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Departments of Endocrinology (K.E.H., J.A., V.B., J.P., F.D.-G., G.A.M.-M., A.C.-B.), Hospital Infantil Universitario Niño Jesús, Universidad Autónoma de Madrid, 28009 Madrid, Spain; Centro de Investigación Biomédica En Red (CIBER) Fisiopatología Obesidad y Nutrición (CB06/03) (J.A., V.B., J.P., G.A.M.-M.), Instituto de Salud Carlos III, 28029 Madrid, Spain; Department of Pediatrics (M.C.), Hospital Universitario Son Dureta, Universidad de Palma de Mallorca, 07014 Palma de Mallorca, Spain; and Department of Pediatric Endocrinology (R.G.), Hospital Universitario La Paz, Universidad Autónoma de Madrid, 28049 Madrid, Spain
Address all correspondence and requests for reprints to: Dr. Ángel Campos-Barros, Department of Medical Genetics, Hospital Universitario La Paz, P° Castellana 261, 28046 Madrid, Spain. E-mail: acamposbarros{at}yahoo.org.
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Abstract |
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Objective: We present the molecular and clinical characterization of two novel IGFALS mutations that caused complete ALS deficiency in three unrelated patients with postnatal growth deficit, low IGF-I and IGFBP-3 levels, and no GH deficiency.
Results: IGFALS mutation screening identified a novel homozygous IGFALS missense mutation, which altered a conserved residue, N276S, in two of the probands. The third proband presented a novel homozygous nonsense mutation, Q320X, that is predicted to generate a severely truncated ALS protein. The affected probands presented a similar phenotype characterized by a moderate postnatal growth deficit associated with undetectable ALS, low IGF-I, IGF-II, and IGFBP-3, and hyperinsulinemia, and, in two cases, delayed puberty.
Conclusions: Primary ALS deficiency due to IGFALS mutations should be considered as a possible cause of postnatal growth deficit in IGF-I-deficient patients in the absence of GH deficiency or insensitivity. Determination of serum ALS levels and basal insulinemia can be helpful in the differential diagnosis of patients with idiopathic IGF-I deficiency.
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Introduction |
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Recently up to four different IGFALS mutations have been reported in patients presenting with postnatal growth deficit (8, 9, 10) associated with undetectable ALS levels, very low IGF-I, IGFBP-3, and normal GH levels as well as a delayed puberty onset in two cases (8, 10), thus supporting a role for ALS in the regulation of the bioavailability of IGFs during postnatal growth.
We report here the detection of two novel inactivating IGFALS mutations and the clinical characteristics of three non-GH-deficient unrelated patients who presented with a postnatal growth deficit associated to undetectable ALS, markedly decreased IGF-I, IGF-II, and IGFBP-3, hyperinsulinemia and, in two cases, delayed puberty. Our present results together with those previously reported (8, 9, 10) indicate that primary ALS deficiency due to IGFALS mutations define a form of autosomal recessive postnatal growth deficit characterized by IGF-I and IGFBP-3 deficiency and hyperinsulinemia in the absence of GHD or GH insensitivity (GHI), which, in some cases, may present with delayed puberty.
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Subjects and Methods |
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and 2. The anthropometric details and relevant clinical history of their relatives were also documented. Ethical approval was obtained from the respective institutions. Informed consent/assent for the undertaken studies was provided by all participants or their legal guardians.
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The first patient was the fourth son of nonconsanguineous Spanish parents. He was born at term with normal birth length (50 cm) and weight (3300 g). Growth retardation was reported since the age of 2 yr (Table 1
). Both parents have normal height for Spanish standards [father 175 cm, –0.1 SD score (SDS); mother 165.5 cm, 0.74 SDS]. The mother had menarche at the age of 15–16 yr and a family history of constitutional delay of growth and puberty. No relevant family history was reported by the father.
Case 2
The second patient was a male subject born at term as the third child of nonconsanguineous Spanish parents with normal birth weight (3300 g; birth height not recorded) consulting for growth retardation at age 4.7 yr. His father, height 161.9 cm (–2.27 SDS), recounted short stature in two older brothers with reported heights of 156 cm (–3.25 SDS) and 160 cm (–2.4 SDS), respectively. The mother, stature 158.3 cm (–0.52 SDS), had menarche at age 10 yr.
Case 3
The third patient was a male referral with idiopathic short stature, born at term (birth weight 2650 g; –1.95 SDS; birth length not recorded) as the first child of nonconsanguineous Spanish parents. At the age of 15 yr, he was referred for endocrinological evaluation due to growth retardation and delayed puberty. His father, final height 173 cm (–0.43 SDS), reported puberty onset at age 14–15 yr and a family history of short stature. His mother, height 159.5 cm (–0.31 SDS) reported menarche at age 9 yr.
In all cases, organic pathology was excluded and hypothalamic-pituitary anomalies were not detected by magnetic resonance imaging.
Serum hormone assays
GH was determined by an automated chemoimmunoluminescence assay (Immulite; Diagnostic Products Corp., Los Angeles, CA). Total IGF-I was determined by immunoradiometric assay (IRMA; Diagnostic Systems Laboratories, Webster, TX) or ELISA (Mediagnost, Tübingen, Germany); free IGF-I, IGF-II, and IGFBP-3 by IRMA (Diagnostic Systems Laboratories); ALS by ELISA (Diagnostic Systems Laboratories); and IGFBP-2 by RIA (Diagnostic Systems Laboratories). Serum IGFBP-1 and GH binding proteins (GHBPs) were determined by ELISA using kits from Medix Biochemica (Kauniainen, Finland) and Diagnostic Systems Laboratories, respectively. Total insulin was determined by IRMA (DRG Diagnostics, Marburg, Germany), testosterone by RIA (Spectria; Orion Diagnostics, Espoo, Finland), and prolactin using the IMX-System Prolactin (Abbott Laboratories, Abbott Park, IL). For the oral glucose tolerance test (OGTT), 1.75 g glucose per kilogram of body weight was administered, and blood samples were obtained from an antecubital vein immediately before and at 30, 60, 90, and 120 min after glucose administration for measurement of plasma glucose and insulin.
Molecular genetic screening
Genomic DNA was isolated from whole blood using the salt precipitation method (11). DNA control samples, obtained from 200 healthy white adult donors, were provided by the National DNA Bank (Cancer Research Center, University of Salamanca, Salamanca, Spain).
The coding sequences, intron/exon boundaries, and known regulatory regions of GHR, IGFBP3, and IGFALS genes were screened for mutations by denaturing HPLC (Wave 3500HT; Transgenomic, Omaha, NE) and subsequent sequencing of any identified heteroduplex or homoduplex variant using the BigDye Terminator version 3.1 kit (Applied Biosystems, Foster City, CA) on an ABI 3100 genetic analyzer. Further information on primer sequences and denaturing HPLC conditions is available on request.
The possibility of common ancestry between families 1 and 2 and between the respective parents for each family was investigated by haplotype analysis of markers flanking the IGFALS locus. Analyzed microsatellites included D16S3024, located approximately 186 kb 5' upstream from IGFALS as well as two novel CA repeat microsatellites, D16S3434 and D16S3435, located approximately 21 and 37 kb 3'downstream from IGFALS, respectively. The new markers were designed and assessed using the chromosome 16 contig (Ensembl Genome Browser; http://www.ensembl.org) and the Tandem repeat finder program (http://tandem.bu.edu/) (12), as previously described (13). Primer sequences are available at the GDB Human Genome Database bank (www.gdb.org).
Western blot analysis
Serum samples (0.9–1.3 µl, i.e. 
10 µg total protein) were electrophoresed on a reducing 10% SDS-polyacrylamide gel. Size-fractionated proteins were blotted onto nitrocellulose and membranes blocked overnight with 5% milk, 1% BSA in 50 mM Tris, 0.05% Tween 20. Primary antibodies for Western blot analysis were goat antihuman ALS (N terminal) or goat antihuman ALS (C terminal) polyclonal antibodies (Diagnostic Systems Laboratories), IGFBP-3 monoclonal (Abcam, Cambridge, UK), and transferrin polyclonal (Sigma Aldrich, St. Louis, MO). Antibody dilutions and incubations were as previously described (9). After incubation with appropriate secondary antibodies, the immunoblots were developed with the ECL system (PerkinElmer Life Sciences, Boston, MA) according to the manufacturer's instructions.
Dual-energy x-ray absorptiometry (DEXA) analysis
Bone mineral density (BMD) and bone mineral content (BMC) were assessed in cases 1 and 2 by lumbar spine (LS) and whole-body DEXA analysis using a QDR Hologic 4500 W System (software version 11.2:5; Hologic, Bedford, MA).
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Results |
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The probands basal and stimulated GH, GHBPs, ALS, IGF-I (free and total), IGF-II, IGFBP-3, IGFBP-1, IGFBP-2, insulin, and glucose serum levels are summarized in Table 2. In all cases, serum IGF-I and IGFBP-3 concentrations were markedly decreased at diagnosis and remained extremely low throughout the follow-up period (Fig. 1 and Table 2). Similarly, ELISA analysis did not detect ALS in the serum of any of the probands at diagnosis or during later examinations (Table 2 and 3).
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). Finally, no abnormalities were observed in prolactin and testosterone levels in any of the probands (data not shown).
Molecular genetic screening
To exclude the possibility of partial GHI, molecular genetic analysis initially focused on the screening of GHR. A nonsynonymous heterozygous variant R386C (c.1156C>T; CGT>TGT) was detected in exon 10 of proband 1. The C>T variant is listed in the single-nucleotide polymorphism database (rs34853905) as a low-frequency (0.013) allelic variant in African-Americans and has not yet been reported in other subpopulation cohorts. However, both proband 1's father and one of his brothers were nonaffected carriers of the same variant. No mutation or nonsynonymous variants were detected in the coding GHR genomic sequences of cases 2 and 3.
Analysis of IGFALS detected a novel homozygous missense mutation (c.827A>G; AAC>AGC; N276S) in probands 1 and 2 (Fig. 2B). The mutation alters a phylogenetically conserved residue, N276, which is located in one of the leucine-rich repeat (LRR) domains, among the seven Asn residues whose N-glycosylation is critical for maintaining ALS affinity for the IGF-I/IGFBP-3 binary complex (14). The parents and two brothers of proband 1 as well as the parents and one sister of proband 2 were heterozygous carriers of the N276S mutation (Fig. 2A), which otherwise was not detected in 200 chromosomes from healthy Spanish donors.
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). Both parents and the proband's brother were heterozygous carriers of the same mutation (Fig. 2A). The mutation was not detected in 200 chromosomes from healthy Spanish controls. Finally, no mutations were detected in the IGFBP3 sequences of the probands. Analysis of IGFALS haplotype
A common haplotype carrying the N276S mutation was observed in the parents of families 1 and 2, supporting the possibility of distant common ancestry (Fig. 2A). Regarding the third family, microsatellite analysis revealed that both parents, natives of the Spanish island of Ibiza, also shared a common haplotype carrying the Q320X mutation (Fig. 2A). Frequency analysis of these two haplotypes in the control Spanish population was determined to be one in 59 and one in 26, respectively.
Western blot analysis of serum ALS
Western immunoblot analysis of serum samples from the three probands confirmed the lack of any detectable serum ALS, whereas a clearly detectable 84- to 86-kDa band corresponding to ALS was recognized by both the N terminus- (Fig. 2C) and C terminus-directed antibodies (data not shown) in sera from the heterozygous carriers or wild-type relatives and controls (Fig. 2D). Western blot analysis confirmed therefore that ALS was absent in the serum samples of the three probands.
Growth and pubertal development
Growth and pubertal development of the three probands are summarized in Fig 3. In all cases the probands showed a growth pattern between –2 and –3 SDS with a bone age (BA) to chronological age (CA) ratio of 1–2.5 yr. Puberty onset and progress was apparently normal in proband 1 (Fig. 3A). In contrast, a decreased prepubertal growth velocity became evident at age 12 yr in proband 2 without having yet reached the pubertal growth spurt peak at age 14 yr, suggesting delayed puberty. (Fig. 3B). Similarly, delayed puberty was also noted in the third proband (Tanner II at age 14.5 yr), who was treated with recombinant human GH (0.2–0.3 mg/kg·wk) from age 15.5 through to 18.8 yr. Due to the treatment overlap with his pubertal onset and progress (Fig. 3C), it is difficult to estimate whether the treatment, which otherwise did not raise the very low IGF-I and IGFBP-3 serum levels (Fig 1), had any effect on his growth. He completed puberty at age 18.9 yr (Tanner V; testicular volume 20 ml) and reached his adult height, 167.6 cm [–1.32 SDS; midparental target height (MPTH) 172.7 cm], at age 20.2 yr (Fig. 3C).
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). Height and serum hormone evaluation of family members
For each family, the individual heights and results of all available serum hormone determinations are summarized in Table 3. We observed a variable effect in the heterozygous carriers: whereas IGFALS haploinsufficiency was evident in the parental and sibling ALS concentrations of all three families, the observed changes in the IGF-I and IGFBP-3 serum concentrations were rather modest. Furthermore, basal insulin levels were normal or in the upper limit of the normal range in all heterozygous carriers (Table 3).
DEXA analysis
DEXA evaluation of LS BMD and whole-body BMC did not detect significant deviations from normality in probands 1 (CA 15.9 yr, BA 15 yr; Tanner stage IV, body weight 52 kg, height 156 cm) and 2 (CA 14.25 yr, BA 12 yr; Tanner stage II, body weight 40.5 kg, height 141 cm). The determined LS BMD was 0.906 and 0.718 g/cm2, for cases 1 and 2, respectively. Using normative data from the literature that accounts for age, gender, and ethnicity (15), after correction for the actual BA, the patients' values corresponded to the 50th percentile. Regarding LS BMC, the values were 34.35 and 19.57 g, respectively, corresponding to the 3rd percentile or less and compatible with their delayed maturation status. No DEXA was performed in proband 3.
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Discussion |
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In all three cases, the nonconsanguineous parents were heterozygous carriers of the mutations, thus indicating that they were transmitted according to an autosomal recessive pattern. Haplotype analysis of the IGFALS locus confirmed that the four nonrelated heterozygous parents, carriers of the N276S mutation, as well as the parents of proband 3, carriers of the Q310X mutation, shared common haplotypes harboring the respective mutant alleles. Hence, haplotype analysis supports the possibility of a distant common ancestor (for families 1 and 2) and/or a founder effect in the parents of the third proband, both natives of the Spanish island of Ibiza.
Primary ALS deficiency generated a similar clinical phenotype in all three probands. This was characterized by a moderate but sustained postnatal growth deficit with a delayed BA vs. CA of 1–2.5 yr, low circulating levels of IGF-I and IGFBP-3, and subnormal levels of IGF-II, in the face of normal or slightly elevated GHBPs and basal GH as well as normal or supranormal GH stimulation tests. Furthermore, delayed puberty was evident in cases 2 and 3 but not in case 1, affected by the same IGFALS mutation as case 2 (N276S), suggesting a certain degree of phenotypic variability, even between patients with the same mutation.
Analysis of the growth charts of the affected probands revealed very similar patterns of postnatal, prepubertal, and pubertal growth patterns, which are, in part, reminiscent of those observed in patients with constitutional delay of growth and puberty. Indeed, a family history of constitutional growth and puberty delay was reported by the parents of cases 1 and 3, respectively.
In contrast, the comparison of the heights achieved by the heterozygous carriers within each family reveals some striking differences. Whereas the heterozygous status does not seem to have affected the growth of the oldest brother (in family 1) and the youngest brother (in family 3), respectively, the second heterozygous brother in family 1 showed a consistent BA delay of approximately 1.5 yr, attaining a height of 168 cm (–1.20 SDS) at age 17.6 yr (Tanner IV). In the second family, the youngest sister, heterozygous for N276S, reached an adult height of 154.5 cm (–1.18 SDS), which is in accordance with the expected MPTH of 152.15 cm. In contrast, the oldest sister, with two wild-type IGFALS alleles, achieved an adult height of 162.7 cm, well above the MPTH, thus indicating that the relative ALS haploinsufficiency may have impaired the growth potential of the heterozygous carriers, i.e. both parents and the younger sister, in this family. A similar observation was noted in a previously described case with a nonsense IGFALS mutation (9).
Despite the markedly decreased circulating IGF-I and IGFBP-3 levels, which are equivalent to those associated with severe growth retardation due to GHD or GHI, all three homozygous probands growth rates were within the normal limits, and final heights or predicted target heights were not severely affected. This indicates that, as previously reported in patients with IGFALS mutations (8, 9, 10) as well as in the Igfals null mouse model, primary ALS deficiency and secondarily the absence of ternary complexes exerts only a moderate but consistent effect on postnatal and prepubertal growth progression, which may associate, in some cases, with delayed puberty. Paracrine and autocrine action of peripheral IGF-I as well as the direct and IGF-I-mediated growth-promoting effects of GH in target tissues have been postulated as a possible explanation for the moderate effects of total ALS deficiency on growth progress and final stature of patients with recessive inactivating IGFALS mutations (8, 9). Indeed, normal liver IGF-I and IGFBP-3 synthesis and diminished half-life due to increased turnover was demonstrated in the ALS null mouse (16, 17, 18) as an explanation for the low IGF-I and IGFBP-3 levels. Moreover, normal growth in the face of a marked increase in basal GH and hyperinsulinemia was also preserved in the liver-specific Igf1-deficient (LID) mouse model despite extremely low circulating IGF-I and IGFBP-3 levels, supporting the hypothesis of peripheral IGF-I and direct GH action as the main promoters of linear postnatal growth.
Interestingly, in all three patients, primary ALS deficiency was associated with chronic hyperinsulinemia of unknown etiology, accompanied by normal fasting glucose and decreased IGFBP-1 and IGFBP-2 levels. Suppressed IGFBP-1 has been associated with hyperinsulinemic/insulin-resistant states (19). Indeed, OGTT confirmed supranormal HOMA index in the two N216S homozygotes, accompanied in proband 2 by glucose intolerance. Both hyperinsulinemia and IR-compatible HOMA values were also observed in the first patient reported with an IGFALS mutation (8) as well as in all family members affected by two new compound heterozygous IGFALS mutations reported by the same group during the preparation of this manuscript (10).
Hyperinsulinemia, IR, and/or alterations in insulin sensitivity have been previously described in patients with primary GHI (20) and the LID and LID/ALS knockout mouse models (18, 21, 22), associated in the latter with markedly elevated mean GH levels. GH secretion was, however, normal or slightly elevated in ALS-deficient patients, suggesting that the observed hyperinsulinemia and IR is unlikely to be due to the elevated GH secretion. Moreover, animal studies showed that impairment of IGF-I signaling through the IGF-I receptor results in impaired glucose tolerance and glucose-stimulated insulin secretion due to a defective glucose-sensing response (23, 24, 25). Although the etiology of hyperinsulinemia and associated IR in ALS-deficient patients remains to be elucidated, ours and others' observations, together with the existing animal studies, strongly suggest that changes in IGF-I bioavailability due to ALS deficiency could, at least in some tissues, significantly impair glucose sensing and insulin sensitivity. This is in support of a hypothetical functional link between IGF-I bioavailability and insulin action that could be influenced or mediated by ALS.
Finally, no changes were observed in whole-body or LS BMD in the two patients affected by the N276S mutation, although a decreased LS, but not whole-body BMC, was found by DEXA analysis, suggesting a certain degree of delayed bone maturation. Decreased LS BMD was observed in the first reported patient with primary ALS deficiency due to a homozygous IGFALS mutation (26) as well as in three male siblings born to consanguineous parents, affected by a novel IGFALS mutation recently presented (27). Similarly, decreased total BMD, cortical thickness, and bone volume were reported in the ALS knockout mouse (18), supporting a role for IGF-I and ALS in the proper acquisition of bone mass and mineralization.
In conclusion, our findings and previous findings by others (8, 9, 10) indicate that complete ALS deficiency due to inactivating recessive IGFALS mutations constitutes a distinct syndrome, for which we propose the term primary ALS deficiency, which causes a moderate postnatal growth deficit consistently associated with markedly decreased IGF-I and IGFBP-3; subnormal IGF-II, IGFBP-1 and IGFBP-2; hyperinsulinemia; IR; and, in some cases, delayed puberty and bone maturation.
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Acknowledgments |
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Footnotes |
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Disclosure Statement: The authors have nothing to declare.
First Published Online February 26, 2008
Abbreviations: ALS, Acid-labile subunit; BA, bone age; BMC, bone mineral content; BMD, bone mineral density; CA, chronological age; DEXA, dual-energy x-ray absorptiometry; GHBP, GH binding protein; GHD, GH deficiency; GHI, GH insensitivity; GHR, GH receptor; HOMA, homeostasis model assessment; IGFBP, IGF binding protein; IR, insulin resistance; IRMA, immunoradiometric assay; LID, liver-specific Igf1 deficient; LRR, leucine-rich repeat; LS, lumbar spine; MPTH, midparental target height; OGTT, oral glucose tolerance test; SDS, SD score.
Received December 4, 2007.
Accepted February 20, 2008.
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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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