0021-972x/92/7404-0743$03.00/0 Journal of Clinical Endocrinology and Metabolism Copyright 0 1992 by The Endocrine Society

Vol.

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74, No. 4

in U.S.A.

Expression of Serum Insulin-Like Growth Factors, Insulin-Like Growth Factor-Binding Proteins, and the Growth Hormone-Binding Protein in Heterozygote Relatives of Ecuadorian Growth Hormone Receptor Deficient Patients* PAUL LENA

J. FIELDER*, CARLSSON**,

JAIME GUEVARA-AGUIRRET, ARLAN L. ROSENBLOOM& RAYMOND L. HINTZ*, AND RON G. ROSENFELD*

Department of Pediatrics (P.J.F., R.L.H., R.G.R.), Stanford University Medical School, Stanford, California 94305; §University of Florida College of Medicine (A.L.R.), Gainesville, Florida 32610; Instituto Endocrinologia Metabolism0 y Reproduction (J.G.A.), Quito, Ecuador; and Genentech Inc. (L.C.),? South San Francisco, California 94080

ABSTRACT. Recently, an isolated population of apparent GH-receptor deficient (GHRD) patients has been identified in the Loja province of southern Ecuador. These individuals presented many of the physical and biochemical phenotypes characteristic of Laron-Syndrome and are believed to have a defect in the GH-receptor gene. In this study, we have compared the biochemical phenotypes between the affected individuals and their parents, considered to be obligate heterozygotes for the disorder. Serum GH, insulin-like growth factor I and II (IGF-I and IGF-II) levels were measured by RIA Insulin-like growth factor binding proteins. (IGFBPs) were measured by Western ligand blotting (WLB) of serum samples, following separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and relative quantitation of serum IGFBPs was performed with a scanning laser densitometer. Serum GH-binding protein (GHBP) levels were measured with a ligand-mediated immunofunctional assay using a monoclonal antibody raised against the GHBP. These values were then compared to values obtained from normal, sex-matched adult Ecuadorian controls, to determine if the above parameters were abnormal in the heterozygotes. The serum IGF-I levels of the GHRD patients were less than 13% of control values for adults and 2% for children. However, the IGF-I levels of both the mothers and fathers were not significantly different from that of the control population. The serum IGF-II levels of the GHRD patients were approxi-

mately 20% of control values for adults and 12% for the children. The IGF-II levels of the mothers were reduced, but were not significantly different from that of the control population. However, IGF-II levels of the fathers were significantly lower than those of controls (64% of control male levels). WLB analysis of serum IGFBP levels of the affected subjects demonstrated increased IGFBP-2 and decreased IGFBP-3, suggesting an inverse relationship between these IGFBPs. The GHRD patients who had the lowest serum IGFBP-3 levels (as measured by WLB) demonstrated a serum protease activity that could proteolyze “‘1-IGFBP-3. GHRD patients who had higher serum IGFBP-3 levels lacked this serum protease activity. There were no differences in the serum IGFBP profiles of the mothers or the fathers for either IGFBP-2 or IGFBP-3, and serum from both groups lacked the ability to significantlyproteolyze ‘Y-IGFBP-3. While GHRD patients had very low levels of serum GHBP, some patients did have measurable GHBP levels. The mean GHBP levels of the mothers (76% of control females) and father (64% of control males), while reduced, did not significantly differ from those of controls. Thus, while serum IGF-II and GHBP levels were somewhat reduced in the parents of the GHRD patients, they do not appear to provide a reliable biochemical marker for heterozygosity of the defective gene. (J Clin Endocrinol Metab 74:

G

H-RECEPTOR deficient (GHRD) patients (LaronSyndrome) were first described by Laron et al. (1, 2). This condition of short stature, is characterized by

743-750,1992)

low serum insulin-like growth factor (IGF) levels, despite normal to increased serum GH levels (1, 2). Patients with this disorder are resistant to the growth promoting actions of GH and have exaggerated responses to GHstimulation tests. It was originally thought that these patients secreted an inactive GH molecule (1, 3), but further studies indicated that these patients lacked functional hepatic GH receptors (4) and serum GH-binding proteins (5, 6). This biochemical phenotype, as well as the physical phenotype of short stature are believed due to a defect in the GH receptor gene, leading to an absence of functional GH receptors (7, 8).

Received April 22, 1991. Address all correspondence and requests for reprints to: Paul J. Fielder, Ph.D., Department of Pediatrics, Stanford University Medical Center, Stanford, California 94305. * This work was supported by NRSA Grant DK08516-01 to P. J. F.; the Instituto Endocrinologia Metabolism0 y Reproduction, Quito, Ecuador; NIH Grant DK28229 to R. G. R.; and a grant from the American Diabetes Association to R. G. R. Presented in part at the 11th International Symposium on Growth and Growth Disorders, Stockholm, Sweden, April 26-27,199l. 743

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744

FIELDER

Recently, a large, isolated population of apparent GHRD patients has been identified in the Loja province of southern Ecuador (9). The genetic defect, although unknown at this time, appears to be inherited in a simple autosomal recessive pattern within the affected families. However, the most surprising finding in this population was the near absence of affected males (1 male us. 19 females). Although an actual growth hormone-receptor (GHR) deficiency has not been proven, these individuals present many of the physical and biochemical phenotypes characteristic of GHRD such as high serum GH, dramatically low serum IGF-I and IGF-II, and a deficiency of serum GH-binding protein (GHBP) (9). Additionally, patients had reduced serum IGF-binding protein-3 (IGFBP-3) and elevated IGFBP-2 levels. These alterations in serum IGFBPs were similar to those found in growth hormone deficient patients (10) and previously reported GHRD patients (10). Because the parents and nonaffected siblings of the GHRD patients appear physically normal, it was important to seek a marker for heterozygosity within the affected families. Both point mutations and gene deletions have been reported in patients with GHRD (7, 8). However, the specific genetic defect causing this disorder in the Ecuadorian population remains to be determined, and genetic screening is not yet possible. Since the biochemical phenotype of this disorder has been determined (9), it is possible that the heterozygote carriers of this disorder may have abnormal levels of GH, GHBP, IGFBPs, or IGFs, which can be employed as biochemical markers. In the present study, we have measured the above biochemical parameters in the parents of the affected individuals, who are obligate carriers of the defective gene, in order to determine if there were any abnormalities which could be used to identify heterozygotes in lieu of a genetic marker. Subjects and Methods Reagents

Pure biosynthetic Thr5’-IGF-I was purchased from Amgen Biologicals (Thousand Oaks, CA) and recombinant IGF-II was the generousgift of Dr. Michele Smith, Lilly ResearchLaboratories (Indianapolis, IN). Recombinant IGFBP-3 was the generousgift of BioGrowth Inc (Richmond, CA). Serum samples

Serum sampleswere obtained from both mothers (n = 9, ages= 25-66) and fathers (n = 10, ages = 23-87) (obligate heterozygotes) of the affected families. For comparison,serum sampleswere also collected from nonrelated adult Ecuadorian men (n = 12, ages= 18-38 yr) and women (n = 10, ages= 2038yr) who werenot of Loja origin (soasto render the possibility of occult heterozygosity less likely), but who also lived in the

ET AL.

JCE & M. 1992 Vol74.No4

Andes. The serum IGF-I, IGF-II, IGFBP, GH, and GHBP values for the Loja GHRD patients (n = 19) have beenreported (9). However, for this study the IGFBP and GHBP levels were reevaluated, using the normal adult Ecuadorian women as controls. Due to differences in the ageof the GHRD patients, the subjectswere separatedaccordingto age(children, 2.2-10.2 yr) (adults, 16.6-49.6yr). RIA of IGF-I

and IGF-I1

The concentrations of both IGF-I and IGF-II were assayed in serum samplesfrom all heterozygotes and sex-matchedcontrols. To separate IGF peptides from their respective binding proteins, serum samples(500 FL) were first chromatographed in 0.25 M formic acid on 0.9 X 100 cm columns containing Sephadex G-50 (11). The fractions eluting between 50 and 67 mL, which contain 90% of the IGF peptide activity, were collected and lyophilized in 1.0 mL 1% BSA. Serum IGF-I concentrations were determined by RIA using lZ51-IGF-I and a polyclonal antisomatomedin-Cantiserum. This antiserum was the generousgift of Drs. L. E. Underwood and J. J. Van Wyk (University of North Carolina at ChapelHill) and is distributed through the Hormone Distribution Program of the National Institute of Diabetes,and Digestive and Kidney Diseasesto the National Hormone and Pituitary Program. Serum IGF-II concentrations were also determined by a similar RIA procedure, using recombinant lz51-IGF-II and a monoclonal antibody against rat IGF-II (Amano Labs, Troy, VA). The inter- and intraassay coefficients of variance for the IGF-I RIA were 8 and 5%, and 10 and 7% for the IGF-II assay.The lower limits of sensitivity for both assaysis approximately 1.0 pg/L in the original sample. Serum

GH measurements

Serum GH measurementswereperformed in the laboratories of Genentech, Inc. (S. San Francisco, CA) with the Hybritech immunoradiometric assay(12). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western ligand blotting of serum IGFBPs

Serum (2 pL) sampleswere applied to a 4% stacking gel and electrophoresedthrough a 12.5%polyacrylamide gel according to the method of Laemmli (13). Prestained molecular weight standardswere run in parallel lanes, and gelswere run under nonreducing conditions overnight at 50 volts/gel. After electrophoresis, size fractionated proteins were transferred to nitrocellulose according to Towbin et al. (14). Briefly, gels were washedfor 15 min in transfer buffer (0.025M Tris-base, 0.192 M glycine, 20% methanol). The proteins were then electroblotted to nitrocellulose membraneswith a semidry electrophoresis transfer unit. Western ligand blotting (WLB) of the filterimmobilized proteins was carried out according to the method of Hossenloppet al. (15). Nitrocellulose membraneswere first washed in Tris-buffered saline (TBS) (0.15 M NaCl, 0.01 M Tris-HCl) containing 3% Nonidet P-40 for 30 min at 4 C. Nonspecific 1251-IGF-IIbinding to membraneswasblocked by preincubation in TBS containing 1% BSA for 2 h and then in TBS containing 0.1%Tween for 15min at 4 C. The membranes

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GH RECEPTOR were then incubated for 12 h with 1.0 X lo6 cpm [1251]IGF-II in 20 ml TBS containing 1% BSA and 0.1% Tween. Finally, the membranes were washed with TBS, air-dried, and exposed to Kodak X-Omat AR film for 3-5 days at -70 C. After development, the autoradiographs were scanned with a laser densitometer and the relative density of the bands corresponding to each IGFBP were expressed as absorbtion units per millimeter. The serum samples were applied to the SDS-gels in the following manner. Four sex-matched adult control samples were loaded in lanes l-4 and the experimental samples were loaded in lanes 5-13. The relative amount of IGFBPs in the experimental samples were expressed as percent of the mean control values (n = 4) for that individual gel. GHBP assay SerumGHBP levelswere measuredin duplicate with a ligand mediated immunofunction assay (LIFA) as previously describedby Carlssonet al. (16). Briefly, 96-well microtiter plates were coated with a monoclonal antibody specific for GHBP (Mab 263,Agen, Australia) by incubating overnight at 4 C with 100pL/well of antibody at 10 pg/mL in 50 mmol/L carbonate buffer, pH 9.6. The coated wells were blocked and washed. Standards(31.2-2000pmol/L of recombinant hGHBP, Genentech Inc.) or samples(50 pL/well) were addedinto coated wells containing 50 pL/well of 9090 pmol/L rhGH and 1.0 mg/mL mouseimmunoglobulin G (Fitzgerald Industries, Chelmsford, MA) in assaybuffer (phosphate-buffered saline containing 5 g BSA, 5.0 mM ethylenediaminetetraacetate, 0.5 mL Tween 20, and 0.1 g Thimerosal per liter. The plates were then sealed, incubated at room temperature for 2 h with gentle agitation, and washed before the addition of a monoclonal anti-hGH antibody (Mab MCB, Genentech Inc.) conjugated to horseradish peroxidase (100 wL/well). After further incubation, for 2 h at room temperature, the plates were washedagain. Freshly prepared substratesolution (0.4 g of o-phenylenediaminedihydrochloride in 1 L of phosphate-buffered saline containing 0.4 mL of 30% hydrogen peroxide) was added to the plates (100 pi/well) and the incubation was carried out in the dark for 15 min at room temperature. The reactions were stopped by the addition of 100 PL of 2.25 mol/L sulfuric acid and absorbance at 490 nm was determined. Data were expressed as GHBP pmol/L. The intra- and interassay coefficients of variation were 7.3 and 11.3%,respectively. IGFBP-3 protease assay Serumsamplesfrom GHRD patients were selectedaccording to the relative amount of IGFBP-3 visualized by WLB. Samples were separated into two subgroups,low and high IGFBP-3. Recombinant-derived (nonglycosylated) IGFBP-3 was iodinated by the Chloramine-T method (17). The recombinantly derived IGFBP-3 migrates with an apparent M, of -29K, as opposedto naturally occurring IGFBP-3 which migrates as a doublet with an M, of =40-45K. Serum IGFBP-3 protease activity was measuredby incubating 30,000 cpm lz51-IGFBP-3 with 2 /IL serum for 5 h at 37 C. Radiolabeledfragments were then separated on a 12.5% SDS-PAGE; the gels were dried, and visualized via autoradiography (17, 18). Intact, nonglyco-

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sylated IGFBP-3 migrates at -29K, and the major fragments produced during proteolysis by serum from pregnant women migrate at ~18 and 14K (19). Additionally, somefragments are present near the dye-front of the gel (