MRI findings and genotype analysis in patients with childhood onset growth hormone deficiency--correlation with severity of hypopituitarism.

AIM: To evaluate the relationship between pituitary size, PIT1 and PROP1 genotype, and the severity of childhood onset growth hormone deficiency (coGHD). PATIENTS: Forty-four patients with coGHD (34 M; 9.7 +/- 4.1 years): severe isolated (SI) GHD (n = 14); partial isolated (PI) GHD (n=13); multiple pituitary hormone deficiencies (MPHD) (n=17). RESULTS: Pituitary abnormalities were found in 7/14 patients with SIGHD (50%), 16/17 patients with MPHD (94.1%), and no patient with PIGHD. Mean pituitary height (PHT SDS) was significantly lower in MPHD than in SIGHD and PIGHD. Pituitary height SDS and pituitary volume (PV) SDS correlated with IGF-I SDS and stimulated GH peaks in the SIGHD and MPHD groups. No PIT1 mutation was identified. The PROP1 AG deletion (301-302) was present in five related patients with MPHD and more severe phenotype than the other patients with MPHD. CONCLUSIONS: Pituitary abnormalities corresponded to the severity of coGHD. Genetic alterations were identified in five related patients with MPHD.

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.

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.

Heterozygous nonsense mutation in exon 3 of the growth hormone receptor (GHR) in severe GH insensitivity (Laron syndrome) and the issue of the origin and function of the GHRd3 isoform.

Mutations in the GH receptor gene (GHR) cause congenital GH insensitivity, a genetic disorder characterized by severe growth retardation associated with high serum concentration of GH and low serum levels of IGF-I. Molecular defects have been identified in all GHR-coding exons, except exon 3, a sequence that encodes part of the extracellular domain of the receptor. In humans, GHR transcripts exist in two isoforms differing by the retention (GHRfl) or exclusion (GHRd3) of this particular exon. As shown recently, such a dimorphic expression pattern, of unknown significance, could result from a retrovirus-mediated deletion event involving exon 3. This model for the generation of those two isoforms, however, leaves open the possibility that GHRd3 transcripts also arise from GHRfl alleles through alternative splicing. Here we report the identification of the first mutation in exon 3 of the GHR (W16X) in a patient with GH insensitivity and who also carries another nonsense mutation in exon 4. Intrafamilial correlation analyses of genotypes (presence of normal or mutant GHRfl and/or GHRd3 alleles), GHR expression patterns, and phenotypes provided direct evidence against an alternative splicing of exon 3. In particular, this exon was retained into transcripts originating from the GHRfl-W16X allele in both the patient and his mother. These observations, given the normal phenotype of the heterozygous parents, revealed also that a single copy of either GHRfl or GHRd3 is sufficient for normal growth.

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.

Mucosal cytology in the determination of the pubertal status in girls.

AIM: To assess and validate the diagnostic value of buccal and vaginal smears in the ambulatory care of girls with potential disorders of puberty. METHODS: Smears were obtained from 77 girls who presented for assessment of their pubertal status, stained as described by Papanicolaou, examined for signs of estrogenization according to the method of Schmitt, and compared with the clinical status. RESULTS: Vaginal but not buccal smears reflect accurately the changes of sexual maturation, even more sensitive than single serum hormone measurements. CONCLUSIONS: Vaginal smears represent a valid, reproducible, and well-tolerated diagnostic tool in the ambulatory care of peripubertal girls to identify their estrogen status with high sensitivity and specificity. The decision to perform confirmative but invasive diagnostic procedures can be based on auxology, physical examination, bone age determination, and analysis of vaginal smears.

Conception and spontaneous delivery after total hypophysectomy.

OBJECTIVE: To report the first case of pregnancy and spontaneous delivery after total hypophysectomy. DESIGN: A case report. SETTING: University hospital. PATIENT(S): A 34-year-old woman with panhypopituitarism after hypophysectomy in childhood for craniopharyngioma. INTERVENTION(S): Successful fertility treatment followed by sufficient hormone substitution therapy during pregnancy. MAIN OUTCOME MEASURE(S): Clinical variables. RESULT(S): The patient had an uncomplicated spontaneous delivery without any substitution of oxytocin before, during, or after delivery. CONCLUSION(S): Normal pregnancy and delivery are possible in women with lack of pituitary function. Maternal pituitary oxytocin release seems to play a limited role in the onset and progression of labor.

Deletions of the homeobox gene SHOX (short stature homeobox) are an important cause of growth failure in children with short stature.

Short stature, with an incidence of 3 in 100, is a fairly frequent disorder in children. Idiopathic short stature refers to patients who are short due to various unknown reasons. Mutations of a human homeobox gene, SHOX (short stature homeobox), have recently been shown to be associated with the short stature phenotype in patients with Turner syndrome and most patients with Léri-Weill dyschondrosteosis. This study addresses the question of the incidence and type of SHOX mutations in patients with short stature. We analyzed the SHOX gene for intragenic mutations by single strand conformation polymorphism, followed by sequencing, in 750 patients and for complete gene deletions by fluorescence in situ hybridization in 150 patients (total, 900 patients). This is the largest group of patients with short stature studied to date for SHOX mutations. All patients had a normal karyotype, and their height for chronological age were below the third percentile or minus 2 SD of national height standards. All were without obvious skeletal features reminiscent of the Leri-Weill syndrome at the time of diagnosis. Silent, missense, and nonsense mutations and a small deletion in the coding region of SHOX were identified in 9 of the 750 patients analyzed for intragenic mutations. Complete gene deletions were detected in 3 of the 150 patients studied for gene deletions. At least 3 of the 9 intragenic mutations were judged to be functional based upon the genotype- phenotype relationship for the parents and normal control individuals. We conclude that SHOX mutations have been detected in 2.4% of children with short stature. The spectrum of SHOX mutations is biased, with the vast majority leading to complete gene deletions. The prevalence of short stature due to SHOX gene mutations among children with short stature appears to be similar to that of GH deficiency or Turner syndrome. Family studies of the children with SHOX mutations often reveal older family members with same mutation who exhibit mild skeletal features reminiscent of the Turner syndrome, such as high-arched palate, short neck, abnormal auricular development, cubitus valgus, genu valgum, short fourth metacarpals, and Madelung deformity.