21-Hydroxylase and 11beta-hydroxylase mutations in Romanian patients with classic congenital adrenal hyperplasia.

CONTEXT: Congenital adrenal hyperplasia (CAH) comprises autosomal recessive disorders mainly due to defects in the 21-hydroxylase (CYP21) gene. OBJECTIVE: The study aimed to perform molecular characterization in 43 Romanian patients with classical CAH forms diagnosed at the Center for Genetic Diseases of the Pediatric Clinic/University Cluj (38 with 21-hydroxylase deficiency, five with 11beta-hydroxylase deficiency), to determine the frequency of mutations in the CYP21A2 gene and attempt a genotype-phenotype correlation in patients with 21-hydroxylase deficiency. DESIGN: Molecular analysis was performed by direct sequencing of PCR amplified products of the CYP21A2 and CYP11B1 genes. RESULTS: The most frequent mutation in Romanian patients with 21-hydroxylase deficiency was I2G (43.9%), followed by deletions and large conversions (16.7%), I172N and the triple mutation (P30L+I2G+del8bp), accounting for 12.1% each, P30L (7.6%) and R356W (1.5%). Genotypes were categorized in three mutation groups (0, A, and B), according to their predicted functional consequences, and compared with clinical phenotype. Positive predictive values were 100, 75, and 100% for groups 0, A, and B, respectively. Overall genotype-phenotype correlation was 87.88%. In the five patients with 11beta-hydroxylase deficiency, the following homozygous mutations were identified: T318R in two related patients; R448H in two unrelated patients; and P94L, a new, yet-undescribed mutation. CONCLUSION: The present study is the first countrywide report of mutational analysis in a Romanian patient population with 21-hydroxylase deficiency. Molecular diagnosis was performed in a small number of CAH patients proved not to suffer from 21-hydroxylase deficiency but from 11beta-hydroxylase deficiency, and a new mutation was identified.

Three new 46,XX male patients: a clinical, cytogenetic and molecular analysis.

BACKGROUND: XX males range phenotypically from completely masculinised individuals to true hermaphrodites and include a subset of SRY negative patients. The correlation between genotype (SRY+/-) and phenotype is still unclear. AIM: To report three new patients with this rare condition, one of whom was diagnosed prenatally and another was SRY negative, and to verify in our patients whether the presence of SRY results in a more masculinised phenotype. PATIENTS AND METHODS: We present two phenotypically normal XX male patients (10 and 13.5 years) and one 3.1 years old XX male with ambiguous external male genitalia Prader IV. The patients were diagnosed by clinical, hormonal, sonographic, genetic and histological criteria. RESULTS: Basal hormonal status was normal for phenotype but an excessive response to GnRH testing was noticed in the second patient together with insufficient hCG stimulation in all three patients. Pelvic ultrasound displayed male structures without Müllerian ducts; testicular biopsy, performed only in the intersex patient, showed Sertoli and Leydig cell hypoplasia. Chromosome analysis confirmed 46,XX karyotype. FISH analysis and molecular analysis by PCR were positive for Yp fragments/SRY gene on Xp in two patients and negative in the patient with ambiguous external genitalia. CONCLUSIONS: In our observation Y chromosome-specific material containing the SRY gene translocated to the X chromosome results in a completely masculinised phenotype. In the intersex patient, incomplete masculinisation without SRY suggests a mutation of one or more downstream non-Y testis-determining genes.

Mutational analysis and genotype-phenotype correlation in patients with classic 21-hydroxylase deficiency from transylvania (north-west Romania).

The regional incidence of 21-hydroxylase deficiency, its mutational spectrum and the correlation of genotype and phenotype has been studied by European, American and Latin-American groups. However, little information is known about the molecular background of the disease in patients from Central-Eastern Europe. The present study aimed to genotype a group of patients from Transylvania, the north-western part of Romania, in order to gain some insight into the molecular pattern and the genotype-phenotype correlation of congenital adrenal hyperplasia (CAH) in this region. We genotyped 17 patients with classic 21-hydroxylase deficiency and, whenever available, their parents in order to verify mutational segregation. The patients came from 13 unrelated families. DNA was prepared from peripheral blood leucocytes and four gene fragments were amplified by PCR. The 21-hydroxylase gene (CYP21B) was completely sequenced and analyzed for point mutations or deletions. Percentage distribution of mutations was as follows: 12G--34.6%, deletions and large conversions (del)--19.2%, P30L--15.4%, 1172N--15.4%, P30L+I2G+del8bp (triple mutation)--11.5%, and R356W--3.8%. Mutational percentage distribution compared to other Latin populations is higher for I2G and I172N and lower for deletions, while the P30L mutation was found at a higher rate than in any other analyzed population. Some differences may arise from the low patient number and from ethnic particularities. The incidence of compound heterozygotes in our group was 76.5%. The genotype seemed to correlate fairly well to phenotype, with a general concordance rate of 82.35%. Clear divergence was found in two patients with the simple virilizing form, exhibiting a homozygous status for I2G and del, respectively. This study offers the first information about the molecular pathology of CAH in Romania and should help to improve management and clinical outcome for patients with CAH in Transylvania. Hopefully, it might also be the first step towards exact and accurate prenatal diagnosis in our country.

Results of interferon treatment in children with chronic hepatitis B.

Many observations report a variable therapeutical response to interferon in children with chronic hepatitis B. In order to evaluate the efficiency of alpha-interferon treatment in the downregulation of viral replication and in the eradication of infection in these patients, we assessed HBeAg/HBeAb and HBsAg/HBsAb seroconversion (as well as with clinical outcome and the changes in the plasma level of aminotransferases) in 61 treated patients. The diagnosis was established by means of the usual clinical, biochemical and histopathological criteria. There was no possibility to viral DNA test and no control group was included. Patients were selected for interferon treatment who displayed at least a two fold rise in the plasma level of aminotransferases as compared to normal values, as well as necroinflammatory activity (score > or = 6) and positive HBeAg as a marker of viral replication. Treatment was carried out with alpha-2a interferon or alpha-2b interferon in a dose of 3 million U/m2/dose in 3 weekly doses for a period of 4-6 months. The monitoring interval was 6.6+/-3 years. HBeAg/HBeAb seroconversion was registered in 77.2% of the patients and mainly occurred during the first year of follow-up (50.9 %). HBsAg/HBsAb seroconversion was revealed in 1.75% of the cases. The therapeutical response was complete, incomplete, transient and absent in 1.75%, 64.9%, 10.5% and 22.8% of the patients, respectively. The results show that the eradication of HBV infection is insignificant, but the downregulation of viral replication and, subsequently the halt of further progression of hepatic lesions is obtained in a high percentage of cases, highlighting the efficiency of this treatment in children with chronic hepatitis B

Growth analysis in patients with 21-hydroxylase deficiency influence of glucocorticoid dosage, age at diagnosis, phenotype and genotype on growth and height outcome.

OBJECTIVE: To evaluate the impact of hydrocortisone dosage, age at diagnosis, compliance, genotype and phenotype on growth and height outcome in 21-hydroxylase-deficient patients. METHODS: We analyzed 37 patients with 21-hydroxylase deficiency (17 had completed growth, 20 still growing). Final (FH)/predicted final height (pFH) and loss of height potential related to target height (TH) were calculated and the impact of 4 hydrocortisone (HC) dosage regimens on height outcome and growth velocities was evaluated. Mean FH SDS and pFH SDS were analyzed in accordance to age at diagnosis, compliance, genotype and phenotype. RESULTS: Mean (FH SDS, pFH SDS) was -1.8+/-1.06 SD, with 35.1% of all 37 patients exhibiting short stature. Doses >20 mg/m2/day during the first year and >15 mg/m2/day during age 1-5 and at puberty resulted in significantly lower FH SDS, pFH SDS and greater height losses. Age at diagnosis, compliance, genotype and phenotype played only a minor role in growth development. CONCLUSIONS: Hydrocortisone substitution in 21-hydroxylase-deficient patients should be kept at the lowest efficient level, if possible <20 during the first year and <15 mg/m2/day until age 5 and during puberty. Normal growth and not complete androgen suppression should be aimed for.