Characterization of two novel homozygous missense mutations involving codon 6 and 259 of type II 3beta-hydroxysteroid dehydrogenase (3betaHSD) gene causing, respectively, nonsalt-wasting and salt-wasting 3betaHSD deficiency disorder.

We identified two homozygous missense mutations in the human type II 3beta-hydroxysteroid dehydrogenase (3/betaHSD) gene, the first in codon 6 of exon II [CTT (Leu) to TTT (Phe)] in a male infant with hyperpigmented scrotum and hypospadias, raised as a male and no apparent salt-wasting since neonatal age, and the second in codon 259 of exon IV [ACG (Thr) to ATG (Met)] in a male pseudohermaphrodite with labial scrotal folds, microphallus, chordee, and fourth degree hypospadias, raised as a female and with salt-wasting disorder since neonatal age. In vitro transient expression of mutant type II 3betaHSD complementary DNAs of L6F, T259M, as well as T259R for comparison was examined by a site-directed mutagenesis and transfection of construct into COS-1 and COS-7 cells. Northern blot analysis revealed expression of similar amounts of type II 3betaHSD messenger ribonucleic acid from the COS-1 cells transfected by L6F, T259M, T259R, and wild-type (WT) complementary DNAs. Western immunoblot analysis revealed a similar amount of L6F mutant protein compared to WT enzyme from COS-1 cells, but neither L6F from COS-7 cells nor T259M or T259R mutant protein in COS-1 or COS-7 cells was detectable. Enzyme activity in intact COS-1 cells using 1 micromol/L pregnenolone as substrate in the medium after 6 h revealed relative conversion rates of pregnenolone to progesterone of 46% by WT enzyme, 22% by L6F enzyme, and 8% by T259M enzyme and less than 4% activity by T259R enzyme. Using 1 micromol/L dehydroepiandrosterone as substrate, the relative conversion rate of dehydroepiandrosterone to androstenedione after 6 was 89% by WT enzyme, 35% by L6F enzyme, 5.1% by T259M enzyme and no activity by T259R enzyme. However, the L6F mutant 3betaHSD activity, despite its demonstration in the intact cells, was not detected in homogenates of COS-1 cells or in immunoblots of COS-7 cells, suggestive of the relatively unstable nature of this protein in vitro, possibly attributable to the decreased 3betaHSD activity. In the case of T259M and T259R mutations, consistently undetectable proteins in both COS cells despite detectable messenger ribonucleic acids indicate severely labile proteins resulting in either no or very little enzyme activity, and these data further substantiate the deleterious effect of a structural change in this predicted putative steroid-binding domain of the gene. In conclusion, the findings of the in vitro study of mutant type II 3betaHSD enzyme activities correlated with a less severe clinical phenotype of nonsalt-wasting and a lesser degree of genital ambiguity in the patient with homozygous L6F mutation compared to a more severe clinical phenotype of salt-wasting and severe degree of genital ambiguity in the patient with homozygous T259M mutation in the gene.

Effects of growth hormone and testosterone on cortical bone formation and bone density in aged orchiectomized rats.

Osteoporosis in men is a disease that is increasing in incidence, and with an increasing elderly population it poses a serious health problem. Since both testosterone (T) and growth hormone (GH) have an anabolic effect on bone and both decrease with aging, we were prompted to test whether the administration of these hormones in combination would increase bone mass in orchiectomized (orx) senile rats more than administration of either agent alone. Twenty-month-old male Wistar rats were divided into five groups with seven animals each: (a) age-matched intact control, (b) orx, (c) orx+GH (2.5 mg/kg/day), (d) orx+T [10 mg/kg, subcutaneous (s.c.), injection given twice a week], and (e) orx+GH+T. Testosterone and GH were given subcutaneously for 4 weeks. Bone histomorphometry of the tibial shaft showed that the orx group had lower cortical bone area than the intact control group. The decrease in cortical bone area was due to increased intracortical porosis as well as decreased periosteal bone formation rate (BFR). Administration of T to the orx animals prevented the development of the porosis and the decrease in periosteal BFR. The bone mineral content (BMC) and bone mineral density (BMD) of the femur as tested by dual-energy X-ray absorptiometry were significantly higher in the orx+T than in the orx group and were not significantly different from that of the intact control group. Administration of GH to the orx rats increased periosteal BFR significantly; however, the BMC and BMD measured were not increased significantly in comparison to the orx group. When GH and T were combined in treatment, the cortical bone area, periosteal BFR, and femoral BMD were all significantly higher than that of the orx and even higher than the intact control rats. Two-way analysis of variance shows that the individual effect of GH and T treatment on the periosteal BFR and cortical bone area was significant. The effect of T, but not GH, on femoral BMC and BMD was also significant; however, there is no synergistic interaction between the two treatments. Four weeks of orx with or without GH or T administration had no significant effect on tibial metaphyseal cancellous bone volume. In conclusion, this short-term study suggests that the combined intervention of GH and T in androgen-deficient aged male rats may have an independent effect in preventing osteopenia. The significant effect of GH+T may be attributed to the prevention of intracortical porosis, and an increase in periosteal bone formation and cortical bone mass.

Macrotestes associated with hyperprolactinemia.

Prolactin secreting pituitary adenomas are a rare finding in prepubertal children /1/. As in adults, their incidence is higher in girls than in boys; however, the macroadenomas are predominant in boys /20-16/. Two prepubertal boys who presented with short stature and linear growth deceleration were diagnosed to have prolactin secreting pituitary macroadenoma associated with growth hormone (GH) deficiency. They were treated with bromocryptine and exogenous recombinant hGH. They achieved a normal adult stature, full sexual maturation and tumor regression on the therapy. In addition, both boys developed macrotestes. Further evaluation ruled out other etiologies for macrotestes. We presume that the elevated prolactin caused local testicular growth factors to induce testicular cell division and/or hypertrophy resulting in an increased testicular volume.

Effect of finasteride on human testicular steroidogenesis.

We studied the testicular function and some androgen-mediated events in 22 males (16-30 years of age) with male pattern baldness that was treated with finasteride (10 mg once daily) for 2 years. Patients were evaluated every 3 months. Prostatic volume was determined in six subjects by endorectal ultrasound scans. Serum gonadotropin, prostate-specific antigen (PSA), and sex hormone levels were determined basally and periodically during the treatment period. Fourteen subjects underwent gonadal stimulation with human chorionic gonadotropin (hCG), and the gonadotropin response to gonadotropin releasing hormone (GnRH) was determined in eight subjects, prior to and after 2 years of therapy. Finasteride treatment resulted in an improvement in the male pattern baldness and prostatic shrinkage that was associated with an increase in serum testosterone levels (17.2 +/- 2.5 vs. 26.3 +/- 1.7 nmol/L) and a decrease in dihydrotestosterone (DHT) levels (1.45 +/- 0.41 vs. 0.38 +/- 0.10 nmol/L), causing a marked increase in that testosterone/DHT ratio. A significant increase in the serum levels of androstenedione (3.67 +/- 0.49 vs. 7.05 +/- 0.70 nmol/L) and estradiol (132 +/- 44 vs. 187 +/- 26 pmol/L) was also noted, whereas androstanediol glucoronide (33.3 +/- 6.4 vs. 10.7 +/- 4.5 pmol) and PSA (1.6 +/- 0.6 vs. 0.4 +/- 0.1 ng/ml) were significantly decreased. No changes in basal or stimulated levels of gonadotropin were observed. There was a significant increase in the testosterone response to hCG during finasteride therapy (delta: 16.7 vs. 35.5 nmol/L) that could be explained, at least in part, by the reduction of testosterone metabolism resulting from the blockage induced by finasteride. The decrease in the androstenedione to testosterone and estrone to estradiol ratios observed after hCG treatment, however, strongly suggests increased activity of the 17-ketosteroid reductase enzyme and an improvement of the testicular capacity for testosterone production.

Growth hormone secretion and effects of growth hormone therapy on growth velocity and weight gain in children with Prader-Willi syndrome.

Obesity, short stature, decreased growth rate and delayed skeletal maturation are common features of children with Prader-Willi syndrome (PWS). In contrast to PWS, children with simple exogenous obesity have normal or increased growth rate and normal or advanced skeletal maturation. Decreased growth hormone (GH) secretion evaluated by pharmacological or physiological testing associated with increased plasma insulin-like growth factor (IGF-I) and GH-binding protein (GH-BP) levels are also characteristic of simple obesity. In order to understand whether the suboptimal GH secretion in PWS is an artifact of the obesity, we studied 33 obese and 11 non-obese PWS children, aged 2-16 years.GH secretion was evaluated with three pharmacological stimuli (insulin, clonidine and L-dopa) and by spontaneous 24-hour GH secretion. Skeletal maturation was delayed in 70% whereas plasma IGF-I and GH-BP were either low or normal. Forty subjects, including ten non-obese children, had GH deficiency by standard testing (failure to respond to two pharmacological stimuli), and all but one had blunted spontaneous 24-h GH secretion. No significant correlation between body mass index (wt/ht2) and spontaneous 24-h GH secretion (r = 0.145), p > 0.06) or GH-BP levels (r = 0.19, p > 0.07) was found. Thirty documented GH deficient children have completed at least two years of GH therapy. With treatment the overall mean height SD and weight SD changed from -2.2 to -0.8 and from 3.5 to 2.4 respectively (p < 0.0001). No patient has developed diabetes mellitus. In conclusion, growth velocity, skeletal maturation, GH secretion and GH dependent proteins in PWS resemble GH deficiency more than simple obesity. Our ongoing study suggests that GH deficiency in PWS is not an artifact of obesity. Although it is unlikely that GH deficiency is the only cause of decreased growth velocity and increased adiposity in PWS, it is a common feature and significant contributory factor. Long term observation will be required until achievement of adult height to determine whether GH therapy actually improves final height.

Characterization of zona glomerulosa function in patients with classic and non-classic forms of congenital adrenal hyperplasia due to 11 beta-hydroxylase deficiency.

The function of the adrenal zona glomerulosa was studied in 18 patients with 11-hydroxylase deficiency confirmed by elevated plasma levels of 11-deoxycortisol. Patients were divided into two groups. Group I (4 males, 7 females; aged 1.2-2.8 yrs) had symptoms at birth or shortly after (classic form), and Group II (4 males, 3 females; aged 7.3-20.1 yrs) had their first clinical manifestation during childhood (non-classic form). To study zona glomerulosa function, patients were given dexamethasone p.o. 2 mg/m2/day x6 days, thus suppressing the zona fasciculata. Six hours after the last dose of dexamethasone, the zona glomerulosa was stimulated by i.v. administration of furosemide 1.0 mg/kg as a single dose. Blood was drawn 2 h later. In the untreated state, all patients had striking elevation of ACTH (Group I: 1,070 +/- 380 pg/ml; Group II: 764 +/- 180 pg/ml), 11-deoxycortisol (Group I: 63,000 +/- 22,000 ng/dl; Group II: 17,200 +/- 5,200 ng/dl) and deoxycorticosterone (Group I: 1,100 +/- 67 ng/dl; Group II: 499 +/- 27 ng%) while plasma renin activity (< 0.5 ng/ml/h in both groups) and aldosterone (Group I: 3.0 +/- 1.8 ng/dl; Group II: 2.3 +/- 1.8 ng/dl) were markedly suppressed. After the administration of furosemide 4 patients in Group I were unable to increase aldosterone (2.8 +/- 0.9 ng/dl) secretion in spite of marked elevation of plasma renin activity (28 +/- 7 ng/ml/h), suggesting an impairment of 11-hydroxylase in the zona glomerulosa.(ABSTRACT TRUNCATED AT 250 WORDS)

Mediastinal teratoma and precocious puberty in a boy with mosaic Klinefelter syndrome.

We describe a boy who developed precocious puberty resulting from chorionic gonadotropin produced by a mediastinal germ cell tumor. Following tumor removal he began spontaneous precocious sexual development which was treated and then arrested spontaneously. Investigation of this arrested puberty established that he had Klinefelter syndrome (KS) mosaicism. He represents the first instance of KS mosaicism reported with a mediastinal germ cell tumor, a neoplasm commonly reported in males with a 47,XXY karyotype. We recommend that all males with KS and early sexual development or with "normal" testicular growth be screened with measurement of germ cell tumor markers including beta-subunit of human chorionic gonadotropin and alpha-fetoprotein.

Male hypogonadism with gynecomastia caused by late-onset deficiency of testicular 17-ketosteroid reductase.

BACKGROUND: 17-Ketosteroid reductase deficiency results in male pseudohermaphroditism because conversion of the weak androgen androstenedione to the more potent androgen testosterone is impaired. If a late-onset form exists, hypogonadism and gynecomastia caused by decreased testosterone production and increased estrogen production, respectively, would be expected as the major clinical manifestations in men. METHODS: We studied 48 male subjects, ranging from 14 to 26 years of age, who had idiopathic pubertal gynecomastia. Serum concentrations of gonadal and adrenal steroid hormones were measured before and after the administration of corticotropin and after the combined administration of chorionic gonadotropin and dexamethasone for three days. RESULTS: We identified three unrelated subjects (ages, 16, 17, and 26 years) with results indicative of a partial deficiency of testicular 17-ketosteroid reductase. The three subjects had gynecomastia as well as decreased libido and impotence. Their mean (+/- SD) base-line serum androstenedione and estrone concentrations were elevated as compared with the levels in the 45 subjects without this enzyme deficiency (androstenedione, 380 +/- 70 vs. 110 +/- 70 ng per deciliter [13 +/- 2 vs. 4 +/- 2 nmol per liter]; estrone, 138 +/- 12 vs. 46 +/- 9 pg per milliliter [511 +/- 44 vs. 170 +/- 33 pmol per liter]). After the administration of chorionic gonadotropin, the mean serum androstenedione concentration in these three subjects was 910 +/- 48 ng per deciliter (32 +/- 2 nmol per liter) and the mean serum estrone concentration was 260 +/- 16 pg per milliliter (962 +/- 59 pmol per liter). The mean serum testosterone concentration at base line was 210 +/- 80 ng per deciliter (7.4 +/- 2.8 nmol per liter) in the 3 subjects, as compared with a value of 410 +/- 12 ng per deciliter (14.4 +/- 0.42 nmol per liter) in the 45 other subjects, and it did not increase in response to the administration of chorionic gonadotropin. The concentrations of androstenedione and estrone in spermatic venous serum were 19 times higher and 73 times higher, respectively, than in normal men. The serum concentrations of follicle-stimulating hormone and luteinizing hormone in these three subjects were inappropriately low, suggesting the presence of hypogonadotropic hypogonadism. CONCLUSIONS: A late-onset form of testicular 17-ketosteroid reductase deficiency can cause gynecomastia and hypogonadism in men.

Elevated serum estradiol associated with increased androstenedione-testosterone ratio in adolescent males with varicocele and gynecomastia.

OBJECTIVE: To investigate the testicular function in adolescents with pubertal gynecomastia associated with varicocele before and after varicocelectomy. DESIGN AND PATIENTS: We have studied six male adolescents 15 to 19 years of age with bilateral gynecomastia. They were selected among other adolescents with gynecomastia because of the presence of visible varicoceles. All of them had normal physical examination and secondary sexual characteristics. This was prospective study of 3 months' duration. All the patients that were included finished the study. SETTING: All the patients were evaluated in the Endocrine Clinic of Winthrop-University Hospital, a tertiary care unit. INTERVENTION: Serum testosterone (T), androstenedione (A), and estradiol (E2) responses to the administration of human chorionic gonadotropin (hCG) 2,000 IU for 3 consecutive days before and 3 months after varicocelectomy were determined. RESULTS: Varicocelectomy did not cause any significant changes in the basal (pre-hCG) levels of the steroid. However, the increase in T levels achieved with hCG was significantly (P less than 0.005) higher after varicocelectomy (before T, 925 +/- 212 ng%; after T, 1,649 +/- 406 ng%). Simultaneously, the stimulated levels of E2 and A were significantly lower (P less than 0.005) after varicocelectomy (E2, 62 +/- 12 pg/mL; A, 326 ng% +/- 80 ng%) than before (E2, 106 +/- 13 pg/mL; A, 580 ng% +/- 95 ng%). CONCLUSION: The reciprocal effect on the levels of T and its immediate precursor, A, suggests an impairment of the 17-ketoreductase enzyme activity. The increased levels of E2 after hCG and its normalization after varicocelectomy suggests that varicoceles may play a pathogenetic role in the development of gynecomastia.

Leydig cell function in adolescent boys with varicoceles.

The incidence of varicoceles in adolescent boys ranges from 5% to 19.5%. We studied five adolescent boys aged 17 to 20 years with visible left-sided varicoceles. All of them had public hair and testicular volumes between 20 to 25 mL and had achieved stage V of pubertal development. Serum gonadotropin response to the intravenous administration of 100 micrograms of gonadotropin-releasing hormone (GnRH) and testosterone response to the administration of 2,000 IU human chorionic gonadotropin (hCG) daily for 3 days before and 3 months after varicocelectomy were measured. Basal levels of both gonadotropins were in the pubertal range, and there was no significant difference between serum levels before and after varicocelectomy. Both gonadotropins, however, showed increased responses to the administration of GnRH (luteinizing hormone [LH]: basal, 12.0 +/- 5.1 mIU/mL; peak, 105.0 +/- 36.0 mIU/mL; follicle-stimulating hormone [FSH]: basal, 11.6 +/- 4.2 mIU/mL, peak, 60.0 +/- 18.0 mIU/ml) that decreased after varicocelectomy (LH: basal, 14.3 +/- 6.0 mIU/mL; peak, 58.6 +/- 12.0 mIU/mL; FSH: basal, 6.8 +/- 4.6 mIU/mL; peak, 38.0 +/- 8.1 mIU/mL). Serum testosterone response to hCG was also significantly improved by varicocelectomy (testosterone peak: before, 780 +/- 210 ng/dL; after, 1850 +/- 170 ng/dL). Testicular biopsy specimens showed no histologic abnormalities and normal spermatogenesis. Endocrine evaluation in adolescent boys with varicoceles could detect an early Leydig cell dysfunction that could be corrected by varicocelectomy.

Failure of sodium benzoate to alleviate plasma and liver ammonia in rats.

The intraperitoneal administration of L-norvaline and L-methionine-SR-sulfoximine to rats caused an increase in the concentration of ammonia in plasma as well as in liver. These compounds interfere with urea and glutamine formation, respectively. Subsequent injection of sodium benzoate failed to alleviate ammonia levels, and on the contrary, caused a further increase. Sodium benzoate itself, when administered, resulted in higher levels of ammonia in plasma and liver of the rats. Administration of glycine to rats treated with benzoate did not lower ammonia levels indicating that other factors besides glycine may also be necessary for the removal of sodium benzoate.

Hypothalamic-pituitary gonadal axis in boys with primary hypothyroidism and macroorchidism.

Nine of 15 boys with severe long-standing primary hypothyroidism were found to have macroorchidism. All 15 patients had elevated thyroid-stimulating hormone levels. However, only those patients with testicular enlargement had striking elevations of serum prolactin and gonadotropin values. The response to gonadotropin-releasing hormone in our patients was blunted, in contradistinction to that of children with true precocious puberty. In spite of the elevated levels of luteinizing hormone, the serum testosterone levels were in the prepubertal range, explaining the lack of peripheral manifestations of androgenic effect. Improvement of testosterone secretion followed decreasing prolactin levels with bromocriptine administration, suggesting an inhibitory effect of prolactin on luteinizing hormone action at the Leydig cell. We conclude that testicular enlargement is the result of continuous follicle-stimulating hormone stimulation and that the term "true precocious puberty" is not appropriate in children with hypothyroidism and macroorchidism unless the hypothalamic-pituitary gonadal axis is shown to be at the pubertal stage.

Effect of prolonged clonidine administration on growth hormone concentrations and rate of linear growth in children with constitutional growth delay.

Sixteen prepubertal children with constitutional growth delay (10 boys and six girls, mean age 7.2 +/- 2.1 years) were administered a daily dose of clonidine (0.15 mg/m2) for a period of 1 year. Growth hormone levels, plasma somatomedin C, and linear growth rate were significantly increased at the end of the treatment. Six of the children maintained the higher growth rate even 6 months after treatment. These and other studies suggest that prolonged stimulation of the hypothalamus by clonidine may ameliorate the impairment of growth hormone release seen in some children with constitutional growth delay. Because of the low cost and the convenience of the oral route, administration of clonidine could be a mode of treatment in some children with poor growth.

Hair zinc, scalp hair quantity, and diaper rash in normal infants.

Hair zinc concentration was determined by atomic absorption spectrophotometry in 308 normal newborn infants and 199 normal infants aged one to twelve months. Hair zinc concentration declined from 204 micrograms/gm at birth to 112 micrograms/gm at age eight months, and then rose to 144 micrograms/gm at age twelve months. Diaper rash was significantly associated with reduced hair zinc, and infants with the least hair had lower zinc levels than infants with the most hair. The data indicate that hair loss and diaper rash found in normal infants is significantly associated with a reduction in hair zinc concentration.

The effects of dietary zinc deficiency on voluntary alcohol drinking in rats.

The effects of dietary zinc deficiency on voluntary alcohol drinking in rats were examined in three separate experiments. Zinc-deficient rats showed a significantly greater voluntary alcohol intake as compared with the pair-fed controls. In the first experiment there were 14 male Sprague-Dawley rats with 7 controls and 7 who were begun on a zinc-deficient diet (below 2 ppm) at body weight 58-81 g. In the second experiment with 11 male Sprague-Dawley rats, the same diet was begun at 103-107 g. In the last experiment, 11 rats were begun at 120-199 g. Hair zinc measurements documented lower levels in the zinc-deficient rats after 6 weeks on the diet. The data indicate that a zinc-deficient state did increase voluntary alcohol intake in rats. In each experiment, the per cent alcohol consumed declined when a normal diet was given after the 6th week, and the difference was significant in experiments 1 and 3.

Endocrine abnormalities in a patient with partial trisomy 4q.

Partial trisomy of the long arm of chromosome 4, usually resulting from a familial segregation of a balanced translocation, has been described in a number of patients. This report describes the genetic and endocrine findings in a 16 year old 46,XY,12q+ mentally retarded male. The banding pattern of the extra chromatin material from this de novo unbalanced translocation shows that the distal segment of the long arm of chromosome 4 is involved. Comparison of the clinical features in this patient with cases of partial trisomy 4q previously reported support the cytogenetic evidence for this translocation involving the distal portion of 4q. Endocrine data suggested an end-organ resistance, characterised by extreme hyperinsulinaemia, primary hypothyroidism, and hypergonadotrophic hypogonadism associated with no signs of autoimmunity. To our knowledge, no endocrine evaluation has been previously reported in patients with partial trisomy 4q.

Growth hormone inhibition causes increased selenium levels in Duchenne muscular dystrophy: a possible new approach to therapy.

Nine children with Duchenne muscular dystrophy were given Sanorex (mazindol), a growth hormone inhibitor, daily for 6 months. There was no significant change in their muscle function, but there was a significant reduction in weight gain and in levels of growth hormone, somatomedin C, hair zinc, serum zinc, and serum LDH. Selenium and glutathione peroxidase in the serum increased significantly. Thirteen other children with growth hormone deficiency had a significant reduction in hair selenium following growth hormone administration. These results show a significant relationship between growth hormone and selenium nutritional status and confirm our previous reports indicating an effect of growth hormone on zinc nutritional status. It is possible that prolonged therapy with a growth hormone inhibitor would attenuate the course and improve the longevity of patients with muscular dystrophy.