[Genotype and allele frequencies of UCP and PPAR gene families in residents of besieged Leningrad and in the control group].

Genotype and allele frequencies of uncoupling proteins 2 and 3 genes (UCP2 and UCP3) and peroxisome proliferator-activated receptors genes (PPARA, PPARD and PPARG) were studied in 206 residents of the siege and in 139 individuals of more than 69 years old (control group). Studied polymorphisms included UCP2 (Ala55Val), UCP3 (C-55T), PPARA (G/C), PPARD (+294T/C), and PPARG (Pro12Ala). The G allele and the G/G genotype (PPARA) were overrepresented in the group of survivors and C/C (UCP3) genotype prevailed in the women of besieged Leningrad compared to relevant control groups of the persons of the same age who did not suffered hungry disaster. Feasible protective effects of PPARA gene allele G and C allele of UCP2 genes are briefly discussed.

[Association of matrix metalloproteinases' polymorphisms of MMP3 and MMP9 with development of genital endometriosis].

We present a comparative analysis of the allelic polymorphism of the matrix metalloproteinase (MMP) gene family, including MMP3 (rs3025058), MMP7 (rs11568818), MMP9 (rs17576, rs2250889), MMP12 (rs2276109), and MMP13 (rs2252070), in patients with external genital endometriosis (EGE) and in a control group of healthy women proven to be free of disease by laparoscopic inspection. We found significant differences in the incidence of particular MMP3 and MMP9 alleles, which substantiate the role of matrix metalloproteinases in EGE pathogenesis. We used the Multifactor Dimensionality Reduction (MDR) analysis to show that 14 allelic combinations of the MMP containing MMP3 (rs3025058) x MMP7 (rs11568818) x MMP9 (rs17576) alleles showed a statistically significant association with an increased risk of EGE, while 10 other combinations correlated with a reduced risk of the disease. MDR analysis produced two statistically significant models for MMP allelic combinations involved in EGE progression, both with 100% penetrance and 83% and 78% accuracy.

[MED12 gene mutations in women with uterine myoma].

Uterine leiomyoma (UL) is a benign and most common tumor that affects 20-45% of women of fertile age. In this study, we analyzed the MED12 second exon nucleotide sequence from 15 DNA samples extracted from LM of 15 subjects with uterine leiomyoma and 15 DNA samples extracted from peripheral blood leukocytes of the same female subjects. It was shown that somatic mutations in the MED12 gene occur in 73% of cases with deletions of varying sizes and missense mutations being most common at codon 44. Mutations in the MED12 gene could play an indirect role in leiomyoma progression by modifying the activity of other genes that encode proteins involved in growth and tumor progression.

[Molecular genetic basis of proximal spinal muscular atrophy and experience in its pharmaceutical treatment].

The review considers the original and published data on the molecular genetic basis of proximal spinal muscular atrophy (SMA), the most common monogenic neuromuscular disease. The structures of the SMN1 gene and SMN2 pseudogene, mutations distorting the SMN1 function, the structure and functions of the Smn neurotrophic protein, its role in biogenesis of small nuclear ribonucleoproteins (snRNPs), and the principles and prdblems of molecular diagnosis in SMA are described. Special consideration is given to the current approaches and prospects of gene and cell therapy of SMA, pharmacogenetic methods to correct the SMN2 function, and original results of long-term treatment of SMA patients with valproic acid drugs.

[Renin-angiotensin and kinin-bradykinin genes polymorphism effects on permanent arterial hypertension in children].

New methods are required for more objective estimation of the polymorphic genes contribution in multifactorial diseases. We suggest new approach based on the calculation of relative "score" as a sum of relevant genetic polymorphisms studied. Application of suggested approach is evaluated in analysis of the genes REN (19-83G>A), AGT (M235T), ACE (I/D), AGTR1 (1166A>C), AGTR2 (3123C>A), BKR2 (-58T>C and I/D) in children with arterial hypertension. The method proved that polymorphism of renin-angiotensin and kinin-bradikynin gene systems renders essential influence on formation of stably raised arterial pressure in girls.

[Biochip development for polymorphism analysis in biotransformation system genes].

Large-scale population researches, diagnostics of genetic predisposition to multifactorial diseases, screening of the polymorphic loci associated with individual sensitivity to pharmaceutical preparations, require the development of effective, exact and rapid methods of analysis for detection of many mutations simultaneously. One of the most perspective methods to solve these problems is a method of allele-specific hybridization with biochips. Taking the analysis of mutations in genes CYP1A1, CYP2D6, GSTM1, GSTT1, NAT2, CYP2C9, CYP2C19 and MTHFR as an example we showed the efficiency of using the approach for identification of individual genetic polymorphism. We believe that the biochips can be also a convenient tool in pharmacogenetics researches.

[The mutation spectrum of the CFTR gene in mucoviscidosis patients from Bashkortostan]. / Spektr mutatsii gena CFTR u bol'nykh mukovistsidozom iz Bashkortostana.

Mutations of CFTR were studied in patients with cystic fibrosis (CF) from Bashkortostan. In total, 15 mutations were observed and 51% of all mutant alleles identified. The most diagnostically significant mutations were delF508 (33.8%), 394delTT (3.52%), CFTRdele2.3(21 kb) (1.41%), R334W (1.41%), 3849+ 10 kbC-->T (1.41%), and N1303K (1.41%). Mutations G542X, 2184insA, S1196X, and W1282X were each found in less than 1% patients. Five new mutations and two neutral substitutions were revealed. These were I488M (exon 10), 1811 + 12A-->C (intron 11), T663S (exon 13), I1226R (exon 19), 4005 + 9A-->C (intron 20), 2097A-->C (A655A, exon 13), and 3996G-->C (V1288V, exon 20). Bashkortostan was shown to differ in CFTR mutation spectrum from other regions of Russia. The results will allow direct DNA diagnostics of CF in far more families. Molecular screening of probands' relatives will contribute to identification and medical genetic counseling of heterozygous carriers, which is essential for CF prevention.

[Suppression of nonsense mutations in the Dystrophin gene by a suppressor tRNA gene]. / Ispol'zovanie gena supressornoi tRNK dlia ispravleniia nonsens-mutatsii v gene distrofina.

Nonsense mutations in the dystrophin gene are the cause of Duchenne muscular dystrophy (DMD) in 10-15% of patients. In such an event, one approach to gene therapy for DMD is the use of suppressor tRNAs to overcome the premature termination of translation of the mutant mRNA. We have carried out cotransfection of the HeLa cell culture with constructs containing a suptRNA gene (pcDNA3suptRNA) and a marker LacZ gene (pNTLacZhis) using their polymer VSST-525 complexes. It was found that the number of cells producing beta-galactosidase depends inversely on the dose of the suptRNA gene. A single in vivo injection of the construct providing for expression of the suptRNAochre gene into mdx mouse muscle resulted in the production of dystrophin in 2.5% of fibers. This suggests that suppressor tRNAs are applicable in gene therapy for hereditary diseases caused by nonsense mutations.

[Polymorphism of nucleotide sequences of human genomic DNA linked to a mucoviscidosis locus]. / Polimorfizm nukleotidnykh posledovatel'nostei genomnoi DNK cheloveka, stseplennykh s lokusom mukovistsidoza.

Polymorphism in the restriction fragments length of human DNA sequences linked to mucoviscidosis locus was studied in the healthy control group and in the families affected by mucoviscidosis. The plasmid clones metH, pJ3.11,XV-2c and pKM.19 were used as hybridization probes. The allelic frequencies of the polymorphic loci were determined for total population and for affected families. The linkage disequilibrium between the disease locus and linked polymorphic loci detectable with XV-2c (TaqI endonuclease) and pKM.19 (PstI endonuclease) was demonstrated. The high informational value of DNA-diagnosis of mucoviscidosis in the family studies with the use of four DNA probes combination has been demonstrated.

[Molecular-genetic analysis of tandem repeats in intron 6B of the CFTR gene in various populations and in cystic fibrosis patients]. / Molekuliarno-geneticheskii analiz tandemnykh povtorov v introne 6B gena TRBM v nekotorykh populiatsiiakh i u bol'nykh mukovistsidozom.

Frequencies of hexa- and heptatandem tetrameric repeats in Russian slavonians from the North-Western part of the country were found to be 0.19 and 0.81, respectively, with the frequency of heterozygotes estimated as 0.31. Similar values of allelic frequencies were found for uzbek and azerbaijan populations (0.24 and 0.76; 0.22 and 0.78, respectively). These data assess the existence of genetic equilibrium of both alleles in the populations studied. Hexa TTR is present twice more often in the CF chromosomes than hepta TTR common for normal chromosomes. Absolute genetic disequilibrium of both TTR alleles was observed for CF chromosomes bearing the F508 mutation. The latter was exclusively detected in a conjunction with hexa TTR allele. Combined application of the F508 test and TTR alleles assay significantly increased the number of totally informative CF-high risk families, and thus might be highly beneficial for a subsequent prenatal diagnosis of CF.

[A simple and reliable method for detection of the R408W mutation in exon 12 of the phenylalanine hydroxylase gene in the molecular diagnosis of phenylketonuria]. / Prostoi i nadezhnyi metod detektsii mutatsii R408W 12-go ékzona gena phenilalaningidroksilazy v molekuliarnoi diagnostike fenilketonurii.

A new method for identification of R408W mutation common in phenylketonuria (PKU) patients in Russia and Eastern Europe is presented. The method is based on restriction of amplified exon 12. Amplification was achieved by PCR and was followed by restriction with StyI endonuclease. This enzyme specifically recognized allele R408W (C-T change in the position 1444 of the PAH gene) but not the normal allele. The method is easily reproduced both in DNA samples and in blood spots on the blotting paper (Gathrie cards) as well as in native cells from chorionic villi samples and amniocytes without preliminary DNA extraction. The method is very reliable and quick and has obvious advantages over other methods (ASO, ARMS) routinely used for identification of R408W mutation in the PKU high risk families.

[Analysis of the association of HLA-DQ1 alleles with mutation of the 21-hydroxylase gene in patients with congenital adrenal hyperplasia]. / Analiz assotsiatsii allelei HLA-DQA1 s mutatsiiami gena 21-gidroksilazy u bol'nykh s vrozhdenno i giperplaziei kory nadpochechnikov.

In 96 patients with congenital adrenal hyperplasia (CAH) and 50 healthy donors from northwestern Russia the distribution of the HLA-DQA1 alleles and the mutation spectrum and frequency at the CYP21B gene were examined. In the patients with nonclassical (NC) CAH, the distribution of the HLA-DQA1 polymorphic alleles was similar to that in the population sample. In the patients with the salt-wasting form of the disease a statistically significant decrease of the *0401 or *501 major allele frequency was observed. The prevalence of certain HLA-DQA1 genotypes, namely, HLA5, HLA3, and HLA4, was observed in the patients with the NC, salt-wasting (SW), and simple virilizing CAH, respectively. Each clinical group was characterized by a specific spectrum of clinically valuable mutations. An association between the CYP21B mutations most frequently found in case of SW and SV CAH (delB, I2splice, and I172N) and certain HLA-DQA1 alleles was demonstrated. The necessity of more precise clinical diagnostics of the NC CAH cases along with detailed examination of this group for determination of the major mutations typical of the NC CAH cases from northwestern Russia is discussed.

[Population and family studies of CAG repeats in the IT-15 gene]. / Populiatsionnyi i semeinyi analiz CAG-povtorov v gene IT-15.

A simple and effective method for typing of CAG repeats in the IT-15 gene has been suggested. This method was applied for examination of the CAG allele distribution in Huntington's disease (HD) patients in five different populations from the Commonwealth of Independent States. A total of 21 normal alleles with the sizes ranging from 9 to 32 triplet repeat units were revealed. Moreover, alleles with the size ranging from 16 to 20 repeats predominated constituting from 54.4 to 74.6% of all alleles in different populations. The number of repeats in one allele in HD patients exceeded 38 units (43 triplets on average). In two families an increase in the CAG repeat units number in the mutant allele upon its paternal transmission was recorded.

[Expression in vivo of the lacZ gene, delivered to mouse lungs using synthetic microspheres]. / Ekspressiia in vivo gena lacZ, dostavlennogo v legkie myshei pri pomoshchi sinteticheskikh mikrosfer.

A capacity of MF-2 synthetic microspheres to serve as the vehicle for transfer of the marker LacZ gene to mouse lung epithelial cells was studied after a single intranasal administration of the MF-2/gene complex. Two types of plasmids carrying marker gene LacZ were used in the experiments: with cytoplasmic (pCMV-LacZ) and nuclear (pCMV-nlsLacZ) localization of the gene product (beta-galactosidase). As early as 7 days after the complexes MF-2/pCMV-LacZ and MF-2/pCMV-nlsLacZ were administered, specific staining for beta-galactosidase revealed this enzyme activity in the epithelial cells of bronchi, bronchioli, and alveoli. The maximum in vivo of the marker gene in the MF-2/pCMV-LacZ complex was observed at day 14 to 21 after administration and the corresponding gene product was detected during the following two months. The MF-2-mediated gene transfer led to a twofold increase in beta-galactosidase activity relative to the case when the "unbound" pCMV-LacZ plasmid was administered. These results suggest that the synthetic microsphere-mediated transfer of alien genes to the lung of experimental animals is promising. Microspheres may be used in gene therapy for pulmonary affections, in particular cystic fibrosis.

[RFLP analysis of Met and D7S23 loci, linked with the cystic fibrosis gene, in the populations of Bashkir and Komi]. / PDRF-analiz lokusov MET i D7S23, stseplennykh s genom mukovistsidoza, v populiatsiiakh Bashkir i Komi.

The allelic polymorphism of MET and D7S23 loci closely linked to the cystic fibrosis gene was studied using polymerase chain reaction of DNA synthesis. Our studies failed to reveal any significant differences in the distribution of the allelic frequencies of these loci in the populations regarded. The distribution of the allelic frequencies of MET locus in Bashkir and Komi populations did not virtually differ from that in the populations of St. Petersburg and Kiev and those of North-West Europe and North America. Similarity in the distribution of allele frequencies between Bashkir, Komi, Lithuanian and Buryatian populations was observed for the D7S23 locus. At the same time, significant difference from the allelic frequencies for this locus was noted in populations of St. Petersburg and Azerbaijan. Genetic distances between the Bashkir and Komi populations under study were specified according to the data on the allelic frequencies of MET and D7S23 loci. Comparative analysis of these distances and those obtained on the basis of the allelic frequencies of biochemically polymorphic systems of AB0, Rh-Hr blood groups, haptoglobin protein (Hp) and the enzyme of acid erythrocyte phosphatase (AcP) was carried out as well.

[Molecular-genetic analysis of certain mutations of the "cystic fibrosis gene" in Moldavia. Characteristics of molecular markers and their linkage with various mutations]. / Molekuliarno-geneticheskii analiz nekotorykh mutatsii "gena mukovistsidoza" v Moldavii. Kharakteristika molekuliarnykh markerov i ikh stsepleniia s razlichnymi mutatsiiami.

Sixty-one patients with cystic fibrosis (CF) from Moldova were tested for mutations delta F508, G551D, and R553X. Frequencies of various alleles of the repeated GATT sequence in intron 6B of the CFTR gene, their linkage to other polymorphic markers, and various mutations were determined. The frequency of occurrence of mutation delta F508 was only 25%. An absolute majority of CF patients (80%) had pancreatic insufficiency. Mutations G551D and R553X were not found in our sample. Each of 31 chromosomes with mutation delta F508 carries the 6-GATT allele. Most "non delta F508" (78%) and normal (80%) chromosomes were marked by 7-GATT allele. Twenty-seven delta F508 chromosomes (96.4%) belong to haplotype B6, and only one to D6. Most chromosomes with "non delta F508" mutations are associated with haplotypes D7 (26.3%) and C7 (21%). In addition, a significant portion of chromosomes from this subgroup were associated with haplotypes A7 (23.7%), A6 (10.5%), and C6 (2.7%), which are not yet described for mutant chromosomes. The results obtained demonstrate that CF in Moldova is mainly associated with mutations other than delta F508, G551D, and R553X. Severe forms of the disease, with pancreatic insufficiency, are more frequently caused by these mutations; moreover, our data provides strong evidence about the presence of at least seven additional CF mutations in Moldova, apart from delta F508, G551D, and R553X. Some of these are probably not described.

[Analysis of deletional damage in SMN1, SMN2, and NAIP genes in patients with spinal muscular atrophy in the northwestern region of Russia]. / Analiz deletsionnykh povrezhdenii v genakh SMN1, SMN2 i NAIP u patsientov so spinal'noi myshechnoi atrofiei severo-zapadnogo regiona Rossii.

Polymerase chain reaction with subsequent SSCP (single-strand DNA conformational polymorphism) and restriction (BselI restriction endonuclease) analyses were used to type the DNA samples of affected individuals and their relatives from 23 Russian families with high risk of spinal muscular atrophy (SMA) residing in the northwestern region of Russia. Deletions of exon 7 of the SMN gene were found in 96% of the individuals examined. The frequency of homozygous deletion of exons 7 and 8 of the SMN1 gene was 65%. The frequency of homozygous isolated deletion of the SMN1 gene exon 7 among the SMA patients was 4.3%. Homozygous deletion of exon 5 of the NAIP gene was found in 22% of SMA patients. In SMA patients, a total of seven deletion types involving the SMN1, NAIP, and SMN2 genes were detected. Deletion of exons 7 and 8 of the SMN1 gene was the most common mutation associated with SMA in patients from the northwestern Russia.

[Analysis of the polymorphic alleles of genes encoding phase 1 and phase 2 detoxication enzymes in patients with endometriosis]. / Analiz polimorfnykh allelei genov, kodiruiushchikh fermenty 1-i i 2-i fazy detoksikatsii, u bol'nykh éndometriozom.

Polymorphysms of the three genes encoding phase 1 (CYP1A1, mEPH1, and CYP2E2) and the three genes encoding phase 2 (NAT2, GSTM1, and GSTT1) xenobiotic detoxication enzymes were typed by use of PCR in 74 patients with extragenital endometriosis. Distribution of the CYP1A1, mEPHX1, CYP2E1, NAT2, and GSTM1 polymorphic alleles in the patient group corresponded to that in the control group. At the same time, functionally defective genotypes GSTM1 0/0, NAT2 S/S; GSTM1 0/0, GSTT1 0/0; and GSTT1 0/0, NAT2 S/S were three, four and eight times more frequent among the patients than in healthy individuals. This observation suggests the existence of a distinct association between the functionally defective alleles of the phase 2 xenobiotic detoxication and endometriosis. Possible mechanisms underlying this association are discussed. It is suggested that typing of the NAT2, GSTM1, and GSTT1 genes can be useful for the assessment of the predisposition to endometriosis.

[Population analysis CTG trinucleotide repeats in the myotonin-protein kinase I gene]. / Populiatsionnyi analiz trinukleotidnykh CTG-povtorov v gene miotonicheskoi proteinkinazy I.

CTG trinucleotide repeat length polymorphism within the 3' region of the myotonin protein kinase I (MP-I) gene was examined with the use of the polymerase chain reaction (PCR) technique. A total of 159 DNA samples from healthy donors from five ethnic groups including Russians (n = 33), Azerbaijanians (n = 29), Uzbeks (n = 31), Moldavians (n = 31), and Georgians (n = 35) were analyzed. The number of CTG repeats varied from 5 to 24, and allele distribution was bimodal in all populations; alleles with 6, 7 and 9 repeats were not found. Statistical treatment of the data was performed by the contingency table collapsing procedure. Allele distribution in the populations of Russians, Azerbaijanians, Uzbeks and Georgians appeared to be statistically homogenous and significantly (P < 0.001) different from that in Moldavians as well as in Japanese, African, and Western European populations. High heterozygosity levels of all populations, studied (72-86%) indicate the usefulness of this polymorphism in population and genome fingerprinting studies.

[Allelic polymorphism of the DNA-loci met and D7S23, linked to the mucoviscidosis gene in human populations of the Volga-Ural region]. / Allel'nyi polimorfizm DNK-lokusov met i D7S23, stseplennykh s genom mukovistsidoza, v populiatsiiakh narodov Volgo-Ural'skogo regiona.

Data on allelic polymorphism of MET and D7S23 DNA loci linked to the human cystic fibrosis gene studied in three Bashkir ethnic groups and some Volga-Ural populations (Tartars, Maris, Mordovians, Udmurts, Chuvashs, and Komis) are presented. Udmurts were found to be substantially different from Bashkirs, Tartars, Mordovians, and Chuvashs by the allele frequency distribution observed for MET, while Komis and Bashkirs differed by this parameter from Mordovians and Maris. Comparative analysis of restriction fragment length polymorphism (RFLP) at the D7S23 locus revealed statistically significant differences in genotype frequencies between Bashkirs of the Arkhangel' skii region and populations of Mordva and Udmurtia. In this respect, the Mordovian population appeared to be notably different from the populations of Bashkortostan, Tatarstan, Marii-El, Udmurtiya, Chuvashiya, and Komis. Genetic distances were calculated and corresponding dendrograms were constructed on the basis of data on Met-H, CS.7, and the ApoB locus hypervariable region allelic frequencies. Three ethnogeographic Bashkir groups belonging to one tree branch were found to be closely related to the populations of Tartars, Maris, Udmurts, and Chuvashs and substantially different from Komis and Mordovians. Thus, the position of Volga-Ural populations on the dendrogram corresponds to the degree of relationship between the Finno-Ugric and Turkic populations, confirming the usefulness of DNA polymorphism analysis for the study of the genetic structure of populations.