Molecular genetic study of congenital adrenal hyperplasia in Serbia: novel p.Leu129Pro and p.Ser165Pro CYP21A2 gene mutations.

PURPOSE: Congenital adrenal hyperplasia (CAH) is an autosomal recessive disease characterized by impaired adrenal steroidogenesis and most often caused by CYP21A2 gene mutations. For the first time, we reported complete spectrum and frequency of CYP21A2 gene mutations in 61 unrelated patients with classical and non-classical CAH from Serbia. METHODS: Direct DNA sequencing of whole CYP21A2 gene and polymerase chain reaction with sequence-specific primers for detection of CYP21A1P/CYP21A2 chimeras were combined. RESULTS: We identified 18 different pathogenic alleles-two of them novel. Mutation detection rate was highest in patients with salt-wasting form of CAH (94.7%). The most prevalent mutation was intron 2 splice site mutation, c.290-13A/C>G (18.5%). Other mutation frequencies were: CYP21A1P/CYP21A2 chimeras (13%), p.P30L (13%), p.R356W (11.1%), p.G110fs (7.4%), p.Q318X (4.6%), p.V281L (4.6%), p.I172N (2.8%), p.L307fs (2.8%), p.P453S (1.9%), etc. Mainly, frequencies were similar to those in Slavic populations and bordering countries. However, we found 6.5% of alleles with multiple mutations, frequently including p.P453S. Effects of novel mutations, c.386T>C (p.Leu129Pro) and c.493T>C (p.Ser165Pro), were characterized in silico as deleterious. The effect of well-known mutations on Serbian patients' phenotype was as expected. CONCLUSIONS: The first comprehensive molecular genetic study of Serbian CAH patients revealed two novel CYP21A2 mutations. This study will enable genetic counseling in our population and contribute to better understanding of molecular landscape of CAH in Europe.

Type 2 diabetes mellitus and impaired glucose regulation in overweight and obese children and adolescents living in Serbia.

An increase in the prevalence of pediatric type 2 diabetes mellitus (T2DM) has been reported by numerous studies in the United States during the past two decades. Available data from Europe are scarce, but also suggest the rising prevalence of this disease in overweight children. The aim of this study was to determine the prevalence of previously undiagnosed T2DM, impaired fasting glucose (IFG) and impaired glucose tolerance (IGT) in a clinic cohort of otherwise healthy overweight and obese Caucasian children and adolescents living in Serbia. The study group consisted of 301 subjects (176 girls, 125 boys) aged 5.2-18.9 years, with body mass index >90th percentile. Oral glucose tolerance test was performed in all subjects. Previously undiagnosed T2DM was discovered in 0.3% (n=1) and impaired glucose regulation in 15.9% (n=48) of the subjects. Isolated IFG was detected in 4.3% (n=13), isolated IGT in 8.3% (n=25) and combined IFG and IGT in 3.3% (n=10) of the subjects. Disturbances of glucose metabolism were present in a substantial number of the subjects, which emphasizes the need for prevention and treatment of childhood obesity.

Antigenic correlation between rat brain synaptic vesicles and rat bone marrow B lymphocytes.

Rabbit anti-rat brain synaptic vesicle serum reacted with thymocytes and B lymphocytes in cytotoxicity and immunofluorescence assays. Quantitative absorption analysis revealed that this antiserum contained antibodies specific for antigenic determinants on the surface membrane of a subpopulation of rat bone marrow B lymphocytes.

Antigenic correlation between rat thymus and superior cervical ganglion.

By using rabbit anti-rat thymocyte and anti-rat superior cervical ganglion sera in cytotoxicity, immunofluorescence and absorption assays, it has been shown that surface membranes of rat thymocytes and cervical ganglion cells (i.e. peripheral nervous tissue cells) contain common antigenic determinants.

Potentiation of experimental allergic thyroiditis in rats adipectomized at birth.

The effect of neonatal extirpation of the interscapular brown adipose tissue (adipectomy) on the development of experimental allergic thyroiditis in 8-week-old inbred Lewis rats was studied. The incidence and intensity of disease were much higher, and delayed skin reactions significantly stronger, in adipectomized rats than in control sham-adipectomized rats.

Antigenic correlations between chicken brain organelles, thymocytes and bursacytes.

Rabbit antisera to chicken whole brain and brain microsomes, synaptic membranes, and synaptic vesicles were used in cytotoxicity and immunofluorescence assays against chicken bursacytes and thymocytes. Brain organelles, bursacytes and thymocytes have shown to possess common antigenic determinants. The common antigen is present in small amounts on thymocytes and can be demonstrated only by immunofluorescence.

In vitro cross-reacting antibodies from rabbit antisera to rat brain synaptic membranes and thymocytes.

Rabbit anti-rat brain synaptic membrane and anti-rat thymocyte antisera were used in cytotoxicity, immunofluorescence, and absorption assays to define the antigenic relationship between the rat brain and thymocytes, and the localizing properties of antibodies. Both antisera cross-reacted with brain tissue and thymocytes. However, anti-synaptic membrane and anti-thymocyte antisera also contained antibodies specific for neurons and thymocytes, respectively. Immunofluorescence showed that antibodies from anti-thymocyte antiserum reacted with antigenic determinants situated on the surface membrane of brain cells.

Bursa lymphocytes and IgM-containing cells in chicken embryos bursectomized at 52-64 hours of incubation.

A technique of surgical removal of the bursal primordium ("bursectomy") of chicken embryos at stage 17, approximately 52-64 hours and 29-32 somites, is described. The survival rate of bursectomized (Bx) embryos approached a level of 50% on the 21st day. About 20% of correctly Bx embryos exhibited malformations of the anal sphincter and the large intestine. Using a rabbit anti-bursacyte serum, which did not react with thymocytes, the specific bursa-derived cell (Bu) marker was detected on the surface of bursa, spleen, bone marrow and thymus lymphocytes. Early embryonic bursectomy caused a moderate depletion of Bu marker-bearing and IgM-containing cells. It has been postulated that embryonic Bu cells can be recruited from sites other than the bursa and in the absence of the bursa.

Preparation of specific anti-thymocyte and anti-bursacyte sera in rabbits.

A method of producing anti-thymocyte and anti-bursacyte sera in rabbits is described. Chickens which served as donors of cells were thoroughly perfused with saline to remove plasma proteins and circulating blood elements. For immunization, thymocytes were obtained from neonatally bursectomized birds, and bursacytes from chickens thymectomized at hatching. The purification of rabbit anti-lymphocyte sera included absorptions with leucocyte-free suspension of chicken erythrocytes, chicken liver cell membranes, thymocytes from bursectomized-irradiated and bursacytes from thymectomized-irradiated chickens, and chicken IgM and IgG immunoadsorbents. Cytotoxicity and fluorescent-antibody assays revealed that anti-thymocyte and anti-bursacyte sera thus produced clearly distinguished the surface antigenic determinants of thymocytes from those of bursacytes.