T Torresani

Isolated Growth Hormone Deficiency Type IA Associated with a 45-Kilobase Gene Deletion within the Human Growth Hormone Gene Cluster in an Italian Family

ABSTRACT: An Italian family with three children presenting with isolated growth hormone (GH) deficiency type IA is described. Restriction endonuclease analysis revealed that the cause of hGH deficiency was a 45-kb gene deletion within the hGH-chorionic somatomammotropin (CS) gene cluster, encompassing the GH-1, CS-L, CS-A, and GH-2 genes. DNA sequence analysis and polymerase chain reaction amplification between two sequences located on each side of the deletion breakpoint accurately identified the deletion breakpoints and indicated that the regulatory sequences located upstream from the TATA box of the mutant CS-B belong to the GH-2 gene. Two of the affected children developed high-titer anti-hGH antibodies after recombinant hGH treatment with secondary growth arrest, whereas the third one maintained normal growth in the presence of very low-titer antibodies. This is the first report of a large deletional mutation within the hGH-CS gene cluster accompanied by phenotypic heterogeneity in terms of growth response and antibody formation in the different patients.

TREATMENT OF PRECOCIOUS PUBERTY IN GIRLS WITH INTRANASAL LHRH AGONIST (BUSERELIN)

Ten girls with idiopathic precocious puberty were treated for 1 - 3 years with Buserelin (B) intranasally at a daily dose of 200 - 900 ug. The data are compared to those obtained previously in 21 untreated and in. 13 girls with precocious puberty treated with cyproterone acetate (Pediat Res 8.248-256, 1974). B was clinically effective in suppressing menses in all but one girl who had low gonadotropins at onset. Breast size did not increase in 8, but pubic and axillary hair development progressed in 8 patients. Peak LH (170.3 ± 39.5 vs 99.1 ± 24.9 ug/1) and FSH (451 ± 73.4 vs 164.8 ± 29.1 ug/1) after LHRH were markedly suppressed during the first months of treatment and remained low thereafter. Basal LH increased intially (18.6 ± 3.8 vs 71.1 ± 17.0 ug/1) and persistently while basal FSH remain unchanged. Prl. E1. E2 levels were unaffected. DHEA gradually increased as seen in normal girls. Differences in height SDS at the beginning (1.72 ± 0.44). at 1 yr (2.01 ± 0.43) and at 2 years of treatment (1.95 ± 0.53) were minimal as were corresponding SDS for bone age (3.27 ± 0.53; 4.15 ± 0.54; 4.19 ± 0.53) and for height prediction related to target height (-1.20 ± 0.61; -1.40 ± 0.48; -173 ± 0.39). The data of statural and skeletal development of B-treated girls are not significantly different from those of untreated girls nor from those treated with cyproterone acetate.Supported by Swiss National Science Foundation grant 3.406.083.

Thyrotropin enzyme-immunoassay in dried blood spots: a spectrophotometric method for neonatal thyroid screening

We describe an enzyme-immunoassay with photometric endpoint determination, suitable for the measurement of thyrotropin (TSH) in dried blood spotted on filter paper. Using reagents of a commercially available test kit provided for the measurement of thyrotropin in 200 microliter serum, we have adapted the method to the determination of thyrotropin in blood spots containing ca. 10 microliter blood. This was achieved by prolongation of the assay time from 3 to 20 hours, and by increasing the amount of enzyme-antibody complex. Precision and sensitivity of the blood spot assay are comparable to those of our in-house thyrotropin RIA, and the RIA/EIA correlation coefficient is 0.987 (n = 150). The advantages of EIA are the simplicity of the photometric end point determination (although strict time control has to be observed in order to avoid drifts in results), the long stability of reagents, and the non-isotopic label. The method therefore appears to be a suitable alternative to thyrotropin RIA for the determination of thyrotropin in neonatal thyroid screening.

SHORT- AND LONG-TERM EFFECTS OF GH IN PATIENTS WITH NORMAL GROWTH DESPITE GH-DEFICIENCY AFTER REMOVAL OF CRANIOPHARYNGIOMA

Removal of craniopharyngioma usually results in panhypopituitarism. Nevertheless, a number of children grow normally or even excessively after extirpation of the tumor despite the proven lack of GH. These children did not undergo GH therapy, We studied the effects of short- and long-term administration of GH on growth and metabolism in 6 patients under regular hormonal replacement therapy. During the short-term GH 15N retention was not significantly stimulated (115.4 ± 9.6 % of basal balance, mean ± SEM) and not different from controls. In contrast, 15N retention was 210.3 ± 20.7 % in children with GH-deficiency of other causes. Long-term administration of GH (2 U/m2 s.c. per day during 12 months) did not influence growth velocity, but increased calf circumference and decreased body mass index and skin fold thickness in prepubertal patients. General well-being and strength improved impressively. IGF-I, IGFBP-3 and the 150 kD IGFBP-complex were decreased before and restored to normal during GH treatment. The reverse was observed for the 50 kD IGFBP-complex. Thus, growth velocity in these patients is not related to an2y of the usual indicators of the growth status and remains unexplained. Although GH therapy does not affect growth, it has other beneficial effects and is recommended for this group of patients with normal growth velocity in the absence of GH.

36 GROWTH HORMONE RESPONSE TO A STANOARDISED EXERCISE TEST RELATED TO PUBERTY IN SHORT, TALL AND NORMAL CHILDREN

To investigate the growth hormone (GH) response in children of varying stature to a physiological stimulus, we measured GH (mU/1) before and 10 min after a standardised bicycle exercise test (15 min) in 34 short (Group I), 14 tall (Group II) and 26 children of normal height (Group III). Mean height SDS (±SD) was different; (Group I -2.7 ±0.55; Group II +2.86 ±0.67; Group III -0.26 ±0.82, p < 0.0005). Mean (±SD) chronological age (yr) was similar (Group I 12.4 ±3.4; Group II 13.6 ±2.0; Group III 13.4 ±3.0), mean (±SD) bone age (BA, yr) however was delayed in Group I (10.4 ±3.7) compared with Group II(14.1 ±1.3) and Group III (13.4 ±3.1) p < 0.0005. Results are expressed as mean ±SEM. Pre-ex. GH was similar (Group I 6.5 ±1.9; Group II 8.9 ±2.6; Group III 9.0 ±2.5). In contrast post-ex. GH was higher in tall (36.6 ±5.5) compared with short (16.6 ±3.1) and normal children (21.2 ±3.5), p < 0.005. However, combining the three groups revealed a high post-ex. GH in relation to the pubertal growth spurt independent of height; GH at BA <10 yrs 13.1 ±3.0; BA >10 yrs 25.5 ±2.8, p < 0.005: GH at P3P4, 34.7 ±4.5; at P1P2 15.2 ±2.4 and at P5 20.7 ±6.3, p < 0.0005). Raised post exercise GH in tall children appears to reflect a normal change in secretion of GH at puberty and this change is also present in short children.Supported by: Smith and Nephew Foundation and Swiss National Science Foundation, Grant No. 3.906.0.83.

Is a decreased LH response to LHRH in prepubertal boys with cryptorchidism predictive of permanent gonadotropin deficiency|[quest]|

Among more than 300 prepubertal boys with uni- or bilateral cryptorchidism, 15 with low LH response to LHRH (peak LH ug/l LER 907: 30.1 ± 7.8 SD) could be followed up to the age of 12 - 16.5 years and had at least 2 further LHRH tests (25 ug/m2 i.v.). At that age, all had signs of spontaneous puberty (testes ⋜3 ml and/or pubes Tanner stage ⋜2); peak LH values were 102.3 ± 40.7 SD (difference first/last LH peak p <0.001 in 14 boys). In 10 of them the rise of LH response appeared at pubes 2 - 3, in 4 at pubes 4 - 5. In one boy with testes of 20 ml and pubes 5 at the age of 14.4 years, LH peak values remained <40, comparable to patients with persistent gonadotropin deficiency. Before puberty, peak FSH values were low in half of these patients; during puberty, 13 boys had normal and 2 increased FSH values.Our results show that LH deficiency - suggested to be a causative factor of cryptorchidism - is present in this group of patients. However, it seems to be of transient nature as demonstrated by the rise of LH response and the occurance of spontaneous puberty.Supported by the Swiss National Foundation Grant No. 3.984.080

In vitro micro-bioassay of plasma LH suitable for serial determinations in children

By modification of an in vitro rat bioassay (1), we have developed a micro-method which allows simultaneous incubation of 12 unknown samples each at 3 dose levels, using 1-10 μl of unextracted plasma. Testes of 8 to 10 weeks old rats are digested with collagenase; the separated interstitial cells are suspended in Medium 199 and incubated under O2/CO2 (95/5%) at 37°C for 3 hours. Testosterone (T) is measured by an 21 hours-RIA using 125-iodine labelled tracer. T production after incubation with 25 ng of the standard pituitary extract hLH LER 907 is about 8 times the basal value produced in the absence of gonadotropins (range of standard curve 0.5 - 25 ngLH; sensitivity 0.5 ngLH; between-assay variation at 25 ng level: 10%). LH values in human plasma determined with our bioassay are higher than RIA values. Our results show that Bio/RIA ratios vary at different RIA LH levels, the lowest so far measured being 2.0. The high bioassay values associated with low immunological LH values observed in some of our patients may help to clarify unexplained discrepancies between RIA results and clinical findings.(1) DUFAU et al, J.Clin Endocrinol Metab 42:958, 1976Supported by the Swiss National Science Foundation Grant No. 3.901.077

Studies in clomiphene (C) treated pubertal boys with gynecomastia (G)

Plasma LH and FSH, basal (b) and after LHRH (p), testosterone (T), estradiol (E2), estrone (E1), androstenedione (Δ4A) and prolactin(PRL) were determined in 5 pubertal boys with G before, during and after 50 mg C daily for 56 days (d). Results (mean, SEM):In conclusion, C increases gonadotropins and testicular steroids. In spite of higher E1 and E2, G is reduced in most patients. This may be due to a higher T/E2 ratio or interference of C with E2 at the receptor site. Supported by the Swiss National Science Foundation (Grants No. 3.883.077 and 3.901.077).

67: PLASMA PROLACTIN (PRL) BEFORE AND AFTER TRH IN 23 CHILDREN WITH CRANIOPHARYNGIOMA

PRL has been determined before and after TRH IV (200 ug/m2) in 23 children (11 ♀/12 ♂) with craniopharyngioma using reagents provided by the NIAMDD, Bethesda USA. As controls served 10 endocrinologically healthy children (4♀/6♂), aged 4-16 yrs, who had TRH tests for other reasons. 13 patients, aged 1-13 yrs, have been studied before surgery, 10 of them and 10 additional patients, aged 1-20 yrs, after surgery.Assuming that PRL secretion is controlled by PIF (PRL inhibiting factor), elevated PRL levels as seen before and after surgery would indicate hypothalamic disorder or disruption of the hypothalamo-pituitary axis. PRL deficiency, observed only after surgery, would reflect pituitary damage.

EFFECT OF α-MSH ON PLASMA GROWTH HORMONE (GH), TSH AND CORTISOL IN CHILDREN

The effect of α-MSH was studied in 19 prepubertal children (mean age 6.85 yrs) with small stature. The tests have been performed in order to rule out GH deficiency.α-MSH was injected intravenously (3 mg during 1–2 min.). Blood samples were taken at 0, 15, 30, 45 and 60 min. In addition, all children had arginine and insulin tolerance tests. GH and TSH were determined by RIA, cortisol by protein binding assay.Results : α-MSH was well tolerated, no untoward or stress reactions could be observed. A normal GH response to α-MSH was seen only in 2 out of 8 children with constitutional small stature and otherwise normal GH secretion. 3 of 11 children suffering from hypopituitarism with documented GH and other hormonal deficiencies, unexpectedly, showed a small but significant rise of GH after α-MSH; all 3 had craniopharyngeoma.α-MSH led to a clear cut increase of cortisol in all but 3 patients with secondary adrenal insufficiency requiring steroid replacement therapy. In each single child, the rise of cortisol after α-MSH and after insulin was of equal size suggesting a common mechanism of action. However, hypoglycemia and stress, as seen in the insulin tolerance test, has not been observed after α-MSH.There was no effect of α-MSH on plasma TSH.