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Successful Long-Term Growth Hormone Therapy in a Girl with Haploinsufficiency of the Insulin-Like Growth Factor-I Receptor due to a Terminal 15q26.2->qter Deletion Detected by Multiplex Ligation Probe Amplification

Departments of Pediatrics (M.J.E.W., H.A.v.D., M.K., J.M.W.) and Endocrinology and Metabolic Diseases (H.A.v.D., M.K.), Center for Human and Clinical Genetics (M.d.M., H.A.v.D., S.G.K., S.J.W., M.L.), and Leiden Genome Technology Center (M.E.K., J.T.D.D.), Leiden University Medical Center, 2300 RC Leiden, The Netherlands; Department of Pediatrics (S.M.P.F.d.M.K.-S.), Erasmus Medical Center-Sophia Children’s Hospital, 3015 GJ Rotterdam, The Netherlands; and Department of Pediatrics (A.M.B.), University Medical Center Groningen, 9700 RB Groningen, The Netherlands

Address all correspondence and requests for reprints to: M. J. E. Walenkamp, Department of Pediatrics J6-S, Leiden University Medical Center, P.O. Box 9600, 2300 RC Leiden, The Netherlands. E-mail: m.j.e.walenkamp{at}lumc.nl.

	Abstract

Top Abstract Introduction Patient and Methods Results Discussion References

 
Context: Microscopically visible heterozygous terminal 15q deletions encompassing the IGF1R gene are rare and usually associated with intrauterine growth retardation and short stature. The incidence of submicroscopic deletions is unknown, as is the effect of GH therapy in this condition.

Objective: The objective of the study was to describe the use of a novel genetic technique [multiplex ligation probe amplification (MLPA)] to detect haploinsufficiency of the IGF1R gene in a patient suspected of an IGF1R gene defect and evaluate the effect of long-term GH therapy.

Patient: A 15-yr-old adolescent, born small for gestational age, showed persistent postnatal growth retardation, microcephaly, and elevated IGF-I levels. She had been treated with GH since the age of 5 yr.

Methods: MLPA and array comparative genomic hybridization (aCGH) were performed to examine gene copy number changes. Dermal fibroblast cultures were used for functional analysis.

Results: With MLPA, a deletion of one copy of the IGF1R gene was detected, defined by aCGH as a loss of 15q26.2->qter. IGF1R mRNA expression was decreased in fibroblasts. IGF-I binding and type 1 IGF receptor protein expression as well as activation of type 1 IGF receptor autophosphorylation and protein kinase B/Akt by IGF-I tended to be lower, but this did not reach statistical significance. GH treatment resulted in a good growth response and a normal adult height.

Conclusions: MLPA and aCGH are useful tools to detect submicroscopic deletions of the IGF1R gene in patients born small for gestational age with persistent growth failure. The phenotype resembles that of a heterozygous inactivating IGF1R mutation. Long-term GH therapy causes growth acceleration in childhood and a normal adult height.

	Introduction

Top Abstract Introduction Patient and Methods Results Discussion References

 
IGF-I is required for normal intrauterine and postnatal growth. The biological functions of IGF-I are mediated through the type 1 IGF receptor (IGF1R), located on the distal long arm of chromosome 15 (15q26.3). Heterozygous inactivating mutations of the IGF1R gene result in a phenotype of intrauterine and postnatal growth failure and microcephaly, with a variable degree of psychomotor retardation (1, 2, 3, 4) and dysmorphic features (1, 3).

Microscopically visible heterozygous terminal 15q deletions encompassing the IGF1R gene have been reported in only a few cases: six patients with a15q26.1->15qter deletion (5, 6, 7, 8, 9) and three patients with a 15q26.2->15qter deletion (10, 11, 12). A low birth size was present in almost all cases, birth weights varying between –1.8 SD score (SDS) and –5.6 SDS and birth lengths between –1.3 SDS and –5.5 SDS.

We report a female patient with pre- and postnatal growth failure and elevated plasma IGF-I levels with a microscopically normal karyotype, in whom multiplex ligation-dependent probe amplification (MLPA) and array-comparative genomic hybridization (aCGH) showed that 15q26.2->qter was deleted, including the IGF1R gene.

	Patient and Methods

Top Abstract Introduction Patient and Methods Results Discussion References

 
The patient and her parents provided written informed consent for clinical and genetic studies. Height, sitting height, weight, and head circumference were measured with standard equipment and expressed as SDS (13). GH reserve was assessed with an arginine test and clonidine test, and GH was measured with Immulite 2000 (Diagnostic Products Corp., Los Angeles, CA) using the World Health Organization National Institute for Biological Standards and Control 1st international standard 80/505 (1 mg = 2.6 IU). Plasma IGF-I, IGF-II, IGF-binding protein (IGFBP)-1 and IGFBP-3 were determined by specific RIAs, as reported earlier (3).

Genetic analysis

Fluorescence in situ hybridization was performed on cultured dermal fibroblasts according to standard procedures. The probes used were the 15q subtelomeric PAC clone 154P1 (GS-154P1, (14) and the BAC clone 342L10 (RP11–342L10, Cancer_1E9, located in 15q26.3, BACPAC Resource Center, http://bacpac.chori.org/order.php).

Total RNA was isolated from cultured fibroblasts and reverse transcribed into cDNA. Genomic DNA was isolated from whole blood, and all coding exons of the IGF1R were PCR amplified and subjected to direct sequencing as described previously (3).

MLPA

MLPA probes for the IGF1R were designed according to White et al. (15) and were directed to exons 2, 8, and 18 of the IGF1R gene, which are conserved exons in different species (detailed information on the probes can be obtained from the authors). The oligonucleotide probes were ordered from Illumina Inc. (San Diego, CA) and used without purification. All reagents for the MLPA were obtained from Medical Research Council Holland (Amsterdam, The Netherlands). Reactions and data analysis were performed according to White et al. (15) and the manufacturer’s instructions.

aCGH

To determine the boundaries of the deletion, we performed aCGH using the 44B human genome CGH microarrays (Agilent, Santa Clara, CA). These arrays contain 43 x 10³ 60-mer oligonucleotide probes (mostly exonic) that span the human genome with an average spacing of 35 kb. aCGH analysis was performed according to the manufacturer’s protocols. After hybridization and washing, slides were dried and scanned using a microarray scanner (Agilent). Images were analyzed with Agilent’s CGH Analytics software.

Functional analysis

Real-time PCR was performed with the Bio-Rad iQ5 multicolor real-time PCR detection system using Hs_IGF1R_SG Quantitect primer assay primers (QIAGEN, Valencia, CA). Fibroblast cultures of the patient and two healthy donors (a 29 yr old female and 48 yr old male) were used for Western blotting (we have not found evidence that IGF1R expression and responsiveness are dependent on age, passage number and sex). Cells were stimulated for 10 min with or without 10 ng/ml IGF-I. Blots were probed with an antiphospho-protein kinase B (PKB)/Akt, total PKB/Akt (Cell Signaling Technology, Beverly, MA), antiphospho-IGF1R (Biosource International, Camarillo, CA), and total IGF1R (Cell Signaling Technology) antibodies as described previously (16). Binding studies were performed using iodinated IGF-I in the presence of an excess of an IGF-I analog that is bound by IGFBPs but not by the IGF1R (Ala³¹Leu⁶⁰-IGF-I; GroPep, Adelaide, Australia) (17). In short, fibroblasts of the patient and six controls (between 5 and 59 yr old) were incubated at 4 C with 30,000 cpm [¹²⁵I]IGF-I, 250 ng/ml Ala³¹Leu⁶⁰-IGF-I, and graded amounts of unlabeled native IGF-I in 250 µl HEPES binding buffer, as previously described (17). After 18 h, cells were washed and solubilized in 1 M NaOH. Radioactivity was determined using a -counter.

	Results

Top Abstract Introduction Patient and Methods Results Discussion References

 
Case report

A 4.5-yr-old girl presented with severe growth retardation (height –3.5 SDS). She was born after 39 wk gestation as the third child of healthy unrelated parents after an uneventful pregnancy, with a weight of 2.1 kg (–3.0 SDS), a length of 47 cm (–1.3 SDS), and head circumference of 33 cm (–2 SDS) (18). Target height corrected for secular trend (4.5 cm per generation) was 171.8 cm (0.2 SDS) (13) and 2 siblings had a normal height (0 and +1.3 SDS). Motor development was normal (she started sitting at the age of 10 months and walking at 17 months), but speech development was slow.

Body proportions were normal and body mass index was 17 kg/m² (1.0 SDS). She had a high-pitched voice, and there was increased abdominal fat distributed in a lobular pattern. She wore strong glasses because of extreme myopia (–10 and –7.5). There were no dysmorphic features.

Celiac disease, hypothyroidism, and other possible causes of growth retardation were excluded and the karyogram was normal (46,XX). IGF-I was 203 ng/ml (+2.5 SDS), IGFBP-3 2.49 mg/l (+0.8 SDS), and IGFBP-1 46.6 ng/ml (normal). Two GH stimulation tests (clonidine and arginine) resulted in peaks of 8.4 and 25 mU/liter. After four daily GH injections (1 mg/m² body surface, Humatrope; Lilly, Indianapolis, IN), IGF-I rose from 224 to 375 ng/ml, and after another 3 d of GH (2 mg/m²), IGF-I was 480 ng/ml. Bone age was delayed by 2.5 yr.

At 5.3 yr GH therapy was started (1 mg/m²·day sc, Humatrope; Lilly), equivalent to 0.26 mg/kg body weight per week). A rapid catch-up growth occurred, followed by a stabilization at a height of –2 SDS, whereas IGF-I was approximately +3.5 SDS. Lean body mass SDS increased to –0.96. Puberty started at the age of 12.8 yr, and at 15 yr, she reached her adult height of 157 cm (–1.6 SDS, 1.8 SD lower than her target height SDS). Head circumference was 53 cm (–1.1 SDS). She had a regular menstrual cycle and has recently completed high school.

Genetic and functional analysis

Sequence analysis revealed no mutation in the coding exons of the IGF1R gene. MLPA showed a heterozygous deletion of all three IGF1R exons (2, 8 and 18) included in the MLPA assay (Fig. 1), which was not present in parental DNA. Fluorescence in situ hybridization confirmed the IGF1R deletion and a deletion of the 15q telomere. aCGH revealed that the deletion comprises 5.2 mb of the terminal part of the long arm of chromosome 15 (15q26.2->15qter), starting from the SPATA8 gene, spanning 34 genes.

View larger version (19K): [in this window] [in a new window]   FIG. 1. Gene scan of the MLPA analysis of the IGF1R gene The arrows in the lower panel show the lower peaks of the three IGF1R probes in the patient, compared with the control in the upper panel, indicating a deletion of IGF1R.

View larger version (13K): [in this window] [in a new window]   FIG. 2. Functional analysis. A, Expression of three IGF1R mRNA fragments. P, Patient; C, control. Cultured dermal fibroblasts showed decreased expression of IGF1R mRNA in the patient vs. a random control. B, Equal amounts of cells of the patient and four controls were seeded in 24-well plates. At confluency, cells were incubated with [125I]IGF-I in the presence of 250 ng/ml Ala31Leu60-IGF-I and increasing amounts of unlabeled native IGF-I. After 18 h, cells were washed and binding of [125I]IGF-I was determined. Data represent the mean of two quadruplicate experiments and are expressed as percentage of total binding in the presence of competition with the lowest concentration IGF-I (0.05 ng/ml), which was set to 100% after correction for nonspecific binding. The displacement curve of the patient’s cells was indistinguishable from four controls (aged 29–59 yr). Scatchard analysis was performed for the calculation of the binding affinity (Kd) and binding capacity (Bmax) of the patient’s cells and six controls (5–59 yr). The Kd and Bmax of our patient showed no significant differences, compared with controls. Values represent the mean of two quadruplicate experiments ± SD. C, Dermal fibroblasts of the patient and two controls (29 and 48 yr) were stimulated with 10 ng/ml IGF-I for 10 min. Protein lysates were collected and 25 µg of protein was used for Western blotting using phosphospecific IGF1R and PKB/Akt (Ser473) antibodies and total IGF1R and PKB/Akt antibodies (picture not shown). Densitometric quantification of the Western blots was performed. Data are expressed as a ratio of phosphor-specific IGF1R or PKB/Akt and total IGF1R or PKB/Akt, respectively. The ratio of the unstimulated lysates was set at 100%. The activation of the IGF1R and PKB/Akt tended to be lower in the patient, but this did not reach significance.

	Discussion

Top Abstract Introduction Patient and Methods Results Discussion References

The clinical presentation of this patient [small for gestational age, persistent short stature, microcephaly, and elevated IGF-I] is similar to that of the rare cases with heterozygous IGF1R mutations (1, 2, 3, 4). Children with a pure terminal deletion of 15q, without the presence of a ring chromosome (5, 6, 7, 8, 9, 10, 11, 12), have a similar growth phenotype. Because deletions of the IGF1R have not been observed in healthy controls, we believe that this phenotype is caused by IGF1R haploinsufficiency. Children with a terminal deletion of IGF1R often have additional abnormalities, and we propose that these are due to the defects in other genes in the deleted region. For example, the normal development in our patient, in the adult woman with a heterozygous missense mutation (3), and three other cases with IGF1R missense mutations (1, 2, 4) suggest that the degree of developmental delay is predominantly determined by the deletion of other genes on 15q26.1->qter. Similarly, the dysmorphic features described in the patients with terminal 15q deletion, such as craniofacial characteristics and anomalies of hand and feet, are uncommon in patients with IGF1R mutations. Our patient had extreme myopia, which have not been reported in other patients with IGF-I resistance.

In all reported cases with 15qter deletion, except one (11), the deletion was detected by regular karyotyping. In our case the deletion was not diagnosed with regular karyotyping, but it was detected through MLPA and further characterized with aCGH. Because the MLPA assay can be easily extended to include as many as 40 different probe sets, this technique is ideally suited for the evaluation of copy number changes of multiple genomic regions simultaneously. We therefore expect that this technique will significantly contribute to the elucidation of genetic causes of unexplained short stature in the future and propose that children presenting with the phenotype of IGF-I resistance should be tested with MLPA, specific for IGF1R, followed by sequencing of the gene.

In vitro, a deletion of one copy of the IGF1R would be expected to result in lower expression of IGF1R mRNA and protein. Indeed, in dermal fibroblasts of the patient, IGF1R mRNA expression was decreased, compared with a panel of controls. However, we did not observe reduced protein expression, and the tendency toward a diminished IGF-I binding and decreased activation of downstream signaling upon a challenge with IGF-I did not reach statistical significance. This is in line with the inconsistent results reported earlier, suggesting that dermal fibroblasts may be a suboptimal model to study the functional consequences of IGF1R haploinsufficiency (6, 10). In contrast, in the family with a missense mutation in the intracellular kinase domain of the IGF1R, we observed a strongly decreased activation of downstream signaling (3). A possible explanation of this discrepancy is a dominant-negative effect of the mutation, resulting in a more pronounced effect in vitro; alternatively the consequences of haploinsufficiency may be cell type dependent, with possibly a relatively strong effect in growth plate chondrocytes, or the fibroblast model may be too insensitive and variable to pick up statistically significant differences.

We have shown that the growth retardation caused by IGF1R haploinsufficiency can successfully be treated with GH, although full catch-up was not reached. This partially confirms earlier reports (6, 10, 19), and we suggest that this positive effect may be due to direct GH effects in combination with strongly elevated plasma IGF-I levels that may partially overcome the decreased IGF-I sensitivity.

In conclusion, the characteristics of a heterozygous terminal 15q deletion are dominated by partial IGF-I resistance due to IGF1R haploinsufficiency. GH treatment considerably improves growth. A normal karyogram in patients with features suggestive for IGF-I resistance does not exclude small deletions, and MLPA is a powerful diagnostic strategy to detect submicroscopic deletions.

	Acknowledgments

 
The authors thank Kerstin Hansson for technical assistance.

	Footnotes

 
Disclosure statement: M.J.E.W. received a lecture fee from Lilly; S.M.P.F.d.M.K.-S. received lecture fees from Pfizer and Lilly; and J.M.W. consults for and receives lecture fees from Ipsen, Pfizer, Novo Nordisk, and Ferring. M.d.M., M.E.K., H.A.v.D., A.M.B., S.G.K., S.J.W., M.L., J.T.D.D., and M.K. have nothing to declare.

First Published Online March 18, 2008

Abbreviations: aCGH, Array-comparative genomic hybridization; IGFBP, IGF-binding protein; IGF1R, type 1 IGF receptor; MLPA, multiplex ligation-dependent probe amplification; PKB, protein kinase B; SDS, SD score.

Received August 9, 2007.

Accepted March 7, 2008.

	References

Top Abstract Introduction Patient and Methods Results Discussion References

 

1. Abuzzahab MJ, Schneider A, Goddard A, Grigorescu F, Lautier C, Keller E, Kiess W, Klammt J, Kratzsch J, Osgood D, Pfaffle R, Raile K, Seidel B, Smith RJ, Chernausek SD 2003 IGF-I receptor mutations resulting in intrauterine and postnatal growth retardation. N Engl J Med 349:2211–2222[Abstract/Free Full Text]
2. Kawashima Y, Kanzaki S, Yang F, Kinoshita T, Hanaki K, Nagaishi J, Ohtsuka Y, Hisatome I, Ninomoya H, Nanba E, Fukushima T, Takahashi SI 2005 Mutation at cleavage site of insulin-like growth factor receptor in a short-stature child born with intrauterine growth retardation. J Clin Endocrinol Metab 90:4679–4687[Abstract/Free Full Text]
3. Walenkamp MJE, Van der Kamp HJ, Pereira AM, Kant SG, van Duyvenvoorde HA, Kruithof MF, Breuning MH, Romijn JA, Karperien M, Wit JM 2006 A variable degree of intrauterine and postnatal growth retardation in a family with a missense mutation in the insulin-like growth factor I receptor. J Clin Endocrinol Metab 91:3062–3070[Abstract/Free Full Text]
4. Inagaki K, Tiulpakov A, Rubtsov P, Sverdlova P, Peterkova V, Yakar S, Terekhov S, LeRoith D 2007 A familial IGF-1 receptor mutant leads to short stature: clinical and biochemical characterization. J Clin Endocrinol Metab 92:1542–1548[Abstract/Free Full Text]
5. Roback EW, Barakat AJ, Dev VG, Mbikay M, Chretien M, Butler MG 1991 An infant with deletion of the distal long arm of chromosome 15 (q26.1-qter) and loss of insulin-like growth factor 1 receptor gene. Am J Med Genet 38:74–79[CrossRef][Medline]
6. Siebler T, Lopaczynski W, Terry CL, Casella SJ, Munson P, De Leon DD, Phang L, Blakemore KJ, McEvoy RC, Kelley RI 1995 Insulin-like growth factor I receptor expression and function in fibroblasts from two patients with deletion of the distal long arm of chromosome 15. J Clin Endocrinol Metab 80:3447–3457[Abstract]
7. Tonnies H, Schulze I, Hennies HC, Neumann LM, Keitzer R, Neitzel H 2001 De novo terminal deletion of chromosome 15q26.1 characterised by comparative genomic hybridisation and FISH with locus specific probes. J Med Genet 38:617–621[Free Full Text]
8. Bhakta KY, Marlin SJ, Shen JJ, Fernandes CJ 2005 Terminal deletion of chromosome 15q26.1: case report and brief literature review. J Perinatol 25:429–432[CrossRef][Medline]
9. Biggio Jr JR, Descartes MD, Carroll AJ, Holt RL 2004 Congenital diaphragmatic hernia: is 15q26.1–26.2 a candidate locus? Am J Med Genet A 126:183–185
10. Okubo Y, Siddle K, Firth H, O'Rahilly S, Wilson LC, Willatt L, Fukushima T, Takahashi SI, Petry CJ, Saukkonen T, Stanhope R, Dunger DB 2003 Cell proliferation activities on skin fibroblasts from a short child with absence of one copy of the type 1 insulin-like growth factor receptor (IGF1R) gene and a tall child with three copies of the IGF1R gene. J Clin Endocrinol Metab 88:5981–5988[Abstract/Free Full Text]
11. Pinson L, Perrin A, Plouzennec C, Parent P, Metz C, Collet M, Le Bris MJ, Douet-Guilbert N, Morel F, De Braekeleer M 2005 Detection of an unexpected subtelomeric 15q26.2-> qter deletion in a little girl: clinical and cytogenetic studies. Am J Med Genet A 138:160–165
12. Rujirabanjerd S, Suwannarat W, Sripo T, Dissaneevate P, Permsirivanich W, Limprasert P 2007 De novo subtelomeric deletion of 15q associated with satellite translocation in a child with developmental delay and severe growth retardation. Am J Med Genet A 143:271–276[Medline]
13. Fredriks AM, van Buuren S, Burgmeijer RJ, Meulmeester JF, Beuker RJ, Brugman E, Roede MJ, Verloove-Vanhorick SP, Wit JM 2000 Continuing positive secular growth change in The Netherlands, 1955–1997. Pediatr Res 47:316–323[Medline]
14. Knight SJ, Lese CM, Precht KS, Kuc J, Ning Y, Lucas S, Regan R, Brenan M, Nicod A, Lawrie NM, Cardy DL, Nguyen H, Hudson TJ, Riethman HC, Ledbetter DH, Flint J 2000 An optimized set of human telomere clones for studying telomere integrity and architecture. Am J Hum Genet 67:320–332[CrossRef][Medline]
15. White SJ, Vink GR, Kriek M, Wuyts W, Schouten J, Bakker B, Breuning MH, den Dunnen JT 2004 Two-color multiplex ligation-dependent probe amplification: detecting genomic rearrangements in hereditary multiple exostoses. Hum Mutat 24:86–92[CrossRef][Medline]
16. Walenkamp MJ, Karperien M, Pereira AM, Hilhorst-Hofstee Y, Van Doorn J, Chen JW, Mohan S, Denley A, Forbes B, van Duyvenvoorde HA, van Thiel SW, Sluimers CA, Bax JJ, de Laat JA, Breuning MB, Romijn JA, Wit JM 2005 Homozygous and heterozygous expression of a novel insulin-like growth factor-I mutation. J Clin Endocrinol Metab 90:2855–2864[Abstract/Free Full Text]
17. Miller SA, Dykes DD, Polesky HF 1988 A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 16:1215
18. Niklasson A, Ericson A, Fryer JG, Karlberg J, Lawrence C, Karlberg P 1991 An update of the Swedish reference standards for weight, length and head circumference at birth for given gestational age (1977–1981). Acta Paediatr Scand 80:756–762[Medline]
19. Raile K, Klammt J, Schneider A, Keller A, Laue S, Smith R, Pfaffle R, Kratzsch J, Keller E, Kiess W 2006 Clinical and functional characteristics of the human Arg59Ter insulin-like growth factor i receptor (IGF1R) mutation: implications for a gene dosage effect of the human IGF1R. J Clin Endocrinol Metab 91:2264–2271[Abstract/Free Full Text]

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