Heterozygous Nonsense Mutation in Exon 3 of the Growth Hormone ...

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The Journal of Clinical Endocrinology & Metabolism 88(4):1705–1710 Copyright © 2003 by The Endocrine Society doi: 10.1210/jc.2002-021667

Heterozygous Nonsense Mutation in Exon 3 of the Growth Hormone Receptor (GHR) in Severe GH Insensitivity (Laron Syndrome) and the Issue of the Origin and Function of the GHRd3 Isoform ¨ RGEN GRULICH-HENN, MARKUS BETTENDORF, JACQUES PANTEL, JU CHRISTIAN J. STRASBURGER, UDO HEINRICH, AND SERGE AMSELEM Institut National de la Sante´ et de la Recherche Me´dicale, Unite´-468 (J.P., S.A.), Hoˆpital Henri Mondor, 94010 Cre´teil, France; Division of Pediatric Endocrinology (J.G.-H., M.B., U.H.), University Children’s Hospital, D-69120 Heidelberg, Germany; and Division of Endocrinology, Department of Internal Medicine (C.J.S.), 10117 Berlin, Germany Mutations in the GH receptor gene (GHR) cause congenital GH insensitivity, a genetic disorder characterized by severe growth retardation associated with high serum concentration of GH and low serum levels of IGF-I. Molecular defects have been identified in all GHR-coding exons, except exon 3, a sequence that encodes part of the extracellular domain of the receptor. In humans, GHR transcripts exist in two isoforms differing by the retention (GHRfl) or exclusion (GHRd3) of this particular exon. As shown recently, such a dimorphic expression pattern, of unknown significance, could result from a retrovirus-mediated deletion event involving exon 3. This model for the generation of those two isoforms, however, leaves open the possibility that GHRd3 transcripts also arise

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ONGENITAL GH INSENSITIVITY (GHI) is a disease condition characterized by postnatal growth retardation, associated with high serum concentration of GH and low serum levels of IGF-I and IGF-binding protein (IGFBP)-3 (reviewed in Ref. 1). This disorder, known also as Laron syndrome (MIM 245590) (2), in its most severe form is transmitted as an autosomal recessive trait resulting from mutations in the GH receptor gene (GHR) (3– 6). Upon binding to GH, the wild-type GHR molecule, an integral cell membrane protein encoded by nine exons and belonging to the cytokine receptor superfamily, can form homodimers that are essential to receptor activation, thereby mediating the well-known biological effects of GH (7–9). A high-affinity binding protein for GH (GHBP) also exists in serum (10, 11); this soluble receptor, which derives from the extracellular domain of the membrane molecule (12), is usually undetectable in patients with Laron syndrome, although patients with normal or high levels of plasma GHBP have also been reported (5). As expected, the GHBP levels in patients with GHI correlate with the location of the underlying GHR mutations: Mutations in the exoplasmic domain are associated with a GHBPnegative phenotype, with the exception of a mutation

Abbreviations: EBV, Epstein-Barr virus; GHBP, binding protein for GH; GHI, GH insensitivity; GHR, GH receptor gene; GHRd3, GHR transcript isoform with the exclusion of exon 3; GHRfl, GHR transcript isoform with the retention of exon 3; hGH, human GH; IGFBP, IGFbinding protein; PTC, premature stop codon.

from GHRfl alleles through alternative splicing. Here we report the identification of the first mutation in exon 3 of the GHR (W16X) in a patient with GH insensitivity and who also carries another nonsense mutation in exon 4. Intrafamilial correlation analyses of genotypes (presence of normal or mutant GHRfl and/or GHRd3 alleles), GHR expression patterns, and phenotypes provided direct evidence against an alternative splicing of exon 3. In particular, this exon was retained into transcripts originating from the GHRfl-W16X allele in both the patient and his mother. These observations, given the normal phenotype of the heterozygous parents, revealed also that a single copy of either GHRfl or GHRd3 is sufficient for normal growth. (J Clin Endocrinol Metab 88: 1705–1710, 2003)

affecting the dimerization site (13), whereas GHBP is detectable in patients carrying a mutation in the transmembrane or intracellular domain. In humans, GHR transcripts exist in several isoforms (4, 12, 14), among which two classes differ by the retention (GHRfl) or exclusion (GHRd3) of exon 3, a sequence that encodes a short region of the extracellular domain of the receptor (4). As shown recently, such a dimorphic expression pattern, the functional significance of which is unknown, could be at least in part explained by a retrovirus-mediated deletion mechanism involving exon 3. (15). This species-specific in-frame deletion, which occurred late during primate evolution, would result from an intrachromosomal recombination event between two similar primate-specific retroelements that flank exon 3 (15). This latter observation, however, leaves open the possibility that GHRd3 transcripts also arise from GHRfl alleles by means of an alternative splice event, a hypothesis raised by several authors in numerous studies (16 –23). The mutations so far identified in patients with GHI involve all coding exons of the GHR gene, except exon 3. As a consequence, if alternative splicing represents a possible mechanism underlying the expression of GHRfl and GHRd3, each of the nearly 50 different mutations already described are expected to alter both isoforms. In the present study, we investigated a patient presenting with a typical Laron syndrome and carrying a nonsense mutation in exon 3 of the GHR gene. The patient was born to unrelated parents who

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were also investigated, making it possible to correlate, in each family member, the genotype at the GHR locus (presence of wild-type and mutant GHRfl and/or GHRd3 alleles) with the expression pattern of these GHR isoforms and the phenotype. Subjects and Methods Subjects The patient with Laron syndrome is the first child of unrelated parents, both of German origin. There was no family history of growth retardation. Parental heights were normal (father’s and mother’s heights are 182 cm and 168 cm, respectively). Pregnancy and delivery were without complications. The patient’s height and weight were in the normal range at birth (51 cm and 3500 g, respectively), but the growth rate declined rapidly within the first months of life, with a height of ⫺5.5 sd score at 6 months (24). The clinical appearance of the boy showed typical features of GH deficiency: obesity, frontal bossing, acromicria, and midface hypoplasia. During the first months of life, he suffered from episodes of hypoglycemia. His GH plasma levels were high, either at baseline (42 ng/ml) or after stimulation with arginine (99 ng/ml), whereas IGF-I and IGFBP-3 plasma levels were low (⬍5 ng/ml and 0.3 mg/liter, respectively), and resistant to a 4-d provocative sc administration of recombinant GH. Finally, plasma GHBP activity was undetectable. For each family member, blood lymphocytes were immortalized through Epstein-Barr virus (EBV) transformation. From these EBV-transformed lymphocytes, genomic DNAs and total RNAs were both extracted for subsequent genetic studies. Informed consent was given by all three family members who participated in the study.

Endocrine evaluation During the arginine test, the patient received arginine 0.5 g/kg body weight as an iv infusion over a period of 30 min. Blood samples were taken before and 15, 30, 45, 60, 90, and 120 min after the arginine infusion. Serum samples were frozen immediately after centrifugation and were stored at ⫺80 C until analysis. Human GH was measured as described below. For the IGF-generation test, the patient also received 0.1 U/kg body weight recombinant human GH sc once daily for a period of 4 d. Blood samples for the determination of IGF-I, IGFBP-3, and GHBP were taken before and 1 d after the human GH (hGH) treatment period. GH and IGFBP-3 were measured using commercially available RIAs (Nichols, Bad Nauheim, Germany). IGF-I and IGFBP-3 were measured using commercially available RIAs (DSL, Sinsheim, Germany). GHBP was measured by ligand-mediated immunofunctional assay as described before (25) as well as by Sephadex gel chromatography.

GHR mutation analysis Each coding exon and intronic flanking sequence of the GHR gene were amplified by PCR, as previously described (26). All amplified products were subsequently directly sequenced using a DNA sequencer Perkin-Elmer ABI prism 377 (Applied Biosystems, Foster City, CA). To determine the parental origin of the two stop mutations identified in the patient, the PCR products spanning the mutated sites and obtained from all three family members were subjected to the following restriction fragment analyses: HinfI allowed the identification of the W16X mutation in exon 3 (loss of restriction site), whereas MaeIII permitted the identification of the C38X mutation in exon 4 (generation of a new restriction site). Briefly, 5 ␮l of each PCR amplification product were digested with 1 U HinfI or MaeIII, according to supplier’s conditions, and each digested product was subsequently analyzed by electrophoresis on a 6% acrylamide gel and visualized after ethidium bromide staining.

Determination of the GHR-exon 3 status at the genomic level To determine the genotype at the GHR-exon 3 locus (i.e. GHRfl/ GHRfl, GHRfl/GHRd3, or GHRd3/GHRd3) of each family member, we

Pantel et al. • GHR Exon 3 Mutation and GHRd3 Origin and Function

performed a multiplex PCR amplification on genomic DNA samples, as described (15). Briefly, this assay, which is based on the use of three primers (two primers bracketing exon 3 and the third one located within this exon), allows discrimination between GHRfl and GHRd3 alleles that are amplified as two products of different size (i.e. 935 bp and 532 bp, respectively).

Transcript analysis To determine the GHR expression pattern of each family member, total RNAs were first isolated from EBV-transformed lymphocytes obtained from the patient and his parents and from a patient’s dermal fibroblast primary culture, by means of the RNAplus protocol (Bioprobe Systems, Montreuil-sous-Bois, France). GHR transcripts were amplified by RT-PCR using primers 5⬘-CCTACAGGTATGGATCTCTGG-3⬘ and 5⬘-CACTGTGGAATTCGGGTTTA-3⬘ located in exons 2 and 10, respectively. To test for the presence of exon 3 in those transcripts, a PCR amplification was subsequently performed using primers 5⬘-GATCAAGTGATGCTTTTTCT-3⬘and 5⬘-CCACCATTGCTCTTAGCTT-3⬘ located in exons 2 and 5, respectively. Amplified products were analyzed by electrophoresis on a 6% polyacrylamide gel and visualized after ethidium bromide staining.

Results Identification of nonsense mutations in exons 3 and 4 of the GHR gene

The screening for mutations of all GHR coding sequences and intronic boundaries led to the identification of two sequence variations in the DNA sample from this patient. The two mutations, which are present in the heterozygous state, introduce a premature termination codon early in the coding sequence. One mutation, which is located in exon 3, is a G-to-A transition, which replaces a tryptophan residue (TGG) by a premature termination signal (TGA) at codon 16 (W16X) (Fig. 1A). The second heterozygous mutation is located in exon 4 (Fig. 1B); it is a C-to-A transversion, which interrupts the coding sequence by replacing a cysteine residue (TGC) by a stop codon (TGA) at position 38 (C38X). Family molecularly-based study: GHR mutations, GHRexon 3 background, GHR transcripts, and phenotypegenotype correlation

The C38X mutation results in the creation of a recognition site for MaeIII, whereas the W16X mutation abolishes a HinfI restriction site. The familial segregation of the two mutant alleles was, therefore, studied by means of restriction enzyme analyses of the PCR fragments spanning exons 3 and 4 of the GHR gene. As depicted in Fig. 1, C and D, this analysis showed the paternal origin of the C38X mutation, whereas the restriction pattern obtained from the maternal genomic DNA appeared consistent with homozygosity for the W16X mutation, an unexpected result given the normal phenotype of this individual. These latter data prompted us to further characterize the genotype at the GHR-exon 3 locus in all three family members (Fig. 2, A and B). A multiplex PCR experiment designed to look for the presence of exon 3 at the genomic level revealed that the patient and his father bear the GHRfl/GHRfl homozygous genotype, whereas the patient’s mother carries an exon 3 deletion in the heterozygous state (GHRfl/GHRd3 heterozygous genotype). Taken together, these data indicate that this latter individual


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FIG. 1. GHR mutations identified in the patient with Laron syndrome and his parents. A, Electrophoregram of the exon 3 fragment containing the W16X nonsense mutation in the heterozygous state. At the 16th codon, R denotes the presence of both an adenine and a guanine residue (on the mutated allele, the TGG codon encoding a tryptophan residue is replaced by a TGA termination codon). B, Electrophoregram of the exon 4 fragment containing the C38X nonsense mutation in the heterozygous state. At the 38th codon, M denotes the presence of both an adenine and cytosine residue (on the mutated allele, the TGC codon encoding a cysteine residue is replaced by a TGA termination codon). C and D, Segregation analysis of the two GHR nonsense mutations within the family. The presence of the W16X mutation in the genomic DNA from all three family members was assessed by means of HinfI digestion of PCR products generated with primers bracketing the mutated site (abolition of a restriction site) (C), whereas MaeIII digestion of exon 4-amplified products was used to study the segregation of the C38X mutation (creation of a restriction site) (D). This analysis showed the maternal origin of the W16X allele (125 bp and 265 bp) and the paternal origin of the C38X allele (85 bp and 135 bp). Lanes M, L, and F correspond to the PCR products generated from genomic DNA samples of the mother, the patient with Laron syndrome, and his father, respectively. The size ladder is the ␾X174 HaeIII-digested fragment purchased from Sigma (L’Isle d’Abeau Chesnes, France).

actually carries one normal GHRd3 allele and a GHRfl allele bearing the exon 3 W16X mutation. To assess both the expression of GHRfl and GHRd3 isoforms and the consequences of these nonsense mutations at the RNA level, GHR transcripts obtained from EBV-transformed lymphocytes of the patient and his parents were reverse transcribed and PCR amplified, as described in Subjects and Methods. These RT-PCR experiments showed the presence of a single 361-bp amplification product in the patient’s lymphocytes, a result consistent with the sole expression of GHRfl isoforms (Fig. 3); a similar GHR expression pattern was observed in the patient’s fibroblasts (data not shown). The father’s RNA sample displayed the same GHR expression pattern, whereas two different amplification products were generated from the mother’s RNA sample: a 361-bp product and a 295-bp amplicon consistent with the expression of GHRfl and GHRd3 isoforms, respectively. Sequencing of those RT-PCR products indeed confirmed those

results, which fit perfectly with the GHR-exon 3 status determined at the genomic level in each individual. Discussion

We have identified a novel GHR mutation in a patient with Laron syndrome, a rare GH-resistant condition usually transmitted as an autosomal-recessive trait. Remarkably, this mutation (W16X), the functional significance of which is unambiguous, lies in exon 3 of the GHR gene, a region of unknown function encoding a 22-amino acid domain of the receptor, and that accounts for the existence of two GHR isoforms, GHRfl and GHRd3, defined by the presence or the absence of this particular sequence. Phenotype-genotype correlation studies provide both in vivo and in vitro evidence against the existence of an alternative splice event involving exon 3, a hypothesis associated with numerous conflicting data (16 – 23). In addition, our family-based investigations reveal that

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FIG. 2. GHR-exon 3 background of the patient with Laron syndrome and his parents. A, GHR-exon 3 multiplex amplification test allowing discrimination between the GHRfl and GHRd3 alleles. This amplification test, based on the use of three primers, two primers bracketing exon 3 and a third one located within exon , is able to reveal the existence of two products: 935 bp and 532 bp, which correspond to the GHRfl and GHRd3 alleles, respectively. The repeated elements bracketing exon 3 are indicated by gray boxes. B, PCR-amplified products generated by all three family members and analyzed on a 1% agarose gel stained with ethidium bromide. Lanes M, L, and F correspond to the PCR products generated from genomic DNA samples of the mother, the patient with Laron syndrome, and his father, respectively. The size marker is the SmartLadder from Eurogentec (Seraing, Belgium).

a single copy of either GHRfl or GHRd3 is sufficient for normal growth. A large number of mutations has already been identified in the GHR gene of patients with GHI. None of these defects, which are spread over the entire GHR gene sequence, involves exon 3. In this study, we have determined the molecular basis of the GH-resistant phenotype of a patient born to unrelated parents and who carries two different nonsense GHR mutations. One of these two unambiguous defects is located in exon 3 (W16X), whereas the second GHR allele bears the C38X mutation located in exon 4, which has already been reported in several independent patients (26, 27). Analysis of the GHR transcripts expressed from either lymphocytes or dermal fibroblasts of the patient revealed the sole presence of two GHR isoforms containing these two premature stop codons (PTCs). Therefore, exon 3 has been retained in both molecular species. This observation reflects the absence of exon 3 splicing, an event that could have rescued the W16X allele. Indeed, the exclusion from the mature transcript of exons that contain PTCs is a wellknown phenomenon referred to as nonsense-associated altered splicing (28). Such a phenomenon, which can be explained by various mechanisms (reviewed in Ref. 29), has been reported in several genetic disorders (30, 31). In some cases, the skipping of exons containing PTCs may preserve the reading frame and lead to the production of a protein with residual function; this is, for instance, the


FIG. 3. GHR transcripts identified in the patient with Laron syndrome and his parents. RT-PCR amplification of the GHR transcripts of the different family members and obtained from total RNA isolated from lymphocytes (lane M, mother; lane L, patient with Laron syndrome; lane F, father; lane B, RT-PCR control performed without RNA). The 361-bp and the 295-bp products correspond to the fulllength transcript (GHRfl) and the exon 3-deleted isoform (GHRd3), respectively.

case for Becker muscular dystrophy, a mild form of Duchenne muscular dystrophy, which is a disease that results from mutations in the dystrophin gene (32, 33). This is clearly in contrast with the present situation that shows that, even in the presence of nonsense mutations, no exon skipping was observed. These results are also in keeping with the severe GH-resistant syndrome documented in the patient and the fact that GHBP was undetectable in this young boy. Our data, therefore, demonstrate the absence of alternative splicing of the GHR exon 3 in this family and, as a result, further confirm the hypothesis that the GHRd3 isoform is transcribed from a GHR allele carrying a genomic deletion of exon 3 (15). To date, the functional importance of the GHR domain encoded by exon 3 is unknown. As shown by means of in vitro studies (16, 17), GHRfl and GHRd3 proteins share similar binding properties toward various isoforms of the pituitary and placentally expressed members of the growth hormone/PRL family (hGH-N, hGH-V, hCS, and PRL). The availability of assays for the determination of each GHBP isoform (i.e. GHBP containing or not the domain encoded by exon 3) has allowed us to show that the two soluble isoforms are released into the peripheral circulation (34). The present observation now provides the opportunity to correlate in each family member, the genotype at the GHR locus (presence of wild-type and mutant GHRfl and/or GHRd3 alleles) with the expression pattern of these GHR isoforms and the phenotype (Fig. 4). In particular, the fact that the two heterozygous parents have a normal phenotype and they carry a null GHR allele in combination with either a normal GHRfl or GHRd3 allele (in the father or the mother, respectively) reveals that, in


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FIG. 4. Genotype, transcript expression pattern, and phenotype correlation in the family. The two GHR alleles are drawn: Boxes correspond to the coding exons and asterisks indicate the two identified nonsense mutations. The two normal GHR alleles are present in this family: the full-length allele (GHRfl-wt) and the exon 3-deleted allele (GHRd3wt). For each transcript isoform, a horizontal black bar figures the length of the coding sequence.

vivo, a single copy of either GHRfl or GHRd3 is sufficient for normal growth. Acknowledgments We are grateful to the family for participating in this study. Received October 24, 2002. Accepted December 27, 2002. Address all correspondence and requests for reprints to: Pr. Serge Amselem, INSERM-U468, Hoˆ pital Henri Mondor, 51 avenue Mare´ chal de Lattre de Tassigny, 94010 Cre´ teil, France. E-mail: serge.amselem@ im3.inserm.fr. This work was supported by the Institut National de la Sante´ et de la Recherche Me´ dicale. J.P. and J.G.-H. contributed equally to this work.

References 1. Savage MO, Burren CP, Blair JC, Woods KA, Metherell L, Clark AJ, Camacho-Hubner C 2001 Growth hormone insensitivity: pathophysiology, diagnosis, clinical variation and future perspectives. Horm Res 55: 32–35 2. Laron Z, Pertzelan A, Mannheimer S 1966 Genetic pituitary dwarfism with high serum concentration of growth hormone: a new inborn error of metabolism? Isr J Med Sci 2:152–155 3. Amselem S, Duquesnoy P, Attree O, Novelli G, Bousnina S, Postel-Vinay MC, Goossens M 1989 Laron dwarfism and mutations of the growth hormonereceptor gene. N Engl J Med 321:989 –995 4. Godowski PJ, Leung DW, Meacham LR, Galgani JP, Hellmiss R, Keret R, Rotwein PS, Parks JS, Laron Z, Wood WI 1989 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 USA 86:8083– 8087 5. Amselem S, Sobrier ML, Dastot F, Duquesnoy P, Duriez B, Goossens M 1996 Molecular basis of inherited growth hormone resistance in childhood. Baillieres Clin Endocrinol Metab 10:353–369 6. Baumann G 2002 Genetic characterization of growth hormone deficiency and resistance: implications for treatment with recombinant growth hormone. Am J Pharmacogenomics 2:93–111 7. Rowlinson SW, Behncken SN, Rowland JE, Clarkson RW, Strasburger CJ, Wu Z, Baumbach W, Waters MJ 1998 Activation of chimeric and full-length growth hormone receptors by growth hormone receptor monoclonal antibodies. A specific conformational change may be required for full-length receptor signaling. J Biol Chem 273:5307–5314 8. Fuh G, Cunningham BC, Fukunaga R, Nagata S, Goeddel DV, Wells JA 1992 Rational design of potent antagonists to the human growth hormone receptor. Science 256:1677–1680 9. 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 10. Baumann G, Stolar MW, Amburn K, Barsano CP, DeVries BC 1986 A specific growth hormone-binding protein in human plasma: initial characterization. J Clin Endocrinol Metab 62:134 –141

11. Herington AC, Ymer S, Stevenson J 1986 Identification and characterization of specific binding proteins for growth hormone in normal human sera. J Clin Invest 77:1817–1823 12. Dastot F, Sobrier ML, Duquesnoy P, Duriez B, Goossens M, Amselem S 1996 Alternatively spliced forms in the cytoplasmic domain of the human growth hormone (GH) receptor regulate its ability to generate a soluble GH-binding protein. Proc Natl Acad Sci USA 93:10723–10728 13. Duquesnoy P, Sobrier ML, Duriez B, Dastot F, Buchanan CR, Savage MO, Preece MA, Craescu CT, Blouquit Y, Goossens M, Amselem S 1994 A single amino acid substitution in the exoplasmic domain of the human growth hormone (GH) receptor confers familial GH resistance (Laron syndrome) with positive GH-binding activity by abolishing receptor homodimerization. EMBO J 13:1386 –1395 14. Goodyer CG, Zogopoulos G, Schwartzbauer G, Zheng H, Hendy GN, Menon RK 2001 Organization and evolution of the human growth hormone receptor gene 5⬘-flanking region. Endocrinology 142:1923–1934 15. Pantel J, Machinis K, Sobrier ML, Duquesnoy P, Goossens M, Amselem S 2000 Species-specific alternative splice mimicry at the growth hormone receptor locus revealed by the lineage of retroelements during primate evolution. J Biol Chem 275:18664 –18669 16. Sobrier ML, Duquesnoy P, Duriez B, Amselem S, Goossens M 1993 Expression and binding properties of two isoforms of the human growth hormone receptor. FEBS Lett 319:16 –20 17. Urbanek M, Russell JE, Cooke NE, Liebhaber SA 1993 Functional characterization of the alternatively spliced, placental human growth hormone receptor. J Biol Chem 268:19025–19032 18. Urbanek M, MacLeod JN, Cooke NE, Liebhaber SA 1992 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 6:279 –287 19. Mercado M, DaVila N, McLeod JF, Baumann G 1994 Distribution of growth hormone receptor messenger ribonucleic acid containing and lacking exon 3 in human tissues. J Clin Endocrinol Metab 78:731–735 20. Esposito N, Paterlini P, Kelly PA, Postel-Vinay MC, Finidori J 1994 Expression of two isoforms of the human growth hormone receptor in normal liver and hepatocarcinoma. Mol Cell Endocrinol 103:13–20 21. Wickelgren RB, Landin KL, Ohlsson C, Carlsson LM 1995 Expression of exon 3-retaining and exon 3-excluding isoforms of the human growth hormonereceptor is regulated in an interindividual, rather than a tissue-specific, manner. J Clin Endocrinol Metab 80:2154 –2157 22. Zogopoulos G, Figueiredo R, Jenab A, Ali Z, Lefebvre Y, Goodyer CG 1996 Expression of exon 3-retaining and -deleted human growth hormone receptor messenger ribonucleic acid isoforms during development. J Clin Endocrinol Metab 81:775–782 23. Stallings-Mann ML, Ludwiczak RL, Klinger KW, Rottman F 1996 Alternative splicing of exon 3 of the human growth hormone receptor is the result of an unusual genetic polymorphism. Proc Natl Acad Sci USA 93:12394 – 12399 24. Reinken L, van Oost G 1992 Longitudinal physical development of healthy children 0 to 18 years of age. Body length/height, body weight and growth velocity. Klin Padiatr 204:129 –133 25. Carlsson LM, Rowland AM, Clark RG, Gesundheit N, Wong WL 1991 Ligand-mediated immunofunctional assay for quantitation of growth hormone-binding protein in human blood. J Clin Endocrinol Metab 73:1216 – 1223

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26. Amselem S, Duquesnoy P, Duriez B, Dastot F, Sobrier ML, Valleix S, Goossens M 1993 Spectrum of growth hormone receptor mutations and associated haplotypes in Laron syndrome. Hum Mol Genet 2:355–359 27. Amselem S, Sobrier ML, Duquesnoy P, Rappaport R, Postel-Vinay MC, Gourmelen M, Dallapiccola B, Goossens M 1991 Recurrent nonsense mutations in the growth hormone receptor from patients with Laron dwarfism. J Clin Invest 87:1098 –1102 28. Hentze MW, Kulozik AE 1999 A perfect message: RNA surveillance and nonsense-mediated decay. Cell 96:307–310 29. Cartegni L, Chew SL, Krainer AR 2002 Listening to silence and understanding nonsense: exonic mutations that affect splicing. Nat Rev Genet 3:285–298 30. Cooper TA, Mattox W 1997 The regulation of splice-site selection, and its role in human disease. Am J Hum Genet 61:259 –266 31. Liu HX, Cartegni L, Zhang MQ, Krainer AR 2001 A mechanism for exon skipping caused by nonsense or missense mutations in BRCA1 and other genes. Nat Genet 27:55–58 32. Koenig M, Beggs AH, Moyer M, Scherpf S, Heindrich K, Bettecken T, Meng


G, Muller CR, Lindlof M, Kaariainen H, de la Chapelle A, Kiuru A, Savontaus M-L, Gilgenkrantz H, Re´can D, Chelly J, Kaplan J-C, Covone AE, Archidiacono N, Romeo G, Liechti-Gallati S, Schneider V, Braga S, Moser H, Darras BT, Murphy P, Francke U, Chen D, Morgan G, Denton M, Greenberg CR, Wrogemann K, Blonden LAJ, van Paassen HMB, van Ommen GJB, Kunkel LM 1989 The molecular basis for Duchenne versus Becker muscular dystrophy: correlation of severity with type of deletion. Am J Hum Genet 45:498 –506 33. Shiga N, Takeshima Y, Sakamoto H, Inoue K, Yokota Y, Yokoyama M, Matsuo M 1997 Disruption of the splicing enhancer sequence within exon 27 of the dystrophin gene by a nonsense mutation induces partial skipping of the exon and is responsible for Becker muscular dystrophy. J Clin Invest 100: 2204 –2210 34. Kratzsch J, Wu Z, Kiess W, Dehmel B, Bosse-Henck A, Reuter W, Pflaum CD, Strasburger CJ 2001 The exon 3-retaining and the exon 3-deleted forms of the growth hormone-binding protein (GHBP) in human serum are regulated differently. Clin Endocrinol (Oxf) 54:61– 68