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.

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.

Ген RASSF1A (3p21.31) относится к группе генов-супрессоров опухолевого роста. Изучен уровень метилирования СрО-островка в промоторном районе этого гена в первичных опухолях почек, молочной железы и яичников в московской популяции (172 случая). Использован метод ПЦР, специфичной к метилированному аллелю (МСП), а также анализ с помощью метилчувствительных рестрикционных эндонуклеаз и последующей ПЦР (МЧРА). Результаты этих методов с высокой достоверностью (согласно ранговой корреляции Спирмана, P < 10 -6) согласуются между собой и с ранее полученными данными бисульфитного секвенирования. По данным МСП и МЧРА частота метилирования промоторного района гена RASSF1A составила 86% (25/29) и 94% (50/53) при почечноклеточном раке, 64% (18/28) и 78% (32/41) при раке молочной железы и 59% (17/29) и 73% (33/45) в эпителиальных опухолях яичников соответственно. Применение набора метилчувствительных рестриктаз (HpaII, HhaI, BshI236I, AciI) повысило чувствительность анализа и позволило изучить статус метилирования 18 СрО-пар промоторного района гена RASSF1A. На основании этих данных удалось оценить плотность метилирования СрО-островка, которая достигала 72% в опухолях почки, 63% при раке молочной железы и 58% при раке яичников (за 100% принято произведение исследованных 18 СрО-пар на число образцов, в которых выявлено метилирование). Метилирование промоторного района гена RASSF1A (11-35%) наблюдали и в образцах ДНК из гистологически нормальной ткани, прилежащей к опухоли, но оно полностью отсутствовало в этих же участках ДНК из крови здоровых доноров (0/15). Частота метилирования СрО-островка в опухолях не зависела от стадии, степени анаплазии и наличия метастазов. С другой стороны, эпигенетическую модификацию гена RASSF1A в опухолях наблюдали существенно чаще, чем геми- и гомозиготные делеции в области этого гена. Согласно этим наблюдениям, метилирование промоторного района гена RASSF1A относится к ранним событиям в онкогенезе и представляет один из главных путей инактивации этого гена в эпителиальных опухолях.

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.

The article is devoted to identification of genetic markers coupled with type 1 diabetes mellitus.