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The Journal of Clinical Endocrinology & Metabolism 87(2):469 – 472 Copyright © 2002 by The Endocrine Society
Poor Reproducibility of IGF-I and IGF Binding Protein-3 Generation Test in Children with Short Stature and Normal Coding Region of the GH Receptor Gene ALEXANDER A. JORGE, SILVIA C. SOUZA, IVO J. ARNHOLD,
AND
BERENICE B. MENDONCA
Unidade de Endocrinologia do Desenvolvimento, Laboratorio de Hormonios e Genetica Molecular LIM/42, Disciplina de Endocrinologia, Hospital das Clinicas, Sa˜o Paulo 05403-900, Brazil The IGF-I generation test has been proposed to select patients with GH insensitivity. Studies have shown that children with idiopathic short stature and GH deficiency, who were expected to be sensitive to GH, presented absent IGF-I and IGF binding protein (IGFBP)-3 responses and/or discordant results at IGF-I and IGFBP-3 generation test for unknown reasons. To assess the reproducibility of the generation test, we studied a group of 12 prepubertal children with short stature and normal GH secretion in whom defects in coding region of GH receptor gene were ruled out. All patients underwent the test twice. Discordant responses between the first and second test were found in five and six patients for IGF-I and IGFBP-3, respectively. When the results of the generation tests were
compared, one notices that IGF-I generation has more concordant positive results than IGFBP-3. In eight generation tests that showed discordant results between IGF-I and IGFBP-3, seven presented a positive IGF-I with a negative IGFBP-3 response. Taking both tests into account, all children presented a normal IGF-I generation. Our findings showed that IGF-I and IGFBP-3 generation test was not reproducible in children that should have responded to GH stimulation. We suggest that, when IGF-I and IGFBP-3 levels fail to respond in the generation test, another test should be performed to confirm GH insensitivity. (J Clin Endocrinol Metab 87: 469 – 472, 2002)
I
N RECENT YEARS, there has been an increasing concept that some children with idiopathic short stature (ISS) have partial insensitivity for GH (GH) action. Therefore, it is reasonable to screen this group of patients through the assessment of IGF-I and IGF binding protein (IGFBP)-3 responses after a short period of GH administration, similarly to what is done to diagnose complete GH insensitivity (GHI or Laron syndrome) (1). Despite the fact that several studies have assessed the use of IGF-I and IGFBP-3 generation test to evaluate children with idiopathic short stature (2– 8), no response criteria for the diagnosis of partial GH insensitivity have been established to date. A previous study compared the responses of the IGF-I and IGFBP-3 generation test between GHD and ISS patients and noticed a lack of response or discordant response of IGF-I and IGFBP3 in these patients (2). A recent study analyzed the specificity of the criteria used to diagnose Laron syndrome, including IGF-I and IGFBP-3 generation test, when applied to children with GH deficiency (GHD) and ISS (3). It disclosed an absence of response in some children who were sensitive to GH. The reason for this failure to respond to GH stimulation and the discordant response of IGF-1 and IGFBP-3 remains unknown. Furthermore, the reproducibility of these tests has not been evaluated to date. The aim of the present study was to investigate the reproducibility of the IGF-I and IGFBP-3 generation test in a group of short stature patients with normal spontaneous or
stimulated GH levels who were found not to harbor defects in the GHR gene. Patients and Methods Patients This study was approved by the Ethics Committee of the University of Sa˜o Paulo School of Medicine and the parents or tutors gave written informed consent. We studied 12 children (7 boys) with short stature, all of them with height less than ⫺2 sd. Nine of them had ISS, characterized by normal response to GH after stimulation tests and absence of any alterations at physical examination and laboratory tests that could account for short stature. As for the other three, one had delayed puberty (a 16-yr-old boy without any clinical and laboratory signs of puberty), one was born small for gestational age, and the other one had Noonan’s syndrome identified through normal karyotype and the use of the criteria established by Van der Burgt et al. (9). During the study, all children were maintained without any therapy and remained prepubertal, assessed by physical examination and prepubertal hormonal levels (basal LH and testosterone for boys and LH and E2 for girls (10). All of the children had adequate nutritional status assessed by interviews with parents or tutors, adequate weight for height, absence of malnutrition signs, and normal laboratory parameters such as whole blood count and albumin measurement. Clinical features of these patients are shown in Table 1.
Molecular studies All patients were submitted to the molecular study of GHR gene to rule out inactivating mutations in this gene that could account for the lack of response at the generation test. Genomic DNA was isolated from peripheral blood leukocytes. We amplified exons 2–9 of the GHR using specific intronic primers, and exon 10 was amplified in three overlapping fragments to cover the entire coding region. PCR products from exons 2–9 were electrophoresed on a polyacrylamide denaturing gel using a specific gradient for each exon according to their melting maps. The samples that presented an abnormal pattern of migration at denaturing gradient gel electrophoresis were
Abbreviations: GHD, GH deficiency; GHI, GH insensitivity (Laron syndrome); GHR, GH receptor; hGH, human GH; IGFBP, IGF binding protein; ISS, idiopathic short stature.
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Jorge et al. • IGF-I and IGFBP-3 Generation Test Reproducibility
TABLE 1. Clinical features of 12 children submitted to IGF-I and IGFBP-3 generation test Patient
Diagnosis
Sex
CA (yr)
1 2 3 4 5 6 7 8 9 10 11 12
ISS ISS SGA ISS DP ISS Noonan S. ISS ISS ISS ISS ISS
M F F F M F M M M F M M
10.0 12.0 2.0 2.0 16.0 11.8 10.0 8.0 7.4 11.1 5.0 3.0
BA (yr)
Height SD
Interval between tests (months)
7.0 10.0 1.0
⫺2.8 ⫺3.9 ⫺6.1 ⫺2.9 ⫺3.2 ⫺3.2 ⫺3.1 ⫺2.1 ⫺2.0 ⫺2.4 ⫺2.7 ⫺2.4
7 2 1 6 5 1 1 19 22 6 15 2
12.5 8.8 4.0 5.0 5.0 6.8 3.0 1.0
CA, Chronological age; BA, bone age; DP, delayed puberty; ISS, idiopathic short stature; SGA, small for gestational age; Noonan S., Noonan’s syndrome.
directly sequenced with dideoxy chain-termination method using a dye terminator kit and an autosequencer. Exon 10 was directly sequenced.
IGF-I and IGFBP-3 generation test All patients underwent IGF-I and IGFBP-3 generation test twice, which involved daily injections of recombinant human GH (hGH) (0.1 U/kg sc) at bed time for 4 d consecutively as standardized by Blum et al. (1). Fasting blood samples were drawn in the morning before the first injection and on d 5 for IGF-I and IGFBP-3 measurements. GH administration was performed at home by parents or tutors who were trained at our outpatient clinic by a registered nurse and a physician. The compliance was checked by interviewing parents or tutors after each test. We used the same cutoff values for the IGF-I and IGFBP-3 generation tests proposed by Blum et al. (1) for the diagnosis of GH insensitivity: an IGF-I increase ⬍15 g/liter and an IGFBP-3 increase ⬍0.4 mg/liter. The response to the first and second test was considered discordant when one was below and other above the cutoff limits. Four of these patients (cases 1– 4) were submitted to a second test because they failed to respond in the first generation test. Considering that the four patients respond in the second generation test we started to perform a second test independently of the result of the first one. The median interval between the first and second test was 6 months (range 1–22 months).
IGF-I and IGFBP-3 serum measurements IGF-I was determined by RIA after extraction with ethanol (Diagnostic Systems Laboratories, Webster, TX) and IGFBP-3 was measured by immunoradiometric method (DSL). The coefficient of variation was ⬍10%, for IGF-I levels from 18 –500 g/liter and for IGFBP-3 levels from 0.14 –12 mg/liter.
Statistical analysis The absolute and percentile increment in IGF-I and IGFBP-3 during the generation test was calculated. The data were analyzed using the two-tailed t test and P ⬍ 0.05 was considered statistically significant. We also used Pearson correlation test to analyze basal and peak IGF-I and IGFBP-3 levels.
Results Molecular studies
Molecular analysis of the GHR gene excluded the presence of any inactivating mutation in the coding region and the splice sites in all patients studied.
IGF-I and IGFBP-3 generation test
Tables 2 and 3 show results of IGF-I and IGFBP-3 generation test respectively, performed on two different occasions. Basal IGF-I and IGFBP-3 values in sd for sex and age showed that almost all patients presented levels between 0 and ⫺2 sd (median: ⫺1.25 sd for IGF-I and ⫺1 sd for IGFBP-3). All the patients had an IGF-I response greater than 15 g/liter in at least one of the tests. Two patients failed to increase IGFBP-3 levels ⱖ 0.4 mg/liter in both generation tests even though they reached an adequate IGF-I generation in either test. Five patients had discordant IGF-I and 6 patients had discordant IGFBP-3 responses between the first and the second test. Most of the patients (n ⫽ 10) presented a greater increase of IGF-I after the second test when compared with the first test and 8 of 12 patients presented a greater increase of IGFBP-3 after the second test. There was no correlation between basal IGF-I and IGFBP-3 levels and ⌬IGF-I or ⌬IGFBP-3. We noticed a great variability between the two generation tests among patients that responded on both occasions. The variability of IGF-I response ranged from 40 –725%, whereas variability of IGFBP-3 ranged from 50 –100%. We observed TABLE 2. IGF-I levels in two generation tests before and after 4 d of hGH administration in 12 children with short stature
Patient
1 2 3 4 5 6 7 8 9 10 11 12
Basal IGF-I (g/liter)
⌬IGF-I (g/liter)
Peak IGF-I (g/liter)
1st test
2nd test
1st test
2nd test
1st test
2nd test
73 207 27 18 96 105 194 69 64 85 109 52
97 81 37 18 94 203 142 145 167 28 88 83
76 204 37 18 241 215 170 158 105 171 125 170
216 298 57 153 396 384 244 344 249 62 220 118
3a ⫺3a 10a 0a 145 110 ⫺24a 89 41 86 16 118
119 217 20 135 302 181 102 199 82 34 132 35
The numbers in italics refer to discordant tests. a Negative response in IGF-I generation test. TABLE 3. IGFBP-3 levels before and after 4 d of hGH administration in 12 children
Patient
1 2 3 4 5 6 7 8 9 10 11 12
Basal IGFBP-3 (mg/liter)
⌬IGFBP-3 (mg/liter)
Peak IGFBP-3 (mg/liter)
1st test
2nd test
1st test
2nd test
1st test
2nd test
2.41 3.2 1.4 2.1 3.3 2.3 3.8 3.1 1.3 2.4 2.4 2.0
2.7 2.8 1.4 1.3 2.1 2.5 2.8 4.0 2.9 2.9 3.0 1.9
2.77 3.5 2.1 1.7 3.3 2.7 3.9 3.2 1.7 2.6 2.8 2.5
3.7 4.5 1.5 3.6 4.1 3.2 2.8 4.4 3.2 3.0 3.7 3.1
0.36a 0.3a 0.7 ⫺0.4a 0.8 0.4 0.1a 0.1a 0.4 0.2a 0.4 0.5
1.0 1.7 0.1a 2.3 1.2 0.7 0.0a 0.4 0.3a 0.1a 0.7 1.2
The numbers in italics refer to discordant tests. Negative response in IGFBP-3 generation test.
a
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Jorge et al. • IGF-I and IGFBP-3 Generation Test Reproducibility
discordant responses between IGF-I and IGFBP-3 generation test at the same test. Eight patients presented a lack of response in IGFBP-3 test but normal IGF-I generation test results. On the other hand, only one patient presented the opposite situation (Fig. 1). Discussion
Partial GHI has recently been found to be involved in the etiology of short stature (11–15). Attie et al. in 1995, showed that 20% of children with idiopathic short stature had low levels of GH binding protein. These children, despite having higher mean 12-h GH levels when compared with children with normal GH binding protein, presented low IGF-I values (11). These findings suggest the presence of some levels of resistance to GH action in those children. In the same year, Goddard et al. described for the first time the presence of mutations in the GH receptor gene in patients with ISS (12). Thereafter, other studies identified GHR gene defects as cause of ISS (13–15). It is a reasonable approach to measure IGF-I and IGFBP-3 increase after the administration of recombinant GH to screen children who have short stature and low levels of IGF-I and/or IGFBP-3 for partial GH insensitivity. In 1994, Blum et al. (1) proposed a scoring system to diagnose complete GHI (Laron syndrome) which included an increase of IGF-I and IGFBP-3, ⬍15 ng/dl and ⬍0.4 mg/liter, respectively, after 4 d of hGH administration (generation test). However, standard responses for IGF-I and IGFBP-3 generation tests have not been established for normal and partial GH insensitivity children to date. Several studies evaluated the response of children with GHD and ISS to generation test using Blum et al. criteria and correlated them with growth responses (2– 6, 8). Thalange et al. investigated the difference of IGF-I and IGFBP-3 responses to the generation test between GHD children and children with ISS suspected to have partial GH insensitivity due to high basal and stimulated GH levels. They found that four children did not reach IGF-I cutoff levels, whereas four did
J Clin Endocrinol Metab, February 2002, 87(2):469 – 472 471
not reach IGFBP-3 cutoff levels at the generation test. However, seven children had discordant responses, with three of them showing a poor IGF-I response but a satisfactory IGFBP-3 response, and four the converse (2). Recently, Blum’s score was evaluated regarding its capacity to differentiate children with ISS and GHD from children with GH insensitivity syndrome (3). The results showed that 21.4% of these children presented an increase of IGF-I below the cutoff values and the same happened for 7.1% regarding IGFBP-3 (3). The reason why some children failed to respond to IGF-I and IGFBP-3 generation tests even though they are sensitive to GH, remains unknown. It is possible that some of these patients have defects in GHR because molecular studies of GHR gene were not performed or perhaps, the test design is not adequate to evaluate GH sensitivity. We studied short children in whom defects in the coding region of GHR gene were ruled out through molecular studies, which should have had a normal response to IGF-I and IGFBP-3 generation test. However, we noticed that a significant number of these patients failed to show adequate responses in the generation test: 42% in the first IGF-I generation, 50% and 33% respectively, in the first and second IGFBP-3 generation. When we analyze both generation tests, all patients showed at least one normal IGF-I response indicating that failure to respond was not due to impaired GH action. When the results of IGF-I and IGFBP-3 generation test are compared, discordant results between the first and second test were found more often in IGFBP-3 than in IGF-I generation. Furthermore, two patients failed to show a normal response to IGFBP-3 in both tests. This fact indicates that IGF-I generation test is a better tool to rule out complete GH insensitivity and is a more reliable index than IGFBP-3 generation test, in opposition to data found in the literature (3). The huge variability in the degree of response in addition to the high number of discordant results between the first and second generation test (42% of discordant results for IGF-I and 50% of discordant results for IGFBP-3) justify the
FIG. 1. Correlation of IGF-I (A) and IGFBP-3 (B) increment between the first and second generation test.
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difficulty to establish a cutoff level for partial GH insensitivity and might explain the discordance between the IGFI/IGFBP-3 generation test and growth responses during hGH therapy (3, 6, 16). Our results suggest that some of the children that failed to reach a normal response at the generation test would not really be resistant to GH because a response could be obtained at a second test. We suggest that, when IGF-I and IGFBP-3 levels fail to respond in the first generation test, a second test should be performed to confirm GH insensitivity. Acknowledgments Received August 10, 2001. Accepted October 22, 2001. Address all correspondence and requests for reprints to: Berenice B. de Mendonca, M.D., Hospital das Clı´nicas, Labarato´rio de Hormonios, Avenida Doutor Eneas de Carvalho Aguiar 155 PAMB, 2 andar Bloco 6, 05403-900 Sa˜o Paulo, Brazil. E-mail:
[email protected]. A.A.J. was supported by Grants 97/12611-0 and 00/14092-4 from Fundac¸a˜o de Amparo a Pesquisa do Estado de Sa˜o Paulo.
Jorge et al. • IGF-I and IGFBP-3 Generation Test Reproducibility
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