HLA DRB1, DQB1 and insulin promoter VNTR polymorphisms: interactions and the association with adult-onset diabetes mellitus in Czech patients.

Both the human leucocyte antigen (HLA) DRB1 and the HLA DQB1 gene loci play a role in the development and progression of autoimmune diabetes mellitus (T1DM). Similarly, the insulin promoter variable number tandem repeats (INS-VNTR) polymorphism is also involved in the pathogenesis of diabetes mellitus (DM). We studied the association between each of these polymorphisms and DM diagnosed in patients older than age 35 years. Furthermore, we analysed possible interactions between HLA DRB1/DQB1 and INS-VNTR polymorphisms. Based on C-peptide and GADA levels we were able to distinguish three types of diabetes: T1DM, latent autoimmune diabetes in adults (LADA) and T2DM. INS-VNTR was genotyped indirectly by typing INS-23HphI A/T polymorphism. The genotype and allele frequencies of INS-23HphI did not differ between each of the diabetic groups and group of healthy subjects. We did, however, observe an association between the INS-23HphI alleles, genotypes and C-peptide secretion in all diabetic patients: A allele frequency was 86.2% in the C-peptide-negative group vs. 65.4% in the C-peptide-positive group (P(corr.) < 0.005); AA genotype was found to be 72.4% in the C-peptide-negative group vs. 42.6% in the C-peptide-positive groups (P(corr.) < 0.01). The HLA genotyping revealed a significantly higher frequency of HLA DRB1*03 allele in both T1DM and LADA groups when compared to healthy subjects: T1DM (25.7%) vs. control group (10.15%), odds ratio (OR) = 3.06, P < 0.05; LADA (27.6%) vs. control (10.15%), OR = 3.37, P < 0.01. The simultaneous presence of both HLA DRB1*04 and INS-23HphI AA genotype was detected in 37.5% of the T1DM group compared to only 9.2% of the healthy individuals group (OR = 5.9, P(corr.) < 0.007). We summarize that in the Central Bohemian population of the Czech Republic, the INS-23HphI A allele appears to be associated with a decrease in pancreatic beta cell secretory activity. HLA genotyping points to at least a partial difference in mechanism, which leads to T1DM and LADA development as well as a more diverse genetic predisposition in juvenile- and adult-onset diabetes. The simultaneous effect of HLA and INS-VNTR alleles/genotypes predispose individuals to an increased risk of diabetes development.

KCNJ11 E23K polymorphism and diabetes mellitus with adult onset in Czech patients.

In this work, we studied the association of the E23K polymorphism of the Kir6.2 ATP-sensitive potassium channels in 212 Czech patients with diabetes mellitus who were diagnosed after the age of 35. Patients were classified into T1DM, LADA and T2DM groups based on C-peptide and GADA levels. Carriers of the predisposing Kir6.2 E23K K allele showed no increased risk of either type of diabetes mellitus development. On the other hand, we found a correlation between E23K SNP of the KCNJ11 gene and C-peptide levels, which may be considered a measure of pancreatic beta-cell activity, although this correlation was not statistically significant. In conclusion, we failed to confirm the Kir6.2 E23K as a genetic marker for T1DM, LADA and T2DM in the Central Bohemian population of the Czech Republic.

PCR in lyme neuroborreliosis: a prospective study.

OBJECTIVES: DNA proof is the only widely available direct diagnostic tool in Lyme borreliosis. Sensitive PCR detecting of spirochetal DNA was prepared and a prospective study in neuroborreliosis was performed. MATERIALS AND METHODS: 57 hospitalised patients with active neuroborreliosis and proved CSF antibodies synthesis were examined. Nested-PCR (utilizing three targets) was used for the detection of specific DNA in plasma, CSF and urine. RESULTS: Before treatment 36 positive patients (63.1%) were found in all tested specimens in parallel, 28 patients (49.1%) were positive in urine, 20 in CSF (35.0%) and 16 in plasma 28.0%). Later only urine was tested and the following results were obtained: 17 positive patients (30.0%) immediately after treatment, 8 (14.0%) after 3 months and one patient persisted positivity after 6 months. CONCLUSIONS: The highest sensitivity of PCR was achieved in the acute period of neuroborreliosis - 63.1% in three body fluids comparing with CSF antibody synthesis.

Apoptosis as an early event in the development of multiple organ failure?

We have recently developed a simple method of plasma free DNA detection, which enables us to distinguish between apoptotic and genomic (necrotic) DNA. After applying this method to the critically ill, we revealed apoptotic DNA on the day of admission to be higher than later when multiple-organ failure developed. Moreover, apoptotic DNA contributed to total plasma DNA much more than DNA from necrotic cells and its increase predicted future development of multiple-organ failure and death.

HLA in Czech adult patients with autoimmune diabetes mellitus: comparison with Czech children with type 1 diabetes and patients with type 2 diabetes.

Type 1 diabetes results from an autoimmune insulitis, associated with HLA class II alleles. The evidence about HLA allele association is not clear in patients diagnosed after 35 years of age. In this study we have analyzed HLA alleles of DQB1 and DRB1 genes by sequence specific primer (SSP)-PCR technique in adult patients with disease onset after 35 years of age. Two hundred and eighty-one patients were divided into three groups according to the insulin therapy, the level of C peptide (CP), and GAD antibodies (anti-GAD). Group 1 (type 1 diabetes in adults) was characterized by CP less than 200 pmol/L and anti-GAD more or less than 50 ng/mL (n = 80). All of them had insulin therapy within 6 months after diagnosis. Group 2 latent autoimmune diabetes mellitus in adults (LADA) was defined by a minimum 6-month-long phase after diagnosis without insulin therapy, and was characterized by CP more than 200 pmol/L and anti-GAD more than 50 ng/mL (n = 70). Group 3 (type 2 diabetes) was characterized by CP more than 200 pmol/L and anti-GAD less than 50 ng/mL (n = 131). None ever had insulin therapy. In group 1, there was increased frequency of DRB1*04 (45.0% vs. controls 14.1%, OR = 5.0, P < 0.0005) and DQB1*0302 alleles (43.3% vs. controls 11.1%, OR = 6.1, P < 0.00005). There was increased frequency of DRB1*03 and DQB1*0201, and decreased frequency of DQB1*0602 (3.3% vs. controls 20.2%), but it was not significant. In group 2, there was a significantly increased frequency of DRB1*03 only (50.0% vs. controls 21.2%, OR = 3.7, P < 0.05). Compared with children with type 1 diabetes and adults with type 2 diabetes (group 3), we conclude that the presence of predisposing DQB1 alleles in adults with type 1 diabetes decreases with the age, probably due to environmental factors. Only the DRB1*03, but not the DQB1 gene, becomes the main predisposing allele in LADA patients. These findings suggest that the presence of HLA-DQB1*0302 identifies patients at high risk of requiring insulin treatment. Type 1 diabetes mellitus (DM) in children or adults may have partly different immunogenetic etiopathogenesis than LADA.

Prostate-specific antigen: current status.

PSA is the most important of all tumor markers because it has significant applications in all aspects of the management of men with prostatic disease. Certainly, the most important utilization of PSA is for early detection of this most ubiquitous of all human neoplasms. In this article the authors describe the molecular forms of PSA and their characteristics, the factors influencing values of serum concentration of PSA, the problems of screening, and particularly the possibility to use PSA for detection of prostate carcinoma. A big problem in prostate carcinoma detection is the low specificity of PSA at the concentrations between 4-10 ng/ml, the so-called diagnostic gray zone, where the incidence of prostate carcinoma is only 25%. The authors evaluate the methods which make it possible to increase the sensitivity and/or specificity of PSA detection, such as PSA density, PSA density of the transition zone, PSA velocity, PSA doubling time, age-specific PSA, free PSA and, prospectively, the use of the RT-PCR technique.

[Hemochromatosis. Determination of the C282Y mutation frequency in the population of the Czech Republic and sensitivity of hemochromatosis detection using Guthrie cards]. / Stanovení frekvence C282Y mutace pro populaci Ceské republiky a Hemochromatosis. Stanovení frekvence C282Y mutace pro populaci Ceské republiky a citlivá detekce hemochromatózy z Guthrie karet.

BACKGROUND: Hereditary haemochromatosis (HH) is a multi-organ disease characterized by increased deposition of iron in some tissues. When discovered early the disease is curable. The Caucasian population is burdened with a high incidence of carriers and homozygotes. The recent discovery of the gene for haemochromatosis (HFE) and of the major mutation C282Y makes direct diagnosis possible. METHODS AND RESULTS: Exploiting a newly-designed polymerase chain reaction system, we determined the frequency of the C282Y mutation of the HFE gene in the Czech population by analysis of 278 chromosomes from anonymous clients of an in vitro fertilization centre (128 male and 150 female karyotypes respectively). There were 14 heterozygotes of the C282Y mutation (6 males and 8 females) and no asymptomatic homozygotes. All 12 patients with classically diagnosed haemochromatosis were homozygous for the C282Y mutation. Twelve Guthrie cards ten years old were tested as a source of the PCR signal for C282Y with a positive result in all cases (100% efficiency). CONCLUSIONS: Our data suggest that the approximate frequency of heterozygotes (carriers) of hereditary haemochromatosis in the Czech population is 1:10 which is comparable with data from the Central European region. The frequency of the major mutation among patients with HH was 100% (12/12), however, the difference with data from neighbouring countries (Austria, Germany) cannot be considered significant due to the small number of patients tested. We also showed the possibility of a retro-diagnosis of the presence/absence of C282Y mutation from Guthrie cards.