Acta PCEdiatr Scand [Suppl] 377: 104-109, 1991
Regulation of Serum Insulin-Like Growth Factors and Their Binding Proteins in Ecuadorean Patients with Growth Hormone Receptor Dysfunction (Growth Hormone Insensitivity) P.J. FIELDER, J. GUEVARA-AGUIRRE2 , A.L. ROSENBLOOM 3, P. COHEN 1, R.L. HINTZ 1 and R.G. ROSENFELD 1 From 1the Depanment of Pediatrics, Stanford University Medical School, Stanford, California, USA, Endocrinologia, Metabolismo y Reproducci6n, Quito, Ecuador, and 3 University of Florida College of Medicine, Gainesville, Florida, USA
2/nstituto
ABSTRACT. Fielder, P.J., Guevara-Aguirre, J,, Rosenbloom, A.L., Cohen, P., Hintz, R.L., Rosenfeld, R.G. (Department of Pediatrics, Stanford University Medical School, Stanford, California, USA, lnstituto Endocrinologia Metabolismo y Reproducci6n, Quito, Ecuador, and University of Florida College of Medicine, Gainesville, Florida, USA). Regulation of serum insulin-like growth factors and their binding proteins in Ecuadorean patients with growth hormone receptor dysfunction (growth hormone Insensitivity). Acta Paediatr Scand [Suppl) 377: 104-109, 1991. Regulation of serum insulin-like growth factors (IGFs), IGF binding proteins (IGFBPs), and growth hormone (GH) binding protein (GHBP) has been investigated In Ecuadorean patients with GH receptor dysfunction (GHRD) and in their heterozygous relatives (parents). Serum IGF-1 and IGF-11 levels were measured by radioimmunoassay (RIA). IGFBPs were Identified by Western ligand blotting of serum samples, following separation by sodium dodecyl sulphate polyacrylamide gel electrophoresis, and relative quantification was performed with a scanning laser densitometer. Serum GHBP levels were measured with a ligand-mediated immunofunctlonal assay (LIFA) using a monoclonal antibody raised against the GHBP. These values were then compared with values obtained from normal, sex-matched Ecuadorean controls. Serum IGF-1, IGF-11, IGFBP-3 and GHBP concentrations were markedly reduced and serum IGFBP-2 values increased in the patients with GHRD. Serum IGF-1 and IGF-11 values were positively correlated in the patients with GHRD, but were not related to the age of the patient. IGFBP-2 and IGFBP-3 concentrations were inversely correlated in the patients with GHRD. When analysed by age, however, IGFBP-2 was related inversely and IGFBP-3 related positively to the age of the patient. The serum IGF-11 levels of the male heterozygotes were significantly reduced when compared with sex-matched controls, but considerable overlap with normal values was found. No other biochemical indices were significantly altered in either male or female heterozygotes. Thus, although GHRD is characterized by dramatic reductions in serum levels of GHBP,IGF-1, IGF-11 and IGFBP-3, none of these assays provides a reliable biochemical marker for heterozygosity. Key words: Growth hormone insensidvily, heterozygotes, growth hormone binding protein, insulin-like growth factors, insulin-like growth factor binding proteins.
Attainment of normal stature is dependent on a complex interaction of growth-promoting hormones, growth factors and their receptors. Genes coding for growth-promoting hormones must be expressed at the appropriate times and the tissues must be competent to respond to these hormones. Failure to produce sufficient amounts of growth-promoting hormones or failure of target tissues to respond can result in growth failure (1). One form of growth failure, first described by Laron eta/. (2-4), is due to defects in the gene encoding the growth hormone (GH) receptor (5, 6). Patients with GH receptor dysfunction (GHRD) have normalto-elevated serum GH, but do not respond to GH. The entity of GHRD is part of the broader clinical syndrome of GH insensitivity, which also includes defects in insulin-like growth factor (IGF) synthesis and/or secretion (Fig. 1). These genetic defects lead to failure of GH to increase production of both IGF-1 and IGF-11, and ultimately to growth failure (2-4, 7). This disruption of the normal GH-GH receptor-IGF axis also results in changes in the serum concentrations of IGF binding proteins (IGFBPs) (7, 8, Fielder PJ et al., unpublished), though the precise factors responsible for the regulation of serum IGFBPs in GHRD are uncertain. The following study attempted to characterize the interrelationships between the IGFs and IGFBPs in a homogeneous cohort of patients with GHRD from Ecuador (7) and their parents, w~o are obligate heterozygotes for the defective gene.
GH insensitivity: regulation of /GFs and JGFBPs
Acta Paediatr Scand [Suppl] 377
HYPOTHALAMUS
PITUITARY
2
~nal transdus;tion ~
TARGET TISSUES
~thesis
Endocrine ~ Autocrine 13IGF Paracrine
[:[:] IGFBPs /'-.... ~ ~ It" Type IIGF receptor
¥~~
fig. I. The GH-IGF axis and the possible defects involved in GH insensitivity. Broad lines indicate possible
mechanistic defects that may lead to GH insensitivity. These defects include failure of GH to bind to its receptor defects in the signal transduction of the GH receptor (2), and defects in IGF synthesis (3) and release (4).
(I),
METHODS Serum samples. Serum samples were obtained from female patients with GHRD (n = 19) of Loja Province origin, and from the mothers (n = 9) and fathers (n = 8) (obligate heterozygotes) of the affected families. For comparison, serum samples were also collected from non-related Ecuadorean men (n = 12) and women (n = 10) who were not of Loja origin (so as to render the possibility of occult heterozygosity less likely), but who also lived in the Andes. The serum IGF-1, IGF-11, IGFBP, GH and GHBP values for the Loja patients with GHRD and their parents have been described (7, Fielder PJ eta/., unpublished). Radioimmunoassay of /GF-1 and /GF-11. The concentrations of both IGF-1 and IGF-11 were assayed in serum samples from all patients, heterozygotes, and sex-matched controls (7, 9, Fielder PJ eta/., unpublished). In order to separate IGF peptides from their respective binding proteins, serum samples of 500 JLI were first chromatographed in methanoic acid, 0.25 mol/1, on 0.9 x 100 em columns containing Sephadex G-50 (9). Sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis and Western ligand blolling of serum /GFBPs. Serum samples, 2 ,.1 each, were applied to a 4% stacking gel and electrophoresed through a 12.5% polyacrylamide gel according to the method of Laemmli (10). Western ligand blotting of the filter-immobilized
105
106
P.J. Fielder et a!.
Acta Paedialr Scand [Suppl] 377
Table 1. Serum /GF concentrations in patients with GHRD and their obligate heterozygote relatives. Values are given as means ± SEM.
Male controls (n = 12) Female controls (n = 9) Patients with GHRD (n = 19) Mothers (n = 9) Fathers (n = 8)
IGF-I (ng/ml)
IGF-II (ng/ml)
197 ± 15 182 ± 14
615 ± 33 536 ± 35
IGFBP-2 (% control)
IGFBP-3 (% control)
GHBP (pmol/1) 230 ± 34 263 ± 35
15 ± 4*
97 ± 10*
162 ± 63
8± 2
47 ± 4*
181 ± 16 182 ± 23
434 ± 55 392 ± 56*
100 ± 9 120 ± 18
77 ± 12 98 ± 9
199 ± 34 146 ± 28
*p < 0.05 relative to sex-matched adult controls. proteins was carried out according to the method of Hossenlopp tt a/. (II). After development, the autoradiographs were scanned with a laser densitometer and the relative densities of the bands corresponding to each IGFBP were expressed as absorption units/mm. The relative amounts of IGFBPs in the experimental samples were expressed as percentages of the mean control values (n = 4) for that individual gel. GH binding protein assay. Serum GHBP levels were measured in duplicate with a ligand-mediated immunofunction assay (LIFA). as previously described (12). Values were expressed as pmol/1. The intra- and interassay coefficients of variation of the LIFA assay were 7.3% and 11.3%, respectively. Statistical analysis. The mean values were compared by Student's r-test and the results were expressed as means ± SEM. Correlations among data were obtained using Spearman's test and regression analysis.
RESULTS The serum levels of IGF-1, IGF-11, IGFBP-3 and GHBP were dramatically reduced in the patients with GHRD, though their serum IGFBP-21evels were increased (Table 1). Although considerable overlap with normal levels was observed, the mean serum IGF-11 was significantly reduced in the fathers of the patients with GHRD (64% of control value, p < 0.05), but not in the mothers (81% of control value). Mean serum GHBP concentrations were also reduced in both the fathers (64% of control value) and mothers (76% of control value), but the differences were not statistically significant. Serum IGF-1, IGFBP-2 and IGFBP-3 levels were close to normal in both sets of parents. Regression analysis was used to determine the relationships among the biochemical parameters. Due to the non-linearity of some of the relationships, however, a polynomial regression line was used and the correlations were calculated with a non-parametric test (Spearman's test). There was a strong positive correlation (r = 0.687, p < 0.05) between serum IGF-1 and IGF-11 in the patients with GHRD (Fig. 2), but neither serum IGF-1 nor IGF-11 was correlated with IGFBP or GHBP. IGFBP-2 and IGFBP-3 were inversely related in the patients with GHRD (Fielder PJ eta/., unpublished) (Fig. 3a), and Spearman's test demonstrated a strong negative correlation (r = -0.745, p < 0.05). When serum IGFBP-2 concentrations were separated according to age, however, there was a strong negative correlation (r = -0.686, p < 0.05) (Fig. 3b). Conversely, IGFBP-3 concentrations were positively correlated with the age of the patient (r = 0.626, p < 0.05) (Fig.3c). IGF-1 (r = 0.332), IGF-11 (r = 0.398) and GHBP (r = 0.082) levels were not significantly correlated with the age of the patients with GHRD. No significant correlations among the above indices were found in the data obtained from either the mothers or fathers. DISCUSSION A large, isolated cohort of patients with GHRD has recently been described in the Loja Province of southern Ecuador (7). This genetic defect appears to be inherited in a simple autosomal recessive pattern. Surprisingly, this cohort was characterized by a near absence of affected males (7). Patients were found to have many of the physical and biochemical features
GH insensitivity: regulation of IGFs and IGFBPs
Acta Paediatr Scand [Suppl) 377
200
r = 0.687
c 0
10
_so
20
60
IGF-1 (ng/ml)
Fig. 2. Regression analysis of the interrelationship between serum IGF-1 and IGF-11 in patients with GHRD. Serum samples were obtained from 19 women of Ecuadorean origin with GHRD, and IGFs were determined by radioimmunoassay. The positive correlation (r = 0.687, Spearman's test) was statistically significant (p < 0.05).
characteristic of GHRD, such as elevated serum GH, low serum IGF-1 and IGF-11, and a near absence of serum GHBP (7, Fielder PJ et al., unpublished). Most pitients also had reduced serum IGFBP-3 and elevated IGFBP-2 levels (Fielder PJ et al., unpublished). The Ecuadorean patients with GHRD provide a unique opportunity to study the ontogeny and regulation of IGFs and their binding proteins jn the complete absence of a functional GH-GH receptor-IGF axis. The present study shows that serum IGFBP-2 and IGFBP-3 levels are inversely correlated in the patients with GHRD, with IGFBP-3 levels increasing with age and IGFBP-2 levels decreasing with age. These age-related changes in IGFBP levels appear to be most striking before puberty, and are not associated with the normal age-related increase in serum IGF-1 levels (13). It is not clear what factors are responsible for the age-related changes in IGFBP. One possibility is that IGFBP-3 is regulated by a protease activity similar to that found in the serum of pregnant women (14) and rodents (15). In an earlier study, serum samples from patients with GHRD with the lowest serum IGFBP-3 (as measured by Western ligand blotting) proteolysed recombinant IGFBP-3 added exogenously (Fielder PJ et al., unpublished). Patients with GHRD with greater amounts of IGFBP-3 lacked this protease activity, as did their heterozygous relatives. Together, these findings suggest that the protease activity found in some of the patients with GHRD may also be age-related, with the increase in IGFBP-3 levels with age resulting in part from a .decrease . in the IGFBP-3 protease activity of the serum. Some of the patients with GHRD had detectable serum 'GHBP in the LIF A a~say. This indicates that the serum GHBP in these patients can bind GH, though to a limited extent (Carlsson LMS et al., unpublished). Serum GHBP levels are believed to provide an indication of hepatic concentrations of GH recceptors ( 16). These data suggest that the Loja patients with GHRD either express a defective GH receptor/GHBP that binds GH with a decreased affinity, or express small amounts of a functional receptor, possibly due to defective regulation of GH receptor gene transcription or translation. Laron et al. suggested that heterozygotes for GHRD had decreased serum GHBP, and that serum GHBP could be a marker for heterozygosity in relatives of the patients (17). Laron's study, however, contained relatives of patients with GHRD who may have not been heterozygous for the defective gene, and some of the parents (presumptive obligate heterozygotes) were found to have normal GHBP concentrations. In our study, parents of the
107
108
P.J. Fielder et al.
Acta Paediatr Scand [Suppl) 377
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Regulation of Serum Insulin-Like Growth Factors and Their Binding Proteins in Ecuadorean Patients with Growth Hormone Receptor Dysfunction (Growth Hormone Insensitivity) P.J. FIELDER, J. GUEVARA-AGUIRRE2 , A.L. ROSENBLOOM 3, P. COHEN 1, R.L. HINTZ 1 and R.G. ROSENFELD 1 From 1the Depanment of Pediatrics, Stanford University Medical School, Stanford, California, USA, Endocrinologia, Metabolismo y Reproducci6n, Quito, Ecuador, and 3 University of Florida College of Medicine, Gainesville, Florida, USA
2/nstituto
ABSTRACT. Fielder, P.J., Guevara-Aguirre, J,, Rosenbloom, A.L., Cohen, P., Hintz, R.L., Rosenfeld, R.G. (Department of Pediatrics, Stanford University Medical School, Stanford, California, USA, lnstituto Endocrinologia Metabolismo y Reproducci6n, Quito, Ecuador, and University of Florida College of Medicine, Gainesville, Florida, USA). Regulation of serum insulin-like growth factors and their binding proteins in Ecuadorean patients with growth hormone receptor dysfunction (growth hormone Insensitivity). Acta Paediatr Scand [Suppl) 377: 104-109, 1991. Regulation of serum insulin-like growth factors (IGFs), IGF binding proteins (IGFBPs), and growth hormone (GH) binding protein (GHBP) has been investigated In Ecuadorean patients with GH receptor dysfunction (GHRD) and in their heterozygous relatives (parents). Serum IGF-1 and IGF-11 levels were measured by radioimmunoassay (RIA). IGFBPs were Identified by Western ligand blotting of serum samples, following separation by sodium dodecyl sulphate polyacrylamide gel electrophoresis, and relative quantification was performed with a scanning laser densitometer. Serum GHBP levels were measured with a ligand-mediated immunofunctlonal assay (LIFA) using a monoclonal antibody raised against the GHBP. These values were then compared with values obtained from normal, sex-matched Ecuadorean controls. Serum IGF-1, IGF-11, IGFBP-3 and GHBP concentrations were markedly reduced and serum IGFBP-2 values increased in the patients with GHRD. Serum IGF-1 and IGF-11 values were positively correlated in the patients with GHRD, but were not related to the age of the patient. IGFBP-2 and IGFBP-3 concentrations were inversely correlated in the patients with GHRD. When analysed by age, however, IGFBP-2 was related inversely and IGFBP-3 related positively to the age of the patient. The serum IGF-11 levels of the male heterozygotes were significantly reduced when compared with sex-matched controls, but considerable overlap with normal values was found. No other biochemical indices were significantly altered in either male or female heterozygotes. Thus, although GHRD is characterized by dramatic reductions in serum levels of GHBP,IGF-1, IGF-11 and IGFBP-3, none of these assays provides a reliable biochemical marker for heterozygosity. Key words: Growth hormone insensidvily, heterozygotes, growth hormone binding protein, insulin-like growth factors, insulin-like growth factor binding proteins.
Attainment of normal stature is dependent on a complex interaction of growth-promoting hormones, growth factors and their receptors. Genes coding for growth-promoting hormones must be expressed at the appropriate times and the tissues must be competent to respond to these hormones. Failure to produce sufficient amounts of growth-promoting hormones or failure of target tissues to respond can result in growth failure (1). One form of growth failure, first described by Laron eta/. (2-4), is due to defects in the gene encoding the growth hormone (GH) receptor (5, 6). Patients with GH receptor dysfunction (GHRD) have normalto-elevated serum GH, but do not respond to GH. The entity of GHRD is part of the broader clinical syndrome of GH insensitivity, which also includes defects in insulin-like growth factor (IGF) synthesis and/or secretion (Fig. 1). These genetic defects lead to failure of GH to increase production of both IGF-1 and IGF-11, and ultimately to growth failure (2-4, 7). This disruption of the normal GH-GH receptor-IGF axis also results in changes in the serum concentrations of IGF binding proteins (IGFBPs) (7, 8, Fielder PJ et al., unpublished), though the precise factors responsible for the regulation of serum IGFBPs in GHRD are uncertain. The following study attempted to characterize the interrelationships between the IGFs and IGFBPs in a homogeneous cohort of patients with GHRD from Ecuador (7) and their parents, w~o are obligate heterozygotes for the defective gene.
GH insensitivity: regulation of /GFs and JGFBPs
Acta Paediatr Scand [Suppl] 377
HYPOTHALAMUS
PITUITARY
2
~nal transdus;tion ~
TARGET TISSUES
~thesis
Endocrine ~ Autocrine 13IGF Paracrine
[:[:] IGFBPs /'-.... ~ ~ It" Type IIGF receptor
¥~~
fig. I. The GH-IGF axis and the possible defects involved in GH insensitivity. Broad lines indicate possible
mechanistic defects that may lead to GH insensitivity. These defects include failure of GH to bind to its receptor defects in the signal transduction of the GH receptor (2), and defects in IGF synthesis (3) and release (4).
(I),
METHODS Serum samples. Serum samples were obtained from female patients with GHRD (n = 19) of Loja Province origin, and from the mothers (n = 9) and fathers (n = 8) (obligate heterozygotes) of the affected families. For comparison, serum samples were also collected from non-related Ecuadorean men (n = 12) and women (n = 10) who were not of Loja origin (so as to render the possibility of occult heterozygosity less likely), but who also lived in the Andes. The serum IGF-1, IGF-11, IGFBP, GH and GHBP values for the Loja patients with GHRD and their parents have been described (7, Fielder PJ eta/., unpublished). Radioimmunoassay of /GF-1 and /GF-11. The concentrations of both IGF-1 and IGF-11 were assayed in serum samples from all patients, heterozygotes, and sex-matched controls (7, 9, Fielder PJ eta/., unpublished). In order to separate IGF peptides from their respective binding proteins, serum samples of 500 JLI were first chromatographed in methanoic acid, 0.25 mol/1, on 0.9 x 100 em columns containing Sephadex G-50 (9). Sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis and Western ligand blolling of serum /GFBPs. Serum samples, 2 ,.1 each, were applied to a 4% stacking gel and electrophoresed through a 12.5% polyacrylamide gel according to the method of Laemmli (10). Western ligand blotting of the filter-immobilized
105
106
P.J. Fielder et a!.
Acta Paedialr Scand [Suppl] 377
Table 1. Serum /GF concentrations in patients with GHRD and their obligate heterozygote relatives. Values are given as means ± SEM.
Male controls (n = 12) Female controls (n = 9) Patients with GHRD (n = 19) Mothers (n = 9) Fathers (n = 8)
IGF-I (ng/ml)
IGF-II (ng/ml)
197 ± 15 182 ± 14
615 ± 33 536 ± 35
IGFBP-2 (% control)
IGFBP-3 (% control)
GHBP (pmol/1) 230 ± 34 263 ± 35
15 ± 4*
97 ± 10*
162 ± 63
8± 2
47 ± 4*
181 ± 16 182 ± 23
434 ± 55 392 ± 56*
100 ± 9 120 ± 18
77 ± 12 98 ± 9
199 ± 34 146 ± 28
*p < 0.05 relative to sex-matched adult controls. proteins was carried out according to the method of Hossenlopp tt a/. (II). After development, the autoradiographs were scanned with a laser densitometer and the relative densities of the bands corresponding to each IGFBP were expressed as absorption units/mm. The relative amounts of IGFBPs in the experimental samples were expressed as percentages of the mean control values (n = 4) for that individual gel. GH binding protein assay. Serum GHBP levels were measured in duplicate with a ligand-mediated immunofunction assay (LIFA). as previously described (12). Values were expressed as pmol/1. The intra- and interassay coefficients of variation of the LIFA assay were 7.3% and 11.3%, respectively. Statistical analysis. The mean values were compared by Student's r-test and the results were expressed as means ± SEM. Correlations among data were obtained using Spearman's test and regression analysis.
RESULTS The serum levels of IGF-1, IGF-11, IGFBP-3 and GHBP were dramatically reduced in the patients with GHRD, though their serum IGFBP-21evels were increased (Table 1). Although considerable overlap with normal levels was observed, the mean serum IGF-11 was significantly reduced in the fathers of the patients with GHRD (64% of control value, p < 0.05), but not in the mothers (81% of control value). Mean serum GHBP concentrations were also reduced in both the fathers (64% of control value) and mothers (76% of control value), but the differences were not statistically significant. Serum IGF-1, IGFBP-2 and IGFBP-3 levels were close to normal in both sets of parents. Regression analysis was used to determine the relationships among the biochemical parameters. Due to the non-linearity of some of the relationships, however, a polynomial regression line was used and the correlations were calculated with a non-parametric test (Spearman's test). There was a strong positive correlation (r = 0.687, p < 0.05) between serum IGF-1 and IGF-11 in the patients with GHRD (Fig. 2), but neither serum IGF-1 nor IGF-11 was correlated with IGFBP or GHBP. IGFBP-2 and IGFBP-3 were inversely related in the patients with GHRD (Fielder PJ eta/., unpublished) (Fig. 3a), and Spearman's test demonstrated a strong negative correlation (r = -0.745, p < 0.05). When serum IGFBP-2 concentrations were separated according to age, however, there was a strong negative correlation (r = -0.686, p < 0.05) (Fig. 3b). Conversely, IGFBP-3 concentrations were positively correlated with the age of the patient (r = 0.626, p < 0.05) (Fig.3c). IGF-1 (r = 0.332), IGF-11 (r = 0.398) and GHBP (r = 0.082) levels were not significantly correlated with the age of the patients with GHRD. No significant correlations among the above indices were found in the data obtained from either the mothers or fathers. DISCUSSION A large, isolated cohort of patients with GHRD has recently been described in the Loja Province of southern Ecuador (7). This genetic defect appears to be inherited in a simple autosomal recessive pattern. Surprisingly, this cohort was characterized by a near absence of affected males (7). Patients were found to have many of the physical and biochemical features
GH insensitivity: regulation of IGFs and IGFBPs
Acta Paediatr Scand [Suppl) 377
200
r = 0.687
c 0
10
_so
20
60
IGF-1 (ng/ml)
Fig. 2. Regression analysis of the interrelationship between serum IGF-1 and IGF-11 in patients with GHRD. Serum samples were obtained from 19 women of Ecuadorean origin with GHRD, and IGFs were determined by radioimmunoassay. The positive correlation (r = 0.687, Spearman's test) was statistically significant (p < 0.05).
characteristic of GHRD, such as elevated serum GH, low serum IGF-1 and IGF-11, and a near absence of serum GHBP (7, Fielder PJ et al., unpublished). Most pitients also had reduced serum IGFBP-3 and elevated IGFBP-2 levels (Fielder PJ et al., unpublished). The Ecuadorean patients with GHRD provide a unique opportunity to study the ontogeny and regulation of IGFs and their binding proteins jn the complete absence of a functional GH-GH receptor-IGF axis. The present study shows that serum IGFBP-2 and IGFBP-3 levels are inversely correlated in the patients with GHRD, with IGFBP-3 levels increasing with age and IGFBP-2 levels decreasing with age. These age-related changes in IGFBP levels appear to be most striking before puberty, and are not associated with the normal age-related increase in serum IGF-1 levels (13). It is not clear what factors are responsible for the age-related changes in IGFBP. One possibility is that IGFBP-3 is regulated by a protease activity similar to that found in the serum of pregnant women (14) and rodents (15). In an earlier study, serum samples from patients with GHRD with the lowest serum IGFBP-3 (as measured by Western ligand blotting) proteolysed recombinant IGFBP-3 added exogenously (Fielder PJ et al., unpublished). Patients with GHRD with greater amounts of IGFBP-3 lacked this protease activity, as did their heterozygous relatives. Together, these findings suggest that the protease activity found in some of the patients with GHRD may also be age-related, with the increase in IGFBP-3 levels with age resulting in part from a .decrease . in the IGFBP-3 protease activity of the serum. Some of the patients with GHRD had detectable serum 'GHBP in the LIF A a~say. This indicates that the serum GHBP in these patients can bind GH, though to a limited extent (Carlsson LMS et al., unpublished). Serum GHBP levels are believed to provide an indication of hepatic concentrations of GH recceptors ( 16). These data suggest that the Loja patients with GHRD either express a defective GH receptor/GHBP that binds GH with a decreased affinity, or express small amounts of a functional receptor, possibly due to defective regulation of GH receptor gene transcription or translation. Laron et al. suggested that heterozygotes for GHRD had decreased serum GHBP, and that serum GHBP could be a marker for heterozygosity in relatives of the patients (17). Laron's study, however, contained relatives of patients with GHRD who may have not been heterozygous for the defective gene, and some of the parents (presumptive obligate heterozygotes) were found to have normal GHBP concentrations. In our study, parents of the
107
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Acta Paediatr Scand [Suppl) 377
(a)
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..
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c: 0
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