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Department of Pediatrics (V.H., K.L.P., B.M.L., R.G.R.), Oregon Health and Science University, Portland, Oregon 97239-3098; Department of Pediatrics (G.H., H.F., D.K.), Medical University of Vienna, Vienna, Austria, A-1090; Lucile Packard Foundation for Children’s Health (R.G.R.), Palo Alto, California 94304; and Department of Pediatrics (R.G.R.), Stanford University, Stanford, California 94305-2038
Address all correspondence and requests for reprints to: Dr. Vivian Hwa, Department of Pediatrics, NRC5, Oregon Health and Science University, 3181 Southwest Sam Jackson Park Road, Portland, Oregon 97239-3098. E-mail: hwav{at}ohsu.edu.
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Abstract |
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Objective: We investigated a case of IGFD in which serum IGF-I and IGFBP-3 were abnormally low, yet growth failure was modest (–2.1 SD score at 15.5 yr of age).
Results: The young male subject, from a consanguineous pedigree, had a postnatal growth profile consistently below the third percentile. The subject had a normal fasting GH level of 3.7 µU/ml and normal serum GH binding protein level (1258 pmol/liter; normal range 431-1892 pmol/liter), but serum IGF-I and IGFBP-3 were profoundly reduced (–5.8 and –7.2 SD score, respectively, at age 12.3 yr), even through puberty. A novel homozygous missense mutation was subsequently identified in the ALS gene, which resulted in severe deficiency of serum ALS (undetectable).
Conclusions: ALS is critical for maintaining normal serum concentrations of IGF-I and IGFBP-3, most likely by prolonging the half-lives of both proteins. ALS deficiency can be associated with moderate growth failure, but in this patient, the onset and progression of puberty appear to be normal. Altogether the results support a modest role for the ternary complex in the regulation of stature.
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Introduction |
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Markedly reduced serum concentrations of IGF-I have also been described in one case of a homozygous frame-shift point mutation in the gene for acid labile subunit (ALS) (7). Because 80–85% of IGF-I in serum circulates as part of a 150-kDa ternary complex (8), comprised of one molecule each of IGF-I, IGF binding protein (IGFBP)-3 or IGFBP-5 (9), and ALS, an inactivating mutation of the gene for ALS results in dramatic reductions of serum levels of both IGF-I and IGFBP-3. Although the reported patient had serum IGF-I concentrations as low as those described in other cases of primary IGFD, growth was only modestly affected, with a height at age 14.6 yr of 145.2 cm (2.04 SD below the mean). Evaluation of this patient’s growth was complicated by the fact that he was adopted as an infant and no record of parental heights was available and by pubertal delay, with Tanner stage 1 sexual development at age 14.6 yr and a bone age of 12.5 yr. The relatively normal growth of this patient, in the face of markedly reduced serum IGF-I concentrations, was presumed to reflect normal serum concentrations of free IGF-I, although assays of free IGF-I or normal production of IGF-I at the level of the growth plate could not be performed at the time of that study. In a follow-up letter (10), the authors reported that by age 19 yr, the patient had spontaneously completed puberty and attained a height of 166.4 cm (0.94 SD below the local mean).
We now report a patient with a novel homozygous mutation of the ALS gene, which resulted in profoundly reduced serum levels of IGF-I and IGFBP-3. In this case, the onset and progression of puberty appear to be normal, but growth is retarded, supporting a modest role for the ternary complex in the regulation of stature.
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Subject and Methods |
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and 2)The male subject is the second child of consanguineous parents of Turkish origin. No data are available regarding birth length and weight, but according to the mother, the pregnancy, delivery, and perinatal period were normal. His childhood medical history was unremarkable; motor development was reported to be normal, but learning disabilities and a mild speech disorder were noted. The parents, who are now divorced, are both modestly short with the father having a height of 161 cm (–2.0 SD score, Turkish standards), and the mother a height of 143.5 cm (–2.8 SD score, Turkish standards). An older sister has a final adult height of 160 cm (0.0 SD score), well above the projected target height of 146 cm. The family, with the exception of the father, lives in Austria where the subject, at age 12.1 yr, was referred for evaluation of short stature. At that time, his height was 130.3 cm (–2.9 SD score, Turkish standards) with a body mass index of 20.0 (75th percentile). Bone age at 12.1 yr of age was read by the standards of Greulich and Pyle: 10 yr for carpal bones and 12 yr for phalanges, radius, and ulna. Physical examination showed normal body proportions, mild tooth irregularities, and mild exophthalmos. Pubertal status was Tanner 1 with testes volume of 2 ml. Routine laboratory analyses ruled out renal, liver, or hematologic diseases. Thyroid function was normal, with LH, FSH, and serum testosterone in the normal, prepubertal range.
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The subject entered puberty at age 13.0 yr and progressed at a normal pace. Height remained consistently –2 to –3 SD score for age (Table 1
), with the most recent measurement of 154.3 cm (–2.1 SD score), taken at age 15.5 yr, at which time the bone age was determined to be 15 yr and puberty had progressed to Tanner V. His predicted adult height, by the standards of Bailey and Pinneau, is calculated to be 159.4 cm (–2.1 SD score, Turkish standards), which is 1.6 cm shorter than his father but 0.65 cm taller than his parental-based target height. Evaluations of serum IGF-I and IGFBP-3 indicated that levels remained abnormally low through puberty (Table 1). Magnetic resonance imaging of the brain indicated normal hypothalamus and pituitary.
Serum assays
Serum samples were analyzed for total IGF-I, free IGF-I, and IGFBP-3 by means of immunoradiometric assays (Diagnostic Systems Laboratories, Webster, TX). ALS and GHBP were measured with ELISAs (Diagnostic Systems Laboratories). Results of assays from Austria were performed by Mediagnost.
Cell cultures
Primary fibroblast cultures were established from skin biopsies taken from the male subject (OTF cells) in compliance with the Institutional Review Board from both Oregon Health and Sciences University (Portland, OR) and the Pediatric Department, Medical University of Vienna (Vienna, Austria). Normal human dermal fibroblasts (CF cells) have been described previously (4). Cell cultures were maintained in 
MEM Earles (Mediatech Cellgro, Herndon, VA) supplemented with 20% fetal bovine serum (Life Technologies, Inc./Invitrogen Corp., Grand Island, NY) at 37 C with 5% CO2. To collect media conditioned by cells, growth media were replaced with serum-free media, and after 24 h, the conditioned media were collected and analyzed as indicated below.
Western immunoblot and ligand analysis
Serum samples (1.5 µl) and conditioned media (40 µl) were solubilized in nonreducing sodium dodecyl sulfate (SDS) sample buffer [0.5 mol/liter Tris (pH 6.8), 2% SDS, 10% glycerol, and 0.003% bromphenol blue] before electrophoresis on 12% SDS-polyacrylamide gels. Size-fractionated proteins were electroblotted onto nitrocellulose and membranes blocked with 1% BSA (RIA grade; Sigma, St. Louis, MO) in Tris-buffered saline-0.1% Tween 20 for ligand blot or with 4% milk-Tris-buffered saline-0.1% Tween 20 for immunoblot. Ligand blot analysis using 125I-IGF-I and 125I-IGF-II (Diagnostic Systems Laboratories) was performed as previously described (11). Primary antibodies for immunoblot analysis were rabbit antihuman human IGFBP-3 polyclonal antibody (11), goat antihuman ALS (N terminal) polyclonal antibody (Diagnostic Systems Laboratories), or goat antihuman ALS (C terminal) polyclonal antibody (Diagnostic Systems Laboratories). Appropriate secondary antibodies (horseradish peroxidase-linked antirabbit IgG or horseradish peroxidase-antigoat IgG antibodies, Amersham Pharmacia Biotech, Uppsala, Sweden) were applied, and proteins of interest were detected with enhanced chemiluminescence system (PerkinElmer Life Sciences, Boston, MA) in accordance with the manufacturer’s protocol.
Genomic DNA
Genomic DNA from either whole blood or primary fibroblast cultures was obtained with Puregene DNA purification system (Gentra Systems, Minneapolis, MN). For the STAT5b gene, each of the 19 exons was PCR amplified and sequenced (oligonucleotides employed are available on request).
For PCR amplification and sequencing of the human igfals gene, the following oligonucleotides were used: exon 1, forward, 5'-ggcacgag ggggttaaca ga-3', reverse, 5'-aaccc cagcagccgc attt-3'; and exon 2 was amplified in three segments: 1) forward, 5'-gcttccg gctgtgctgg tat-3' and reverse, 5'-taggccag cctgttgccc-3'; 2) forward, 5'-gcagcct ctgggacctc aa-3' and reverse, 5'-cgag aggccggtgaaggt-3'; and 3) forward, 5'-t ctctgggaac tgtctccgga ac-3' and reverse, 5'-gggccg ccatgccttc-3'. The mutation is in segment 3 of exon 2.
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Results |
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The diagnoses of classical or nonclassical GHI syndromes were excluded on the basis of normal sequencing of the genes for the GHR and STAT5b. The strikingly low serum concentrations of IGF-I and IGFBP-3, the failure to raise serum levels of IGF-I and IGFBP-3 after administration of GH, and the modest growth failure were similar to those recently reported in a patient carrying a mutation in the human acid labile subunit, ALS, gene (7). Analysis of serum ALS in the subject was therefore undertaken. ALS was not detected in serum obtained from the subject when 14 yr of age (Table 2) or from samples obtained when 15.2 yr of age (<0.4 µg/ml). Both the mother and sister of the subject, in contrast, had normal to elevated levels of serum ALS (Table 2).
Western immunoblot analysis of serum samples confirmed lack of detectable ALS in serum of the subject (Fig. 1). In pooled normal sera, an immunoreactive band at approximately 85 kDa corresponding to ALS was readily detected by antibodies that recognize either the N terminus or C terminus of ALS (Fig. 1). The same immunoreactive bands were observed in sera from the mother and the sister. In contrast, in serum from the subject, neither intact nor truncated forms of ALS were detected (Fig. 1). Serum ALS therefore was clearly lacking in the subject.
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Abnormally low levels of serum IGF-I and IGFBP-3 in the subject had been noted throughout adolescence (Table 1), and these observations were confirmed by independent analysis, using a different RIA (Table 2). The sister, as expected, had normal serum levels of IGF-I and IGFBP-3, but, interestingly, the mother was found to have serum concentrations of IGF-I and IGFBP-3 just below the normal ranges (Table 2). Serum samples from the father were not available for analysis.
The strikingly low concentrations of serum IGF-I and IGFBP-3 in the absence of ALS in the subject may result in an increased availability of free IGF-I, as has been suggested (7). This could therefore account for the surprisingly modest growth retardation of the subject. To evaluate this possibility, free IGF-I was determined. As shown in Table 3, in pooled normal sera from adults, free IGF-I was determined to be 0.9 ng/ml, which is within the range determined by Juul et al. (12). By comparison, sera from a GH-deficient (GHD) subject had, as expected, abnormally low levels of total IGF-I and concentrations of free IGF-I (baseline) well below –2 SD. GH treatment of the GHD subject subsequently restored total IGF-I to normal levels, with free IGF-I (0.79 ng/ml) correspondingly increased to within the normal range (Table 3). Similar to untreated GHD, free IGF-I determined in serum for a GHI subject lacking STAT5b (4) also fell well below –2 SD. The free IGF-I determined in sera obtained from the mother and sister of the current subject correlated with their total IGF-I. Interestingly, although the subject had abnormally low total IGF-I levels, free IGF-I appeared to be consistently higher than those detected in sera from untreated GHD and GHI cases but remained significantly lower than normal (Table 3).
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The abnormally low concentrations of serum IGFBP-3 could be the consequence of aberrant expression and production of IGFBP-3 or could result from rapid turnover of IGFBP-3 because of the absence of serum ALS (8). The low IGFBP-3 levels detected in the serum (Tables 1 and 2) were confirmed by immunoblot and Western ligand blot analyses (Fig. 2A, upper and lower panels, respectively). Unlike normal serum or sera from the mother and sister of the subject, steady-state, intact (40–45 kDa doublet) and truncated (28 kDa) IGFBP-3 (13) were poorly detected in serum from the subject. In stark contrast, intact IGFBP-3 was readily, and normally, detected in media conditioned by primary dermal fibroblast cells established from the subject (Fig. 2B, OTF, compared with normal fibroblasts, CF). These results suggested that IGFBP-3 expression was not aberrant in the subject. Furthermore, the results were consistent with the assumption that, in the absence of ALS, normal levels of ternary complexes were not maintained and, as a consequence, serum IGFBP-3 was rapidly turned over.
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The combination of undetectable ALS in the subject’s serum and evidence suggesting normal IGFBP-3 expression pointed to an IGFD due to a defect in ALS expression. The human igfals gene (3.3 kb) consists of two exons, with exon 1 encoding the first five amino acids of the signal sequence and the remainder of the prepeptide encoded by exon 2. DNA extracted from either OTF cells or whole blood from the subject revealed a homozygous substitution at nucleotide 1318 of the prepeptide, which altered the codon encoding aspartic acid, D (gac), to asparagine, N (aac), at residue 440 (Fig. 3). The parents were heterozygous at nucleotide 1318, whereas the sister, who was of normal stature and exhibits normal to elevated levels of ALS, was homozygous wild type (Fig. 3). DNA sequencing of a 400-bp region in exon 2 encompassing nucleotides 1180–1581 of the ALS cDNA indicated that no polymorphisms exist at nucleotide 1318 [more than 300 DNAs sequenced, single nucleotide polymorphism (SNP) database, http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?CMD=search&DB=snp
]. Hence, the homozygous missense mutation identified in the subject is recessive and was transmitted autosomally.
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Discussion |
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The mutation in the igfals gene was autosomal recessive because the parents were heterozygous for the nucleotide (1318 of the prepeptide) and the sister of the subject was homozygous wild type. No polymorphism has been reported at this site, although a total of nine SNPs in the human igfals gene have been recorded by GenBank. The DNA sequence of the igfals gene in the subject was otherwise unremarkable. Unlike the first reported case of ALS deficiency in which a frameshift mutation led to early termination of ALS expression (7), in the present case, the missense mutation resulted in a D440N substitution of the ALS prepeptide. Based on molecular modeling of the ALS protein, D440 is within one of the 20 leucine-rich repeat (LRR) motifs (15) that are arranged in a donut-like shape (16, 17). The N substitution at residue 440, interestingly, generated a consensus motif for N-glycosylation (Fig. 4) in a protein normally highly glycosylated (16). Overall, the effect(s) of D440N on ALS structure and function remains unclear; the consequence clearly was undetectable levels of circulating ALS.
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In both rodents and humans, postnatal growth was only mildly affected by the lack of ALS, despite levels of IGF-I and IGFBP-3 usually associated with clinical phenotypes of severe growth retardation due to GHD, GHI, or other causes of IGFD. One hypothesis proposed is that in the absence of normal levels of stable ternary complexes, free IGF-I may still be normal and thus account for the relatively mild growth retardation (7). Our analysis of free IGF-I, although not definitive, suggested that, whereas steady-state free IGF-I in our subject may be higher than those observed in cases of GHD, GHI, and IGFD, levels were still low in comparison with normal. These observations would be consistent with the possibility of high IGF flux into tissues. The mild growth retardation phenotype also suggested the possibility that paracrine and autocrine effects of peripheral IGF-I was critical for normal, human, postnatal growth. Indeed, preliminary analysis of IGF-I expression in primary OTF cells suggested that peripheral GH-induced regulation of IGF-I was normal (data not shown). Furthermore, because GH production may be increased in subjects with ALS deficiency, local production of IGFs may be even higher than normally observed, and thereby compensate for any deficiency of circulating IGF-I.
It is of note that the height of the current subject, consistently 2.0–3.0 SD below the Turkish standard, is similar to that of his parents, who are heterozygous for the ALS mutation. His final predicted height of 159.4 cm is –2.1 SD score, considerably shorter than the final height of –0.9 SD score reported for the first case of an ALS mutation. On the other hand, the predicted adult height of the current case is consistent with the parental target height. Whether the mild short stature of the parents is the consequence of a haplo-insufficiency due to the heterozygous ALS mutation or some as-yet-unidentified etiology is not clear, although it is noteworthy that the sister, who is homozygous for the wild-type ALS, is of normal stature and well above her parental target height.
Finally, in addition to modest growth retardation, delayed onset of puberty was noted in the previously reported case of ALS mutation (7) and in a new case recently identified by the same group (20). The current subject, in contrast, had a normal onset of puberty at age 13 yr, with normal progression through age 15.5 yr. Hence, delayed puberty does not appear to be a consistent characteristic of ALS deficiency. Indeed, the phenotype of ALS deficiency appears to consist, at most, of mild growth retardation, and this may result in an underestimate of its prevalence. With increasing use of IGF-I and IGFBP-3 assays in the evaluation of children with growth retardation, it appears likely that additional cases will be identified, thus providing an opportunity for a more comprehensive analysis of the associated clinical characteristics. The combination of a mild growth phenotype in the face of markedly low serum IGF-I and IGFBP-3 concentrations should, in fact, suggest the possibility of an underlying ALS deficiency.
In conclusion, we observed the following: 1) ALS is critical for maintaining normal serum concentrations of IGF-I and IGFBP-3, most likely by prolonging the half-lives of both proteins; 2) ALS deficiency can result in moderate growth failure, characterized by biochemical evidence suggestive of GHI and IGFD, in the presence of normal or elevated serum GHBP; 3) the surprisingly modest growth retardation observed in ALS deficiency supports an important role for free IGF-I and/or peripheral IGF-I in statural growth; and 4) in the evaluation of short stature, ALS abnormalities should be considered when low circulating levels of IGF-I and IGFBP-3 persist on IGF generation studies.
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Acknowledgments |
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Footnotes |
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V.H., G.H., K.L.P., B.M.L., H.F., D.K., and R.G.R. have nothing to declare.
First Published Online February 28, 2006
Abbreviations: ALS, Acid-labile subunit; GHBP, GH binding protein; GHD, GH deficient; GHI, GH insensitivity; GHR, GH receptor; IGFBP, IGF binding protein; IGFD, IGF deficiency; LRR, leucine-rich repeat; SDS, sodium dodecyl sulfate; SNP, single nucleotide polymorphism; STAT, signal transducer and activator of transcription.
Received December 30, 2005.
Accepted February 17, 2006.
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