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An Intronic Growth Hormone Receptor Mutation Causing Activation of a Pseudoexon Is Associated with a Broad Spectrum of Growth Hormone Insensitivity Phenotypes

Centre for Endocrinology (A.D., C.C.-H., F.M.-M., S.A.A., A.J.L.C., M.O.S., L.A.M.), William Harvey Research Institute, Barts and the London, Queen Mary, University of London, London EC1M 6BQ, United Kingdom; Pediatric Endocrinology Division (A.B., S.T.), Infant’s and Children’s Hospital of Brooklyn at Maimonides, Brooklyn, New York 11219; Department of Paediatrics (S.J.R.), Heartlands Hospital, Birmingham B9 5SS, United Kingdom; Department of Paediatrics (G.E.B.), Leeds General Infirmary, Leeds LS1 3EX, United Kingdom; and Endocrine Science Research Group (P.E.C.), School of Medicine, University of Manchester, Manchester M13 9PT, United Kingdom

Address all correspondence and requests for reprints to: Dr. L. A. Metherell, Centre for Molecular Endocrinology, First Floor North, John Vane Building, Charterhouse Square, London EC1M 6BQ, United Kingdom. E-mail: l.a.metherell{at}qmul.ac.uk.

	Abstract

Top Abstract Introduction Patients and Methods Results Discussion References

 
Context: Inherited GH insensitivity (GHI) is usually caused by mutations in the GH receptor (GHR). Patients present with short stature associated with high GH and low IGF-I levels and may have midfacial hypoplasia (typical Laron syndrome facial features). We previously described four mildly affected GHI patients with an intronic mutation in the GHR gene (A_-1G_-1 substitution in intron 6), resulting in the activation of a pseudoexon (6) and inclusion of 36 amino acids.

Objective: The study aimed to analyze the clinical and genetic characteristics of additional GHI patients with the pseudoexon (6) mutation.

Design/Patients: Auxological, biochemical, genetic, and haplotype data from seven patients with severe short stature and biochemical evidence of GHI were assessed.

Main Outcome Measures: We assessed genotype-phenotype relationship.

Results: One patient belongs to the same extended family, previously reported. She has normal facial features, and her IGF-I levels are in the low-normal range for age. The six unrelated patients, four of whom have typical Laron syndrome facial features, have heights ranging from –3.3 to –6.0 SD and IGF-I levels that vary from normal to undetectable. We hypothesize that the marked difference in biochemical and clinical phenotypes might be caused by variations in the splicing efficiency of the pseudoexon.

Conclusions: Activation of the pseudoexon in the GHR gene can lead to a variety of GHI phenotypes. Therefore, screening for the presence of this mutation should be performed in all GHI patients without mutations in the coding exons.

	Introduction

Top Abstract Introduction Patients and Methods Results Discussion References

 
GH INSENSITIVITY (GHI) of genetic origin may be caused by a range of mutations in genes coding for proteins with specific roles in the GH-IGF-I axis (1). So far more than 60 different mutations have been identified in the GH receptor (GHR) gene that affect GH binding or GHR signaling (2). The disorder is characterized by resistance to the physiological actions of GH and patients present with severe short stature, elevated GH levels, and low IGF-I (3, 4). Analysis of a large cohort of GHI patients has shown that GHI may be associated with different clinical and biochemical phenotypes, exemplified by short stature ranging from –4 to –10 SD below the mean and IGF-I levels that range from just below normal to undetectable. GHI patients may also have dysmorphic facial features characterized by midfacial hypoplasia and prominent forehead [typical Laron syndrome (LS) facial features] (2, 5, 6).

The majority of molecular defects in GHI have been identified in the region encoding the GHR extracellular domain, responsible for GH binding. Patients with such mutations have absent or extremely low GH binding protein (GHBP) levels and a severe GHI phenotype (7). In a few cases, a severe GHI phenotype is also seen with normal or even high GHBP levels (8, 9, 10).

Two mutations that affect the intracellular domain producing a truncated GHR that heterodimerizes with the wild-type GHR and act in a dominant negative manner were reported in the late 1990s (11, 12). Both result in a GHBP-positive phenotype with normal faces and a relatively mild phenotype (13).

In 2001, our group described a novel intronic point mutation located between exons 6 and 7 of the GHR gene (13). This mutation was present in four GHI siblings with normal facial appearance and normal GHBP levels. Potential exons (pseudoexons) are frequently found within introns but are normally not included in the mature mRNA because the splicing machinery fails to recognize them (14). The described mutation (A_-1G_-1) is at the 5' donor splice site of one such pseudoexon (6), and leads to recognition of the pseudoexon and inclusion of an additional 108 bases between exons 6 and 7. This translates into the addition of 36 amino acids in the receptor extracellular domain (13).

We have now identified the pseudoexon mutation (6) in seven additional patients and report the molecular, endocrine, and phenotypic characteristics of this population.

	Patients and Methods

Top Abstract Introduction Patients and Methods Results Discussion References

 
Patients

Clinical and auxological details. Eleven patients with severe short stature were referred to us with a possible diagnosis of GHI. Clinical and biochemical details for seven patients are presented in Table 1. Data for the remaining four patients were previously published (15).

Biochemical investigations. Serum IGF-I, IGF binding protein-3 (IGFBP-3), acid-labile subunit (ALS), and GHBP were measured from venous blood samples using an enzyme-linked immunosorbent assay (ELISA kit; Diagnostic System Laboratories, Inc., Webster, TX). For IGF-I, the assay sensitivity was 0.01 ng/ml. The intraassay and interassay coefficients of variation were 8.6 and 6.8% for mean serum concentrations of 104 and 90 ng/ml, respectively. For IGFBP-3, the assay sensitivity was 0.04 ng/ml. The mean intraassay and interassay coefficients of variation were 7.2 and 8.3%, respectively. For ALS, the assay sensitivity was 0.7 ng/ml, and the interassay coefficient of variation was 8%. For GHBP, the assay sensitivity was 1.69 pmol/liter. The intraassay and interassay coefficients of variation were 5.6 and 8.4% at 20 pmol/liter, respectively.

Genetic analysis

Genomic DNA was extracted from peripheral blood leukocytes, and each exon of the GHR, plus the pseudoexon (6), including their intronic boundaries, were amplified by PCR using specific primers (primer sequences available on request). The complex single polymorphic region in intron 9 of the GHR was also studied. Specific primers (F 5'-CCCAGTTCCAGTTCCAAAGA-3' and R 5'-CACTGTGGAATTCGGGGTTTA-3') were used to amplify intron 9. Roman numerals indicate genotypes of this region, as defined (16). Cycling conditions were: 95 C for 5 min (1 cycle); 95 C for 30 sec, 55 C for 30 sec, and 72 C for 30 sec (30 cycles); and 72 C for 5 min. PCR products were visualized on 1% agarose gel, and sequenced using the ABI Prism Big Dye Sequencing kit and an ABI 377 automated DNA sequencer (Applied Biosystems, Foster City, CA) in accordance with the manufacturer’s instructions.

Genotype analysis

To identify the presence of a common ancestor in the unrelated patients with the pseudoexon mutation, six markers spanning 17.10 Mb around the GHR gene on chromosome 5 were genotyped in the affected members of the five families. Dinucleotide repeat markers d5s2021 d5s2022, d5s430, d5s2082, d5s2087, d5s474 were used. These were amplified by PCR, and resultant fragments were electrophoresed on a 5% PAGE gel on an ABI 377 automated sequencer; analysis was performed using Genescan software (Applied Biosystems).

	Results

Top Abstract Introduction Patients and Methods Results Discussion References

 
Sequencing analysis

Sequencing analysis of patients revealed the presence of the previously published mutation (A_-1G_-1 at the 5' pseudoexon 6 splice site) in a homozygous state in all cases (Fig. 1). Sequencing of parental DNA for families A and B revealed that both sets of parents are heterozygous for the 6 mutation. Sequencing of the GHR coding exons, flanking splice junctions, and branch point sequences showed no other mutations.

View larger version (18K): [in this window] [in a new window]   FIG. 1. A, Partial sequences of GHR in patients and controls. Arrows indicate the base change from A to G. B, Schematic diagram of the splicing mechanism. The A–G mutation in intron 6 activates the 5' splice site, resulting in the inclusion of the pseudoexon sequence in the mutant GHR protein.

Biochemical data showed normal GHBP levels in all patients but differing degrees of GH insensitivity, as demonstrated by the wide range of growth failure and serum IGF-I, IGFBP-3, and ALS levels. Biochemical and auxological data for seven patients are shown in Table 1. Data for the remaining four patients were previously published (15).

Patient A1 was a 7-yr-old female related to the previously described highly consanguineous Pakistani family (13). Her height was –5.0 SD. Like the other affected family members, she had normal facial features. Biochemically, she had a mild GHI phenotype.

Patient B1 was an 8-yr-old female, and her grandparents are cousins of Pakistani origin. Her height was –6.0 SD. She had a younger sibling with growth retardation and elevated GH levels (DNA not available for analysis). She had typical LS facial features, with a prominent forehead and midfacial hypoplasia. Biochemically, she had severe GHI, as shown by the almost undetectable levels of GH-dependent proteins (IGF-I, IGFBP-3, and ALS).

Patient C1 was a 21-yr-old male from first-degree cousins of Palestinian-Arab origin. His height was –5.0 SD. He has two other siblings with short stature (DNA not available for analysis), and they all had typical LS facial features. Biochemically, he had a mild GHI phenotype. His IGF-I and IGFBP-3 values were normal and are likely to reflect his pubertal status.

Patients D1 and D2 were from a consanguineous Pakistani marriage. D1 was a 6-yr-old male whose height SD was –3.4, and IGF-I, IGFBP-3, and ALS levels were low, whereas his brother, D2, aged 11, had low ALS levels, but his IGF-I and IGFBP-3 levels were normal, reflecting his pubertal status. His height SD was –4.6. Both had typical LS facial features.

Patients E1 and E2 were sisters from a consanguineous Pakistani family. They had similar height SD scores of –3.3 and –3.5, respectively. Both displayed a normal facial phenotype, but E1 had subnormal IGF-I and IGFBP-3 levels, whereas her sister’s values were normal for her age, again reflecting her pubertal status.

Genotype analysis

Analysis of a 17.10 Mb region on chromosome 5 surrounding the GHR gene revealed the presence of the same genotype in the affected members of three unrelated families (A, B, and C), suggesting a common ancestor. A recombination event has occurred in family B downstream of the GHR locus, between markers D5S2087 and D5S474. Families D and E are not known to be related but have the same haplotype being identical by descent upstream of the GHR and homozygous, both at the GHR locus and downstream. The analysis of the single nucleotide polymorphisms in intron 9 of the GHR and the marker D5S2087 show the same genotype in all families (Fig. 2) over a conserved region of at least 1.8 Mb, suggestive of a common ancestor.

View larger version (27K): [in this window] [in a new window]   FIG. 2. Family pedigrees and genotype analysis. Affected family members are shown in black, possibly affected in gray. In family A, the five cousins had identical genotypes. In family D, the brothers had identical genotypes, and in family E, the sisters had identical genotypes. The genotypes of the affected members of each family are indicated below the family trees in boxes. All families had an identical genotype at the GHR locus and marker D5S2087, shown in bold.

	Discussion

Top Abstract Introduction Patients and Methods Results Discussion References

The functional consequences of this insertion have been studied in detail (18). Maamra et al. (18) have shown that the pseudoexon containing GHR has significantly impaired cell surface trafficking, as shown by cell surface binding, fluorescence-activated cell sorter analysis, and confocal immunofluorescence studies. The signaling ability of the receptor, as judged by JAK2 and STAT5 phosphorylation and lactogenic hormone response element reporter gene analysis, was reduced in proportion to the reduction in cell surface receptor, implying that the signaling properties of the mutant protein were unimpaired.

Analysis of a large cohort of GHI patients has permitted the identification of the pseudoexon mutation in a total of 11 patients from five apparently unrelated families. It is likely that the siblings of the two patients (B1 and C1) also carry the pseudoexon mutation because they are reported to have growth retardation, short stature, and typical LS facial features. To date, no heterozygote effect has been shown for any GHR mutation, and, in keeping with this, the parents in family A and B show no atypical growth phenotype, despite heterozygosity for the pseudoexon mutation.

The five unrelated families originate from different geographical locations. However, genotyping of a region spanning 17.10 Mb around the GHR gene on chromosome 5 and analysis of the complex single polymorphic region in intron 9 of the GHR showed the same genotype for all affected members, suggesting the presence of a common ancestor.

Phenotypical variability in GHI patients with the same mutation is known to occur (4, 5, 19), and this is particularly striking in these patients. The five members of family A have normal facial features and a mild degree of GHI, with low but detectable IGF-I levels and heights ranging from –3.3 to –5.6 SD. The other six patients have phenotypes varying from the mild phenotype (patients E1 and 2) like that seen in family A to a very severe GHI coupled with typical LS facial features (patient B1). Hence, we have demonstrated that the pseudoexon mutation can cause more severe GHI phenotypes than we originally reported, and it is tempting to speculate that there could also be a milder phenotype associated with it. The possibility arises that the pseudoexon mutation is responsible for a small number of idiopathic short stature patients.

We suggest that the different clinical phenotypes observed in patients carrying the pseudoexon mutation may be caused by the presence of transcript heterogeneity. The presence of a splice mutation may result in a competitive use of the normal and mutant splice sites, and the production of different transcripts. Analysis of cDNA from patients carrying splice mutations has shown that multiple abnormal splicing events can occur alongside the production of varying amounts of the normal splice product (20, 21). Therefore, it is possible that mildly affected subjects in the pseudoexon cohort might have a GHR transcript ratio in favor of the normal protein, while severely affected patients have a ratio in favor of the mutant GHR.

Alternative explanations for the clinical heterogeneity over and above that observed with typical missense and nonsense mutations of the GHR can be envisaged. As outlined previously, the inclusion of the pseudoexon leads to impaired trafficking of the GHR. Genetic variability in components of GHR processing and trafficking might conceivably have a greater influence than they do with the wild-type GHR, as would components of receptor degradation pathways.

Both genetic and environmental factors could be involved in defining the ratio of spliced transcripts. Mechanisms driving mRNA splicing are still far from being fully understood. The spliceosome, a complex of small nuclear ribonucleoproteins and multiple other proteins, catalyzes splicing of pre-mRNAs. In particular, the small nuclear RNPs U1 and U2 are implicated in recognition of the 5' splice site and branch site, respectively (22, 23). The A–G base change at the 5' splice site responsible for GHI in patients with the pseudoexon mutation does not create a new splice site but does increase the bp match of the pre-mRNA with the U1 small nuclear RNA, resulting in the recognition by the spliceosome of the pseudoexon sequence and its inclusion in the mature mRNA (13).

In conclusion, sequencing of the pseudoexon should be considered in all patients with GHI, where no coding exon mutations are found, regardless of the severity of their disease.

	Footnotes

 
Disclosure Statement: The authors have nothing to disclose.

First Published Online December 5, 2006

Abbreviations: ALS, Acid-labile subunit; GHBP, GH binding protein; GHI, GH insensitivity; GHR, GH receptor; IGFBP-3, IGF binding protein-3; LS, Laron syndrome.

Received July 14, 2006.

Accepted November 28, 2006.

	References

Top Abstract Introduction Patients and Methods Results Discussion References

 

1. Laron Z 2004 Laron syndrome (primary growth hormone resistance or insensitivity): the personal experience 1958–2003. J Clin Endocrinol Metab 89:1031–1044[Abstract/Free Full Text]
2. Savage MO, Attie KM, David A, Metherell LA, Clark AJL, Camacho-Hübner C 2006 Endocrine assessment, molecular characterization and treatment of growth hormone insensitivity disorders. Nat Clin Pract Endocrinol Metab 2:395–407[CrossRef][Medline]
3. David A, Metherell LA, Clark AJ, Camacho-Hubner C, Savage MO 2005 Diagnostic and therapeutic advances in growth hormone insensitivity. Endocrinol Metab Clin North Am 34:581–595[CrossRef][Medline]
4. Rosenfeld RG, Rosenbloom AL, Guevara-Aguirre J 1994 Growth hormone (GH) insensitivity due to primary GH receptor deficiency. Endocr Rev 15:369–390[CrossRef][Medline]
5. Woods KA, Dastot F, Preece MA, Clark AJ, Postel-Vinay MC, Chatelain PG, Ranke MB, Rosenfeld RG, Amselem S, Savage MO 1997 Phenotype: genotype relationships in growth hormone insensitivity syndrome. J Clin Endocrinol Metab 82:3529–3535[Abstract/Free Full Text]
6. Woods KA, Savage MO 1999 IGF-Deficiency. In: Rosenfeld RG, Roberts CT, eds. IGF in health and disease. Totowa, NJ: The Humana Press; 651–674
7. Burren CP, Woods KA, Rose SJ, Tauber M, Price DA, Heinrich U, Gilli G, Razzaghy-Azar M, Al-Ashwal A, Crock PA, Rochiccioli P, Yordam N, Ranke MB, Chatelain PG, Preece MA, Rosenfeld RG, Savage MO 2001 Clinical and endocrine characteristics in atypical and classical growth hormone insensitivity syndrome. Horm Res 55:125–130[CrossRef][Medline]
8. 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[Medline]
9. Woods KA, Fraser NC, Postel-Vinay MC, Savage MO, Clark AJ 1996 A homozygous splice-site mutation affecting the intracellular domain of the growth hormone (GH) receptor resulting in Laron syndrome with elevated GH-binding protein. J Clin Endocrinol Metab 81:1686–1690[Abstract]
10. Silbergeld A, Dastot F, Klinger B, Kanety H, Eshet R, Amselem S, Laron Z 1997 Intronic mutation in the growth hormone (GH) receptor gene from a girl with Laron syndrome and extremely high serum GH binding protein: extended phenotypic study in a very large pedigree. J Pediatr Endocrinol Metab 10:265–274[Medline]
11. Ayling RM, Ross R, Towner P, Von Laue S, Finidori J, Moutoussamy S, Buchanan CR, Clayton PE, Norman MR 1997 A dominant-negative mutation of the growth hormone receptor causes familial short stature. Nat Genet 16:13–14[CrossRef][Medline]
12. Iida K, Takahashi Y, Kaji H, Nose O, Okimura Y, Abe H, Chihara K 1998 Growth hormone (GH) insensitivity syndrome with high serum GH-binding protein levels caused by a heterozygous splice site mutation of the GH receptor gene producing a lack of intracellular domain. J Clin Endocrinol Metab 83:531–537[Abstract/Free Full Text]
13. Metherell LA, Akker SA, Munroe PB, Rose SJ, Caulfield M, Savage MO, Chew SL, Clark AJ 2001 Pseudoexon activation as a novel mechanism for disease resulting in atypical growth-hormone insensitivity. Am J Hum Genet 69:641–646[CrossRef][Medline]
14. Sun H, Chasin LA 2000 Multiple splicing defects in an intronic false exon. Mol Cell Biol 20:6414–6425[Abstract/Free Full Text]
15. Bjarnason R, Banerjee K, Rose SJ, Rosberg S, Metherell LA, Clark AJ, Albertsson-Wikland K, Savage MO 2002 Spontaneous growth hormone secretory characteristics in children with partial growth hormone insensitivity. Clin Endocrinol (Oxf) 57:357–361[CrossRef][Medline]
16. Amselem S, Duquesnoy P, Attree O, Novelli G, Bousnina S, Postel-Vinay MC, Goossens M 1989 Laron dwarfism and mutations of the growth hormone-receptor gene. N Engl J Med 321:989–995[Abstract]
17. Savage MO, Blum WF, Ranke MB, Postel-Vinay MC, Cotterill AM, Hall K, Chatelain PG, Preece MA, Rosenfeld RG 1993 Clinical features and endocrine status in patients with growth hormone insensitivity (Laron syndrome). J Clin Endocrinol Metab 77:1465–1471[Abstract]
18. Maamra M, Milward A, Esfahani HZ, Abbott LP, Metherell LA, Savage MO, Clark AJ, Ross RJ 2006 A 36 residues insertion in the dimerization domain of the growth hormone receptor results in defective trafficking rather than impaired signaling. J Endocrinol 188:251–261[Abstract/Free Full Text]
19. Rosenbloom AL, Guevara-Aguirre J, Rosenfeld RG, Fielder PJ 1990 The little women of Loja: growth hormone receptor deficiency in an inbred population of southern Ecuador. N Engl J Med 323:1367–1374[Abstract]
20. Lemahieu V, Gastier JM, Francke U 1999 Novel mutations in the Wiskott-Aldrich syndrome protein gene and their effects on transcriptional, translational, and clinical phenotypes. Hum Mutat 14:54–66[CrossRef][Medline]
21. Zhu Q, Watanabe C, Liu T, Hollenbaugh D, Blaese RM, Kanner SB, Aruffo A, Ochs HD 1997 Wiskott-Aldrich syndrome/X-linked thrombocytopenia: WASP gene mutations, protein expression, and phenotype. Blood 90:2680–2699[Abstract/Free Full Text]
22. Michaud S, Reed R 1993 A functional association between the 5' and 3' splice-site is established in the earliest prespliceosome complex (E) in mammals. Genes Dev 7:1008–1020[Abstract/Free Full Text]
23. Faustino NA, Cooper TA 2003 Pre-mRNA splicing and human disease. Genes Dev 17:419–437[Free Full Text]

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