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Original Article |
Department of Genetics (C.K.B.), Stanford University, Stanford, California 94305; Department of Pediatrics (K.A.S., K.L.P., E.T., R.G.R.), Oregon Health & Science University, Portland, Oregon 97239; and Institute of Endocrinology, Metabolism, and Reproduction (J.G.-A.), Quito, Ecuador
Address all correspondence and requests for reprints to: Karin A. Selva, M.D., Department of Pediatrics, Oregon Health & Science University, 707 SW Gaines Road, Child Development and Rehabilitation Center-Pediatrics (CDRC-P), Portland, Oregon 97201. E-mail: selvak{at}ohsu.edu.
Abstract
A total of 198 subjects were randomized to either high-dose (0.05 mg/kg·d) or low-dose (0.025 mg/kg·d) GH for 7 d; the alternate dose was then received after a 2-wk washout period. Groups included in the study were: normal, GH-insensitive (GHI; homozygous for the E180 splice mutation); heterozygous GHI (carriers of the E180 splice mutation); GH-deficient; and idiopathic short stature.
Serum IGF binding protein-3 (IGFBP-3) concentrations (collected on d 1, 5, and 8 of treatment weeks) were GH-dependent, with significant elevation by d 5 of treatment, regardless of dose, in all normal subjects. GHI subjects had low baseline IGFBP-3 and poor or no response to either low- or high-dose GH. Heterozygous subjects, however, did not differ from age-matched normals with regard to IGFBP-3 generation. All GH-deficient subjects had subnormal baseline concentrations of IGFBP-3; most, but not all, were able to generate levels into normal ranges by 8 d of therapy. Children with idiopathic short stature showed a better response in IGFBP-3 generation compared with that previously observed with IGF-I, reaching concentrations in normal range with either dose of GH, suggesting that any GHI in this group is relatively limited to IGF-I production.
For the diagnosis of GHI, the highest sensitivity (100%) and specificity (92%) was found on d 8 of the high-dose GH-IGFBP-3 generation test. Failure to raise both IGF-I and IGFBP-3 lowered sensitivity to 82–86% with low-dose GH, and 86–91% with high-dose GH.
DIFFERENTIATING BETWEEN GH deficiency (GHD), GH insensitivity (GHI), and idiopathic short stature (ISS) based on serum levels of IGF-I and IGF binding protein-3 (IGFBP-3) has been problematic, because these biochemical parameters may overlap in these diagnoses. In addition, partial GHI has been hypothesized to be an etiology of some forms of ISS, further complicating the spectrum of diagnostic entities for short stature (1, 2, 3, 4, 5). Mutations in the GH receptor (GHR) gene, such as the E180 splice mutation in the Ecuadorian subjects of this study, cause classic GHI (Laron syndrome) in homozygous patients. Persons heterozygous for this mutation have not been shown to have a phenotypic effect (6). Other mutations of the GHR, however, have been reported to have a dominant negative effect in heterozygous subjects; yet, it is unclear how often heterozygosity for such mutations results in ISS (7, 8, 9, 10, 11).
The IGF generation test is a classic endocrine stimulation test used to assess GH responsiveness for over 30 yr (12, 13, 14). Since the initial descriptions, the utility of the IGF-I generation test has been investigated in the evaluation and management of various forms of short stature, including the diagnosis of GHI and partial GHI, and for growth rate prediction in patients receiving GH therapy (14, 15, 16, 17, 18). Despite multiple studies, however, there is no consensus on how the test should be performed, including what dosage of GH should be employed and for how long a time (2).
Although initial investigations were limited to IGF-I, IGFBP-3 generation has been proposed to provide additional insight into the diagnosis of partial GHI, especially when used in conjunction with IGF-I generation. Blum et al. (19) reported IGFBP-3 as a more accurate discriminator of GHI from GHD than was IGF-I in such generation tests. Thalange et al. (4) found the difference between basal and stimulated IGFBP-3 more significant than the difference between basal and stimulated IGF-I levels when distinguishing partial GHI from GHD. Cotterill et al. (5), on the other hand, found no significant differences in IGF-I or IGFBP-3 generation in children with short stature, although they did use the test to diagnose individual patients with partial GHI, based on high GH levels and low basal or GH-stimulated IGF-I and IGFBP-3 values.
The current study reports IGFBP-3 generation data in 198 Ecuadorian patients who received courses of both low- and high-dose GH. We have reported recently on IGF-I generation data from this cohort (20). A specific aim of this study is to examine the GH responsiveness of subjects with GHI and their heterozygous relatives, as well as individuals with GHD or ISS, by assessing the stimulated levels of IGFBP-3 in response to exogenous GH relative to that of age- and sex-matched normal controls. Concurrently, the study permits evaluation of several protocols (4 or 7 d of either low- or high-dose GH) for IGF-I/IGFBP-3 generation tests, and the development of normative data for these biochemical markers for children and adults.
Subjects and Methods
Subjects (Table 1
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One hundred males and 98 females, all from Ecuador, were enrolled in the study. Normal male and female adults and children comprised 72 of the subjects; 22 were subjects with classic autosomal recessive GHI and, in this population, all due to the E180 splice mutation of the GHR gene; 65 were their heterozygous relatives; 23 were subjects with GHD; and 16 were children with ISS. This is the largest cohort of GHI patients in the world, in a geographically isolated area in the southern region of Ecuador. The E180 splice mutation affects the extracellular portion of the GHR and results in marked reduction of GH binding (21, 22). All 198 subjects were genotyped for the E180 splice mutation, as previously described, and all GHD and ISS subjects were shown to lack the E180 splice mutation (23, 24). One GHD subject had had a craniopharyngioma, but the remainders were idiopathic. Some subjects with GHD also had other hormone deficiencies, primarily TSH, but all were appropriately replaced during the study period. GHD patients were documented to have a GH level below 10 ng/ml upon provocation (insulin and clonidine). All GHD patients were naive to GH therapy at the time of this study. Children with ISS were defined as having a height less than -2 SD with a peak GH level greater than 10 ng/ml.
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). Informed consent was obtained from all subjects or from their parents if the subject was under 18 yr of age. This study was approved by the institutional review board of the Institute of Endocrinology, Metabolism, and Reproduction in Quito, Ecuador.
Study design
Within each diagnostic group at enrollment, the 198 subjects were randomized into two groups, differing only by the dosage of GH used initially. Either a low dose (0.025 mg/kg·d) or a high dose (0.05 mg/kg·d) of GH was given for 7 d. Subjects were taught to self-administer sc injections of GH, given each evening. Fasting blood samples were drawn on the mornings of d 1, 5, and 8. A 2-wk washout period followed, and the subjects then received the other dose of GH, with fasting blood samples again drawn on the mornings of d 1, 5, and 8. Recombinant human GH was kindly provided by Genentech, Inc. (South San Francisco, CA).
Assays
The specimens were centrifuged within hours after collection from subjects’ homes or doctors’ offices in Quito, Ecuador, and the sera were removed. They were then shipped on dry ice to the Rosenfeld laboratory (Oregon Health & Science University, Portland, OR), where they were stored at -70 C until assayed. IGFBP-3 assays were performed using IRMA kits, kindly provided by Diagnostic Systems Laboratories, Inc. (Webster, TX). Samples with values below the lowest or above the highest standard at the dilution recommended by the kit instructions were assayed again at appropriate dilutions, to assure that values fell within the most appropriate portion of the curve. The kit has an intra-assay coefficient of variation of 1.8–3.9% and an inter-assay coefficient of variation of 0.5–1.9%. The sensitivity of the IGFBP-3 assay is 90 ng/ml. Normal adult human serum from a pool of an equal number of 20- to 40-yr-old males and females was used as a standard in all assays.
IGF- I and IGFBP-3 values are expressed in nanograms per milliliter. To convert to Systeme International (SI) units for IGF-I, multiply by 0.1307 to find nanomoles per liter.
Statistical analyses
The normative data were examined for statistical significance by a two-way, repeated measure ANOVA for both males and females of the following age groups: younger than 10 yr, 10–18 yr, 18–40 yr, and older than 40 yr. To compare specific differences between diagnostic groups, we used an unpaired, two-tailed t test. Values were considered significant if P was less than 0.05.
Results
Normative data (Figs. 1A and 2 and Table 2)
An equal number of male and female normal subjects, totaling 72, were enrolled; 38 subjects were younger than 18 yr of age, and 34 subjects were older than 18 yr of age. In these and subsequent patient groups, subjects were divided by gender and by age. Thus, even with a total of 198 subjects studied, the subject number in some groups was not sufficient to represent an accurate normal range. We do, however, use these values as the normal ranges for our data, to permit comparisons with the recognition that considerable additional data are required to generate useful normal ranges. In all normal subgroups, serum IGFBP-3 concentrations were GH dependent. Significant elevation occurred by d 5 and 8 compared with baseline, using either the low or high dose of GH (all P < 0.001, except males 18–40 yr of age, P = 0.014; females < 10 yr of age, P = 0.007; and females 10–18 yr of age, P = 0.027). The degree of elevation of IGFBP-3 at d 8 relative to d 5 was not significantly different in any age group, nor was the elevation seen with the high dose relative to the low dose in any age group, with the exception of females 18–40 yr (P = 0.027). Although the rise in IGFBP-3, on a percentage of baseline level, is less than that of IGF-I, the trends seen in these data are quite similar to those reported for the IGF-I values from the same samples (20). Table 2 provides the mean IGFBP-3 levels for each age and gender group, whereas data are presented graphically showing ranges, ±1 SD, and means of the IGFBP-3 levels in Fig. 2. When comparing molar concentrations of IGF-I and IGFBP-3, normal children maintain a similar ratio in baseline and post GH, whereas, in adults, more IGF-I is generated relative to IGFBP-3, as evidenced by the change in slope (Fig. 1A).
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and 3 and Tables 2 and 3)
Twenty-two GHI subjects, 7 male and 15 female, were confirmed to be homozygous for the E180 splice mutation, affecting the extracellular portion of the GHR. As previously reported, baseline serum IGFBP-3 concentrations were quite low and appeared to show little response to either low- or high-dose GH (26). Of these 22 subjects, only one had a rise in IGFBP-3 greater than 400 ng/ml on d 5 of low-dose GH. This equates to a sensitivity of 95% for IGFBP-3 in the diagnosis of GHI at this time mark and dose (21 of 22 subjects; Table 3; Ref. 19). By comparison, IGF-I generation (using a 
<15 ng/ml) had a sensitivity of only 86% (19 of 22) on d 5 of low-dose GH. On d 8 of low-dose GH or d 5 or 8 of high-dose GH, IGFBP-3 generation had 100% sensitivity, whereas IGF-I generation had a sensitivity of only 82–91%. Both at baseline and after GH, molar concentrations of IGF-I remained low relative to IGFBP-3 (Fig. 1B).
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and 4 and Table 2)
Twenty-three patients were enrolled, and of these, 17 were under the age of 18 yr. All GHD subjects had low baseline serum IGFBP-3 concentrations for age (314–2784 ng/ml). Most GHD subjects, but clearly not all, were able to generate IGFBP-3 levels into the normal baseline ranges after a week of low- or high-dose GH. After 7 d of high-dose GH, however, four subjects still had not achieved stimulated IGFBP-3 concentrations equivalent to baseline levels observed in normal subjects. Graphs of the IGF-I/IGFBP-3 ratios show an increase of IGF-I relative to IGFBP-3 by d 8 of GH, although molar ratios are still not normalized (Fig. 1B). In the results reported previously on IGF-I levels, six of the subjects were considered poor responders to GH, defined as an inability to generate IGF-I levels above 100 ng/ml on either d 5 or d 8 of the treatment weeks. However, all of these subjects did have an IGFBP-3 response to GH, with IGFBP-3 values ranging from 301-1826 ng/ml.
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and 5 and Table 2)
Sixty-five subjects heterozygous for the E180 splice mutation, confirmed by molecular diagnosis, completed the study. As seen in Fig. 5, the majority of baseline and stimulated IGFBP-3 concentrations lie within the observed ranges for the normal individuals in this population. There were no significant differences found when comparing the changes in IGFBP-3 generation of the heterozygotes to the age-matched normals. A few males in the older than 40 yr group had lower IGFBP-3 responses to both low- and high-dose GH than the age-matched normal subjects; these same subjects had demonstrated low IGF-I responsiveness, as well. Noncompliance with the study regimen or an undiagnosed comorbid condition, such as liver disease, cannot be excluded. These results may also reflect the relatively small number of normal subjects in these groups and, furthermore, may be an explanation for a greater IGF-I response to GH seen in some heterozygous males between 18 and 40 yr of age and some females over age 40 yr relative to their age-matched normal subjects. When comparing the ratios of IGF-I and IGFBP-3 in normal adults and children and in heterozygous individuals, the slopes of the curves are similar in all cases (Fig. 1A).
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and 6 and Table 2)Sixteen children with ISS participated in the study; all lacked the E180 splice mutation of the GHR gene and had stimulated GH concentrations greater than 10 ng/ml. Baseline and stimulated IGFBP-3 levels fell generally within the normal range. This IGFBP-3 responsiveness contrasts with IGF-I results (20), in which many ISS subjects had subnormal IGF-I concentrations and demonstrated a poor to modest response to GH.
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)
Table 3 shows the relative sensitivity and specificity of IGF-I and IGFBP-3 generation in the diagnosis of GHI, defining a positive test as a failure to raise IGF-I by at least 15 ng/ml, or IGFBP-3 by at least 400 ng/ml after a course of GH (19). IGFBP-3 generation on d 8 of high-dose GH resulted in the best combination of sensitivity (100%) and specificity (91%). Failure to raise both IGF-I and IGFBP-3 resulted in 100% specificity on d 8, but sensitivity was 82% on low-dose GH and 91% on high-dose GH. Thus, some confirmed GHI subjects were able to raise IGF-I by more than 15 ng/ml or IGFBP-3 by more than 400 ng/ml. Conversely, no GHD subject failed to raise either IGF-I or IGFBP-3.
Discussion
The IGF generation test is a measure of the stimulated response of an individual to GH, and can be used to assess levels of GH-dependent peptides, such as IGF-I, IGFBP-3, and acid-labile subunit. Although the test has been available for over 20 years, we still lack an adequate reference base of normative, gender-specific values for all age groups (27). Studies in the past have examined the test for diagnostic utility in differentiating etiologies of short stature (2), for predicting the response to GH treatment (16, 28, 29), or for examining GH responsiveness in aging, ß-Thalassemia (30, 31), or other conditions, but conclusions have been limited and varied. Initially, investigators examined serum levels of IGF-I as the endpoint (32, 33), but now most include IGFBP-3, as well (4, 5, 18, 19, 34).
We recently reported our results for IGF-I generation in these subjects, of whom almost 200 completed the study (20). In the 72 normal subjects, the IGFBP-3 serum levels mirror the IGF-I levels; they are GH-dependent at all ages. The rise in the levels is significant by d 5 of treatment on both the low dose and the high dose of GH, and again, as with IGF-I, there appears to be little advantage in using the high dose over the low dose of GH or in extending the test to 8 d. However, the longer administration and higher dose provided the most sensitive and specific test in the establishment of GHI within this population. The doses of GH were arbitrarily chosen, reflecting doses commonly used in children, but much higher than those used in adults. Compared with IGF-I levels, however, the relative rise in the levels of IGFBP-3 is less dramatic. This is evident when one examines the molar ratio of IGF-I and IGFBP-3 (Fig. 1A). The decrease in the slope from d 1 to d 8 of normal adults shows the larger increase in IGF-I levels compared with IGFBP-3. Interestingly, the children maintain a proportional rise in both parameters.
Blum et al. (19) published criteria for the diagnosis of GHI, including the following: height less than -3 SD, elevated GH, low basal IGF-I and IGFBP-3, and, in the IGF generation test, an IGF-I increase of less than 15 ng/ml and IGFBP-3 increase of less than 400 ng/ml. In our analysis of IGF-I levels, we found 5 of 22 GHI subjects (all proven to have the same E180 splice mutation) had stimulated IGF-I levels that increased more than 15 ng/ml. Of these same five subjects, only one, however, had a rise in IGFBP-3 greater than 400 ng/ml. Thus, using the Blum criteria, the sensitivity of IGFBP-3 to select for GHI is 100% on d 8 of high-dose GH, in comparison to 91% for IGF-I. Each marker is specific on d 8 of high-dose GH (97% for IGF-I, 92% for IGFBP-3), and in our cohort, their combined use was 100% sensitive and 90% specific, when analyzing the 61 subjects with a height less than -2 SD (all GHI, GHD, and ISS subjects included) on d 8 of high-dose GH. Thus, 22 of 22 GHI subjects failed to raise either IGF-I or IGFBP-3 on d 8 of high-dose GH; 35 of 39 GHD and ISS subjects raised either IGF-I or IGFBP-3 or both. We recommend, therefore, analyzing GH-stimulated levels of both IGF-I and IGFBP-3 if one is suspicious of GHI in a patient with significant short stature. Recently, the utility of the generation of IGF-I/IGFBP-3 in short stature has been challenged, due to concerns about reproducibility (35). These authors performed two separate IGF generation tests, spaced 1–22 months apart, in 12 children with ISS. They found poor reproducibility in terms of IGF-I and IGFBP-3 generation, with several of their subjects failing the Blum criteria on one or both of the IGF generation tests. Examination of data from our study, however indicates significant correlation between the two different generation tests in the normals, heterozygotes, GHI, and GHD subjects. Only in patients with ISS was poor reproducibility in IGF-I and IGFBP-3 generation observed, a subject which clearly warrants further study and analysis (manuscript in preparation).
Of the GHD subjects, there were six who had a poor IGF-I response to GH, failing to raise their serum levels above 100 ng/ml. IGFBP-3 levels, in contrast, were substantially elevated in all of these subjects, with the exception of one subject on low-dose GH (IGFBP-3 of 301 ng/ml). The one previously reported GHD subject, with a IGF-I level less than 15 ng/ml, had a IGFBP-3 value of 617 ng/ml by d 5 and of 1010 ng/ml by d 8 on high-dose GH. The six GHD subjects who had relatively poor IGF-I responses are examples of patients who may require close monitoring of GH therapy, with consideration given to changes in dose based on their growth velocity and IGF-I levels. In these patients, IGFBP-3 may prove to be a useful marker of compliance, and IGF-I a measure of optimal hormone replacement. In general, the molar ratio graphs of IGF-I to IGFBP-3 nicely show a trend to normal in GHD subjects while they were receiving GH replacement. It is of interest that IGFBP-3 more quickly normalizes than does IGF-I in response to GH; only four subjects had values less than normal baseline values by d 8 on the high-dose of GH.
No biochemical or phenotypic effect is seen in the E180 splice heterozygous subjects. The IGF-I levels clearly overlapped the normal profiles, as do the IGFBP-3 concentrations shown in this study. Given this biochemical profile, the slight difference in height seen in this cohort of E180 splice carriers compared with normal Ecuadorian controls may be due simply to chance, rather than reflect an effect of heterozygosity for the mutation. Importantly, however, the conclusions drawn from these data should only be applied to the effect of heterozygosity for the E180 splice mutation. Although classic GHI due to homozygosity or compound heterozygosity for mutations of the GHR gene is rare, over 30 different mutations and gene deletions have been identified in such patients (22). Some of these mutations clearly can act in a dominant negative manner, because dimerization of the GHR is required for transmission of the GH-stimulated signal. Partial GHI may, furthermore, produce a variable phenotypic effect (7, 8, 9, 10). The IGF generation test (both for IGF-I and IGFBP-3) is necessary to identify and to characterize patients with GHI, complete or partial, classic or atypical.
The combined IGF-I and IGFBP-3 generation test may be particularly useful in distinguishing patients with ISS from GHD or GHI. The 16 children with ISS who completed this study had basal and generated IGF-I levels in the lower half of normal or below the normal range. In comparison, their baseline and stimulated IGFBP-3 levels were completely within normal ranges. These subjects are obviously not classically GHD, because they passed provocative GH tests and have normal basal concentrations of IGFBP-3; on the other hand, they may be relatively GHD, because they do not have enough GH to elevate IGF-I levels to entirely age-appropriate values. Alternatively, their relative IGF-I deficiency may represent a form of partial GHI characterized by poor IGF-I production, possibly reflecting a post-receptor defect. ISS is a heterogeneous group, and hopefully, over the next few years, we will begin to unravel some of the etiologies, such as post-receptor signaling defects and other gene mutations (36, 37, 38).
Use of the IGF generation test, including both IGF-I and IGFBP-3, will aid us in the diagnosis, management, and understanding of patients with disorders of the GH-IGF axis. It is safe and relatively simple to perform. Although the low-dose (0.025 mg/kg·d) GH for 4 d with fasting blood samples drawn at baseline and the morning after the fourth dose appears to be adequate in most instances, high-dose GH stimulation for 7 d appears to improve test sensitivity and specificity. The utility of longer tests (e.g. 2–4 wk) warrants evaluation. Furthermore, these results would suggest that monitoring of serum concentrations of IGF-I and IGFBP-3 in patients receiving GH might be useful in optimizing therapy. Future research should investigate the IGF-I response, the IGFBP-3 response, and ratios of IGF-I to IGFBP-3 in response to GH, with correlation of these parameters to growth velocity while on GH therapy.
Acknowledgments
Footnotes
This work was funded by grants from the Genentech Foundation for Growth and Development (to R.G.R.) and National Institutes of Health Grants CA-58110 and DK-51513 (to R.G.R.) and 5T32-HD07497 (to C.K.B. and K.S.).
Abbreviations: GHD, GH-deficient or GH deficiency; GHI, GH-insensitive or GH insensitivity; GHR, GH receptor; IGFBP-3, IGF binding protein-3; ISS, idiopathic short stature.
Received January 16, 2002.
Accepted July 10, 2002.
References
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