Mutant luteinizing hormone receptors in a compound heterozygous patient with complete Leydig cell hypoplasia: abnormal processing causes signaling deficiency.

Over the past 5 yr several inactivating mutations in the LH receptor gene have been demonstrated to cause Leydig cell hypoplasia, a rare autosomal recessive form of male pseudohermaphroditism. Here, we report the identification of two new LH receptor mutations in a compound heterozygous case of complete Leydig hypoplasia and determine the cause of the signaling deficiency at a molecular level. On the paternal allele of the patient we identified in codon 343 a T to A transversion that changes a conserved cysteine in the hinge region of the receptor to serine (C343S); on the maternal allele a T to C transition causes another conserved cysteine at codon 543 in trans-membrane segment 5 to be altered to arginine (C543R). Both of these mutant receptors are completely devoid of hormone-induced cAMP reporter gene activation. Using Western blotting of expressed LH receptor protein with a hemagglutinin tag, we further show that despite complete absence of total and cell surface hormone binding, protein levels of both mutant LH receptors are only moderately affected. The expression and study of enhanced green fluorescent protein-tagged receptors confirmed this view and further indicated that initial translocation to the endoplasmic reticulum of these mutant receptors is normal. After that, however, translocation is halted or misrouted, and as a result, neither mutant ever reaches the cell surface, and they cannot bind hormone. This lack of processing is also indicated by reduced presence of an 80-kDa protein, the only N-linked glycosylated protein in the LH receptor protein profile. Thus, complete lack of signaling by the identified mutant LH receptors is caused by insufficient processing from the endoplasmic reticulum to the cell surface and results in complete Leydig cell hypoplasia in this patient.

The influence of prenatal dexamethasone treatment on urinary excretion of adrenocortical steroids in newborns.

UNLABELLED: Congenital adrenal hyperplasia due to 21-hydroxylase deficiency suspected in 14 newborns (5 F, 9 M), was treated prenatally with dexamethasone from weeks 7-9 of gestation. The 24 h urinary excretion of selected adrenocortical steroids derived from fetal and definitive adrenal zones was evaluated in these newborns at the age of 3 9 days. Among 11 babies born healthy, in one of six treated until confirmation of male karyotype in gestational weeks 12-17 and in four of five treated until delivery, suppression of fetal adrenal zone steroids was observed, accompanied additionally in three by a diminished excretion of tetrahydrocortisone. In three babies born affected (2 male, 1 female), excretion of 17alpha-hydroxyprogesterone and 21-deoxycortisol metabolites did not differ from 12 affected, age-matched controls, not treated prenatally. However, some influence on suppression of the fetal adrenal zone metabolite 16alpha-hydroxypregnenolone was observed in two newborns treated until delivery. CONCLUSIONS: Heterogeneity in the fetal adrenal response to maternal dexamethasone treatment was confirmed. Suppression of fetal adrenals, especially within the fetal adrenal zone, can be observed in some babies born healthy until at least 1 week after birth.

Luteinizing hormone secreting adrenal tumour as a cause of precocious puberty.

A boy aged 6 years presented with genital precocity, enlarged testes and advanced linear growth. An ovoid mass 3-4 cm in diameter was identified by MRI scan in the right adrenal gland. Serum concentrations of LH, testosterone, alpha-subnuit, dehydroepiandrosterone sulphate, androstenedione and oestradiol were persistently elevated. LH was unresponsive to bolus i.v. injection of GnRH or to GnRH analogue therapy. Serum FSH was normal. After removal of the adrenal tumour, serum LH, alpha-subunit, testosterone and adrenal androgen levels fell to normal. In incubation medium of cultured disaggregated tumour cells, LH concentrations were greater than twice the mean serum concentration and 4-5-fold higher than in the medium of cultured non-neoplastic adrenal cells. Specific immunostaining of the tumour was positive for LH and alpha-subunit in many areas and these were not found in the adjacent non-neoplastic adrenal. Testicular biopsy showed almost complete spermatogenesis although germinal cell types were numerically lower than in normal men. These findings are consistent with an adrenocortical adenoma secreting LH being the cause of the patient's precocious puberty.

Definitive diagnosis of enzymatic deficiencies of steroidogenesis in at-risk newborns and infants by urinary marker analysis using GC/MS-SIM.

A simplified urinary marker analysis for diagnosis of congenital adrenal hyperplasia (CAH) and 5alpha-reductase deficiency in infancy by GC/MS-SIM is introduced. The analysis was performed in 161 patients aged 3-90 days, 99 females and 62 males. CAH due to 21-hydroxylase deficiency was diagnosed in 61 patients (42 females and 19 males; in 10 cases simple virilizing form and in 51 patients salt-wasting form) and CAH induced by 3beta-hydroxysteroid dehydrogenase deficiency without salt loss in 1 female patient. In 2 full-term newborns and 6 preterm infants, a false-positive diagnosis of CAH, which had been based on serum steroid evaluation, was made. In these cases, increased excretion of fetal adrenal zone steroids was confirmed as a possible source of false-positive serum 11-deoxycortisol and 17alpha-hydroxyprogesterone values. Lack of fetal adrenal zone steroid metabolites in 2 male newborns with salt loss symptoms led to the diagnosis of adrenal insufficiency due to X-linked adrenal hypoplasia and adrenal hemorrhage. A single analysis of urinary CAH markers by the very sensitive and selective GC/MS-SIM method can replace numerous assays of various steroids that must be carried out for positive diagnosis of abnormal steroidogenesis in infancy.

[Precocious maturation of the testis associated with excessive secretion of estradiol and testosterone by Leydig cells]. / Przedwczesne dojrzewanie jadra przy nadmiernym wydzielaniu estradiolu i testosteronu przez komórki Leydiga.

We present the quantitative description of spermatogenesis in a 4.5-year-old boy with precocious puberty, where Leydig cell hyperplasia was associated with excessive secretion of testosterone (T), but predominantly with estradiol (E). The results were compared with data obtained from an age-matched group and adult men without hormonal abnormalities. We showed, that excessive secretion of T and E with relative deficiency of FSH is sufficient to induce testicular tubule maturation and qualitatively complete spermatogenesis, although with a poorer quantitative aspect.

Molecular study of the 5 alpha-reductase type 2 gene in three European families with 5 alpha-reductase deficiency.

The molecular basis of 5 alpha-reductase (5 alpha R) deficiency was investigated in four patients from three European families. In the French family, the first patient was raised as a female, and gonadectomy was performed before puberty. The second sibling, also raised as female, differed in that gonadal removal was performed after the onset of pubertal masculinization. The other two patients, both from Polish families, developed masculinization of external genitalia during puberty. All patients developed a female sexual identity. In all cases, no known consanguinity or family history of 5 alpha R deficiency was reported. The genomic DNAs of the patients were sequenced after polymerase chain reaction amplification of the five exons of the 5 alpha R type 2 gene. We found two homozygous mutations responsible for glutamine to arginine and histidine to arginine substitution in families 1 and 3, respectively. In family 2, we found a heterozygous mutation responsible for an asparagine to serine substitution at position 193. The glutamine/arginine 126 mutation in the French family was previously reported in a Creole ethnic group, and the Polish histidine/arginine 231 mutation was previously reported in a patient from Chicago. Moreover, all of the mutations created new restriction sites, which were used to determine the kindred carrier status in the three families. Because 5 alpha R deficiency is known to be a heterogenous disease in terms of clinical and biochemical expression, our data suggest that molecular biology analysis of the type 2 gene could be an essential step in diagnosing 5 alpha R deficiency.

Heterogeneity of urinary steroid profiles in children with adrenocortical tumors.

The excretory patterns of urinary steroids determined by capillary gas chromatography in 11 children (aged 0.8-16.5 years) with adrenocortical tumors were established. In 8 patients the predominant clinical feature was virilization, in 3 others, Cushing's syndrome. In 5 patients (3 carcinoma, 2 adenoma) very high excretion of 3 beta-hydroxy-5-ene steroids was observed. In 2 others (adenomas) only moderately elevated excretion of 11 beta-hydroxyandrosterone was found. In 1 patient (adenoma) pregnanediol dominated in the steroid profile, accompanied by moderately elevated 3 beta-hydroxy-5-ene steroids. Out of 3 Cushingoid patients (1 carcinoma, 2 adenomas), 1 presented an atypical urinary steroid pattern for hypercortisolemia, without 5 alpha-reductase and 11 beta-hydroxysteroid dehydrogenase deficiencies. Neither the urinary steroid pattern nor tumor size alone were reliable indicators of tumor malignancy, as evaluated by a pathological examination and subsequent metastasis-free survival.

Growth hormone-releasing hormone reverses secondary somatotroph unresponsiveness.

Twenty severely GH-deficient prepubertal children aged 10.7 +/- 2.1 yr (mean +/- SD) and with a height SD of -4.92 +/- 1.02 were treated with sc injections of GHRH 1-44 (10 micrograms/kg BW) for 6 months either daily (11 patients) or 3 times/week (nine patients). An acute iv GHRH test (2 micrograms/kg BW) was performed before and after 2 and 6 months of treatment. Mean (+/- SD) peak GH responses to these tests were 2.92 +/- 3.01, 4.57 +/- 4.91, and 7.56 +/- 8.14 micrograms/L, respectively (P less than 0.05, pretreatment vs. 6 months). The mean growth velocity (GV) during treatment was only 2.99 +/- 1.67 cm/yr and only two patients increased their GV by more than 2 cm/yr. A correlation was found between GV during treatment and the peak serum GH response to GHRH acute test before treatment (r = 0.68, P less than 0.005) as well as between GH response to the acute test and patient's bone age (r = -0.46, P less than 0.05). The results indicate that in some severely GHD patients with no response to GHRH even after a 2-month priming period, 6 months of treatment with GHRH can evoke pituitary responsiveness. We speculate that the duration of the GHRH deficiency and its severity plays a role in the ability of somatotrophs to respond to this stimulus.