Marcelo Cidade Batista

No evidence of the inactivating mutation (C566T) in the follicle-stimulating hormone receptor gene in Brazilian women with premature ovarian failure

OBJECTIVEnTo investigate the presence of FSH receptor gene mutations in women with premature ovarian failure (POF).nnnDESIGNnClinical and molecular studies.nnnSETTINGnResearch laboratory in a university setting.nnnPATIENT(S)nFifteen 46,XX women with POF and 42 normal fertile controls.nnnINTERVENTION(S)nExon 7 was amplified and digested with BsmI to screen for the previously described inactivating mutation C566T. Exon 10 was screened for mutations by denaturing gradient gel electrophoresis and direct sequencing.nnnMAIN OUTCOME MEASURE(S)nPolymerase chain reaction followed by restriction enzyme analysis, denaturing gradient gel electrophoresis, and direct sequencing.nnnRESULT(S)nNo inactivating mutations were identified in exons 7 and 10 of the FSH receptor gene in women with familial or sporadic POF. Exon 10 had two polymorphisms, G919A and G2039A, whose allelic frequencies were 46.7% and 56.6%, respectively, in women with POF. The allelic frequency of both polymorphisms was 59.5% in normal fertile controls.nnnCONCLUSION(S)nNo inactivating mutations in exons 7 and 10 of the FSH receptor gene were identified in Brazilian women with POF. A high frequency of two polymorphisms that are in linkage disequilibrium was found in exon 10 of this gene.

GH Values after Clonidine Stimulation Measured by Immunofluorometric Assay in Normal Prepubertal Children and GH-Deficient Patients

Objective: To establish the cut-off values of GH measured by immunofluorometric assay, a more sensitive and specific assay, in normal prepubertal children and compare their values with those of proven GH-deficient patients. Methods: 30 normal children (20 males) and 26 patients with known causes of GH deficiency were submitted to the clonidine test and their GH values were compared. A powdered clonidine tablet (0.1 mg/m2) was given orally and blood samples for GH measurements were drawn at times –30, 0, 60, 90 and 120 min. Results: GH peak values presented a wide variation ranging from 1.7 to 25 µg/l (mean ± SD = 12.87 ± 5.8 µg/l) in the normal group. The cut-off values for the 5th and 10th percentiles of the distribution curve were 3.3 and 5.5 µg/l, respectively. In the GH deficiency group, maximum GH levels after clonidine stimulation ranged from <0.1 to 2.1 µg/l (0.56 ± 0.58 µg/l). Conclusions: The cut-off values obtained with the immunofluorometric method are lower than the ones obtained by radioimmunoassay. We suggest a cut-off value of 3.3 µg/l (5th percentile) that ensures 100% of sensitivity along with 93% of specificity to exclude the diagnosis of GH deficiency when using this immunofluorometric method.

Male pseudohermaphroditism due to nonsalt-losing 3β-hydroxysteroid dehydrogenase deficiency: Gender role change and absence of gynecomastia at puberty

Adrenal and gonadal functions were evaluated on two adult cousins with male pseudohermaphroditism due to congenital 3 beta-hydroxysteroid dehydrogenase deficiency (3 beta-HSD) without clinical salt-losing. Both patients had been reared as females since birth. Case 1 presented at age 17 with perineal hypospadias virilization without gynecomastia and a female to male gender role change at puberty. Case 2 had previously undergone bilateral orchidectomy in childhood and presented primary amenorrhea, absence of virilization and a female gender role at the age of 24. In the basal state, as well as after ACTH and hCG stimulation, 3 beta-hydroxy-5-ene-steroid levels were disproportionately elevated, resulting in abnormal 3 beta-hydroxy-5-ene: 3-oxi-4-ene steroids ratios. Normal basal serum cortisol with inadequate cortisol response to ACTH was observed in both patients. Elevated basal plasma renin activity (PRA) and normal basal serum aldosterone (ALDO) were present in both subjects. After ACTH stimulation serum ALDO rose adequately in Case 1 but subnormally in Case 2. Salt restriction resulted in an increase in serum ALDO and no salt loss in Case 1 whereas in Case 2 the substantial rise in PRA and serum ALDO were unable to prevent slight urinary sodium loss. Case 1 had normal basal serum testosterone with subnormal response to hCG stimulation. Incubation of testicular tissue in vitro with [3H]DHEA resulted in large Androstenediol production but diminished testosterone conversion confirming the 3 beta-HSD deficiency in the testes. We conclude that (1) absence of gynecomastia and a female to male gender role change may be observed in the male pubertal presentation of nonsalt-losing 3 beta-HSD deficiency and (2) the different functional behavior of zona glomerulosa in our patients suggests the presence of variable degrees of 3 beta-HSD deficiency in the zona glomerulosa of the nonsalt-losing form.

Menstrual disorders and infertility caused by inactivating mutations of the luteinizing hormone receptor gene

OBJECTIVEnTo review clinical findings, hormone levels, and DNA analyses in genetic males and females with inactivating mutations of the LH receptor gene.nnnDESIGNnReview of reported cases.nnnSETTINGnA university hospital.nnnPATIENT(S)nGenetic males and females with inactivating mutations of the LH receptor gene.nnnRESULT(S)nThe clinical presentation in genetic males ranged from female genitalia to male genitalia with micropenis caused by Leydig cell hypoplasia. Genetic females presented with amenorrhea or oligomenorrhea, enlarged cystic ovaries, and infertility. Both males and females had elevated LH levels and LH/FSH ratios. Sequencing of genomic DNA revealed homozygous or compound heterozygous deletions, nonsense mutations, or missense mutations in the LH receptor gene.nnnCONCLUSION(S)nThis study of patients with inactivating mutations of the LH receptor indicates that in genetic males, the action of hCG and LH is necessary for the normal development of primary and secondary sexual characteristics. In contrast, secondary sexual characteristics develop in genetic females in the absence of LH action, but they fail to ovulate.

Spironolactone-reversible rickets associated with 11β-hydroxysteroid dehydrogenase deficiency syndrome

A 7-year-old girl had growth retardation, hypertension, and hypokalemic alkalosis. Baseline serum aldosterone concentration and plasma renin activity were low and unresponsive to sodium deprivation and to orthostatic changes. Baseline serum progesterone, 17-hydroxyprogesterone, 11-deoxycortisol, and cortisol levels were normal and adequately responsive to ACTH stimulation. No steroid was found abnormally elevated. A diagnosis of 11 beta-hydroxysteroid dehydrogenase deficiency was established on the basis of elevated urinary tetrahydrocortisol plus allotetrahydrocortisol/tetrahydrocortisone ratio, determined by gas chromatography-mass spectrometry. Evaluation of bone mineral metabolism and parathyroid function, and skeletal radiographs, revealed the presence of rickets and secondary hyperparathyroidism. Treatment with spironolactone alone for 2 months corrected hypertension, hypokalemic alkalosis, and all laboratory and radiologic evidence of rickets and hyperparathyroidism, resulting in acceleration of growth rate. The response to spironolactone suggests that a hypermineralocorticoid state is responsible for the hypertensive syndrome and that rickets and hyperparathyroidism could be a consequence of excess mineralocorticoid activity.

Mutation analysis of the follicle-stimulating hormone receptor gene in girls with gonadotropin-independent precocious puberty resulting from autonomous cystic ovaries.

OBJECTIVEnTo search for germline activating mutations of the FSH receptor in girls with gonadotropin-independent precocious puberty.nnnDESIGNnMolecular studies in human tissue.nnnSETTINGnFour girls with polycystic ovaries and gonadotropin-independent isosexual precocious puberty without clinical and molecular features of McCune-Albright syndrome.nnnINTERVENTION(S)nPeripheral blood was used for DNA extraction. The alpha-subunit of the Gs gene and the entire exon 10 of FSH receptor gene were amplified by polymerase chain reaction (PCR). Gs-alpha mutations characteristic of McCune-Albright syndrome were excluded by denaturating gradient gel electrophoresis (DGGE) and allele-specific PCR. Exon 10 of the FSH receptor gene was analyzed by DGGE and direct sequencing.nnnMAIN OUTCOME MEASURE(S)nResults of DGGE and direct sequencing.nnnRESULT(S)nNo germline activating mutations were detected in exon 10 of our patients. Instead, two previously described polymorphisms were found, leading to the substitution of alanine for threonine at position 307 and of serine for asparagine at position 680 of the FSH receptor molecule.nnnCONCLUSION(S)nGermline activating mutations were not found in exon 10 of the FSHR gene in any of our patients. Further studies, preferably in ovarian tissue, will be required to exclude the presence of somatic activating mutations of the FSH receptor in these patients.

Prepubertal male pseudohermaphroditism due to 17-ketosteroid reductase deficiency: diagnostic value of a hCG test and lack of HLA association.

Most patients with male pseudohermaphroditism (MPH) due to 17-ketosteroid reductase (17-KSR) deficiency were diagnosed at or after puberty when significant virilization occurred. We report 2 prepubertal sibs (Case 1, 4 yr and Case 2, 10 yr) unambiguously raised as females, with clitoral enlargement, separate urethral and vaginal orifices and gonads palpable at the inguinal canal bilaterally. Basal serum LH, FSH, 17-hydroxyprogesterone, testosterone (T), Dihydrotestosterone and dehydroepiandrosterone (DHEA) were normal for age. †4-Androstenedione (†4-A) was slightly elevated in Case 2 but nondiagnostic. Steroid measurements after human chorionic gonadotropin (hCG) stimulation were compared with those of boys with male external genitalia submitted to the same hCG protocol: peak T was subnormal (Case 1, 80, Case 2, 91, vs normal 329 ± 129 ng/dl, mean ± 1SD), peak †4-A elevated (Case 1, 477, Case 2,264, vs normal 44 ± 26 ng/dl) resulting in an abnormally elevated †4-A/T ratio (Case 1, 6.0, Case 2,2.9, vs normal 0.12 ± 0.09) and establishing the diagnosis of 17-KSR deficiency. This diagnosis was confirmed in vitro by minimal T production when testicular tissue of both patients was incubated with tritiated †4-A. The 2 sibs did not share a single haplotype for the HLA complex indicating lack of association between HLA and the locus of the gene for 17-KSR. In conclusion, in 2 sibs with MPH the subnormal T and elevated †4-A response to the hCG test indicated the diagnosis of 17-KSR deficiency followed by orchiectomy to avoid later virilization at puberty.

Validation of an immunoassay for anti-Müllerian hormone measurements and reference intervals in healthy Brazilian subjects

Background Anti-Müllerian hormone is marker of ovarian and testicular reserve. The clinical use of this hormone requires proper standardization of reference intervals. The aims of this study were to validate the Anti-Müllerian hormone Gen II immunoassay, to establish Anti-Müllerian hormone reference intervals in healthy subjects, and to evaluate the influence of hormonal contraceptives, smoking, and body mass index on Anti-Müllerian hormone. Methods The validation of the Anti-Müllerian hormone Gen II assay (Beckman Coulter Company, TX, USA) was performed using a simplified protocol recommended by Clinical Laboratory Standard Institute. One-hundred and thirty-three healthy females and 120 males were prospectively selected for this study. Results The analytical and functional sensitivities of the Anti-Müllerian hormone Gen II immunoassay were 0.02 and 0.2u2009ng/mL, respectively. Intra-assay coefficients ranged from 5.2 to 9.0%, whereas inter-assay precision ranged from 4.6 to 7.8% at different concentrations. In females, Anti-Müllerian hormone showed progressive decline with increasing age (ru2009=u2009−0.4, pu2009<u20090.001), whereas in males, age showed no influence on Anti-Müllerian hormone concentrations. In females, Anti-Müllerian hormone concentrations did not differ between users and non-users of hormonal contraceptives, smokers, and non-smokers and obese and lean individuals. However, there was a negative and significant correlation between Anti-Müllerian hormone and body mass index in males (ru2009=u2009−0.3, pu2009=u20090.008). Conclusions Anti-Müllerian hormone Gen II assay was reliable for determining serum Anti-Müllerian hormone concentrations. Anti-Müllerian hormone concentrations declined with aging and presented a wide inter-individual variability. The lack of influence of hormonal contraceptives, smoking, and obesity on Anti-Müllerian hormone in both sexes allowed us to refine the normative concentrations for the Brazilian population.

Definition of reference ranges for β-isomerized carboxy-terminal telopeptide collagen type I for children and adolescents

Abstract Background: Bone metabolism involves many complex pathways that are disturbed by several bone diseases. The literature shows some limitations concerning pediatric reference intervals to bone markers, mainly because of the low number of patients included in the studies, the heterogeneity of methods, beyond the fact that it is time-consuming and expensive. The aim of this study was to determine reference values for β-isomerized carboxy-terminal telopeptides collagen type I (β-CTX), a marker of bone resorption, for children and adolescents. Methods: Blood samples from 246 patients were collected and β-CTX was measured using an electrochemiluminescence immunoassay (ECLI). Results and conclusions: We propose reference ranges for β-CTX concentration from the 2.5 percentile and 97.5 percentile for each age group. The reference values obtained, concerning children and adolescents, might be useful in the evaluation of diseases such as osteosarcoma and anorexia in both childhood as adolescence.

Clinical and Molecular Features of Testicular and Ovarian Resistance To Luteinizing Hormone 14

Clinical and Molecular Features of Testicular and Ovarian Resistance To Luteinizing Hormone 14