, V.
Right arrow Articles by Mullis, P. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Petkovic, V.
Right arrow Articles by Mullis, P. E.
Related Collections
Right arrow Neuroendocrinology and Pituitary
Right arrow Pediatric Endocrinology
The Journal of Clinical Endocrinology & Metabolism Vol. 92, No. 11 4427-4435
Copyright © 2007 by The Endocrine Society

Exon Splice Enhancer Mutation (GH-E32A) Causes Autosomal Dominant Growth Hormone Deficiency

Vibor Petkovic, Didier Lochmatter, James Turton, Peter E. Clayton, Peter J. Trainer, Mehul T. Dattani, Andrée Eblé, Iain C. Robinson, Christa E. Flück and Primus E. Mullis

Department of Pediatric Endocrinology, Diabetology, and Metabolism (V.P., D.L., A.E., C.E.F., P.E.M.), Inselspital, University Children’s Hospital, CH-3010 Bern, Switzerland; National Institute for Medical Research (J.T., I.C.R.), Mill Hill, London NW7 1AA, United Kingdom; Department of Pediatric Endocrinology, Royal Manchester Children’s Hospital (P.E.C.), Manchester M27 4HA, United Kingdom; Department of Endocrinology (P.J.T.), Christie Hospital, Manchester M20 4BX, United Kingdom; and Biochemistry, Endocrinology, and Metabolism Unit and Developmental Endocrinology Research Group (M.T.D.), Clinical and Molecular Genetics Unit, Institute of Child Health, London WC1N 1EH, United Kingdom

Address all correspondence and requests for reprints to: Professor Dr. Primus E. Mullis, University Children’s Hospital, Pediatric Endocrinology, Inselspital CH-3010 Bern, Switzerland. E-mail: primus.mullis{at}insel.ch.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Context and Objective: Alteration of exon splice enhancers (ESE) may cause autosomal dominant GH deficiency (IGHD II). Disruption analysis of a (GAA) (n) ESE motif within exon 3 by introducing single-base mutations has shown that single nucleotide mutations within ESE1 affect pre-mRNA splicing.

Design, Setting, and Patients: Confirming the laboratory-derived data, a heterozygous splice enhancer mutation in exon 3 (exon 3 + 2 A->C) coding for GH-E32A mutation of the GH-1 gene was found in two independent pedigrees, causing familial IGHD II. Because different ESE mutations have a variable impact on splicing of exon 3 of GH and therefore on the expression of the 17.5-kDa GH mutant form, the GH-E32A was studied at the cellular level.

Interventions and Results: The splicing of GH-E32A, assessed at the protein level, produced significantly increased amounts of 17.5-kDa GH isoform (55% of total GH protein) when compared with the wt-GH. AtT-20 cells coexpressing both wt-GH and GH-E32A presented a significant reduction in cell proliferation as well as GH production after forskolin stimulation when compared with the cells expressing wt-GH. These results were complemented with confocal microscopy analysis, which revealed a significant reduction of the GH-E32A-derived isoform colocalized with secretory granules, compared with wt-GH.

Conclusion: GH-E32A mutation found within ESE1 weakens recognition of exon 3 directly, and therefore, an increased production of the exon 3-skipped 17.5-kDa GH isoform in relation to the 22-kDa, wt-GH isoform was found. The GH-E32A mutant altered stimulated GH production as well as cell proliferation, causing IGHD II.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
GH IS ESSENTIAL FOR postnatal somatic growth in humans, and GH deficiency (GHD) causes metabolic alterations and growth failure. Short stature associated with isolated GHD (IGHD) has been estimated to occur in about 1:4,000 to 1:10,000 births, with 5–30% of patients having affected first-degree relatives (1, 2, 3). The autosomal-dominant form of IGHD type II (IGHD II) is mainly caused by mutations at the donor splice site in intervening sequence 3 (5'IVS-3), i.e. within the first 6 bp in intron 3 (4), resulting in missplicing at the mRNA level and the subsequent loss of exon 3 leading to the production of a 17.5-kDa human GH isoform (5). This isoform lacks amino acids 32–71, which is the linker domain between the first two helices of GH, disrupting an internal disulfide bridge and thereby overall protein structure (6, 7). At the functional level, it exhibits a dominant-negative effect on the secretion of the 22-kDa isoform in both tissue culture and transgenic animals (8, 9, 10). The 17.5-kDa isoform is initially retained in the endoplasmic reticulum, disrupts the Golgi apparatus, impairs GH and other hormonal trafficking (11), and partially reduces the stability of the 22-kDa isoform (8). Furthermore, transgenic mice overexpressing the 17.5-kDa isoform exhibit a defect in the maturation of GH secretory vesicles and anterior pituitary gland hypoplasia due to a loss of the majority of somatotropes (8, 9, 12).

Skipping of exon 3 caused by GH-1 gene alterations other than those at the donor splice site, including the mutations that are located within purine-rich sequences in the exon splice enhancer (ESE1m1 and ESE1m2 in ESE1, respectively), and in the intron splice enhancer (ISEm1 and ISEm2 in ISE, respectively), has also been reported in other patients with IGHD II (4, 12, 13, 14, 15, 16, 17). The first seven nucleotides in exon 3 seem to be crucial for the splicing of GH mRNA (18) such that some nonsense mutations cause a missplicing at the mRNA level and result in the translation of different GH isoforms. After the molecular analysis of a Swiss family suffering from IGHD II caused by a disruption of a (GAA) (n) ESE motif within exon 3 (exon 3 + 5 A->G), Moseley et al. (16) introduced single-base mutations by mutagenesis to study the role of each single nucleotide within ESE1 of GH1 and described their effect on pre-mRNA splicing. It was shown that the presence of an exon 3 + 5 A->G mutation (ESE1m1) increased significantly the production of mRNA for the 17.5-kDa GH isoform relative to the 22-kDa isoform. Furthermore, another artificially introduced mutation in ESE1, exon 3 + 2 A->C, evoked even higher production of the 17.5-kDa GH isoform, showing that exon 3 skipping is favored over use of the exon 3 + 45 cryptic splice site, which is the next available 3' splice site after the weak IVS2 3' splice site.

Importantly, we are now in a position to describe the clinical phenotype of this heterozygous exon 3 + 2 A->C mutation in ESE1 of the GH-1 gene found in two families with IGHD II. This mutation causes a glutamic acid to alanine amino acid change at position 32 in the human GH protein and leads to missplicing at the mRNA level, producing large amounts of the 17.5-kDa GH isoform. Therefore, the aims of the study were to both describe the phenotype and investigate the molecular defect evoked by the GH-E32A mutation causing familial IGHD II. First, the mRNA splicing of GH-E32A was compared with wt-GH at the protein level by Western blot. Furthermore, the impact of the mutant GH peptide on the intracellular and extracellular production of wt-GH as well as on cell viability and proliferation on transfection of GH mutant in AtT-20 cells was investigated. In addition, by using confocal microscopy, colocalization of the GH-E32A with different organelles (endoplasmic reticulum, Golgi and secretory granules) within the secretory pathway was studied in detail.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Family 1 (Swiss family)

We describe a nonconsanguineous three-generation family from Turkey. Basically, all members depicted were auxologically examined (19), whereas five individuals were studied in detail, namely the grandmother, father, and mother as well as the two offspring (Fig. 1AGo). The analysis of the pedigree suggested an autosomal dominant growth disorder; and standard pharmacological stimulation tests (insulin-induced hypoglycemia, arginine stimulation) were performed (20). Auxological and laboratory data are presented in Table 1Go. Furthermore, genetic analysis revealed a familial E32A heterozygous mutation of the GH-1 gene in the affected members of the family.


Figure 1
View larger version (8K):
[in this window]
[in a new window]

  		FIG. 1. A three-generation pedigree of the Swiss patient’s family (A) and English patient’s family (B) are shown, with affected individuals indicated in open symbols containing smaller solid symbols (black: GH-E32A mutant confirmed; gray: phenotype suggests the GH-E32A genotype). The height (centimeters) and height SD scores are reported (19 ). DNA sequencing revealed that the affected subjects available for genetic screening presented with heterozygosity for the E32A transition mutation of the GH-1 gene.

 

View this table:
[in this window]
[in a new window]

  		TABLE 1. Auxological and laboratory data (Swiss family)

 
Patient III:2 in Pedigree 1 (Fig. 1AGo) was born at term (39 3/7 wk of pregnancy); birth weight and length were 2620 g [–2.1 SD score (SDS)] and 46.2 cm (–1.7 SDS), respectively (19). Growth retardation became obvious at the age of 5 3/12 yr. At the age of 6 yr, the patient was referred and presented with short stature (–3.3 SDS for age and sex), and IGHD was diagnosed (Fig. 2AGo and Table 1Go) (19). Subsequently recombinant human GH (rhGH)-replacement therapy (Norditropin; Novo-Nordisk, Copenhagen, Denmark; at a dose of 25 µg/kg) was administered daily sc.


Figure 2
View larger version (48K):
[in this window]
[in a new window]

  		FIG. 2. Growth charts of Swiss patient III-2 (A) and English patients II-1 and III-1 (B and C). The solid circles indicate the height measurements, the open circles the bone ages (21 ). The arrow pointing up indicates the beginning of rhGH treatment; arrows pointing down indicate stopping treatment. A, rhGH treatment started at age 7 yr. B, Pituitary GH (pitGH) was started at age 8.2 yr and was withdrawn at age 9.5 yr. rhGH therapy was introduced at 11 yr and discontinued when the patient reached the approximate final height. C, rhGH was started at age 6.3 yr with an initial period of catch-up growth. Between ages 7 and 9 yr and 10 and 11.7 yr, he had very poor compliance as depicted in the growth chart.

 
Her brother (III:1) was born at term (38 6/7 wk of pregnancy) after a normal pregnancy. The delivery and peri- and postnatal course were uncomplicated. His birth weight and length were 3160 g (–1.2 SDS) and 49.4 cm (–0.6 SDS), respectively (19). His growth and development were in the normal range, and no GH-1 gene mutation (E32A) was found.

The Ethical Committee of the University Children’s Hospital approved the experimental protocol. Informed consent was obtained from parents as well as family members involved.

More detailed information has been uploaded as supplemental data, published as supplemental data on The Endocrine Society’s Journals Online Web site at http://jcem.endojournals.org.

Family 2 (English family)

The index case (II:1 in Fig. 1BGo) was referred for evaluation of short stature in 1982 at the age of 7.2 yr (height 99 cm: –5.9 SDS) (19). His birth weight at term was 3100 g (–1.3 SDS) (19). His mother was short at 147 cm (height SDS –2.9) but had never undergone any investigations (Fig. 1BGo, gray symbol). Patient II:1 had pituitary function testing, which revealed low peak levels of GH during sleep (3.2 ng/ml) and an arginine stimulation test (2.4 ng/ml), with the other hormones all normal (20). Computed tomography scan revealed a small anterior pituitary gland occupying 50% of the fossa. Human pituitary-derived GH was started but discontinued on its withdrawal in 1985. rhGH was then introduced and stopped at age 16 yr (Fig. 2BGo). At retesting off GH at 16.6 yr, peak GH levels to arginine and exercise were 5.8 and 2 ng/ml, respectively (20). Further assessment with a glucagon stimulation test at age 21 yr then showed a normal peak GH level of 12.3 ng/ml (20). However, an IGF-I level at age 25 yr was low at 101 ng/ml (normal range 127–360 ng/ml; –3.1 SDS).

He has had four children to three mothers (Fig. 1BGo). His first son, patient III:1, presented for evaluation of short stature at age 5.7 yr (height 92 cm, –5.2 SDS) (19). His birth weight at term was 3100 g (–1.3 SDS). Peak GH level to arginine stimulation was 2.4 ng/ml with a hypoplastic anterior pituitary on magnetic resonance imaging (20). Treatment with rhGH was started at age 6.3 yr with an initial catch-up of growth, but compliance, and thus response, has been poor (Fig. 2CGo). Both patients II:1 and III:1 were heterozygous for the GH-E32A mutant (Fig. 1BGo).

Patient III:2 has not been assessed at our unit but has recently been diagnosed to have IGHD and is due to start rhGH replacement. DNA is not available from this child; however, heterozygosity for GH-E32A is likely, indicated in gray in Fig. 1BGo.

Patient III:3’s perinatal course had been normal and his birth weight was 3900 g (+0.8 SDS). His growth velocity from 2 months of age was poor, and in view of the family history and early signs of GHD, he had an endocrine evaluation. His peak GH level to arginine was 2.3 ng/ml and his IGF-I less than 25 ng/ml (20). His height SDS at 1.9 yr of age before starting on rhGH replacement therapy was –3.9 SDS (76 cm) (19). In addition to a homozygosity for {Delta}F508 in the CFTR gene, he is heterozygous positive for the GH-E32A mutant.

Patient III:4 also had an uneventful perinatal period with a birth weight of 4200 g (+1.38 SDS) but was screened for cystic fibrosis and the GH-1 gene mutation in view of the family history. His height SDS at age 7 months was +0.89. He has a normal GH-1 gene.

Cell culture and treatment

Mouse pituitary (AtT-20/D16v-F2) cells and Chinese hamster ovary (CHO-K1) cells were cultured as previously described (22).

Expression vectors

Wild-type GH (wt-GH, full-length, accession no. J03071) was cloned in pXGH5 (Nichols Institute Diagnostics, San Juan Capistrano, CA) as previously described (16). To generate the GH mutant studied (GH-E32A), site-directed mutagenesis was performed using the QuickChange site-directed mutagenesis kit from Stratagene (Basel, Switzerland) and mutant oligonucleotide primers: forward, 5'-TCC TTC TCC TAG GCA GAA GCC TAT ATC C-3' and reverse, 5'-GGA TAT AGG CTT CTG CCT AGG AGA AGG A-3'.

Western blot analysis

CHO cells were transiently transfected with wt-GH, GH-E32A, or cotransfected with wt-GH and GH-E32A using nucleofection technology (Nucleofector, Amaxa Biosystems GmbH, Cologne, Germany) in an electroporation cuvette with nucleofector solution T using the program U-30. Cells were transferred into fresh prewarmed medium containing 10% fetal calf serum. Six hours after transfection, the cells were stimulated with 50 µM forskolin (Sigma Aldrich, Buchs, Switzerland) for 2 h. Untransfected CHO cells stimulated with forskolin were used as a negative control. After 24 h of growth, cellular proteins were extracted using radioimmunoprecipitation assay lysis buffer, and 35 µg of total cell lysates were separated on 15% SDS-PAGE gel and blotted on Immobilon P transfer membranes (Millipore, Bedford, MA) by semidry transfer using the Trans-Blot semidry apparatus (Bio-Rad Laboratories, Hercules, CA). Membranes were probed with polyclonal rabbit antihuman GH antibodies (ICN Pharmaceuticals, Inc., Eschwege, Germany). As a secondary antibody, antirabbit immunoglobulin (DakoCytomation, Glostrup, Denmark) was used. Protein bands were visualized by enhanced chemiluminescence substrate reagent and exposed on ECL Plus films (Amersham Pharmacia Biotech, Dübendorf, Switzerland). These experiments were repeated five times, and the data are given accordingly (Fig. 3Go).


Figure 3
Figure 3
View larger version (46K):
[in this window]
[in a new window]

  		FIG. 3. Splicing of wt-GH vs. GH-E32A in CHO cells analyzed by Western blot. The cells were transiently transfected with either wt-GH or GH-E32A or cotransfected with wt-GH and GH-E32A. Untransfected CHO cells were used as a negative control. Different GH isoforms were quantitated using ImageQuant software comparing different GH isoforms normalized to ß-actin control. Data are given as mean ± SD after five independent quantification experiments.

 
GH secretion/production after forskolin stimulation

AtT-20 cells were cultured in DMEM+5% fetal calf serum in six-well plates and transfected using FuGene 6 (Roche Diagnostics AG, Rotkreuz, Switzerland). The cells were transiently transfected with 1 µg plasmid in total, containing 1 µg of wt-GH, 0.5 µg of wt-GH and pcDNA3.1, 0.5 µg of wt-GH and GH-E32A, 0.5 µg of GH-E32A and pcDNA3.1, or 1 µg of pcDNA 3.1 as a negative control. Twenty-four hours after transfection, the cells were stimulated with 50 µM forskolin (Sigma Aldrich). Aliquots of culture medium were collected before stimulation (basal) and 15, 30, 60, 90, 120, and 150 min after stimulation, when the cells were washed with PBS and lysed using radioimmunoprecipitation assay buffer. GH was measured in the aliquots of culture medium (extracellular) and in lysed cell extracts (intracellular) by the DSL-10–1900 Active human GH ELISA kit (Diagnostic Systems Laboratories, Webster, TX). These experiments were performed five times in triplicates. Furthermore, intracellular GH content was analyzed in stimulated as well as nonstimulated cells.

Hormonal measurement

Human GH immunochemiluminometric assays and DSL-GH ELISA measurements were performed as previously described (23).

Confocal microscopy analysis/nucleofection and AtT-20 cell proliferation assay

The preparation of the AtT-20 cells, immunofluorescence staining, and the antibodies used have all been previously reported (22, 24)

Statistical analysis

The statistical significance of results from GH production, confocal studies, and cell proliferation assays in AtT-20 cells was assessed using ANOVA one-way test plus Dunnet’s multiple comparison tests, comparing GH-E32A with wt-GH.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Production of different splice variants (protein) in cells expressing wt-GH vs. GH-E32A

To investigate the differences in the splice products of wt-GH vs. GH-E32A after forskolin stimulation in CHO cells, we performed Western blot analysis (Fig. 3Go). As expected, transfected wt-GH produced mostly the 22-kDa GH isoform (88.2%), only small amounts of 20-kDa (10.1%) and almost undetectable amounts of 17.5-kDa (1.7%). However, the transfected GH-E32A mutant yielded a reduced proportion of 22-kDa GH isoform (24.7%) but at the same time an increase in proportion of 20-kDa (19.5%) and especially the 17.5-kDa isoform (55.8%). When CHO cells were cotransfected with wt-GH and GH-E32A, the amounts of 22, 20, and 17.5 kDa produced were 64.4, 13.5, and 22.1%, respectively. These data contrast with the amount of 88.2% (22 kDa), 10.1% (20 kDa), and 1.7% (17.5 kDa) found whenever wt-GH/wt-GH was transfected into CHO cells (Fig. 3Go). Data are indicated in Fig. 3Go as mean ± SD of five independent experiments.

The secretion of GH in the supernatant of AtT-20 cells

Having analyzed the production of different GH isoforms in the cell derived from the GH-E32A mutant through aberrant mRNA splicing, we investigated the possible impact of the increased production of the 17.5-kDa isoform on the secretion of the wt-GH. AtT-20 cells were transfected individually with either wt-GH (wt-GH/wt-GH or wt-GH/pcDNA 3.1) or GH-E32A (E32A/E32A or E32A/pcDNA 3.1) or cotransfected with both wt-GH and GH-E32A (wt-GH/ E32A) (Fig. 4AGo). Cells transfected with pcDNA 3.1 were used as a negative control. The transfection efficiency was checked by using an enhanced green fluorescent protein (EGFP)-N1 plasmid; cells were analyzed by fluorescent microscopy and efficiency was found to be consistent among independent experiments.


Figure 4
View larger version (34K):
[in this window]
[in a new window]

  		FIG. 4. A, GH secretion/production after forskolin stimulation 24 h after transfection, AtT-20 cells were stimulated with 50 µM forskolin for 2.5 h. Aliquots of culture medium were taken for GH measurement before stimulation (basal release) and at the time points shown after stimulation. These experiments were performed five times in triplicate. The amount of GH measured in the medium (8.9 ng/ml) in which the wt-GH transfected AtT-20 cells (wt/wt) were grown was set as 100%, and the other measurements were calculated with respect to this. Overall, a significant increase (P < 0.01) of GH release was found after 120 min. Importantly, GH-E32A showed a significant reduced release effect (P < 0.05 at 120 min) on wt-GH when double transfected (wt/pcDNA3 vs. wt/E32A). *, P < 0.05; **, P < 0.01. B, Intracellular GH production after forskolin stimulation 24 h after transfection; AtT-20 cells were stimulated with 50 µM forskolin for 2.5 h. Stimulated as well as nonstimulated cells were lysed, and intracellular GH content was measured at the time points given. These experiments were performed five times in triplicate. The amount of basal GH measured (21.5 ng/ml), in which the wt-GH transfected AtT-20 cells (wt/wt) were grown, was set as 100%, and the other measurements were calculated accordingly. An increase of intracellular GH was found after 60 min, which became statistically significant after 90 min (P < 0.05) except in the experiments in which wt/E32A was tested. Importantly, the increase of GH content appeared 30–60 min before the GH release into the medium. *, P < 0.05. S, Stimulated; NS, nonstimulated.

 
Twenty-four hours after transfection, AtT-20 cells were stimulated with 50 µM forskolin for 2.5 h. Aliquots of culture medium were taken for GH measurement before stimulation (basal release) and at given time points after stimulation (30 min interval up until 150 min). These experiments were performed five times in triplicate. The basal amount of GH measured in the medium (8.9 ng/ml), in which the wt-GH transfected AtT-20 cells (wt/wt) were grown, was set as 100%, and the other measurements were compared against this. Overall, a significant increase (P < 0.01) of GH release was found after 120 min. The GH-E32A mutant presented a significantly reduced GH release (P < 0.05) vs. wt-GH when stimulation in double-transfected (wt/pcDNA3 vs. wt/E32A) AtT20 cells was assessed.

However, because the basal GH concentration in medium varied, depending on the transfected plasmid containing either wt-GH or GH-E32A, the differences ({Delta}) between basal and peak GH concentrations were assessed. {Delta} values were 53.3 ± 2.5, 37.0 ± 3.1, 30.0 ± 2.2, 21.8 ± 1.8, and 21.7 ± 2.0% in wt/wt, wt/pcDNA3, wt/E32A, E32A/E32A, and E32A/pcDNA3, respectively. Furthermore, GH-E32A significantly affects not only basal GH release (Fig. 4AGo; wt/pcDNA3 vs. E32A/pcDNA3: P < 0.01) but also has an impact on its stimulated production and later release (at 120 min wt/pcDNA3 vs. E32A/pcDNA3: P < 0.05). Note also that GH-E32A has a major impact in reducing GH release when double transfected with wt-GH (wt/E32A) and compared with wt/wt (P < 0.01 at 120 min).

Intracellular GH content after forskolin stimulation

Twenty-four hours after transfection, GH was measured in AtT-20 cells stimulated with 50 µM forskolin for 2.5 h. Stimulated as well as nonstimulated cells were lysed and intracellular GH content was measured following the time frame given. These experiments were performed five times in triplicate (Fig. 4BGo). The amount of basal GH measured (21.5 ng/ml) in the wt-GH transfected AtT-20 cells (wt/wt) was set as 100%, and the other measurements were calculated accordingly. A mean increase of intracellular GH was seen after 60 min, which became statistically significant after 90 min (P < 0.05) except in the experiments in which wt/E32A was tested. Importantly, the increases of GH content were observed 30–60 min before the increased GH release into the medium. These data underline the dominant-negative impact of GH-E32A on wt-GH production.

Intracellular localization of wt-GH and GH-E32A analyzed by confocal microscopy

To analyze the impact on the secretory pathway, colocalization of the wt-GH or GH-E32A within different compartments of the secretory pathway was studied in AtT-20 cells, applying quantitative confocal microscopy analysis (Fig. 5Go). AtT-20 cells were transiently transfected with either wt-GH or GH-E32A, and the expression pattern was examined 24 h after transfection. Again, we checked the transfection efficiency using EGFP-N1 and found it to be consistent among different experiments. Panel I of Fig. 5Go shows the overall distribution of wt-GH and GH-E32A, with a perinuclear region having the heaviest staining, which is consistent with accumulation in the endoplasmic reticulum and Golgi complex, and in a punctate staining near the plasma membrane, being consistent with accumulation of GH peptide in secretory granules. Costaining with antibodies against markers for endoplasmic reticulum (anti-Grp94), Golgi (anti-ßCOP), and/or granules (anti-Rab3a) were performed, and confocal microscopy images of independent cells were analyzed. Fluorescent intensities of colocalized areas were measured for wt-GH and GH-E32A with each organelle, and the degree of colocalization of two fluorochrome signals was measured from a single cell. The average Pearson correlation coefficients did not reveal any significant difference in the extent of subcellular colocalization between wt-GH and GH-E32A with endoplasmic reticulum or Golgi. However, we observed significantly lower colocalization (P < 0.01) between wt-GH and GH-E32A with secretory granules.


Figure 5
View larger version (26K):
[in this window]
[in a new window]

  		FIG. 5. Subcellular colocalization of wt-GH and GH-E32A with different organelle markers in fixed cells. A, GH and Grp94 (endoplasmic reticulum marker) colocalization. B, GH and ßCOP (Golgi marker). C, GH and Rab3a (secretory granules). Row I, Specific staining for wt-GH or GH-E32A is indicated by fluorescent green color (fluorescein isothiocyanate labeled). Row II, Positive staining for cell organelles is shown by fluorescent red color (Cy3 labeled) in the same microscopic field as in row I. Row III, Merged images. Row IV, Masked areas, in which the intensity of the green and red fluorescence was measured in the colocalized area. Below each set of confocal images, the degree of colocalization between GH and that given organelle is presented as the average of Pearson correlation coefficients for 20 independent cells (means ± SD). **, P < 0.01.

 
Cell proliferation assay

To support and complement the secretion studies, which clearly showed a dominant-negative effect of GH-E32A on the secretion of the wt-GH, cell proliferation assays were performed. Cell proliferation was compared after 24, 48, and 72 h after transfection of AtT-20 cells with either wt-GH or GH-E32A or cotransfected with wt-GH and GH-E32A (Fig. 6Go). Transfection efficiency in viable AtT-20 cells was checked using EGFP-N1 as a positive control and was found to be 75% ± 5% (average of three independent experiments). Throughout the first 2 d of the time course, we did not observe any significant difference in the proliferation rate of cells expressing wt-GH or GH-E32A or coexpressing wt-GH and GH-E32A (Fig. 6Go). In contrast, at d 3 the cells expressing GH-E32A or coexpressing wt-GH and GH-E32A exhibited significantly lower cell proliferation rates (P < 0.05) than those expressing wt-GH. These results support the idea that GH-E32A not only reduces the proliferation capability of AtT-20 cells but also may exert a dominant-negative effect on wt-GH in cell viability and/or proliferation rate in these cells. However, a dose-dependent stimulation by GH cannot fully be excluded.


Figure 6
View larger version (8K):
[in this window]
[in a new window]

  		FIG. 6. Cell proliferation was assessed using the MTS assay. AtT-20 cells transiently transfected with wt-GH, wt-GH and GH-E32A, GH-E32A, and pUC18 vector as control. Proliferation was estimated at 24, 48, and 72 h after transfection. Results are expressed as mean OD of three independent experiments in triplicate (means ± SD).

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
We report two families presenting with the exon 3 + 2 A->C mutation leading to the substitution of glutamic acid by alanine at position 32 in the GH molecule, resulting in the production of a mutant form of GH (GH-E32A). This mutation was found in the heterozygous state in all affected individuals with this familial IGHD II, available for GH-1 gene screening.

Clinically, these two families demonstrate clearly that the E32A mutation can generate a variable range of growth retardation. Adult heights of untreated individuals (height SDS –2.9 to –4.5), however, are on average greater than those seen with IGHD due to homozygous mutations in the GH-1 gene (25). In addition, the severity of the GHD defined by stimulation tests may be variable and even minimally different from normal after GH replacement therapy, at least for a short period of time before newly relapsing.

Because this mutation alters the coding sequence, one possibility is that the GH-E32A mutant has an impact on binding or signaling of the GH receptor in a dominant-negative fashion. We made and tested this mutant and found normal binding as well as signaling, compared with wt-GH (Besson, A., and P. E. Mullis, unpublished data). A more likely possibility is that it alters splicing, giving rise to a mutant allele product. Thus, the expression of studies in CHO cells revealed that wt-GH splicing almost exclusively produced the 22-kDa isoform with barely detectable amounts of 17.5-kDa GH isoform. However, GH-E32A led to a significant increase in missplicing and production of the 17.5-kDa GH isoform (55% of total GH protein). These data are in agreement with the data of Moseley et al. (16) obtained at the mRNA level. These data are therefore of vital importance to our understanding of GH gene splicing and suggest that, depending on the location of the mutation within the GH-1 gene, not only with respect to the splice sites (26) but also to the ESE, the splicing of exon 3 varies and therefore impacts to differing extents on the quantity of 17.5 kDa GH produced, ultimately leading to variable phenotypic expression of IGHD II (27, 28, 29, 30, 31, 32, 33).

The production and secretion of abundant quantities of peptide hormones is the major task of peptide-secreting neuroendocrine cells. A dominant-negative effect of the 17.5-kDa GH isoform on the secretion of 22-kDa GH has been demonstrated by several independent studies (9, 10, 12). Because the GH-E32A mutant generated an increased amount of 17.5-kDa GH isoform, compared with wt-GH, we investigated the possible dominant-negative effect of this mutant on the secretion of the wt-GH.

Forskolin-stimulated release of GH was studied in either single- or double-transfected (wt-GH and GH-E32A) AtT-20 cells. Although GH-E32A cells already showed a significantly different basal GH production (P < 0.01) when compared with wt-GH cells, the stimulated GH release peaked overall at 120 min (Fig. 4AGo). Furthermore, cells coexpressing both wt-GH and GH-E32A (as in our heterozygote subjects) showed a significant reduction in GH release after forskolin stimulation when compared with those expressing wt-GH (wt/wt: P < 0.01; wt/pcDNA3: P < 0.05). These results suggest that GH-E32A exerts a dominant-negative effect on the release of the wt-GH and correlate with the impaired GH secretion observed in the patients carrying the heterozygous GH-E32A mutation. Although the intracellular GH production peaked 30–60 min before the GH release into the medium and paralleled those secretory data obtained, the mechanism leading to a dramatically reduced release is most likely explained by an increased production and accumulation of the 17.5-kDa GH isoform (26).

With GH-E32A, some wild-type as well as some 17.5 kDa (exon 3 skipped GH isoform) is produced (22 kDa: 64.4%; 17.5 kDa: 22.1%; Fig. 3Go), so the E32A mutant form of GH might not have a fully cytotoxic effect on the somatotroph cells as has been suggested in other autosomal dominant endocrine disorders (28, 34, 35). Furthermore, the mutant form might rather have a time-dependent impact on the regulated secretory pathway, blocking its secretion over time. However, this seems to be fully reversible as has been shown in subjects presenting with the R183H mutant GH form (24, 36). The somatotroph cells might recover after the removal of the constant endogenous GHRH stimulation after exogenous GH replacement to normalize IGF-I levels. This hypothesis is underlined by the fact that the patient (II-1, English family; Fig. 1BGo), when retested after GH replacement, appeared GH sufficient but developed GHD again later. Similar results could be obtained when testing the Swiss family for GHD before, during, and after a trial of human GH treatment. From the practical point of view, it is important to remember this mechanism of IGHD II as a potential cause of temporary reversibility of GHD after the cessation of rhGH treatment at the time of transition.

The secretory pathway consists of a series of membrane-bound compartments through which proteins targeted for secretion are moved (37, 38, 39). We compared the association of either wt-GH or GH-E32A with different compartments within the secretory pathway, namely endoplasmic reticulum, Golgi, and secretory vesicles, by costaining with antibodies against GH and against markers of these organelles. Our results revealed that in the case of both wt-GH and GH-E32A, no significant difference was observed in colocalization with endoplasmic reticulum or Golgi. These findings suggest that both GH variants are successfully folded, sorted, and exported from the endoplasmic reticulum and that they pass through the Golgi apparatus in a similar way. However, GH-E32A presented a significant reduction in colocalization with secretory granules, compared with wt-GH. These results correlate with the secretion results obtained in AtT-20 cells in which a slight reduction in secretion of GH-E32A, compared with wt-GH, was observed.

The variations in the levels of the 17.5-kDa GH isoform may be correlated with the severity of clinical phenotypes observed in some patients with IGHD II (27, 28, 40). Transgenic mice having high copy numbers of the {Delta}3 allele presented a more severe phenotype than mice with low copy numbers (9). Moreover, the data from the same study suggested that due to the inability to be secreted, the complexes of the 17.5- and 22-kDa isoforms accumulate in the endoplasmic reticulum, Golgi, and cytosol, eventually proving toxic to the cell and causing cell death. Therefore, we attempted to investigate the proliferation rate and viability of AtT-20 cells expressing wt-GH and GH-E32A singly or coexpressed together. The data obtained clearly demonstrated the dominant-negative effect of GH-E32A on wt-GH in proliferation and/or viability of AtT-20 cells.

In conclusion, we report a novel E32A mutation in the GH molecule found in two different pedigrees with IGHD II. A single-nucleotide change (exon 3 + 2 A->C) leads to aberrant mRNA splicing generating high concentrations of the 17.5-kDa GH isoform, compared with wt-GH. Due to increasing production of the 17.5-kDa isoform relative to the 22-kDa isoform, the GH-E32A mutant exhibited a dominant-negative effect on wt-GH on both secretion and cell proliferation, as investigated in AtT-20 cells.


   Footnotes
 
This work was supported by Grant 3200BO-105853 from the Swiss National Science Foundation (to P.E.M.). In addition, P.E.M. is recipient of a Sabbatical Leave Grant sponsored by the European Society for Pediatric Endocrinology and is currently at the National Institute for Medical Research at Mill Hill (London, UK).

Disclosure Statement: The authors have nothing to disclose.

First Published Online August 28, 2007

Abbreviations: EGFP, Enhanced green fluorescent protein; ESE, exon splice enhancer; GHD, GH deficiency; IGHD, isolated GHD; IGHD II, IGHD type II; rhGH, recombinant human GH; SDS, SD score; wt, wild-type.

Received April 16, 2007.

Accepted August 21, 2007.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

  1. Lacey KA, Parkin JM 1974 Causes of short stature. Lancet 1:42–45[CrossRef][Medline]
  2. Rona RJ, Tanner JM 1977 Aetiology of idiopathic growth hormone deficiency in England and Wales. Arch Dis Child 52:197–208[Abstract/Free Full Text]
  3. Vimpani GV, Vimpani AF, Lidgard GP, Cameron EHD, Farquhar JW 1977 Prevalence of severe growth hormone deficiency. Br Med J 2:427–430[Abstract/Free Full Text]
  4. Mullis PE, Deladoey J, Dannies PS 2002 Molecular and cellular basis of isolated dominant-negative growth hormone deficiency, IGHD type II: insights on the secretory pathway of peptide hormones. Horm Res 58:53–66[CrossRef][Medline]
  5. Binder G, Ranke MB 1995 Screening for growth hormone (GH) gene splice-site mutations in sporadic cases with severe isolated GH deficiency using ectopic transcript analysis. J Clin Endocrinol Metab 80:1247–1252[Abstract]
  6. De Vos AM, Ultsch M, Kossiakoff AA 1992 Human growth hormone and extracellular domain of its receptor: crystal structure of the complex. Science 255:306–312[Abstract/Free Full Text]
  7. Cunningham BC, Ultsch M, De Vos AM, Mulkerrin MG, Clauser KR, Wells JA 1991 Dimerization of the extracellular domain of the human growth hormone receptor by a single hormone molecule. Science 254:821–825[Abstract/Free Full Text]
  8. Lee MS, Wajnrajch MP, Kim SS, Plotnick LP, Wang J, Gertner JM, Leibel RL, Dannies PS 2000 Autosomal dominant growth hormone (GH) deficiency type II: the Del32–71-GH deletion mutant suppresses secretion of wild-type GH. Endocrinology 141:883–890[Abstract/Free Full Text]
  9. McGuinness L, Magoulas C, Sesay AK, Mathers K, Carmignac D, Manneville JB, Christian H, Phillips 3rd JA, Robinson IC 2003 Autosomal dominant growth hormone deficiency disrupts secretory vesicles in vitro and in vivo in transgenic mice. Endocrinology 144:720–731[Abstract/Free Full Text]
  10. Hayashi Y, Yamamoto M, Ohmori S, Kamijo T, Ogawa M, Seo H 1999 Inhibition of growth hormone (GH) secretion by a mutant GH-I gene product in neuroendocrine cells containing secretory granules: an implication for isolated GH deficiency inherited in an autosomal dominant manner. J Clin Endocrinol Metab 84:2134–2139[Abstract/Free Full Text]
  11. Graves TK, Patel S, Dannies PS, Hinkle PM 2001 Misfolded growth hormone causes fragmentation of the Golgi apparatus and disrupts endoplasmic reticulum-to-Golgi traffic. J Cell Sci 114:3685–3694[Medline]
  12. Ryther RC, McGuinness LM, Phillips 3rd JA, Moseley CT, Magoulas CB, Robinson IC, Patton JG 2003 Disruption of exon definition produces a dominant-negative growth hormone isoform that causes somatotroph death and IGHD II. Hum Genet 113:140–148[Medline]
  13. Cogan JD, Prince MA, Lekhakula S, Bundey S, Futrakul A, McCarthy EM, Phillips 3rd JA 1997 A novel mechanism of aberrant pre-mRNA splicing in humans. Hum Mol Genet 6:909–912[Abstract/Free Full Text]
  14. Cogan JD, Ramel B, Lehto M, Phillips 3rd J, Prince M, Blizzard RM, de Ravel TJ, Brammert M, Groop L 1995 A recurring dominant negative mutation causes autosomal dominant growth hormone deficiency—a clinical research center study. J Clin Endocrinol Metab 80:3591–3595[Abstract]
  15. Takahashi I, Takahashi T, Komatsu M, Sato T, Takada G 2002 An exonic mutation of the GH-1 gene causing familial isolated growth hormone deficiency type II. Clin Genet 61:222–225[CrossRef][Medline]
  16. Moseley CT, Mullis PE, Prince MA, Phillips 3rd JA 2002 An exon splice enhancer mutation causes autosomal dominant GH deficiency. J Clin Endocrinol Metab 87:847–852[Abstract/Free Full Text]
  17. McCarthy EM, Phillips 3rd JA 1998 Characterization of an intron splice enhancer that regulates alternative splicing of human GH pre-mRNA. Hum Mol Genet 7:1491–1496[Abstract/Free Full Text]
  18. Ryther RC, Flynt AS, Harris BD, Phillips 3rd JA, Patton JG 2004 GH1 splicing is regulated by multiple enhancers whose mutation produces a dominant-negative GH isoform that can be degraded by allele-specific small interfering RNA (siRNA). Endocrinology 145:2988–2996[Abstract/Free Full Text]
  19. Prader A, Largo RH, Molinari L, Issler C 1989 Physical growth of Swiss children from birth to 20 years of age. Helv Paediatr Acta 52(Suppl):1–125
  20. Ranke MB 2003 Diagnosis of growth hormone deficiency and growth hormone stimulation tests. In: Ranke MB, ed. Diagnosis of endocrine function in children and adolescents. Basel, Switzerland: Krager
  21. Greulich WW, Pyle SI 1959 Radiographic atlas of skeletal development of the hand and wrist. 2nd ed. Stanford, CA: Stanford University Press
  22. Petkovic V, Besson A, Thevis M, Lochmatter D, Eble A, Fluck CE, Mullis PE 2007 Evaluation of the biological activity of a GH mutant (R77C) and its impact on GH responsiveness and stature. J Clin Endocrinol Metab 92:2893–2901[Abstract/Free Full Text]
  23. Besson A, Salemi S, Deladoey J, Vuissoz JM, Eble A, Bidlingmaier M, Burgi S, Honegger U, Fluck C, Mullis PE 2005 Short stature caused by a biologically inactive mutant growth hormone (GH-C53S). J Clin Endocrinol Metab 90:2493–2499[Abstract/Free Full Text]
  24. Salemi S, Yousefi S, Baltensperger K, Robinson IC, Eble A, Simon D, Czernichow P, Binder G, Sonnet E, Mullis PE 2005 Variability of isolated autosomal dominant GH deficiency (IGHD II): impact of the P89L GH mutation on clinical follow-up and GH secretion. Eur J Endocrinol 153:791–802[Abstract/Free Full Text]
  25. Besson A, Salemi S, Gallati S, Jenal A, Horn R, Mullis PS, Mullis PE 2003 Reduced longevity in untreated patients with isolated growth hormone deficiency. J Clin Endocrinol Metab 2003 88:3664–3667[Abstract/Free Full Text]
  26. Salemi S, Yousefi S, Lochmatter D, Eble A, Deladoey J, Robinson IC, Simon HU, Mullis PE 2007 Isolated autosomal dominant growth hormone deficiency: stimulating mutant GH-1 gene expression drives GH-1 splice-site selection, cell proliferation, and apoptosis. Endocrinology 148:45–53[Abstract/Free Full Text]
  27. Procter AM, Phillips III JA, Cooper DN 1998 The molecular genetics of growth hormone deficiency. Hum Genet 103:255–272[CrossRef][Medline]
  28. Mullis PE, Robinson IC, Salemi S, Eble A, Besson A, Vuissoz JM, Deladoey J, Simon D, Czernichow P, Binder G 2005 Isolated autosomal dominant growth hormone deficiency: an evolving pituitary deficit? A multicenter follow-up study. J Clin Endocrinol Metab 90:2089–2096[Abstract/Free Full Text]
  29. Missarelli C, Herrera L, Mericq V, Carvallo P 1997 Two different 5' splice site mutations in the growth hormone gene causing autosomal dominant growth hormone deficiency. Hum Genet 101:113–117[CrossRef][Medline]
  30. Phillips 3rd JA, Cogan JD 1994 Genetic basis of endocrine disease. 6. Molecular basis of familial human growth hormone deficiency. J Clin Endocrinol Metab 78:11–16[CrossRef][Medline]
  31. Fofanova OV, Evgrafov OV, Polyakov AV, Poltaraus AB, Peterkova VA, Dedov, II 2003 A novel IVS2–2A>T splicing mutation in the GH-1 gene in familial isolated growth hormone deficiency type II in the spectrum of other splicing mutations in the Russian population. J Clin Endocrinol Metab 88:820–826[Abstract/Free Full Text]
  32. Millar DS, Lewis MD, Horan M, Newsway V, Easter TE, Gregory JW, Fryklund L, Norin M, Crowne EC, Davies SJ, Edwards P, Kirk J, Waldron K, Smith PJ, Phillips 3rd JA, Scanlon MF, Krawczak M, Cooper DN, Procter AM 2003 Novel mutations of the growth hormone 1 (GH1) gene disclosed by modulation of the clinical selection criteria for individuals with short stature. Hum Mutat 21:424–440[CrossRef][Medline]
  33. Mullis PE 2005 Genetic control of growth. Eur J Endocrinol 152:11–31[Abstract/Free Full Text]
  34. Ito M, Jameson JL, Ito M 1997 Molecular basis of autosomal dominant neurohypophyseal diabetes insipidus. Cellular toxicity caused by the accumulation of mutant vasopressin precursors within the endoplasmic reticulum. J Clin Invest 99:1897–1905[Medline]
  35. Arnold A, Horst SA, Gardella TJ, Hisamitsu B, Levine MA, Kronenberg HM 1990 Mutation of the signal peptide-encoding region of the preproparathyroid hormone gene in familial isolated hypoparathyroidism. J Clin Invest 86:1084–1087[Medline]
  36. Deladoëy J, Stocker P, Mullis PE 2001 Autosomal dominant GH deficiency due to an Arg183His GH-1 gene mutation: clinical and molecular evidence of impaired regulated GH secretion. J Clin Endocrinol Metab 86:3941–3947[Abstract/Free Full Text]
  37. Dannies PS 1999 Protein hormone storage in secretory granules: mechanisms for concentration and sorting. Endocr Rev 20:3–21[Abstract/Free Full Text]
  38. Dannies PS 2000 Protein folding and deficiencies caused by dominant-negative mutants of hormones. Vitam Horm 58:1–26[CrossRef][Medline]
  39. Dannies PS 2001 Concentrating hormones into secretory granules: layers of control. Mol Cell Endocrinol 177:87–93[CrossRef][Medline]
  40. Binder G, Keller E, Mix M, Massa GG, Stokvis-Brantsma WH, Wit JM, Ranke MB 2001 Isolated GH deficiency with dominant inheritance: new mutations, new insights. J Clin Endocrinol Metab 86:3877–3881[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Clin. Endocrinol. Metab.Home page
K. S. Alatzoglou, J. P. Turton, D. Kelberman, P. E. Clayton, A. Mehta, C. Buchanan, S. Aylwin, E. C. Crowne, H. T. Christesen, N. T. Hertel, et al.
Expanding the Spectrum of Mutations in GH1 and GHRHR: Genetic Screening in a Large Cohort of Patients with Congenital Isolated Growth Hormone Deficiency
J. Clin. Endocrinol. Metab., September 1, 2009; 94(9): 3191 - 3199.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow Submit a related Letter to the Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Petkovic, V.
Right arrow Articles by Mullis, P. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Petkovic, V.
Right arrow Articles by Mullis, P. E.
Related Collections
Right arrow Neuroendocrinology and Pituitary
Right arrow Pediatric Endocrinology

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals