Pubertal timing after neonatal diethylstilbestrol exposure in female rats: neuroendocrine vs peripheral effects and additive role of prenatal food restriction.

We studied the effects of neonatal exposure to diethylstilbestrol (DES) on pubertal timing in female rats. We examined associated neuroendocrine changes and effects of prenatal food restriction. Age at vaginal opening was advanced after exposure to 10 µg/kg/d of DES and delayed after 1 µg/kg/d (subcutaneous injections). Using this lower dose, pulsatile GnRH secretion was slower at 25 days of age. Both doses reduced KiSS1 mRNA levels at 15 days of age. Using functional Kisspeptin promoter assay, 1 or 10 µM DES reduced or increased KISS1 transcription, respectively. Leptin stimulatory effect on GnRH secretion in vitro (15 days of age) was reduced after prenatal food restriction and neonatal DES exposure (higher dose), both effects being cumulative. Thus, alterations in pubertal timing by DES neonatally are not unequivocally toward precocity, the level of exposure being critical. We provide evidence of neuroendocrine disruption and interaction with prenatal food availability.

KCNJ11 activating mutations cause both transient and permanent neonatal diabetes mellitus in Cypriot patients.

Heterozygous mutations of the KCNJ11 gene encoding the Kir6.2 subunit of the ATP-sensitive potassium channel (K(ATP) channel) of the pancreatic ß-cell cause diabetes in about 30-60% of all permanent neonatal diabetes mellitus cases diagnosed before 6 months of age. The K(ATP) channel plays an essential role in the regulation of the electrical status of the membrane through which the secretion of insulin is activated. Transient neonatal diabetes mellitus due to KCNJ11 mutations is less frequent than abnormalities affecting the imprinted region of chromosome 6q24. We studied the genetic basis of two Cypriot patients who developed diabetes before 6 months of age. They both carried mutations of the KCNJ11 gene. The R201H mutation was identified in a patient who developed hyperglycemia and ketoacidosis at the age of 40 d and was successfully transferred to sulphonylureas which activate the channel through an ATP independent route. The R50Q mutation was identified in a child diagnosed at day 45 after birth with remission of his diabetes at 9 months of age. The same defect was identified both in his asymptomatic mother and the recently diagnosed 'type 2' diabetic maternal grandmother. The remission-relapse mechanism in cases of transient neonatal diabetes is not known. Nevertheless, it is possible that the residue of the mutation within the Kir6.2 molecule is associated with the sensitivity to ATP reflecting to the severity of the diabetic phenotype.

Molecular defects of the CYP21A2 gene in Greek-Cypriot patients with congenital adrenal hyperplasia.

BACKGROUND/AIM: To determine the mutations in the CYP21A2 gene in Greek-Cypriots with congenital adrenal hyperplasia (CAH) and attempt a genotype-phenotype correlation. SUBJECTS AND METHODS: Molecular analysis was performed by multiplex ligation-dependent probe amplification and direct sequencing of PCR products of the CYP21A2 gene in 32 CAH patients. RESULTS: The most frequent genetic defect in the classic salt-wasting and simple virilizing forms was the IVS2-13A/C>G (55%) mutation, followed by Large lesion (20%) and in the non-classical form, the p.V281L (79.5%). Genotypes were categorized in 4 mutation groups (null, A, B and C). All 3 patients in the null group manifested the salt-wasting form and all 6 patients in mutation group A presented with the classical form. One patient in group B had the simple virilizing form and 22 patients in group C exhibited the non-classical form. CONCLUSION: The spectrum of mutations of the CYP21A2 gene in our population is comparable to the most common reported in similar ethnic groups. The knowledge of the ethnic specificity of the CYP21A2 mutations represents a valuable diagnostic tool for all forms of CAH.

Rare mutations in the CYP21A2 gene detected in congenital adrenal hyperplasia.

OBJECTIVES: The purpose of this study was to identify and determine the frequencies of rare CYP21A2 gene mutations in patients with 21-hydroxylase deficiency (21-OHD) in the Cypriot population. DESIGN AND METHODS: Direct sequencing and MLPA analysis of the CYP21A2 gene. RESULTS: A group of families with 21-OHD were screened for the presence of rare CYP21A2 gene mutations. The rare V304M missense mutation was detected as compound heterozygous in two females with the nonclassical (NC) form of congenital adrenal hyperplasia (CAH). The rare F306insT was also detected in a female with severe salt wasting in the homozygous state and in cis in both alleles with the V281L mutation. Lastly, the rare A391T missense mutation was reported in a female patient with NC-CAH. A carrier rate of 2.1% for the V304M was also observed in a cohort of healthy controls. CONCLUSIONS: The frequency of V304M mutation among Cypriots is high and the first reported so far and patients characterized as compound heterozygotes or heterozygotes are most readily identified by a mild phenotype of CAH. Thus, V304M should be included in the panel of mutations associated with the NC forms of 21-OHD.

Gender differences in the prevalence and severity of bone disease in thalassaemia.

BACKGROUND: The pathogenesis of bone disease in thalassaemia major (TM) is multifactorial and remains unclear, although gonadal dysfunction probably has the most dominant role. OBJECTIVE: The aim of the study is to investigate the impact of several factors on the development of reduced bone mass in patients of both sexes with TM, treated in our Center. SUBJECTS AND METHODS: 76 thalassaemic patients (26 males, 50 females) of Greek Cypriot origin with a mean age of 31.4 (17-53) years were included in the study. All patients were on the standard treatment protocol for thalassemia at our Center. Bone mineral density (BMD) of lumbar spine and femoral neck was measured by dual-energy X-ray absorptiometry. Factors known to be associated with low bone mass (gender, endocrine disorders, iron overload) were included in the analysis to ascertain possible associations. Iron overload calculation was based on the mean ferritin level over a 6-year period and T2* MRI of the liver and the heart. Statistical analysis was performed with the SPSS program. RESULTS: Bone disease was present in the spine of 89.5% of the patients and in 84.2% at the femoral site. Male patients were more frequently affected than females (92.3% vs. 88.0% in the spine and 88.5% vs. 82% at the femoral neck). Hypogonadal patients were found to be more frequently affected compared to eugonadal patients (94.1% in spine and 88.2% cyat the femoral neck compared to 89.5% and 81.6% respectively). Males with normal gonadal function were more severely affected in the lumbar spine than eugonadal women (male mean BMD z-score was -3.0 vs. -2.037 in women, p=0.004). Low BMD values were found to be more common in the presence of endocrinopathies. No correlation was found between ferritin status and severity of bone disease. Patients with severe liver iron overload seemed to be more affected in the spine. Heart T2* MRI measurements showed that patients with severe and moderate iron overload in the heart were more affected with bone disease in both the spine and femoral neck. CONCLUSIONS: This study demonstrates a gender difference not only in the prevalence of osteoporosis/osteopenia in patients with TM, but also in the severity of the disorder, as males are more frequently and severely affected than females. Moreover, hypogonadism may have a greater impact on spine BMD in females than in males. The underlying pathogenic mechanisms contributing to the development of bone disease in thalassaemia are multiple and complicated, indicating the necessity of further investigation in order to understand the pathophysiology of this highly prevalent complication. Further research in this field will allow the design of preventive and therapeutic measures.

Effect of bisphosphonate treatment on bone mineral density in patients with thalassaemia major.

BACKGROUND: Bisphosphonates are potent inhibitors of osteoclastic bone resorption and have been recently used in thalassaemia major (TM) osteoporosis with encouraging results. OBJECTIVE: The aim of the study is to investigate the effect of two Bisphosphonate drugs, Alendronate and Pamidronate on bone mass in patients of both genders with TM, treated in our Center. SUBJECTS AND METHODS: 53 (22 males, 31 females) Thalassaemic patients of Greek Cypriot origin were randomly divided into two groups. 29 patients in group A with a mean age of 33, 32 years were treated with alendronate and 24 patients in group B with a mean age of 34, 36 years received pamidronate for a period of 2 years. The effectiveness of both drugs was estimated based on the change of Bone mineral density (BMD) values of lumbar spine and femoral neck. Bone mineral density (BMD) of lumbar spine and femoral neck was measured by dual-energy X-ray absiorptiometry. All patients were on the standard treatment protocol of Thalassaemia. Statistical analysis was performed with the SPSS program. RESULTS: After completion of treatment with pamidronate the mean lumbar spine BMD has improved from -2.813 to -2.174 (p<0.001) and the mean hip BMD from -2.138 to -2.078 (p=0.018). The change of spine BMD in patients who received alendronate was from -2.720 to -2.602 (p=0.059) and the changes in BMD at the femoral neck from -2.035 to -2.007 (p=0.829). CONCLUSIONS: This study demonstrates the efficacy of two bisphosphonate drugs in improving BMD values in patients with TM and osteoporosis. Since the origin of bone disease in TM is multifactorial and some of the underlying pathogenic mechanisms are still unclear, further research in this field is needed, which will allow the design of optimal therapeutic measures.