Recurrent nonsense mutations in the growth hormone receptor from patients with Laron dwarfism.

In addition to its classical effects on growth, growth hormone (GH) has been shown to have a number of other actions, all of which are initiated by an interaction with specific high affinity receptors present in a variety of tissues. Purification of a rabbit liver protein via its ability to bind GH has allowed the isolation of a cDNA encoding a putative human growth hormone receptor that belongs to a new class of transmembrane receptors. We have previously shown that this putative growth hormone receptor gene is genetically linked to Laron dwarfism, a rare autosomal recessive syndrome caused by target resistance to GH. Nevertheless, the inability to express the corresponding full-length coding sequence and the lack of a test for growth-promoting function have hampered a direct confirmation of its role in growth. We have now identified three nonsense mutations within this growth hormone receptor gene, lying at positions corresponding to the amino terminal extremity and causing a truncation of the molecule, thereby deleting a large portion of both the GH binding domain and the full transmembrane and intracellular domains. Three independent patients with Laron dwarfism born of consanguineous parents were homozygous for these defects. Two defects were identical and consisted of a CG to TG transition. Not only do these results confirm the growth-promoting activity of this receptor but they also suggest that CpG doublets may represent hot spots for mutations in the growth hormone receptor gene that are responsible for hereditary dwarfism.

Specific assay for insulin-like growth factor (IGF) II using the IGF binding proteins extracted from human cerebrospinal fluid.

A protein-binding assay for insulin-like growth factor II (IGF II) is described. The assay uses IGF binding proteins extracted from human cerebrospinal fluid which have selective affinity for IGF II. IGF I was 9 times less potent than IGF II in displacing [125I]IGF II, and when mixtures of the IGFs were assayed at IGF I/IGF II ratios of 2, 5, and 10, interference from IGF I in the assay was 0%, 5%, and 9%, respectively. Given the serum concentrations of IGF I and IGF II estimated by RIA and by this protein-binding assay, IGF I can be said to have had no cross-reaction when IGF II was assayed in human serum and at most 5% cross-reaction in the case of rat serum. After separation of IGFs from their binding proteins by acidic gel filtration, serum IGF II levels (mean +/- SE) measured by this method were 1322 +/- 66 ng/ml in normal adults, 500 +/- 65 ng/ml in patients with total GH deficiency, 1327 +/- 69 ng/ml in untreated acromegalic patients, and 1817 +/- 145 ng/ml in uremic patients undergoing chronic hemodialysis. In postpubertal young rats, the mean serum IGF II level was 43 +/- 2.6 ng/ml and after hypophysectomy it was 16 +/- 2.4 ng/ml. Although the IGF II levels in man and in the rat were different, they appeared to be similarly GH dependent, although less so than IGF I. In view of the sensitivity (0.03 ng IGF II) and the specificity of this assay, the small quantities of cerebrospinal fluid required (1 mu leq/assay tube) and its applicability for IGF II measurement in several species, the use of this assay for measuring IGF II in a variety of biological media can be envisaged.

Effects of nutrient intake on growth, insulin-like growth factors, and their binding proteins in a Laron-type dwarf.

In Laron-type dwarfism, a basal growth rate independent of GH and insulin-like growth factor-I (IGF-I) is maintained. This represents a unique model to further assess the relationship between growth and nutritional status. In a child aged 3 yr, 7 months with severe anorexia, growth was followed in relation to his caloric intake. While receiving 496 Cal/day with 11.6 g/day protein, he grew at a rate of 2 cm/yr (period I). The mean plasma IGF-I level was below 0.07 U/mL, insulin was 3.8 +/- 0.2 microU/mL, and blood glucose was 2.9 +/- 0.3 mM/L. During moderate hyperalimentation with 1280 Cal/day and 38.3 g/day protein (period II) for 7 months, growth rate increased to 9 cm/yr with no significant change in plasma IGF-I and persistence of relative hypoinsulinemia (low response to oral glucose tolerance test). IGF-binding proteins, analyzed by Western ligand blotting, showed that 41.5- and 38.5-kilodalton forms, which were initially low, increased to form a pattern similar to that observed in hypopituitarism. These results suggest that catch-up growth did not require normal circulating GH and/or IGF-I activity. Therefore, nutrition contributes to catch-up growth and achievement of potential statural growth by a distinct cellular effect.

Preferential measurement of insulin-like growth factor (IGF) I-related peptides in serum with the aid of IGF-binding proteins (IGF BPs) produced by rat liver in culture. Estimation of serum IGF BP levels.

A protein-binding assay for insulin-like growth factor (IGF) was developed which preferentially measures IGF I-related peptides in serum. Binding proteins (BPs) extracted from the culture media of livers from approximately 4-week-old rats were used in the assay, with pure IGF I as tracer and a partially purified IGF preparation as standard. Serum samples were gel filtered in acetic acid to separate the IGFs from their BPs. IGFs could be detected with this assay in extracts corresponding to as little as 0.2 microliter normal serum. The affinity of these liver BPs was greatest for IGF I. IGF II, somatomedin A, and multiplication-stimulating activity were found to be 2, 3, and 10 times less potent, respectively, than IGF I in displacing [125I]IGF I. There was no cross-reaction with insulin and proinsulin, structurally the most closely related peptides. There was a highly significant correlation (r = 0.98, P less than 0.001) between IGF values obtained from simultaneous assays, using either 1) somatomedin C/IGF I antibodies, or 2) the liver BPs, for acromegalic, normal, and hypopituitary serum extracts. Nevertheless, the protein-binding assay yielded values 1.7-fold those of the RIA. The BPs therefore do not possess the specificity of the antibodies for IGF I, but they do have the advantage of being less species specific in that they permit measurement of IGFs in the rat (among others) with the same sensitivity as in man. The validity of the assay was demonstrated both by the studies of IGF levels as a function of age, which yielded a profile characteristic of IGF I, and by the GH-dependence of the IGF levels measured. Mean IGF levels (+/- SEM) were the following: 1.03 +/- 0.03 U/ml in normal adults; 2.62 +/- 0.10 in acromegalic patients; 0.19 +/- 0.01 in patients with total GH deficiency; 0.58 +/- 0.04 in patients with partial GH deficiency (the reference serum being assigned a potency of 1 U IGF/ml). Human GH administration (6 mg/m2, im) to untreated hypopituitary patients on average provoked a 2-fold increase in IGF levels within 24 h. In hypophysectomized rats there was a close relation between the IGF level attained and the dose of GH administered (P less than 0.001). IGF BPs were titrated by incubating different concentrations of the serum extracts with [125I]IGF I and comparing these with a BP preparation obtained from the reference serum used in the IGF assay and arbitrarily assigned a value of 1 U IGF BP/ml.(ABSTRACT TRUNCATED AT 400 WORDS)

Angiotensin and adrenal steroidogenesis: study of 21-hydroxylase-deficient congenital adrenal hyperplasia.

The effects of angiotensin have been studied in four adult patients with the simple virilizing form of congenital adrenal hyperplasia due to 21-hydroxylase deficiency. They were treated with hydrocortisone (25 mg/day) throughout this investigation. Plasma ACTH was normal in three cases, and androstenedione was normal in all cases. However, urinary pregnanetriol, plasma 17-hydroxyprogesterone (17 OHP), aldosterone, and renin activity was increased. The patients were then submitted to three protocols: sodium depletion (10 meq Na/day) for 5 days, sodium repletion (200 meq Na/day) for 5 days, and angiotensin infusion (sufficient to maintain a pressor response) for 60 min. Urinary pregnanetriol, plasma 17 OHP, and androstenedione levels increased in all patients after sodium depletion and decreased after sodium repletion. Plasma ACTH levels were not modified by changes in the sodium balance. Furthermore, angiotensin infusion increased aldosterone and 17 OHP plasma concentrations without any change in the plasma ACTH level. This study shows the direct action of angiotensin on adrenal steroidogenesis, at least in 21-hydroxylase deficiency. It confirms that even in the simple virilizing form, combined treatment with glucocorticoids and mineralocorticoids helps to normalize plasma 17 OHP levels.

Plasma lipotropin increase in man after growth hormone administration. Comparison between extractive and biosynthetic hormones.

The action of human growth hormone (hGH) on plasma lipotropins (beta-and gamma-LPH) in 15 GH deficient patients was studied by comparing the effects induced by the acute administration of the extracted and biosynthetic molecules. The purified extracted preparation (6 mg/m2 im) induced a dramatic rise in plasma LPH: basal (49 +/- 12 pg/ml (mean +/- SEM); peak 1,658 +/- 262 pg/ml. The same dose of biosynthetic methionyl-hGH (met-hGH) induced no significant change in plasma LPH. Both preparations caused identical plasma GH increases. Six different commercially available extracted hGH preparations (Choay, France; Serono, Italy; France Hypophyse, France; Kabi, Sweden; Nordisk, Denmark; International Standard, Great Britain) all showed definite cross-reactivity in the LPH radioimmunoasay, varying from 0.1 to 1.0%, on a weight basis. No cross reactivity was found with met-hGH (less than 0.0001%). On gel exclusion chromatography, the LPH immunoreactivity of the purified preparations was dissociated from the GH immunoreactivity and eluted at the position of beta-and gamma-LPH. These data show that extracted hGH preparations are all contaminated with LPH and raise the question of the possible consequences of chronically elevated plasma LPH in treated patients. The use of biosynthetic met-hGH should prevent this occurence.

Early paradoxical decrease in serum somatomedin activity following injection of growth hormone.

The early effects have been investigated of an intra-muscular growth hormone injection (6 mg/m2) on serum somatomedin activity in 21 children with growth hormone deficiency and in 5 children with constitutional short stature. In cases of hGH total deficiency, there was an early and significant decrease in SM activity, which reached a minimum level 2 hours after the injection (-34% p less than 0.01). After 24 hours, SM activity increased to +64% above basal levels. In cases of hGH partial deficiency and in short, non-hGH deficient children, no similar early changes were observed. Neither was any correlation found between the variations in SM activity and those in hGH or free fatty acid levels. The unexpected early decrease in SM activity following hGH administration seems to be related to GH deficiency.

Serum levels of insulin-like growth factor (IGF) and IGF binding protein in constitutionally tall children and adolescents.

Serum insulin-like growth factor (IGF) and IGF binding protein (BP) levels were studied in 89 constitutionally tall children and adolescents (height greater than mean for age + 3 SD in 90% of the subjects). After separation by acidic gel filtration, the samples were assayed for IGF using a protein-binding assay (which measures mainly IGF I-related peptides) and for IGF BP by titration, in both cases using IGF I as tracer. The reference standard was a pool of normal adult serum with an assigned potency of 1 U IGF and 1 U IGF BP/ml. IGF levels increased with age in a manner similar to that in normal subjects, but at higher concentrations (P less than 0.02). The values were 0.77 +/- 0.05 (SE) U/ml in 1- to 5-yr-old children, 0.96 +/- 0.08 U/ml in 5- to 11-yr-old prepubescent children and 1.51 +/- 0.07 U/ml during puberty. BP levels developed in a different way from the above in that the increase with age was slight (0.70 +/- 0.07, 0.67 +/- 0.09, and 0.97 +/- 0.07 U/ml, respectively, for the three periods considered) and the levels were lower than those of normal subjects (P less than 0.005). After fusion of the epiphyses both IGF and BP levels were within the range of normal values. Thirteen girls underwent prolonged treatment (mean, 26 months) with ethinyl estradiol (250-300 micrograms/day). Deceleration in growth was accompanied by a progressive decrease in IGF levels throughout the period of therapy and a rise in BP levels during the first 6 months, after which they stabilized within the range of normal values. Although it is possible that excessive secretion of GH (which was not demonstrated by the usual tests) may be the cause of the elevated IGF levels during growth in these constitutionally tall subjects, it seems likely that the high IGF levels combined with the abnormally low levels of BP were responsible for their excessive growth.

Pituitary-adrenal axis activity in treated congenital adrenal hyperplasia: static and dynamic studies.

The pituitary-adrenal axis activity was evaluated in 43 patients, treated for congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency, by measuring plasma ACTH, 17-hydroxyprogesterone (17-OHP), testosterone, and aldosterone. Dynamic studies were performed by injecting 250 micrograms synthetic ACTH im and collecting blood samples 1 h later for steroid analysis. Twelve to fourteen hours after the last hydrocortisone dose given the evening before, plasma ACTH fluctuated widely from less than 10-475 pg/ml, 17-OHP exceeded normal values and varied from 1-275 ng/ml, while testosterone ranged from 3-151 ng/100 ml. The correlations between ACTH and 17-OHP (n equal 61, r equal 0.665, P less than 0.001) and between 17-OHP and testosterone (n = 43, r = 0.761, P less than 0.001) were good, while that between 17-OHP and aldosterone (n = 64, r = 0.512, P less than 0.001) was rather poor. One hour after ACTH injection, the mean level of 17-OHP was significantly increased as compared to the mean basal level [96.8 ng/ml +/- 10.6 (SE) as compared to 67.0 ng/ml +/- 8.1 (SE)]. However, only 12 out of the 48 tests showed a positive response equal to or greater than 100%, and the majority of these responses (10 out of 12) occurred when basal levels of 17-OHP were between 10-70 ng/ml. This suggests that when basal levels fall outside these values, the pituitary-adrenal axis is either too inhibited or too stimulated to react to exogenous ACTH. Of the 48 tests where 17-OHP was measured, 23 had basal level values within these limits, the mean being 40.3 ng/ml. The corresponding mean ACTH level was 99 pg/ml with a wide range (1-230 pg/ml). On the other hand, in prepubertal children who exhibited 17-OHP concentrations between 10-70 ng/ml, testosterone varied from 3-30 ng/100 ml, with a mean of 16.0 ng/100 ml +/- 1.9 (SE) which is not different from the mean level found in normal children [14.0 ng/100 ml +/- 1.3 (SE)]. Thus, under the influence of endogenous ACTH which is moderately increased, 17-OHP concentrations far exceed normal values, whereas plasma testosterone seems to be unaffected.

Studies of ACTH secretion control in 116 cases of Cushing's syndrome.

Plasma ACTH (normal value: 0.16 plus or minus mU/100 ml) was measured in 116 patients with Cushing's syndrome, using a bioassay including dynamic tests and sequential determinations. In 10 patients with adrenal tumors ACTH levels were nondetectable (ND) or low, and usually nonstimulatable. In 10 patients with ectopic ACTH secretion high levels (0.42 plus or minus 0.07 mU/100 ml) were measured. The extracts of 6 tumors yielded an ACTH-like substance. Forty-three patients with Cushing's disease (without pituitary tumor) had, before treatment, a mean ACTH level of 0.18 plus or minus 0.01 mU/100 ml, accompanied by high levels of plasma cortisol (32.1 plus or minus 1.9 mug/100 ml). Irregular nycthemeral variations occurred. ACTH rose to 0.30 mU/100 ml after incomplete adrenalectomy (20 patients) and to 1.14 mU/100 ml after total adrenalectomy (21 patients). Dexamethasone (8 mg per day) suppressed ACTH levels. Metyrapone induced a normal ACTH rise, but at abnormal times. Lysine-vasopressin (LVP) induced an ACTH mean relative increase of 120% before, and of 140% after adrenalectomy (i.e., within the normal range). Six nonadrenalectomized patients with pituitary tumors showed similar abnormalities of ACTH regulation. However, the ACTH rise after LVP was above 500%. When pituitary tumors occurred after adrenalectomy (12 patients) the mean basal ACTH level was 18 mU/100 ml. Dexamethasone induced a 90% decrease, and LVP a 416% increase in ACTH levels. In 6 patients with nodular adrenal hyperplasia, ACTH was undetectable before treatment. After adrenalectomy, ACTH rose to 0.4 mU/100 ml (11 patients) and the increase after LVP was 90%. Five additional patients developed pituitary tumors. These data confirm the abnormalities of ACTH feedback regulation in Cushing's disease. However, even when pituitary tumors occur, ACTH levels can be altered by metyrapone, dexamethasone and LVP. This last test is of particular interest for the detection of pituitary tumors. The follow-up pattern of treated nodular adrenal hyperplasia appears to be very similar to that of Cushing's disease.

Comparison of basal and adrenocorticotropin-stimulated plasma 21-deoxycortisol and 17-hydroxyprogesterone values as biological markers of late-onset adrenal hyperplasia.

Plasma 21-deoxycortisol (21-DOF) and 17-hydroxyprogesterone (17-OHP) concentrations were assayed before (basal) and 1 h after ACTH stimulation in 4 groups of normal subjects (35 follicular phase women, 22 luteal phase women, 33 adult men, and 15 prepubertal children) and in a group of 31 patients with the late-onset form of congenital adrenal hyperplasia (LOCAH) due to 21-hydroxylase deficiency as well as in 31 LOCAH) heterozygotes. The mean basal plasma 21-DOF concentrations in each of the 4 groups of normal subjects were between 8 ng/dL (0.23 nmol/L) and 11 ng/dL (0.31 nmol/L), and they increased significantly after ACTH stimulation to between 36 ng/dL (1.04 nmol/L) and 44 ng/dL (1.27 nmol/L). There were no differences in basal or ACTH-stimulated plasma 21-DOF levels in these 4 groups, whereas their basal and post-ACTH plasma 17-OHP levels did vary. Among the LOCAH patients, 83.8% had basal plasma 21-DOF levels and 61.2% had basal plasma 17-OHP levels higher than the highest basal 21-DOF [30 ng/dL (0.86 nmol/L)] and 17-OHP [450 ng/dL (13.61 nmol/L)] concentrations in the normal subjects, and all individual 21-DOF and 17-OHP levels after ACTH stimulation [greater than or equal to 404 ng/dL (11.67 nmol/L) and greater than or equal to 1040 ng/dL (31.47 nmol/L), respectively] were markedly higher than the highest 21-DOF [76 ng/dL (2.19 nmol/L)] and 17-OHP [580 ng/dL (17.55 nmol/L)] levels in the normal subjects. The mean post-ACTH/basal plasma level ratios among the LOCAH patients were 19.75 for 21-DOF and 8.03 for 17-OHP. In LOCAH heterozygotes, basal 21-DOF values were higher than normal in 48.3%, and post-ACTH values were higher than normal in 93.5% of the cases. In contrast, basal plasma 17-OHP levels were similar in LOCAH heterozygotes and normal subjects, and only 16.1% of the LOCAH heterozygotes had post-ACTH plasma 17-OHP levels higher than the highest normal value. If sex and phase of the menstrual cycle are taken into account, along with the incremental responses (post-ACTH minus baseline value) of plasma 21-DOF and 17-OHP, to compare LOCAH heterozygotes and normal subjects, the discriminating power for detection of heterozygocity was somewhat increased for 21-DOF (to 100%) and appreciably increased for 17-OHP (to 30%).(ABSTRACT TRUNCATED AT 400 WORDS)

Molecular forms of serum insulin-like growth factor (IGF)-binding proteins in man: relationships with growth hormone and IGFs and physiological significance.

Insulin-like growth factor-I (IGF-I) and IGF-II are associated in the blood with specific binding proteins (BPs), forming complexes that elute in gel filtration with estimated mol wt around 40 and 150 kD. The latter appears to be under GH control. Five molecular forms of BP (41.5, 38.5, 34, 30, and 24 kD) have been identified by Western blotting using 125I-labeled IGF. All five forms are present in the smaller complexes, but only the 41.5- and 38.5-kD forms are found in the larger complexes. In this study immunoblotting showed that the 41.5- and 38.5-kD forms were recognized by antibodies directed against the GH-dependent BP purified from human plasma, and the 30-kD form was recognized by antibodies directed against the BP purified from amniotic fluid. The 34- and 24-kD forms proved to be immunologically unrelated to the other three. In sera with large quantities of the 41.5- and 38.5-kD forms, an additional band was often observed immediately ahead of the migration front of the 30 kD band. This was recognized by the anti-GH-dependent BP antibody and probably corresponds to a degradation product of the 41.5- and 38.5-kD BPs. Serum 41.5- and 38.5-kD BPs have been found to be elevated in acromegaly, where GH hypersecretion causes increased IGF-I levels, and diminished in cases of genetic or idiopathic GH deficiency and defects of the GH receptor (Laron's syndrome), where both IGF-I and IGF-II are decreased, as well as in Pygmy adults and children who have isolated IGF-I deficiency. In all of these conditions, the proportions of the 34- and 30-kD forms were inversely related to those of the 41.5- and 38.5-forms. Under treatment, the BP profiles tended to return to normal. In cases of GH deficiency caused by a tumor, the BP profiles resembled those of hypopituitary or normal serum, depending on whether IGF levels were diminished or normal. It, therefore, seems that BP synthesis is coordinated with IGF-I synthesis and may not be directly GH dependent. The results of neutral pH gel filtration analysis of hypopituitary (idiopathic and tumoral) and normal sera point to a relationship between the levels of circulating IGFs and those of the 150-kD IGF-BP complex whose binding units are the 41.5- and 38.5-kD BPs. It, therefore, seems that the 150-kD complex controls the bioavailability of IGF-I and IGF-II.(ABSTRACT TRUNCATED AT 400 WORDS)

Genetic, biochemical, and clinical studies of patients with A328V or R213C mutations in 11betaHSD2 causing apparent mineralocorticoid excess.

Apparent mineralocorticoid excess is a recessively inherited hypertensive syndrome caused by mutations in the 11beta-hydroxysteroid dehydrogenase type 2 gene, which encodes the enzyme normally responsible for converting cortisol to inactive cortisone. Failure to convert cortisol to cortisone in mineralocorticoid-sensitive tissues permits cortisol to bind to and activate mineralocorticoid receptors, causing hypervolemic hypertension. Typically, these patients have increased ratios of cortisol to cortisone and of 5alpha- to 5beta-cortisol metabolites in serum and urine. We have studied 3 patients in 2 families with severe, apparent mineralocorticoid excess and other family members in terms of their genetic, biochemical, and clinical parameters, as well as normal controls. Two brothers were homozygous for an A328V mutation and the third patient was homozygous for an R213C mutation in the 11beta-hydroxysteroid dehydrogenase type 2 gene; both mutations caused a marked reduction in the activity of the encoded enzymes in transfection assays. The steroid profiles of the 7 heterozygotes and 2 other family members studied were completely normal. The results of a novel assay used to distinguish 5alpha- and 5beta-tetrahydrometabolites suggest that 5beta-reductase activity is reduced or inhibited in apparent mineralocorticoid excess. In 1 patient undergoing renal dialysis for chronic renal insufficiency, direct control of salt and water balance completely corrected the hypertension, emphasizing the importance of mineralocorticoid action in this syndrome.

11 beta-Hydroxysteroid dehydrogenase deficit: a rare cause of arterial Hypertension. Diagnosis and therapeutic approach in two young brothers.

We report the clinical history and results of endocrine investigations in two brothers born to consanguineous parents, who presented with hypokalemia and arterial hypertension when they were aged 2 and 6 years. The hormonal serum assay results, including extremely low values for aldosterone and plasma renin activity, favored the existence of apparent mineralocorticoid excess. A diagnosis of 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) deficiency was made, based on assays of the hydrogenated urinary metabolites of cortisol and cortisone, as well as of corticosterone and dehydrocorticosterone. Indeed we found a very low rate of urinary elimination of cortisone metabolites: tetrahydrogenated cortisone was reduced to between 0.10 and 30 mumol/24 h, which is 15-100 times lower than the normal rate; hexahydrogenated cortolones alpha and beta were found to be 7- to 20-fold lower than normal levels; and the 11-keto-17-ketosteroid derivatives of cortisone were also reduced. Urinary elimination of the cortisol-reduced metabolites 5 beta- and 5 alpha-tetrahydrogenated cortisol were slightly reduced or normal. These results argue in favor of a deficit in the enzyme 11 beta-HSD, which oxidizes cortisol into cortisone. A moderate defect in the conversion of cortisol into 5 beta-THF compared to normal conversion into 5 alpha-THF was also found. With respect to corticosterone metabolism, we demonstrated the presence of a defect in the oxidation of that steroid into dehydrocorticosterone, also due to the deficit in 11 beta-HSD. Arterial hypertension and hypokalemia were corrected by treatment with dexamethasone, concomitantly with correction of the low aldosterone and plasma renin activity levels. On the other hand, during this treatment, urinary concentrations of the metabolites of cortisol, cortisone and corticosterone were only moderately affected.

Plasma neurotensin levels in humans: relation to hormone levels in diseases involving the hypothalamo-pituitary-thyroid axis.

This study was aimed to investigate, in humans, the possible relationship between plasma neurotensin (NT) levels and the activity of the hypothalamo-pituitary-thyroid axis. Neurotensin was measured by radioimmunoassay in 14 healthy adult volunteers and in 41 patients among whom 10 were considered as controls and 31 had thyroid dysfunction according to free thyroxine and thyrotropin plasma values. Basal NT levels were not significantly different in healthy adults and in control patients: 9.7 +/- 1.1 fmol/ml (mean +/- SEM) vs 13.3 +/- 2.9 fmol/ml, respectively. In patients with central hypothyroidism the NT level was significantly lower (5.7 +/- 1.2 vs healthy volunteers and controls; p < 0.05) and in patients with peripheral hypothyroidism and hyperthyroidism the NT level was significantly higher (35.9 +/- 12.8 and 29.9 +/- 9.5 fmol/ml, respectively, vs healthy adults (p < 0.01) and vs controls (p < 0.05)). After thyrotropin-releasing hormone (TRH) injection (250 micrograms iv) in nine subjects (two control patients, five patients with hypothyroidism and two patients with hyperthyroidism), NT levels decreased independently of the endocrine status from mean values of 13.4 +/- 8.4 at basal level to 7.3 +/- 0.8 fmol/ml 30 min after injection (p < 0.01 on paired percentage decrease values). These data suggest that plasma NT levels in humans depend upon the pituitary-thyroid status and indicate that TRH could exert a negative regulation on circulating NT levels.

IGFs and their binding proteins.

Insulin-like growth factors (IGFs), initially known as somatomedins, and their specific, high-affinity binding proteins (IGFBPs) are synthesized in most tissues, but principally in the liver. The interest of measuring their circulating levels, which reflect liver production, is to obtain indications as to their endocrine function and regulation. IGF-I plays a pivotal role in post-natal growth. Its half-life is significantly increased by its association with IGFBPs and its serum levels reflect somatotropic status, unlike growth hormone (GH) which has a much shorter half-life and whose secretion comes in pulses. Since investigation of growth retardation must include the most finely tuned appreciation possible of somatotropic secretion, assays of IFG-I and electrophoretic analysis of IGFBP profile can be useful tools, both diagnostically and therapeutically, and can help in determining the need or otherwise for GH treatment, especially in view of the growing demand for such therapy.

The application of a new highly-sensitive radioimmunoassay for plasma 21-deoxycortisol to the detection of steroid-21-hydroxylase deficiency.

21-deoxycortisol (21-DF) is a steroid of strictly adrenal origin formed by the 11-hydroxylation of 17-hydroxyprogesterone. This metabolic pathway is minor in normal subjects, in whom basal plasma concentrations range from 0.03 to 0.63 nmol/L and from 0.865 to 1.50 nmol/L after adrenocorticotrophic hormone (ACTH; Synacthène Immédiat, Ciba/Geigy, France). However, this metabolic pathway becomes major in 21-hydroxylase-deficient patients: in those who have the classical form of congenital adrenal hyperplasia (CAH) basal plasma 21-DF levels can attain more than 144 nmol/L. The synthesis of two isomers, E and Z, of the 21-deoxycortisol-3-carboxymethyloxime (CMO) hapten enabled us to prepare the corresponding E and Z immunogens by coupling them to bovine serum albumin (BSA), as well as the corresponding iodinated E and Z 21-DF-3-CMO-histamine tracers. We developed a very sensitive radioimmunoassay for 21-DF in plasma by associating an anti-21-DF-3-CMO-BSA-E isomer antibody to an iodinated 21-DF histamine-Z isomer (standard curve IC 50 = 8 pg/tube). This plasma 21-DF radioimmunoassay allowed diagnosis of the classical form of CAH in untreated newborn (basal 21-DF levels greater than 144 nmol/L), as well as the late-onset form (post-ACTH 21-DF levels greater than 11.54 nmol/L), and also permitted detection of 21-hydroxylase-deficient heterozygotes of both forms of CAH among the general population (post-ACTH 21-DF levels between 2.02 and 9.52 nmol/L).

Hypogonadotrophic hypogonadism associated with prelingual deafness due to a connexin 26 gene mutation.

In Mediterranean countries, almost half the incidence of non-syndromic congenital hearing loss is caused by mutations in the gap junction (GJ) connexin 26 gene (GJB2/DFNB1 locus). In this form of deafness the cochlear defect is usually isolated. We describe here the first case of hypogonadotrophic hypogonadism in association with this particular cochlear defect. The male patient had moderate deafness inherited from his deaf parents. All family members had a homozygous 35delG mutation in the connexin 26 gene. This mutation accounts for 70% of all connexin 26 gene mutations. The patient was referred to a paediatric endocrinology unit at 11 years of age for moderate growth retardation. Growth rate was normal until 11 years. The patient then presented delayed puberty (testicular volume 4 ml, penis length 4 cm) and did not undergo the usual pubertal growth spurt. LH and FSH secretory responses to GnRH at the age of 14.5 years (bone age 13.5 years), were: LH baseline level 1.1 IU/l, peak 34 IU/l; FSH baseline level 1.8 IU/l, peak 5.7 IU/l. Testosterone concentration was <0.11 ng/ml. From 11 to 14 years old, testosterone concentration ranged from 0.11 to 0.2 ng/ml. Anti-Mullerian hormone (AMH) level was 38.6 ng/ml (normal for Tanner stage I), cortisol 109 ng/ml, and ACTH 37 pg/ml., Karyotype was 46 XY. On MRI analysis, the anterior pituitary and olfactory bulbs were normal. These data were consistent with partial hypogonadotrophic hypogonadism of hypothalamic origin, and the patient was treated with testosterone. This report supports the possible involvement of connexins in puberty initiation. Connexins may play a part in the co-ordination and synchronisation of GnRH release.

Pharmacokinetics of recombinant human insulin-like growth factor I given subcutaneously to healthy volunteers and to patients with growth hormone receptor deficiency.

The pharmacokinetics of recombinant human insulin-like growth factor I (rhIGF-I) were studied in healthy volunteers and in patients with growth hormone receptor deficiency (GHRD; Laron syndrome). Following single subcutaneous injections of rhIGF-I, 40 and 80 micrograms/kg, to healthy volunteers, the peptide was absorbed slowly, with a maximum concentration reached after about 7 hours. Following daily multiple subcutaneous injections of rhIGF-I, 40 micrograms/kg, trough concentrations of IGF-I were increased by 277 +/- 50 micrograms/l (mean +/- SD) from baseline. IGF-I was thus characterized as a low-clearance peptide, with a clearance and half-life estimated at about 0.20 ml/minute/kg and 20 hours, respectively, in healthy volunteers. The volume of distribution was low, about 0.20-0.36 litres/kg, the bioavailability of subcutaneously administered rhIGF-I was 100%, and the rate of production of IGF-I was estimated to be about 50 micrograms/kg/day (3.5 mg/day). Patients with GHRD had low baseline IGF-I concentrations (30-50 micrograms/l) and a much more rapid turnover of IGF-I compared with that in healthy volunteers. The clearance and half-life of IGF-I were estimated to be about 0.60 ml/minute/kg and 6 hours, respectively. The volume of distribution was about the same as in healthy subjects. Due to the rapid turnover of IGF-I, trough IGF-I concentrations were increased to just above baseline during subcutaneous injections of 40 micrograms/kg once daily for 7 days. The maximum increase in IGF-I levels was 111 +/- 12 micrograms/l and 150 +/- 3 micrograms/l following daily subcutaneous injections of 40 x 1 and 40 x 2 micrograms/kg for 7 days, respectively.

[Creutzfeldt-Jakob disease in 4 children treated with growth hormone]. / Maladie de Creutzfeldt-Jakob chez quatre enfants traités par hormone de croissance.

Creutzfeldt-Jakob disease was diagnosed in four growth hormone recipients at the age of 10, 11, 18 and 19 years. To our knowledge, the two first cases are the first instances of Creutzfeldt-Jakob disease recorded in children. Three of them were still being treated with synthetic hormone at the onset of the disease. Neurological disorders: ataxia and diplopia, appeared first, dementia and myoclonus appeared later. Eighteen cases of Creutzfeldt-Jakob disease in growth hormone recipients are now recorded, and the present risk of Creutzfeldt-Jakob disease in pituitary growth recipients is estimated to be 1/300. Because of the long incubation period, new cases are to be feared. Other causes of iatrogenic Creutzfeldt-Jakob disease are reviewed. These facts incite to consider carefully using products of human origin in human therapy. The interactions between growth hormone, prion and host's genomic make-up are still not clear.