[The use of DYS14 marker for sex determination].

The possibilities of real-time PCR amplification of DYS14 marker located on Y chromosome for sex determination were studied. Samples of plasma of 30 men and 30 women were investigated for this aim. Real-time PCR amplification of DYS14 marker (located inside gene coding TSPY1 protein) was used for sex determination. According to the obtained results, 30 samples belonged to men and 30--to women. In all our experiments the results were confirmed by use of marker SRY, widely used in forensic examination. Detection limit of DNA region containing DYS14 in reaction mixture was established after experiment with dilution of male DNA and is equal to 6.7 pg of DNA (two copies of genome), which corresponds to 6.7 ng of DNA (2000 copies of genome) in 1 ml of blood. Sex determination with small amounts of genetic material in investigated sample becomes possible with such characteristics. Method can be used for noninvasive prenatal diagnostics for the timely detection of congenital diseases associated with sex and in forensic medical examination.

[Association of the polymorphisms of the ERBB3 and SH2B3 genes with type 1 diabetes].

To study the association with diabetes mellitus type 1 we performed analysis of the distribution of frequencies of alleles and genotypes of polymorphic marker rs2292239 of ERBB3 gene, encoding epidermal growth factor receptor type 3 and polymorphic marker rs3184504 of SH2B3 gene, encoding adaptor protein LNK. The study included groups of T1DM patients and unrelated controls of Russian origin. Genotyping was performed using methods of RFLP and real-time amplification. For the polymorphic marker rs2292239 of ERBB3 gene was not found statistically significant associations with type 1 diabetes, while analysis of the distribution of frequencies of alleles and genotypes of the polymorphic marker rs3184504 of SH2B3 gene showed the presence of association with T1DM in Russian population.

[Association of the C1858T polymorphism of the PTPN22 gene with type 1 diabetes].

To study the association with diabetes mellitus type 1 (T1DM) we performed TDT analysis and analysis of the distribution of frequencies of alleles and genotypes of polymorphic marker C1858T of the PTPN22 gene, encoding tyrosine phosphatase of non-receptor type (LYP). Groups of concordant (27 families) and discordant (62 families) sibpairs and groups of T1DM patients and unrelated controls of Russian origin were recruited in Endocrinology Research Center, Moscow and Center of Diabetes, Samara. For a given polymorphic marker was not found statistically significant associations with type 1 diabetes in the transmission disequilibrium test, while analysis of the distribution of frequencies of alleles and genotypes showed the association with T1DM. Thus, the polymorphic marker C1858T of the PTPN22 gene is associated with T1DM in Russian patients.

[Molecular genetics of type 1 diabetes mellitus: achievements and future trends].

Type 1 diabetes mellitus (T1DM) is a widespread, severe disease which results from the immunologically mediated destruction of the beta-cells of pancreatic Langerhans islets. To date the several loci involved to the T1DM development have been reliably identified by means of a number of approaches: MHC locus, VNTR within 5'-nontranscibed region of insulin (INS) gene, CTLA4 gene, encoding surface receptor of T cells, PTPN22 and PTPN2 genes, encoding tyrosine phosphatases of T lymphocytes, interleukin 2 (IL2) gene and alpha-chain of its receptor gene (IL2RA), as well as KIAA0350 gene (unknown function) and IFIH1 gene, encoding receptor of double-stranded DNA generated during viral infections. The functional analysis of proteins encoded by the genes, which are involved to the T1DM development, performed to confirm the hypothesis that on the one hand the origin of T1DM development is founded on the some deregulation of mechanisms of the immune tolerance formation and on the other hand the cause is founded on the formation of destructive immune response against own proteins of organism after virus infection or some other immune stress. Thus the protein products of MHC, INS, PTPN22 and PTPN2 genes involve in the formation in thymus of T-lymphocyte repertoire, which provides the immune defense of organism. On the other hand the nonspecific activation of T cells, from that starts the autoimmune destruction of beta-cells of Langerhans islets of pancreas, in all probability, connects with the protein products of CTLA4, IL2, IL2RA genes, and, perhaps, PTPN22 and PTPN2 genes. The only exception, if not considering the genes with unknown function,--is the IFIH1 gene, but its association with T1DM confirms that fact, that the certain types of virus infection can lead to the activation of autoreactive T cells and T1DM development.

[Methylation of the promoter region of the RASSF1A gene, a candidate tumor suppressor, in primary epithelial tumors]. / Uroven' metilirovaniia promotornoi oblasti gena RASSF1A v pervichnykh épitelial'nykh opukholikh.

Methylation of the promoter CpG-islands of the candidate tumor suppressor gene RASSF1A (3p21.31) was studied in primary tumors of kidney, breast and ovary (172 cases). Methylation-specific PCR (MSP) and methyl-sensitive restriction endonuclease digestion followed by PCR (MSRA) were applied. Statistically significant correlation (P << 10(-6)) was shown for the results of the MSP and MSRA, and the data of bisulfite sequencing reported earlier. The frequency of RASSF1A methylation according to MSP and MSRA was 86% (25/29) and 94% (50/53) in renal cell carcinoma (RCC) and 64% (18/28) and 78% (32/41)--in breast carcinoma (BC) samples, and 59% (17/29) and 73% (33/45) in ovarian epithelial tumors (OET), respectively. The use of several methyl-sensitive restriction enzymes (HpaII, HhaI, Bsh12361, AciI) enhanced the sensitivity of MSRA and allowed to analyze methylation status of 18 CpG-pairs in the RASSF1A CpG-island. Density of methylation of the RASSF1A CpG-island was 72% (644/900) in RCC, 63% (361/576) in BC, and 58% (346/594) in OET samples (18 CpG-pairs multiplied to the number of samples shown methylation were assumed as 100%). The RASSF1A gene methylation was also observed in samples of morphologically normal tissues adjacent to corresponding tumors (11-35%), but it was not detected in blood DNAs of healthy donors (0/15). The RASSF1A methylation frequency did not show significant correlation to tumor stage, grade and metastases (P = 0.3-1.0). The RASSF1A gene methylation was observed more frequently than other investigated aberrations--hemi- and homozygous deletions inside or around this gene. These observations are consistent with the hypothesis that the RASSF1A gene methylation is an early event in the carcinogenesis and one of the dominant ways of its inactivation.

[Optimal local hypothermia regimen in the experimental resection of the single kidney]. / Optimal'nyi rezhim mestnoi gipotermii pri rezektsii edinstvennoi pochki v éksperimente.

The morphological and functional changes of the single kidney were investigated in experiment on 133 dogs after removal of 45--50% of its tissue. Resection of the single kidney under normothermal conditions was established to result in the animal's death within 1--14 days due to acute renal insufficiency. The operation executed under conditions of cooling to 25 degrees C is of high risk. Resection under 5 degrees C hypothermia fails to cause an acute renal insufficiency but considerably disturbs the renal function. The use of local hypothermia to 15 degrees C in resection of the single kidney gives the optimal temperature regimen since it prevents the development of acute renal insufficiency and the compensatory-restorative processes in the kidney can develop successfully.