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A Novel Deletion in the GH1 Gene Including the IVS3 Branch Site Responsible for Autosomal Dominant Isolated Growth Hormone Deficiency

Unit of Pediatrics (D.V., G.B.), Department of Medical Sciences, Eastern Piedmont University, 28100 Novara, Italy; Department of Pediatrics (L.G., D.F., B.d.N.), Luigi Sacco Hospital, University of Milan, 20157 Milan, Italy; and Laboratory of Human Genetics (M.Go., P.M.-R., M.Gi.), Department of Medical Sciences, Eastern Piedmont University and Interdisciplinary Research Center on Autoimmune Diseases, 28100 Novara, Italy

Address all correspondence and requests for reprints to: Mara Giordano, Ph.D., Dipartimento di Scienze Mediche, Via Solaroli 17, 28100 Novara, Italy. E-mail: giordano{at}med.unipmn.it.

	Abstract

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Context: The majority of mutations responsible for isolated GH type II deficiency (IGHD II) lead to dominant negative deleteriously increased levels of the GH1 exon 3 skipped transcripts.

Objective: The aim of this study was the characterization of the molecular defect causing a familial case of IGHD II.

Patients: A 2-yr-old child and her mother with severe growth failure at diagnosis (–5.8 and –6.9 SD score, respectively) and IGHD were investigated for the presence of GH1 mutations.

Results: We identified a novel 22-bp deletion in IVS3 (IVS3 del+56–77) removing the putative branch point sequence (BPS). Analysis of patients’ lymphocyte mRNA showed an excess exon 3 skipping. The mutated allele transfected into rat pituitary cells produced four differently spliced products: the exon 3 skipped mRNA as the main product and lower amounts of the full-length cDNA and of two novel mRNA aberrant isoforms, one with the first 86 bases of exon 4 deleted and the other lacking the entire exon 4. A mutagenized construct lacking exclusively the 7 bp of the BPS only generated the exon 4 skipped and the full-length isoforms. The presence of the full-length transcript in the absence of the canonical BPS points to an alternative BPS in IVS3.

Conclusion: The IVS3 del+56–77 mutation, causing IGHD II in this family, has two separate effects on mRNA processing: 1) exon 3 skipping, analogous to most described cases of IGHD II, an effect likely caused by the reduction in size of the IVS3, and 2) partial or total exon 4 skipping, as a result of the removal of the BPS.

	Introduction

Top Abstract Introduction Subjects and Methods Results Discussion References

 
APPROXIMATELY 75% OF the circulating GH is a 191-amino-acid peptide with a molecular mass of 22 kDa. Alternative splicing of the original transcript gives rise to minor amounts of several smaller isoforms. The most represented of these alternative spliced transcripts lacks the first 45 bp of exon 3 through activation of an in-frame cryptic splice site and produces a 20-kDa peptide missing amino acids 32–46 (1). Complete skipping of exon 3, accounting for 1–5% of the total GH1 transcripts, results in a 17.5-kDa form lacking amino acids 32–71. Two isoforms lacking exons 3–4 and exons 2–4 and encoding 11.3- and 7.4-kDa peptides have also been detected (2).

Mutations in the GH1 gene have been identified both in type I (autosomal recessive) and type II (autosomal dominant) familial isolated GH deficiency (IGHD) (2, 3). IGHD II is caused mainly by mutations affecting GH mRNA splicing, falling within the first six bases of the IVS3 5' donor splice site (4, 5, 6, 7, 8) or disrupting splicing enhancer elements. The latter includes two mutations in an exon 3 splicing enhancer, ESEm1 + 1GT (9) and ESEm2 + 5AG (10), and two mutations in an IVS3 splicing enhancer, ISEm1(IVS3 + 28 GA) and ISEm2(IVS328–45) (11, 12). All the reported splicing mutations lead to deleteriously increased levels of the exon 3 skipped transcripts encoding the 17.5-kDa isoform. This isoform lacks the protein linker domain between helix 1 and helix 2 of the mature GH and a cysteine residue, Cys⁵³, involved in the interaction between helix 1 and helix 4 (13). Thus, without the amino acids encoded by the third exon, the molecule cannot fold normally. This altered GH structure exhibits a dominant negative effect on secretion of the 22-kDa isoform. The 17.5-kDa isoform is initially retained in the endoplasmic reticulum, disrupts the Golgi apparatus, impairs both GH and other hormonal trafficking (14), and decreases the intracellular stability of the wild-type GH (15). Moreover, the 17.5-kDa isoform causes a dose-dependent disruption of GH secretory vesicles when expressed in GC cells and transgenic mice (16).

In addition to the above cited splicing mutations, three missense mutations, V110F, P89L, and R183H, are reported (8, 17, 18).

We here describe a novel splicing mutation in an IGHD II family, a 22-bp deletion in the IVS3 of the GH1 gene encompassing the branch point sequence (BPS) (11). This mutation generates at least three aberrant isoforms: the more abundant exon 3 skipped isoform and two minor isoforms lacking the first 86 nucleotides of exon 4 and the entire exon 4, respectively.

	Subjects and Methods

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Subjects

The proband, a 2-yr-old girl, came to our observation for severe growth failure at the age of 7 months. She was born by spontaneous delivery after a normal pregnancy at 36 wk gestation with a weight of 2720 g (25th percentile) and a length of 44 cm (3rd percentile). Her phenotype had the IGHD characteristic features: slow but harmonious body development with regular segment proportions, prominent frontal bones, and saddle nose. After biochemical evaluations that excluded organic diseases, two stimulation tests for GH secretion study were performed showing a complete GH deficiency (immunoradiometric assay; DiaSorin, Stillwater, MN) confirmed by the IGF-I dosage (chemiluminescent enzyme-labeled immunometric assay; Medical System, Los Angeles, CA) (Table 1). Analysis of TSH (electrochemiluminescence immunoassay; Roche, Indianapolis, IN) and cortisol (RIA; Adaltis, Montreal, Canada) excluded other anterior pituitary hormone deficiencies (Table 1). The magnetic resonance imaging of the pituitary region performed with narrow scanning and gadolinium injection reported a normal anatomy of the region with the sagittal T1 images showing a mild hypoplasia of the pituitary gland with a height at –2.0 SD score (SDS) for age (21). Therapy with human recombinant GH, started at the age of 9 months at the dose of 0.033 mg/kg·d, yielded a very good response; the growth velocity reached 20 cm/yr (Table 1).

PCR amplification and sequencing of the GH1 gene

Genomic DNA was amplified by PCR using primers flanking the GH1 gene and a proofreading Taq polymerase (Finnzymes, Espoo, Finland). The resulting 2.7-kb product was used as template for a series of nested PCRs using internal primers for the proximal promoter, the five exons, the four introns, and the untranslated regions of the GH1 gene. PCR conditions and primer sequences are available upon request.

The resulting PCR products were visualized on a 2% agarose gel, purified from the gel using a Perfectprep Gel Cleanup system (Eppendorf, Hamburg, Germany) and used as template for sequencing in both senses with the Big-dye terminator cycle sequencing reaction kit (Applied Biosystems, Foster City, CA) and an ABI 3100 automated sequencer.

The PCR product containing the IVS3 deletion in the heterozygous state was then cloned by the pMOSBlue T-vector kit (Amersham Biosciences, Little Chalfont, UK) following the manufacturer’s instructions, and the two alleles were separately sequenced.

Synthesis and amplification of GH cDNA

Total RNA was extracted from lymphocytes by RNAwiz (Ambion, Austin, TX). cDNA was obtained by RT-PCR using the RETROscript Kit (Ambion) and amplified with three different couples of primers designed to amplify different parts of the wild-type cDNA (Fig. 1).

View larger version (11K): [in this window] [in a new window]   FIG. 1. Scheme of GH1 pre-mRNA splicing showing the full-length mRNA and the RT-PCR products. RT-PCR1 performed with primers GH1 (5'-TTCCCAACCATTCCCTTATC-3') and GH6 (5'-TCTTCCAGCCTCCCCATCAGC-3) amplifies part of exon 2, exon 3, and exon 4 allowing detection of exon 3 skipped products. RT-PCR2 performed with primers GH5 (5'-TACCAGGAGTTTGAAGAAGC-3') and GH31 (5'-TCCAGGGCCAGGAGAGGCACTGGGG-3') amplifies exon 3, exon 4, and exon 5 allowing detection of exon 4 skipped products. RT-PCR3 performed with primers GH2 (5'-CGTCTGCACCAGCTGGCCTTT-3') and GH7 (5'-AAGCCACAGCTGCCCTCCACAGA-3') amplifies part of exon 2, exon 3, exon 4, and part of exon 5 allowing detection of both exon 3 and exon 4 skipped products.

Plasmid preparation and cell transfection

The 2700-bp PCR product encompassing the entire GH1, obtained from the proband’s genomic DNA, was cloned into the pMOS plasmid (pMOSBlue T-vector kit; Amersham). Two clones containing the wild-type and the mutated allele, respectively, were selected by sequencing. A fragment of 2 kb containing the whole GH1 sequence was released by digestion with BamHI, purified from gel by the Qiaquick PCR purification kit (QIAGEN, Hilden, Germany), and inserted into the pcDNA3.1(+) expression vector (Invitrogen, Chatsworth, CA) previously digested with BamHI.

The in vitro mutagenized mut7 was generated from the wild-type GH1-carrying plasmid by the QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) using two mismatch complementary oligonucleotides.

DH101F' competent cells were then transformed with the different constructs and grown on LB/ampicillin media.

Plasmid DNA was extracted (Plasmid Midiprep; QIAGEN), sequenced, and used for transient transfection of the rat pituitary GH4C1 cell line (American Type Culture Collection, Rockville, MD). Cells were grown to approximately 80% confluence in 35-mm dishes and transfected with 2 µg of each construct using the Fugene 6 transfection reagent (Roche), following the manufacturer’s instructions. Total RNA was purified from rat cells 48 h after transfection by RNeasy Mini Kit (QIAGEN) and reverse transcribed.

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Sequencing of the GH1 gene

Sequencing of the GH1 gene in the proband of an IGHD II family revealed the presence of a heterozygous 22-bp deletion in IVS3 (Fig. 2). The deletion is flanked by a stretch of five Cs (nt 53–57 of the IVS3) at the 5' side and of three Cs (nt 75–77) at the 3' side. Because three of these Cs are retained in the deleted allele, it is not possible to exactly define the limits of the deletion. We arbitrarily named the mutation as IVS3 del+56–77. The deletion includes the IVS3 BPS, which extends from nt 67 to nt 73 of IVS3 (between –25 and –20 from the 3' splice site) (11). The same deletion was detected in the proband’s mother (subject II-2, Fig. 3A), whereas it was absent in all the tested unaffected family members.

View larger version (11K): [in this window] [in a new window]   FIG. 2. Sequence of GH1 IVS3 and of part of exons 3 and 4. The intron splicing enhancer (ISE) and the BPS are in bold. The putative cryptic splice site is boxed. In IVS3 and exon 4, the cryptic BPSs are underlined.

View larger version (53K): [in this window] [in a new window]   FIG. 3. A, Pedigree of the IGHD II family. Black symbols indicate affected individuals. For each member are indicated the stature (expressed in centimeters; only in unaffected individuals), the GH1 promoter haplotypes resulting, from top to bottom, from the allelic combinations of single-nucleotide polymorphisms –278G/T, –75A/G, –57T/G, –6A/G, +3G/C, and the IVS3 sequence (wt, wild-type; del, IVS3 del+56–77 mutation). From family segregation, it was possible to reconstruct both GH1 haplotypes (indicated with a broken line) for the deceased individual I-1. The haplotype in which the de novo mutation occurred is boxed. The proband is indicated by an arrow. B, RT-PCR1 performed on lymphocyte mRNA; C, RT-PCR2 performed on lymphocyte mRNA. The gel lanes are positioned under the corresponding individual. All the visible bands were extracted from the gel and sequenced.

Analysis of GH1 transcripts in lymphocytes

Ectopic transcript analysis was performed on cDNA from lymphocyte mRNA of all the family members. Two couples of primers were specifically designed to detect an aberrant splicing involving either exon 3 (RT-PCR1) or exon 4 (RT-PCR2), immediately 5' and 3' of the intron with the deletion (Fig. 1).

After amplification with RT-PCR1 primers (Fig. 3B), a band of 389 bp corresponding to the wild-type full-length cDNA was detected in all the individuals. In addition, the affected subjects III-1 and II-2 showed a smaller band of 269 bp with approximately the same intensity of the full-length band. This 269-bp band was scarcely detectable in the normal-height members and corresponded to the 17.5-kDa isoform lacking the entire exon 3. A faint 344-bp band, corresponding to the 20-kDa isoform, was visible in all the subjects.

Most of the previously reported BPS mutations in humans lead to either the partial or total skipping of the exon that follows the mutated intron (25, 26, 27, 28) or to the retention of the intron carrying the mutated BPS (29). The presence of aberrant splicing products involving exon 4 was therefore investigated by RT-PCR2 (Fig. 3C). A unique band of 548 bp, corresponding to the full-length transcript, was detected both in the affected and unaffected family members.

Thus, the information deriving from lymphocyte mRNA analysis was that the IVS3 deletion induces the production of a remarkable amount of mRNA lacking exon 3.

In vitro expression of the GH1 allele harboring the IVS3 del+56–77 mutation

To assess whether the skipping of exon 3 was the only aberrant product generated by the del+56–77 mutation, we analyzed the transcripts of the mutated allele in pituitary cells in vitro. To this purpose, we transfected GH4C1 rat pituitary cells with an expression vector containing either the normal GH1 allele (wt-GH1) or the GH1 allele with the IVS3 del+56–77 mutation (del-GH1). Because the two constructs were independently transfected, this analysis also allowed us to discriminate the products of the mutant and of the wild-type allele, which were coamplified in the lymphocyte cDNA.

The mRNAs from transfected cells were reverse transcribed and amplified with RT-PCR1 and RT-PCR2 primers. These are specific for the human GH1 and did not amplify the rat mRNA from untransfected GH4C1 cells (Fig. 4, lane 2).

View larger version (45K): [in this window] [in a new window]   FIG. 4. RT-PCR performed on the mRNA extracted from GH4C1 pituitary cells: untransfected (rat cDNA; lane 2), transfected with the wild-type GH1 gene (wt-GH1; lane 3), transfected with the GH1 allele harboring the IVS3 del+56–77 mutation (del-GH1; lane 4), and transfected with the mutagenized GH1 gene harboring the 7-bp BPS deletion (mut7; lane 5). The RT-PCR was performed with the three couples of primers of RT-PCR1 (A), RT-PCR2 (B), and RT-PCR3 (C). Each PCR product was eluted from the gel and characterized by sequencing. The result is outlined on the right of each band. *, Heteroduplex molecules formed by mispairing of the 398- and 233-bp fragment.

When amplified with RT-PCR2 primers (Fig. 4B), which do not anneal to the exon 3 skipped product, the cDNA from cells transfected with wt-GH1 only contained the full-length transcript (lane 3), whereas del-GH1 generated three differently spliced mRNAs (lane 4) corresponding to 1) the full-length mRNA (548 bp), 2) a 462-bp transcript in which the first 86 bp of the exon 4 were deleted, and 3) a 383-bp transcript in which the 165 bp of exon 4 were absent. Thus, in pituitary cells transfected with the deleted allele, it was possible to detect two novel aberrantly spliced isoforms involving exon 4 that were not visible in the patients’ lymphocytes and in pituitary cells transfected with the wild-type allele. Moreover, the RT-PCR2 primers showed that the mutant allele also produces a small amount of the full-length mRNA.

To investigate which of the above described alternatively spliced fragments was the direct consequence of the BPS deletion, the wt-GH1 plasmid was mutagenized to obtain a sequence lacking exclusively the 7 bp of the BPS consensus (mut7). The cDNA produced by mut7 showed only the aberrant exon 4 skipped fragments as expected for a BPS mutation (Fig. 4, A and B). As observed for del-GH1, the mut7 construct also produced a visible amount of the full-length cDNA.

A third set of primers (RT-PCR3) located within exons 1–2 and exon 5 was designed to amplify all the isoforms in the same reaction (Fig. 1). Because we did not perform a quantitative PCR, we can only roughly estimate the relative amount of the different isoforms, with the limitation that smaller fragments are in any case preferentially amplified. Four fragments were visible on a 2% agarose gel in correspondence of the deleted allele (Fig. 4C, lane 4). The two bands of higher intensity corresponded to the 17.5-kDa isoform (398 bp) and to a smaller transcript lacking both exons 3 and 4 (233 bp), encoding the normal 11.3-kDa isoform (2). The latter is detectable only under the RT-PCR3 conditions (Fig. 1) and was visible with a similar intensity also among the wt-GH1 transcripts (Fig. 4C, lane 3). The two fainter bands corresponded to heteroduplex molecules formed by mispairing of the 398- and 233-bp fragments. The full-length cDNA and the transcripts with aberrant exon 4 skipping were not visible in this PCR. The wt allele (Fig. 4C, lane 3) yielded, besides the 233-bp fragment, a strong band corresponding to the full-length cDNA and two fainter bands corresponding to the 17.5- and 20-kDa isoforms.

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Mutations in the GH1 splicing elements that result in an increased level of exon 3 skipped transcripts encoding the 17.5-kDa isoform are a common cause of autosomal dominant type II IGHD and severe growth failure (4, 5, 6, 7, 8, 9, 10, 11, 12).

Here we report a new mutation, consisting of a 22-bp deletion in the IVS3 (IVS3 del+56–77) causing IGHD II in a child and her mother. The deletion completely removes the reported BPS (AGCCAAT) (11) located between –20 and –25 upstream of the intron 3/exon 4 junction (Fig. 2) matching five of the seven residues of the weakly defined mammalian BPS (Y₈₁NY₁₀₀T₈₇R₈₁A₁₀₀Y₉₄, where Y represents pyrimidine, R represents purine, and N represents any base) (30). The A at position –21 (underlined) likely represents the highly conserved adenine involved in the lariat formation with the donor splice site.

Lymphocyte mRNA analysis showed that this mutation induced a splicing pattern analogous to that observed in most cases of IGHD II; the proband and her mother showed two principal mRNA species approximately in equal amount, namely the full-length mRNA (encoded by the normal allele) and an aberrant splicing product with the skipping of exon 3 (encoded by the mutant allele) (Fig. 3B).

Accordingly, the clinical phenotype determined by the 22-bp IVS3 deletion correlated with that observed in other IGHD II patients harboring splice site mutations. The phenotype of IGHD II patients is heterogeneous. On average, patients carrying splice site mutations show earlier age of onset of the growth failure that is progressive and more severe than patients carrying missense mutations (8, 31, 32). However, phenotype variability is present also among affected members of the same family (8, 31, 33) showing that the GH-dependent growth during childhood is individually different and modified by several factors, most of them still unknown. Both our patients showed an early postnatal onset of the growth failure that was progressive and severe. Patient III-1 showed an arrest of growth at the age of 4 months and was diagnosed at 7 months (–5.8 SDS); her mother was diagnosed at 4.6 yr (–6.9 SDS) and showed a height of –5 SDS at the age of 2 yr (no information is available at an earlier age). The clinical phenotype was somewhat less severe in the mother; GH secretion after provocative test, completely absent in the daughter, was severely but not completely reduced in the mother at the childhood age diagnosis, and the size of the pituitary gland was normal in the mother at the adult age, whereas it was at the lower limit size in the child. When retested in adulthood, the mother had no GH response to the strong GHRH plus arginine stimulation test, suggesting an exhausted function of the somatotropic cells along the years. Conversely, she did not develop other pituitary hormone deficiencies as described in some adult IGHD II patients (32).

The mutation described here is different from other intronic mutations detected in IGHD II families (4, 5, 6, 7, 8, 9, 10, 11, 12) because it leaves intact the splice junctions and the enhancers, whereas it completely removes the BPS. On the basis of what has been observed in other human diseases (25, 26, 27, 28, 29), we predicted that a mutation involving the BPS would induce the total or partial skipping of exon 4. However, lymphocyte mRNA analysis as well as analysis on the mRNA extracted from rat pituitary cells transfected with del-GH1 revealed that the major transcript was the exon 3 skipped. Two aberrantly spliced isoforms expected for a BPS mutation were detectable exclusively in experimental conditions where the exon 3 skipped mRNA was excluded from the possible targets (Fig. 4B, lane 4). The first isoform, lacking the entire exon 4, encodes for a putative mature peptide depleted of amino acids 72–126. The other was an mRNA devoid of the first 86 bases of exon 4, and it causes a frameshift leading to a putative protein differing from the wild type from amino acid 72 and prematurely truncated at residue 77. It likely results from the use of a cryptic acceptor splice site at position 85–86 of exon 4. The recognition of this splice site might be mediated by the presence of a putative cryptic BPS (CGCCAAC), matching six of the seven bases of the consensus sequence, located between nt 78 and 84 of exon 4 (Fig. 2).

The content of the abnormal mRNA lacking exon 4, or a part of it, is likely very low in the patients’ lymphocytes compared with the normal-sized and to the exon 3 skipped mRNA and can therefore be visualized only under specific experimental conditions. One of the reasons for the low content, at least for the prematurely truncated isoform, might be its rapid degradation owing to nonsense-mediated mRNA decay (34).

The two aberrant splicing events involving exon 4 are the direct consequence of the missing BPS, as demonstrated by the mutagenized construct lacking exclusively the 7 bp of the BPS consensus (Fig. 4B, lane 5). Conversely, the high content of the exon 3 skipped isoform remains to be explained. In a recent report, Ryther and colleagues (35) demonstrated that the overall size of IVS3 is crucial for exon 3 inclusion in the mRNA and showed that deletions of 12–14 bp are sufficient to increase exon 3 skipping. They suggested that this is because the decreased IVS3 size rather than the deletion of specific sequences. We can thus speculate that the main effect of the 22-bp deletion, i.e. the increased exon 3 skipping with production of the IGHD II-specific pathological amount of the 17.5-kDa isoform, is the consequence of the remarkably decreased size of IVS3 that overwhelms the effect caused by the BPS deletion.

However, in the deletion mutants reported by Ryther et al. (35), the IVS3 BPS was unaltered and mediated the lariat formation with the IVS2 donor splice site. In our case, it is not clear how the two mutants lacking the IVS3 branch site (namely the del+56–77 allele and the mutagenized mut7) can correctly process the IVS3 splicing necessary for the exon 3 skipped and the full-length mRNA (36). A similar situation was reported for other disease-causing BPS mutations where the mutated allele also produced the wild-type isoform in addition to the isoform with an aberrant splicing (29, 37, 38). An obvious explanation for these observations is the use of an alternative BPS elsewhere in the IVS3 efficiently replacing the deleted one. Two sequences located between –40/–46 and –61/–67 from the GH1-IVS3 acceptor splice site in the deleted allele, within a distance compatible with a BPS function (39), match the consensus more than others. However, the del-GH1 plasmid mutagenized at the highly conserved A of either or both of these two cryptic BPSs yielded the same splicing pattern (data not shown) as the nonmutagenized construct (Fig. 4). It is thus conceivable that other sequences with less homology to the consensus might act as a cryptic BPS. In fact, although several reported BPS mutations cause severe phenotypes, it has also been demonstrated that a mutated BPS can be replaced by a cryptic BPS with no resemblance to the consensus branch site, apart from the A residue necessary for the lariat formation (40).

In conclusion, despite the absence of the canonical BPS, the exon 3 skipped is the principal transcript and the cause of IGHD II in our patients. Thus, the context in which the BPS mutation is located, i.e. the 22-bp deletion, has a greater phenotypic influence than the BPS mutation itself. It is unlikely that the two isoforms with an aberrant splicing of exon 4 also contribute to the IGHD II phenotype because they are presumably poorly expressed.

	Acknowledgments

 
We are grateful to the patients and their relatives. We thank Dr. T. Vago and Dr. G. Baldi from the Endocrine Laboratory of Luigi Sacco Hospital for hormonal dosage and Dr. M. Bevilacqua from the Endocrinology Department of Luigi Sacco Hospital for cooperation on adult patient management and scientific support.

	Footnotes

 
This work was supported by grants from Pfizer, Italian Network for Molecular Biology in Pediatric Endocrinology, Eastern Piedmont University, the Italian Ministry for University and Research (Cofin 2003), and Cariplo Foundation. M.G. and D.V. are Ph.D. fellows of Dottorato in Medicina Molecolare, Eastern Piedmont University.

First Published Online December 20, 2005.

Abbreviations: BPS, Branch point sequence; IGHD, isolated GH deficiency; SDS, SD score.

Received July 29, 2005.

Accepted December 13, 2005.

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