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Centro de Investigaciones Endocrinológicas (H.M.D., P.A.S., P.B., M.G., H.G.J.), Hospital de Niños "R. Gutiérrez," 1425 Buenos Aires, Argentina; Pediatric Endocrinology and Metabolism (A.L., S.K.), Mayo Clinic, Rochester, Minnesota 55095; Endocrinology (F.H.M.), Children’s Hospital of Western Ontario, London, Canada N6A 4G5; and Medical Research Laboratories (J.F.), Clinical Institute, Aarhus University Hospital, DK-8000 Aarhus, Denmark
Address all correspondence and requests for reprints to: Horacio M. Domené, Centro de Investigaciones Endocrinológicas, Hospital de Niños "R. Gutiérrez," Gallo 1330, 1425 Buenos Aires, Argentina. E-mail: hdomene{at}cedie.org.ar.
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Abstract |
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Objective: Our objective was to study the characterization, at biochemical and molecular levels, of the cause for severe circulating IGF-I and IGFBP-3 deficiency in a male patient with mild growth retardation.
Patients: We report an adolescent male with delayed growth and pubertal development (Tanner stage I, –2.00 SD score for height at the age of 15.3 yr), profound circulating IGF-I and IGFBP-3 deficiency, and poor response to GH treatment.
Results: The index case, as well as one of his brothers, and his sister were found to be compound heterozygotes for two novel IGFALS gene mutations: C540R, a missense point mutation; and S195_197Rdup, a 9-bp duplication. The parents and youngest brother were found to be carriers for one of these two mutations. The three affected siblings had marked reduction of IGF-I and IGFBP-3 levels, undetectable serum levels of ALS, inability to form ternary complexes, and moderate insulin resistance. All of them attained a normal near-adult height (between –1.0 and –0.5 SD score), which was nonetheless lower than that of their heterozygous brother. The IGF system was only modestly affected in the heterozygous carriers.
Conclusions: This study confirms the critical role of ALS in forming ternary complexes and the maintenance of normal levels of IGF-I and IGFBP-3. Insulin resistance, pubertal delay in male patients, and poor GH responsiveness seem to be frequent findings in ALS deficiency. However, haploinsufficiency of the IGFALS gene has no discernible clinical effects with only modest impact on the IGF system.
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Introduction |
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The disruption of the IGFALS gene by targeted inactivation in the mouse (6) as well as mutational inactivation of the gene in humans (7, 8) result in a marked reduction in circulating levels of both IGF-I and IGFBP-3, probably the result of a more rapid clearance, due to either an increased efflux or an accelerated degradation. Despite the profound reduction in circulating IGF-I and IGFBP-3 levels, postnatal growth is only mildly affected if at all. Pubertal delay and insulin resistance were found in one ALS-deficient patient (7). However, because that was the first reported case, it was not clear if these abnormalities were related to ALS deficiency per se, or merely coincidental findings. Thus, a recently reported new ALS-deficient patient presented with pubertal development at an appropriate age (8).
We now report a family that was found to have three affected siblings with complete ALS deficiency, in association with two novel IGFALS gene mutations. The parents and one other sibling were found to be heterozygous carriers for one of the mutations. Thus, the effects of complete and haploinsufficiency of the IGFALS gene are described.
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Subjects and Methods |
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The B2 was initially examined elsewhere in the United States at the age of 15.3 yr for delayed growth and pubertal development. He was the second of four siblings from nonconsanguineous, American, white parents of primarily Norwegian and German descent. Both parents had normal stature [father (F) 188.0 cm, +1.50 SD score (SDS); mother (M) 157.5 cm, –0.93 SDS]. At the initial examination, his height was 156.2 cm (–2.00 SDS), weight 56 kg (body mass index 22.9 kg/m2, +1.23 SDS), bone age 14 yr, testicular volume 2 cm3, and pubic hair development was at Tanner stage I. Preliminary studies showed a normal GH response of 10 µg/liter to an arginine test and low IGF-I levels of 12 µg/liter (1.56 nmol/liter) (reference values 109–485 µg/liter or 14.2–63.0 nmol/liter). With the diagnosis of non-GH deficient short stature, recombinant human GH (rhGH) treatment was started at the age 15.5 yr at a dose of 0.3 mg/kg·wk. The dose was gradually increased to 0.8 mg/kg·wk due to the poor clinical and IGF-I responses to treatment. Pretreatment growth velocity was 6.2 cm/yr, whereas posttreatment velocity was 8.0 cm/yr, and IGF-I level only increased to 54 µg/liter (7.02 nmol/liter). At the age of 16.1 yr, while the patient was on GH treatment, he was admitted to the Mayo Clinic, Rochester, Minnesota. At that time he had a height of 163.4 cm (–1.0 SDS), weight 64.8 kg (0.0 SDS), genital and pubic hair Tanner stage I, and testicular volume 2.1–2.4 cm3. Laboratory evaluation showed: IGF-I, 57 µg/liter (7.41 nmol/liter) (237–601 µg/liter or 30.8–78.1 nmol/liter); IGFBP-3, 0.3 µg/liter (10.5 nmol/liter) (2.5–4.8 µg/liter or 87.5–168 nmol/liter); GHBP, 504 pmol/liter (431–1892); negative anti-GH antibodies; and prepubertal testosterone levels of 0.28 ng/ml (0.97 nmol/liter). The dose of rhGH was reduced to 0.5 mg/kg·wk, and im testosterone enanthate was added at 50 mg/month. Total length of rhGH treatment was 17 months. The association of mild growth retardation with marked reduction of IGF-I and IGFBP-3 levels, and the inability of GH to improve growth and serum IGF-I levels suggested the diagnosis of ALS deficiency, which was confirmed by undetectable serum levels of ALS (<0.5 mg/liter, reference values 20–35 mg/liter).
Subsequently, blood samples were obtained from the B2 and all his immediate family members for the study of the GH-IGF axis and for molecular characterization of the IGFALS gene.
Serum levels of T4, free T4, T3, TSH, antithyroperoxidase antibodies, LH, FSH, estradiol, and prolactin were determined by electrochemoluminescence (Elecsys; Roche, Indianapolis, IN), levels of GH and insulin by immunofluorometric assay (Immulite; Diagnostic Products Corp., Los Angeles, CA), and testosterone by RIA (Diagnostic Systems Laboratories, Inc., Webster, TX). IGF-I levels were determined by RIA after serum extraction by the acid-ethanol method followed by cryoprecipitation (9), IGFBP-3 by immunoradiometric assay, and ALS by ELISA (10) using commercial kits (Diagnostic Systems Laboratories, Inc.).
Serum IGFBPs were evaluated by Western ligand blot (11), and ALS protein by Western immunoblot using antibodies against the amino terminal (hALS1–34) and the carboxy terminal regions (hALS551–578; Diagnostic Systems Laboratories, Inc.) of the ALS protein (7).
Ternary complex formation was evaluated by neutral size-exclusion chromatography (7). A total of 100 µl serum was incubated overnight at 22 C with 3.5 x 106 counts per minute of 125I-IGF-I, and then cross-linked with the addition of disuccinimidyl suberate (Sigma-Aldrich, St. Louis, MO). Samples were chromatographed on a HiPrep 16/60 Sephacryl S-200HR column (Amersham Pharmacia Biotech AB, Uppsala, Sweden). A total of 500-µl aliquots was loaded onto the column, and 1-ml fractions were collected and counted.
Genomic DNA from all family members was isolated from peripheral leukocytes based on the use of cetyltrimethylammonium bromide lysis buffer and isoamyl alcohol-chloroform extraction (12). Exons 1 and 2 and contiguous intron sequences, corresponding to the IGFALS gene (GenBank accession no. AF192554), were amplified by PCR using oligonucleotide primers flanking both exons (exon 1: F1 5'-ccgcagtggggaaatccaga-3'; and R1 5'-gggtggaggacgggacagag-3'. Exon 2 was amplified in two fragments: fragment A: F2A, 5'-gagccaagccctgagcgtctgt-3'; and R2A 5'-ccttgaggaagagtcggcgg-3'; and fragment B: F2B 5'-aacgtgttcgtgcagctgcc-3' and R2B 5'-tccccagcacaaggtgagcc-3'). PCR products were purified using Wizard Clean up (Promega, Madison, WI). Afterward, the purified products were sequenced in both directions in an ABI 3730xl automatic sequencer (Macrogen, Seoul, South Korea) using the same oligonucleotides primers used for amplification and/or internal oligonucleotide primers (for exon 1: INT-1 reverse 5'-agcgggctcagggtcacaga-3'; for exon 2 fragment A: INT-A1 reverse 5'-tcgtcatcgtagctgcagac-3'; INT-A2 reverse 5'-ctgctcagacggttgttgct-3'; INT-A3 reverse 5'-gcagcgatgaggttgcggtcca-3'; and INT-A1 forward 5'-agcaacaaccgtctgagcag-3'; for fragment B: 2A reverse 5'-ccttgaggaagagtcggcgg-3'; INT-B forward 5'-agctgcctgggacgcatccg-3'; and 2B reverse 5'-tccccagcacaaggtgagcc-3').
The presence of a missense point mutation (c.1618T>C; C540R) and a 9-bp duplication (c.583_591dup9; p.S195_197Rdup) was examined in all family members by restriction fragment length polymorphism, taking advantage of the fact that each mutation introduces a new restriction site for the Btg I and Fsp I enzymes (New England BioLabs, Inc., Ipswich, MA), respectively.
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Results |
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The B2 had delay of growth and pubertal development (Tanner stage I at 16.1 yr). After a 6-month course of testosterone treatment, puberty developed normally, and he reached Tanner stage V for both sexual development and pubic hair at the age of 18.0 yr. Although his height was below normal at 16 yr, he had a normal growth spurt while receiving rhGH plus testosterone, reaching a normal near-adult height of 172.6 cm (–0.5 SDS) at the age of 18.0 yr (Fig. 1
), close to his midparental target height (MPTH) of 179.2 cm (Table 1). Retrospectively collected data showed a similar pubertal development and growth history for the oldest brother, who entered puberty at 16 yr of age. He was found to be at Tanner stage V and had a normal adult height of 174.0 cm (–0.5 SDS) at the age of 19.6 yr. In contrast, the pubertal development was normal in the youngest sister (G1), who had menarche at the age of 13 yr, and a normal height of 156.2 cm (–1.00 SDS) at 15.4 yr of age. There was no history of pubertal delay in either of the parents, or in the youngest brother (B3) (Table 1).
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Total, high-density lipoprotein-, and low-density lipoprotein-cholesterol, as well as triglycerides, were normal except for the B2 [total cholesterol 205 mg/dl (5.3 mmol/liter), reference values < 200 mg/dl (5.17 mmol/liter); and triglycerides 183 mg/dl (2.07 mmol/liter), reference values < 160 mg/dl; (1.81 mmol/liter)]. Levels of calcium, phosphorous, and PTH were normal in all family members. Thyroid function evaluated by TSH, and total, free T4 and T3 levels was also normal in all family members (data not shown).
Gonadal function evaluation
All subjects studied had normal LH, FSH, prolactin, and estradiol levels according to their sex and pubertal stage (data not shown). Testosterone levels were normal in the males (7.0, 5.1, 5.8, and 6.1 ng/ml, or 24.3, 17.7, 20.1, and 21.2 nmol/liter for the F and three brothers, respectively).
Glucose–insulin
Although glucose levels were normal in all the children and their parents, insulin levels, and consequently the homeostasis model of assessment (HOMA) index, were elevated in the B2, his older brother (B1), and younger G1 (Table 2).
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Basal GH levels were elevated only in the B2 (6.8 µg/liter). Circulating levels of IGF-I and IGFBP-3 were markedly decreased in the B2, his B1, and G1, whereas IGF-I levels were subnormal in the F, low normal in the M, and normal in the B3 (Table 2
). ALS was undetectable in the B2, his B1, and G1 (all < 0.5 mg/liter), low in the F and B3 (–3.1 and –3.0 SDS, respectively), and low normal in the M.
Western ligand and Western immunoblot studies
Western ligand blot of the IGFBPs showed that the B2, his B1, and G1 had a marked reduction in the density of the 40- to 43-kD doublet corresponding to intact IGFBP-3, and subnormal densities of the bands corresponding to IGFBP-1 and -2. A normal IGFBP profile was observed in the M, F, and B3 (Fig. 2). Western immunoblot using antibodies against both the amino (Fig. 3, panel A) and carboxy terminals (Fig. 3, panel B) showed that no 84- to 86-kD ALS protein band was detected in the B2, his B1, and G1. An apparently normal ALS-protein band was observed in the M, whereas a less intense band was observed in the B3 and F.
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Size-exclusion chromatography revealed no ternary complex formation in the B2, his B1, and G1, whereas normal ternary complexes were observed in the B3, F, and M (Fig. 4).
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The complete sequencing of the IGFALS gene revealed that the B2 harbored two different mutations: a transition at the first base of codon 540 (c.1618T>C) resulting in a missense point mutation (p.C540R); and a 9-bp duplication (c.583_591dup9) predicting the insertion of three extra amino acids in the seventh leucine-rich repeat (LRR) (p.S195_197Rdup) (Fig. 5, panel A). Because both mutations created new enzymatic restriction sites (Btg I for p.C540R and Fsp I for p.S195_197Rdup, respectively), restriction fragment length polymorphism analysis could be performed. This analysis showed that the B1 and G1 were also compound heterozygotes for the same two mutations. The other family members turned out to be carriers for one IGFALS gene mutation. The F and B3 carried the duplication mutation, whereas the M carried the missense point mutation (Fig. 5, panel B).
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Discussion |
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By extending the endocrine and genetic studies to the siblings and parents of the B2, we found that two of the patient’s siblings were also compound heterozygotes for the same IGFALS gene mutations and had similar endocrine profiles. The phenotype for ALS deficiency is mild, and as illustrated by this report, it could remain unnoticed, even within a family with a known affected child.
This study expands the spectrum of IGFALS mutations, showing for the first time that this condition could be present in nonconsanguineous families, adding the three newly diagnosed patients to the two male ALS-deficient patients previously published (7, 8). Moreover, this is the first description of ALS deficiency in a female. Another interesting finding of this study is the association of insulin resistance with ALS deficiency. Confirming our previous findings, the three ALS-deficient siblings had normal glucose levels at the expense of increased levels of insulin. The consistent finding of insulin resistance and markedly reduced levels of IGF-I suggests that normal IGF-I levels have important insulin-like action, probably by stimulating glucose transport, particularly at skeletal muscle (17). Insulin levels were not reported in the recently described ALS-deficient patient (8). A delayed onset of puberty was observed in the two affected males, as it was in our previously described ALS-deficient male patient, whereas the female patient had a normal menarche. Because no history of pubertal delay was present in this family, and the sibling carrying only one ALS-mutated allele had a normal climbing of puberty, it is possible that circulating IGF-I would be involved in the activation of the onset of puberty in males. Considering that delayed puberty is usually observed in patients with reduced levels of IGF-I (e.g. malnutrition, GH deficiency, GH resistance, primary IGF-I deficiency, and type 1 diabetes), it has been postulated that the increasing levels of circulating IGF-I represents one of the metabolic signals involved in the initiation of puberty (18). Increased levels of IGF-I could improve gonadotropin secretion (19) and increase the sensitivity of the gonads to their action (20). However, circulating IGF-I remained unchanged during the follow-up of the B2, as well as in the two affected siblings. Furthermore, no pubertal delay was observed in the patient described by Hwa et al. (8), indicating that the delay of puberty is not a consistent finding in ALS deficiency.
The heterozygous carriers (the parents and B3) had ALS levels below or in the lower limits of the normal range, suggesting that in normal subjects, both alleles of the IGFALS gene are expressed. Similar low-normal levels of ALS have been recently reported in the haploinsufficient M of the ALS-deficient patient described by Hwa et al. (8). In heterozygous carriers, IGF-I levels were low normal or even below normal. In these subjects, the finding of normal IGFBP-3 levels and capability to form ternary complexes may indicate that the presence of one normal IGFALS gene allele may be sufficient to maintain ALS levels above a certain threshold to form ternary complexes, preserving IGFBP-3 from early degradation.
The segregation of the biochemical phenotype, only present in compound heterozygous patients, is compatible with an autosomal recessive inheritance pattern. In addition, the complete lack of ALS in compound heterozygous patients, as well as the low or low-normal levels of ALS in heterozygous carriers, suggest that the novel mutations each inactivate one of the IGFALS gene alleles. The p.C540R predicts the loss of Cys 540, a highly conserved residue in the C-terminal domain of LRR proteins, most likely involved in a disulfide bond. Thus, we speculate that this mutation precludes formation of disulfide bridges or leads to erroneous pairing of cysteines, thereby disturbing the integrity of the spatial arrangement of the ALS protein, which is normally a donut-shaped molecule (21), and consequently its structure and function. Inactivating mutations in the two highly conserved Cys residues at the carboxy terminus have been described in other LRR proteins (22), such as nyctalopin (23), LH receptor (24), TSH receptor (25), and glycoprotein Ib
(26). The p.S195_197Rdup mutation, encoding for three extra amino acid residues at the seventh LRR, may disrupt the alignment of hydrophobic residues. Similar mutations involving insertions or deletions of 3–8 amino acids have been reported in the gene encoding for nyctalopin (23), associated with congenital stationary night blindness type 1, presumably changing or disrupting the loop structure. Although only the in vitro expression and determination of ternary complex formation of the mutant proteins will allow the characterization of the biological impact of these mutations, the resulting effect of the joint presence of both mutations is the complete lack of immunological and functional ALS protein.
The three affected siblings had normal, near-adult heights, close to their expected target height (–0.5, –0.5, and –1.0 SDS, or 5.2, 6.6, and 10 cm below MPTH for B1, B2, and G1, respectively). However, their heights were reduced when compared with the heterozygous brother (B3: +0.5 SDS at the age of 14 yr). Moreover, both parents reached an adult height equal or greater than their own MPTH (Table 1), suggesting that IGF-I transport by ternary complexes is required to fulfill completely the growth potential.
Based on the five diagnosed ALS-deficient patients, the two previously reported boys (7, 8, 27) and the two boys and one girl described in the present study, it is possible to glean similarities as well as differences. First, all affected patients attained a height within or slightly below population-specific growth standards. In those cases in which parents’ heights were available, patients’ heights were close to the calculated MPTH but consistently below their normal or heterozygous carrier siblings. In the patient reported by Hwa et al. (8), his predicted adult height was 159.4 cm, or –2.1 SDS for Turkish standards, shorter than our previously reported patient and the three affected siblings from the present study, but it coincided with the parental height of his parents. We can only speculate that pubertal retardation and, consequently, a longer period of prepubertal growth may result in a higher adult stature. Because only subjects either compound heterozygous or heterozygous carriers for mutations in the IGFALS gene are present in the studied members of the family, and no subject carried two wild-type alleles, it is not possible to address the issue of the effect of heterozygous status on postnatal growth.
Finally, this report also confirms that despite the marked deficiency of circulating IGF-I, postnatal growth is only mildly affected. It is not known how almost normal growth is maintained in this condition. This may result from preserved peripheral IGF-I production acting at the cartilage level. Increased IGF-I efflux to the peripheral tissues at the expense of the circulating IGF-I pool and IGF-I-independent GH growth promoting effects may also have an effect.
In summary, this family illustrates the main phenotypic features of ALS deficiency: 1) marked circulating IGF-I deficiency, insulin resistance, and delayed puberty in male patients, with a mild effect on linear growth in patients with biallelic deficiency; and 2) no clinical effects and only moderate impact on the IGF system in subjects carrying one mutated allele of the IGFALS gene.
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Acknowledgments |
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Footnotes |
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Disclosure Statement: F.H.M. has received lecture fees less than $10,000, and J.F. has received consulting fees less than $10,000. H.M.D., P.A.S., A.L., S.K., P.B., M.G., and H.G.J. have nothing to declare.
First Published Online August 28, 2007
Abbreviations: ALS, Acid-labile subunit; B1, older brother; B2, index case; B3, youngest brother; F, father; G1, sister; HOMA, homeostasis model of assessment; IGFBP, IGF binding protein; LRR, leucine-rich repeat; M, mother; MPTH, midparental target height; rhGH, recombinant human GH; SDS, SD score.
Received May 24, 2007.
Accepted August 17, 2007.
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References |
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)-subunit of the high molecular weight insulin-like growth factor-binding protein complex. J Clin Endocrinol Metab 70:1347–1353This article has been cited by other articles:
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  I. Ueki, S. L. Giesy, K. J. Harvatine, J. W. Kim, and Y. R. Boisclair The Acid-Labile Subunit Is Required for Full Effects of Exogenous Growth Hormone on Growth and Carbohydrate Metabolism Endocrinology, July 1, 2009; 150(7): 3145 - 3152. [Abstract] [Full Text] [PDF]
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 S. Yakar, C. J. Rosen, M. L. Bouxsein, H. Sun, W. Mejia, Y. Kawashima, Y. Wu, K. Emerton, V. Williams, K. Jepsen, et al. Serum complexes of insulin-like growth factor-1 modulate skeletal integrity and carbohydrate metabolism FASEB J, March 1, 2009; 23(3): 709 - 719. [Abstract] [Full Text] [PDF]
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 H A van Duyvenvoorde, M J E Kempers, T. B Twickler, J van Doorn, W J Gerver, C Noordam, M Losekoot, M Karperien, J M Wit, and A R M M Hermus Homozygous and heterozygous expression of a novel mutation of the acid-labile subunit Eur. J. Endocrinol., August 1, 2008; 159(2): 113 - 120. [Abstract] [Full Text] [PDF]
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I Banerjee, D Hanson, R Perveen, A Whatmore, G C Black, and P E Clayton Constitutional delay of growth and puberty is not commonly associated with mutations in the acid labile subunit gene. Eur. J. Endocrinol., April 1, 2008; 158(4): 473 - 477. [Abstract] [Full Text] [PDF]
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