W. Nicolau

Low Frequency of CYP21B Deletions in Brazilian Patients with Congenital Adrenal Hyperplasia Due to 21-Hydroxylase Deficiency

The frequency of large mutations was determined in 131 Brazilian patients with different clinical forms of 21-hydroxylase deficiency, belonging to 116 families. DNA samples were examined by Southern blotting hybridization with genomic CYP21 and C4cDNA probes after TaqI and BglII restriction. Large gene conversions were found in 6.6% and CYP21B deletions in 4.4% of the alleles. The breakpoint in these hybrid genes occurred after exon 3 in 92% of the alleles. All rearrangements involving CYP21B gene occurred in the heterozygous form, except in a patient with simple virilizing form who presented homozygous CYP21B deletion. Our data showed that in these Brazilian patients, CYP21B deletions were less frequent than in most of the large series previously reported.

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.

Identification of nonclassical 21-hydroxylase deficiency in girls with precocious pubarche

Recent studies have described mild adrenal enzymatic defects in patients presenting with precocious pubarche. In order to identify these defects we have evaluated basal and ACTH — (25 IU iv) stimulated serum adrenal steroid levels in 19 girls, 2 - to 8.3-year-old, with precocius pubarche (pubic hair Tanner II–III). Two patients had clitorial enlargement. Bone age was moderatly advanced in 10 patients and 2 to 3.7 yr in four others. Four patients had high basal serum levels of 17-hydroxyprogesterone (17OHP) (525+202 ng/dl, mean +SD), compatible with the diagnosis of nonclassical congenital adrenal hyperplasia due to 21-hydroxylase deficiency (NCCAH-21OH), which was confirmed by an increased response of 17OHP to ACTH (3425±953 ng/dl). Fifteen patients had moderatly elevated basal 17OHP levels (56+38 ng/dl) but a normal 17OHP response (191±71 ng/dl) to ACTH, compatible with the diagnosis of idiopathic precocious pubarche (IPP). The Cortisol response to ACTH was normal in both groups. Basal values of DHEA-S were 651 ±256 and 506+462 ng/ml and of DHEA 380±24 ng/dl and 205±102 ng/dl, in NCCAH- 21OH and IPP, respectively. We conclude that: i) clinical findings and baseline levels of DHEA-S and DHEA in IPP can be indistinguishable from the late onset 21 hydroxylase deficiency; ii) baseline levels of 17OHP are sufficient for the diagnosis of NCCAH-21OH; iii) the ACTH stimulation test is indicated only when baseline levels of 17OHP are moderately elevated (100–300 ng/dl).

Effect of glycemic control on growth hormone and IGFBP-1 secretion in patients with type I diabetes mellitus

Growth hormone (GH) secretion disorders have been reported in poorly controlled type I diabetes mellitus patients. Our work was aimed to evaluate GH secretion in 9 type I young diabetes mellitus patients as well as the low molecular weight IGF-binding protein secretion (IGFBP-1) in 5 of them. The patients did not show any signs of malnutrition or neurovascular complications, neither were they on any medication except for insulin. The study protocol included blood samples collection during a 24-h period for measurement of glucose, glycated hemoglobin, GHIGF-I and IGFBP-1 levels under two situations: on poor glycemic control and after 2–3 months on better control through systematic diet, low in carbohydrates and increase in insulin dosage. GH secretion data were analyzed by Cluster algorithm for pulsatility parameters; for rhythm assessment Cosinor method was used. The first study (poor control) reported significant increase of GH maximal and incremental amplitude and duration pulse values, when compared to the second study (better control). Mean 24-h secretion values as well mean GH for interpulse intervals (valleys) decreased, although not statistically significant. The fraction of pulsatile GH/24 h GH did not change significantly with better glycemic control. No changes in pulse frequency were observed. Mean IGF-I concentrations were significantly higher when patients were on better glycemic control. An ultradian variation for GH secretion was noticed in the first study (poor control) and a circadian variation in the second one (better control). IGFBP-1 analysis showed significant decrease of the mean 24-h values under better glycemic control. Linear regression analysis demonstrated a correlation between IGFBP-1 levels and fasting glucose levels. A circadian variation was present in IGFBP-1 secretion, irrespective of glycemic control. Therefore, we concluded that for type I diabetic patients: 1. GH secretion is increased on poor control, through maximal, incremental amplitude and pulse duration values; 2. IGFBP-1 values were significantly reduced and IGF-1 levels significantly higher after better glycemic control; 4. GH ultradian secretion is reported on poor control, and circadian on the better one, 5. IGFBP-1 circadian secretion occurred irrespective of glycemic control.

Characterization of 8 Point Mutations in the Cyp21b Gene in Brazilian Patients With Congenital Adrenal Hyperplasia (Cah) Due To 21-Hydroxylase Deficiency 31

Congenital adrenal hyperplasia due to 21-hydroxylase deficiency (CAH) is an autosomal recessive disorder due to mutations in its structural gene (CYP21B). These mutations can be deletions, large gene conversions or point mutations. We have studied 99 Brazilian patients with different clinical forms of CAH representing 180 non-related chromosomes. Using Southern blot with these 180 alleles we found 16 alleles (9.7%) with large gene conversions and 7 alleles(3.2%) with deletions in the CYP21B gene, showing that deletions are an uncommon cause of the disease in our series. We have studied the frequency of 8 point mutations in the remaining 157 alleles through allele-specific polymerase chain reaction (method and controls kindly provided by Robert Wilson). Table The most frequent mutation in SW form was 12 splice, in SV form was I172N and in LO was V281L, although the latter showed a lower frequency than those found in literature. One allele resulted from a de novo I172N mutation that was found on the paternal haplotype of the patient but not in the father. Paternity was confirmed by multiple polymorphic markers. In the alleles that presented point mutations, 95% had a single mutation and 5% had two, with the following most common associations: 12 Splice+V281L and Q318X+R356W. Mutations were identified in 77% of the 180 alleles, with no identification in 13% of the SW and SV forms and 39% of the alleles in the LO form.