GROWTH WITHOUT GROWTH HORMONE: THE "INVISIBLE" GH SYNDROME T. BISTRITZER J. C. LOVCHIK
S. A. CHALEW A. A. KOWARSKI
Department of Pediatrics, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA Growth hormone (GH) deficiency, diagnosed by radioimmunoassay (RIA) measurements of GH in blood after provocation or in continuous 24 h samples of venous blood, is usually associated with growth failure. 4 non-obese boys have been identified who had normal linear growth despite apparent GH deficiency by standard RIA. All 4 patients had normal GH concentrations as measured with an IM-9 cell radioreceptor assay (RRA). The RRA/RIA ratio of the 4 patients significantly exceeded that of controls. Thus, these patients secrete a molecule with normal GH receptor binding and bioactivity which is "invisible" to the standard GH RIA. This variant GH is possibly expressed from the human GH-V gene or a mutant allele.
Introduction GROWTH hormone (GH) has a crucial role in promoting normal linear growth during childhood. Deficient growth hormone secretion leads to growth failure. The GH radioimmunoassay (RIA) is the standard method for determination of growth hormone levels in blood samples. Subnormal concentrations of GH by RIA in samples obtained after provocation or in 24 h blood collections are the established diagnostic tests for GH deficiency. 1-5 All children who appear to have GH deficiency are expected to show growth impairment. Growth without GH is difficult to reconcile with our present understanding of the role of GH in promoting growth. Several children with craniopharyngioma have been reported to show normal or even excessive growth after surgical removal of the tumour and subsequent documentation of GH deficiency.6-9 The growth of these patients has been attributed to hyperinsulinism secondary to obesity induced by hypothalamic damage.8 In another child lacking GH normal growth was attributed to a putative growth factor that was not detected with a GH radioreceptor assay. 10 We describe here 4 non-obese healthy children with normal growth despite GH deficiency by standard diagnostic tests in which the GH levels were measured with RIA. In all 4 patients, however, GH levels were normal
Fig 1-Standard curve of the IM-9 cell hGH radioreceptor assay.
when measured with a radioreceptor assay (RRA). We postulate that the 4 children secrete a variant GH that binds to the GH receptors, mimics the biological activity of GH, but is invisible
the standard RIA.
Patients and Methods
Subjects 4 patients were referred to the endocrine diagnostic unit at the University of Maryland for diagnostic GH testing. The referring diagnosis in case 1 was acromegaly; the other 3 children were thought to have growth hormone deficiency. The growth velocity of all 4 patients was found to have been consistently normal for several years before the study. Patient 1 was growing slightly above the 95th percentile, patient 2 was growing along the 5th percentile, patient 3 along the 25th percentile, and patient 4 along the 10th percentile. A 24 h integrated concentration test (IC-GH) was done in all 4 children. In addition, an insulin-arginine tolerance test (IATT) was carried out in patients 3 and 4. Blood samples for GH RRA were taken from venous blood obtained during the GH provocative tests of patients 3 and 4, and during the IC-GH tests of patients 1 and 2. For each patient the samples selected for RRA were those with the highest GH levels by RIA. In the light of the apparent GH deficiency of these children, patients 2-4 were started on a trial of GH therapy, 011 mg/kg three times a week.
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morphological damage of brain tissue. Stroke 1983; 14: 329-31. 2. Thomas DJ. Haemodilution in acute stroke. Stroke 1985; 16: 763-64. 3. Grotta J, Ackerman R, Correia J, Fallick G, Chang J. Whole blood viscosity parameters and cerebral blood flow. Stroke 1982; 13: 296-301. 4 Wood JH, Kee DB. Hemorheology of the cerebral circulation in stroke. Stroke 1985; 16: 765-72 5. Italian Acute Stroke Study Group. The Italian hemodilution trial in acute stroke. Stroke 1987; 18: 670-76. 6. Rankin J. Cerebrovascular accidents in patients over the age of 60 II, prognosis Scott Med J 1957; 2: 200-15. 7. Peto R, Pike MC, Armitage P, et al. Design and analysis of randomized clinical trials requiring prolonged observation of each patient. I. Introducton and design. BrJ Cancer 1977; 35: 1-39. 8. Yusuf S, Peto R, Lewis J, Collins R, Sleight P. Beta blockade during and after myocardial infarction: an overview of the randomised trials. Prog Cardiovasc Dis 1985; 27: 335-71. 9 Gilroy J, Barnhart M, Meyer J S. Treatment of acute stroke with dextran 40. JAMA 1969; 210: 293-98 10. Spudis EV, De La Torre E, Pikula L. Management of completed strokes with dextran 40. A community hospital failure. Stroke 1973, 4: 895-98.
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322 TABLE I-CLINICAL AND LABORATORY DATA
= Body mass index. ND = Not done. SMC = Somatomedin-C
Stimulated GH samples from 18 children and young adults of normal stature and history of normal growth rate were used as controls.
Diagnostic Studies Two provocative GH stimulation tests (using insulin-induced hypoglycaemia followed by administration of arginine") were done after an overnight fast. A plasma GH level 10 ng/ml at any time during the procedure was considered a normal response.1 The IC-GH was measured by’continuous withdrawal of venous blood for 24 h with a Cormed minipump and a non-thrombogenic blood withdrawal system. The range of IC-GH in healthy children of normal
Assay Methods GH RIA.-Plasma GH levels
antibody method.12 The specificity and accuracy of our RIA has been reported elsewhere.2 The coefficient of variation was 16 % and the intra-assay coefficient of variation 7-4%. IM-9 Cell GH RRA.-Purified 125I-labelled hGH was obtained from Hazleton Biotechnologies Corp. Polyclonal rabbit anti-GH antibody was obtained from the National Hormone and Pituitary
Program, Baltimore, Maryland, and is routinely used in this laboratory. IM-9 cells were obtained from American Type Culture Collection. RPMI-1640 medium, glutamine, and fetal bovine serum were obtained from W. M. A. Bioproducts (Walkersville, Maryland). IM-9 cells were grown in continuous culture in RPMI-1640 medium with 25 mmol/1 ’Hepes’ buffer (Sigma), supplemented with 10% fetal bovine serum and L-glutamine (4 mmot/1). The cells were grown in 150 ml Falcon flasks (VWR) at 37°C in 5%
COz, At 72 h, the cells were divided into 3, and fresh medium was added to each flask. We modified a previously described two-stage binding assay" by increasing the volume of plasma from 01 ml to 0-5 ml. The standard curve (fig 1) was prepared with plasma from patients with surgical hypopituitarism. The sensitivity of the standard curve was improved by increasing the number of cells in each test from approximately 15 million to 35 million. All samples were run in triplicate. The intra-assay coefficient of variation was 15%. The inter-assay coefficient of variation, measured in 16 consecutive assays, was 26%. Statistical Analysis Results are reported as the mean between the control group and the Student’s t-test.
(SD). Statistical significance patients was evaluated with
Results Clinical and laboratory data from the patients are in table I. Fig 2 shows the individual RRA/RIA ratios for GH from controls and patients. The RRA/RIA ratios represent the mean of assays from two different samples except for patient 4. There was no overlap between the RRA/RIA ratios of patients and controls. RRA/RIA ratio of the control group (1-3 [0-3]) was significantly lower than that of patients (7-8 [5-1], p < 0-0001). The GH levels from the 4 patients, as measured by RIA and RRA, are presented in table II. Patient 2 was treated with human GH (hGH) for 6 months. His growth rate before treatment was 7-5 cm/year and during therapy 8-6 cm/year, and he entered Tanner stage II during the treatment period. Patient 3 was treated with hGH for 9 months. His growth rate before treatment was 7 cm/year, and on therapy 10 cm/year. Patient 3 progressed from Tanner stage II to III during therapy. Patient 4 was treated with hGH for 9 months. His growth before treatment was 5-8 cm/year and on therapy 6 cm/year. His sexual maturation reached Tanner stage II during that time.
TABLE II-GH LEVELS AS MEASURED BY RRA AND RIA FROM PATIENTS AND CONTROLS
Fig 2-Ratio of the concentration of hGH, as measured by the IM-9 cell RRA, divided by its concentration, as measured by the RIA, in control subjects and patients with the "invisible" GH syndrome. The ratio represents the mean of two separate from whom 1 sample was tested).
samples (except patient 4,
Discussion Our group has previously reported the first 2 cases of bioinactive growth hormone syndrome.14 These 2 children displayed all the clinical manifestations of GH deficiency and subsequently responded vigorously to GH therapy. Despite normal plasma GH levels by standard RIA,14 their GH showed greatly reduced binding to pregnant rabbit liver membrane. We postulated that the 2 patients secreted a variant GH which was biologically inactive but had normal
immunoreactivity. 14 The present study describes 4 boys in whom the opposite clinical picture was manifested. Their growth rate was normal, but they all had an apparent deficiency of GH as measured by RIA. However, normal GH concentrations in the same plasma samples were demonstrated with an IM-9 cell radioreceptor assay. In all 4 patients the GH RRA/RIA ratio, which is the ratio of bioactivity to immunoreactivity of the circulating GH, exceeded the range of controls. Our results indicate that the plasma of the 4 patients contained an unknown hormone which could not be detected with standard GH RIA but which is capable of binding to the GH receptors on IM-9 cells. We conclude that the putative hormone mimics the biological activity of GH, since all 4
patients were growing normally. 2 of the 3 patients who were treated with GH did not have a significant increase in growth rate. Patient 3 grew 3 cm/yr better on GH therapy, but the growth improvement was at the time of pubertal progression and might have been due to coincidental increase in sex steroids. The general lack of increased growth of our patients treated with a replacement dose of GH also suggests that they did not have GH deficiency. The "invisible GH" in our patients is different from the "growth factor" described by Geffner et al,lO which had no affinity for the hGH receptor on IM-9 cells. The growth of the child in Geffner’s report was independent of GH and other known growth factors. Several reports have suggested the presence of an "invisible" GH. A number of acromegalic patients have been reported to have significantly higher concentrations of GH by radioreceptor binding assay than by RIA.15 It has been suggested that the excessive receptor binding of GH in these acromegalic patients may be due to secretion of a variant GH from an alternative gene at a different level. This gene, hGH-V, was discovered during studies on cloning and sequencing of the hGH gene. 16 This variant gene codes for a polypeptide which is nearly homologous to hGH. The hGH-V differs at 15 of the 218 aminoacids which form the pre-hGH molecule (hGH plus its leader peptide).16 The hGH-V gene was reported to be unexpressed, since no mRNA derived from it could be detected in human cells .17,11, In-vitro expression of a recombinant hGH-V gene generated a polypeptide which exhibited only a very weak binding affinity to polyclonal hGH antibodies." However, the hGH-V polypeptide had 50% of the binding activity of hGH in a radioreceptor assay with human lymphocytes and 100% binding activity in a radioreceptor assay with pregnant rabbit liver membranes.18 Thus if the hGH-V gene were expressed, it would be "invisible" by the RIA but biologically active. The structure of our putative hormone is unknown. Since the aminoacid sequence of GH is genetically determined, an aberrant form of hGH, which is immunologically inert but metabolically active, could arise either from genetic errors in the biosynthesis of hGH or from an expression of the
hGH-V gene.19 Consequently, patients who would secrete the normally unexpressed hGH-V gene could account for a hormone that exhibits significant GH biological activity but a low affinity for GH antibodies. We found considerable variability in the ratio of RRA/ RIA among our controls of normal stature (fig 2). Some of this variability might be accounted for by the inherent variability of the two assay techniques. However, minor structural variants of the GH molecule with altered affinity to the GH receptor2O-23 may be present in subjects of normal stature, accounting for the observed variability in their
RRA/RIA. We thank Dr S. B. Biswas for review of the manuscript, Teresa Palese, Baiba Pironis, and Jackie Newhouse for technical assistance, and Diane Ogorzalek for preparing the manuscript. This work was supported by the Bressler Research Fund of the University of Maryland.
Correspondence should be addressed to A. A. K., Department of Pediatrics, University of Maryland School of Medicme, BRB 10-047, 655 West Baltimore Street, Baltimore, Maryland 21201, USA. REFERENCES 1. Zadik 2.
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