Immune System and Regeneration.

The review article is devoted to a role of pluripotent stem cells and immune system in renewal of tissues (regeneration). Cell-precursors (progenitor cells) and differentiated cells can be divided a limited number of times and aren't capable of ensuring regeneration of tissues during the whole process of ontogenesis. The renewal of tissues during the whole long period is impossible without the participation of a specialized system which is responsible for regeneration. The given system is made up of pluripotent stem cells which are capable of differentiating themselves into all types of somatic cells, and into a line of genital cells. These stem cells are also capable of reproducing themselves over the whole lifespan of the organism. The participation of pluripotent stem cells and the possible mediation of antigen-presenting cells and T-helpers/T-suppressors in the complex with molecules of the MHC I class/II class make it possible to consider that exactly this immune system is responsible for regeneration of tissues in the organism. The participation in the regeneration process is the most important (and perhaps the leading) function of the immune system. With age the quantity of pluripotent stem cells gradually decreases. It leads to violation of renewal of tissues at people over 35-40 years old. Transfusion of mononuclear fraction of peripheral blood procured from young donors 18-23 years old with the same blood groups and sex as the recipient (RF patent number 2350340), allows people over 40-50 years old to reestablish the pool of pluripotent stem cells and the process of tissue renewal.

[Analysis of polymorphism of bone metabolism genes and evaluation of the risk of osteopenia in cosmonauts].

Densitometry of cosmonauts following long-duration missions shows reduction of bone mineral density (BMD). On the average, post-flight BMD remains within the normal range and the broad variability of individual BMD values sometimes is qualified as local osteopenia. Individual reactions are typed by similarity of amount and rate of BMD loss. At present, analysis of functionally significant polymorphism of bone metabolism genes is the most effective instrument for diagnostics of susceptibility to osteopenia and osteoporosis. The investigation was aimed to analyze polymorphism of genes of vitamin-D and (VDR) and calcitonin (CALCR) receptors, and of collagen-1 alpha-1-chain (Col1a-1) in candidate cosmonauts and cosmonauts returned from 5 to 7-mo. missions. According to the results of analysis, in the majority of cosmonauts rapid BMD loss correlated with TT genotype by VDR gene but not with genotypes Tt and tt and associated with carriage of incomplete s-allele in the Col1a1 gene. Yet, in several instances high BMD loss rates were personified with carriers of VDR gene alleles (homo- and heterozygote states--tt and Tt) and heterozygote by Col1a1 gene (Ss).

[Effect of TT genotype of the vitamin D receptor gene on bone mineral density in dialysis patients].

AIM: to evaluate the effect of Taqi genotypes of vitamin D receptor (VDR) gene polymorphism on bone mineral density (BMD) and the risk for secondary osteoporosis (OP) in patients on programmed hemodialysis. SUBJECTS AND METHODS: Eighty-two patients treated with long-term hemodialysis were examined. Along with physical examination, osteodensitometry was carried out and VDR gene polymorphism was studied in all the patients. RESULTS: OP in the study skeletal parts was more common in the TT-genotype group. The serum concentration of intact parathyroid hormone and the activity of alkaline phosphatase were also higher in the TT-genotype group. However, the diferences were statistically insignificant between the TT-, Tt-, and tt- genotype groups. BMD was lowest in each study skeletal parts in the TT-genotype group and this difference was statistically significant in the proximal femur, which was estimated by the Z index (p = 0.02). CONCLUSION: The results of the present study confirm the hypothesis that the VRD gene is a genetic determinant of bone metabolism in patients with chronic renal disease who receive long-term hemodialysis.

[Some aspects of the regeneration process carried out by means of pluripotent stem cells].

This work is devoted to the vital topic of regeneration by stem cells. Cells-predecessors and differentiated cells can divide a limited number of times (Alberts et al., 1994) and are not capable of providing tissue regeneration throughout the ontogenesis. The tissue renewal during such a long period is impossible without participation of a specialized system responsible for regeneration. The given system is submitted by stem cells which are capable of being differentiated in all types of somatic cells and in a line of germ cells, and also have ability to self-renew during the whole life of an organism. Results of our research suggest that stem cells make up a universal mechanism of regeneration which has been formed during evolution.

[Analysis of association of Col1a1 gene alleles with the development of osteoporosis]. / Analiz assotsiatsii allelei gena Col1a1 s razvitiem osteoporoza.

Allele frequencies of the G-->T polymorphism at the regulatory region of the Collal gene in the population of the northwestern Russia (control group) and in osteoporotic patients were estimated by the RFLP method based on PCR-mediated site-directed mutagenesis. Three patient groups with radiologically confirmed osteoporosis were examined. Group 1 consisted of 64 patients with severe osteoporosis complicated by fractures (SO); group 2 included 15 children with idiopathic osteoporosis (IO); group 3 consisted of 98 women with postmenopausal osteoporosis developed at the background of estradiol-deficiency state (PMO). The frequency of functionally defective allele s in the control group was 16.7%. It was statistically different from that in the SO patients (48.4%) (P < 0.01) and in the IO children (40%) (P < 0.01). The frequency of allele s in the PMO patients constituted 23.0% and it was similar to that in the control group (P > 0.05). Analysis of the Collal alleles provides early detection of the individuals with hereditary predisposition to osteoporosis and prophylaxis of the disease at the presymptomatic stage.

[Molecular-genetic analysis of polymorphism of the DXS532 locus in people from the Volga-Ural region]. / Molekuliarno-geneticheskii analiz polimorfizma lokusa DXS52 u narodov volgo-ural'skogo regiona.

The DXS52 polymorphic locus mapping to the 5'-region of the blood-clotting factor VIII gene on the X chromosome was genotyped in seven Volga-Ural ethnic groups (Bashkirs, Tatars, Chuvashes, Maris, Mordovians, Udmurts, and Komis). A total of 47 different genotypes and 15 allelic variants of this locus were described. Substantial intra- and interpopulation heterogeneity of the ethnic groups studied in respect to frequency and distribution of the DXS52 alleles and genotypes was demonstrated. The unimodal DXS52 allele frequency distribution pattern with the peak at 1690 bp was typical to Mordovians and Komis. Chuvashes and Maris, as well as Udmurts, were characterized by bimodal frequency distribution patterns, with the peaks at 1690 and 670 bp, and 1690 and 1390 bp, respectively. Moreover, Bashkirs and Tatars displayed trimodal DXS52 allele frequency distribution patterns with the peaks at 1690, 1390, and 670 bp. The DXS52 allele frequency distribution patterns described in populations of the Volga-Ural region were found to be remarkably different from those established for the mixed Moscow population and the population of Western Europe. These data indicate that the DXS52 locus is highly informative, and this polymorphic system can serve as a molecular marker for population genetic studies.

[Molecular genetic study of the factor VIII gene in families from Bashkir with hemophilia A]. / Molekuliarno-geneticheskoe izuchenie gena faktora VIII v sem'iakh s gemofiliei A iz Bashkirii.

The distribution of hemophilia A was studied in Bashkortostan. The factor VIII gene of the blood coagulation system was analyzed in 34 patients with hemophilia A and 48 of their close relatives. Inversion of intron 22 of the factor VIII gene was revealed in nine cases, which comprised 30% of the total sample analyzed. The type II and type III of this mutation occurred at a relatively high frequency, which may be explained by the founder effect and genetic drift. The allelic frequencies of the polymorphic locus HindIII at intron 19 were similar; a substantial allelic heterogeneity of both microsatellite (CA)-repeats at intron 13 and the DXS52 locus were found on normal and mutant X chromosomes. The molecular genetic analysis of (CA)-repeats and the loci HindIII and DXS52 in families with hemophilia A makes it possible to reveal up to 89% of the informative families.

[Molecular genetic analysis of polymorphism of the HindII and (CA)-repeat of the factor VIII gene loci in people of the Volgo-Ural region]. / Molekuliarno-geneticheskii analiz polimorfizma lokusov HindIII i (CA)-povtorov gena gaktora VIII u narodov volgo-ural'skogo regiona.

Molecular genetic analysis of polymorphism of the factor VIII gene at loci HindIII (intron 19) and (CA)-repeats (intron 13) was performed in seven ethnic groups of the Volga-Ural region (Bashkirs, Tatars, Chuvashes, Maris, Mordovians, Udmurts, and Komis). The allelic frequency of the HindIII locus was shown to be similar in these ethnic groups. The ethnic groups were more heterogeneous with regard to the genotype and allele frequencies of (CA)-repeats, which indicates that this polymorphic system can be used to characterize the genetic structure of the Volga-Ural human populations.

[Comparative analysis of allele polymorphism of three short tandem repeats in the Russian, Uzbek and Georgian populations]. / Sravnitel'nyi analiz allel'nogo polimorfizma trekh korotkikh tandemnykh povtorov v populiatsiiakh russkikh, uzbekov, gruzin.

The allele polymorphism of the AGC short tandem repeat (STR) of exon 1 of the androgen receptor (AR) gene located in Xq11-12, ATCT STR of intron 40 of the von Willebrand factor (vWF) gene located in chromosome 12p12, and AGAT STR of an anonymous DNA sequence (STRX1) from the short arm of the X chromosome was analyzed in the Georgian, Uzbek, and Russian populations. Polymerase chain reaction (PCR) with DNA of unrelated persons revealed 14 AR, 7 vWF, and 7 STRX1 alleles in Georgians; 14, 8, and 6 alleles, respectively, in Uzbeks; and 16, 8, and 9 alleles, respectively, in Russians. The heterozygosity at these STR was 0.61, 0.78, and 0.46 in Georgians; 0.60, 0.83, and 0.44 in Uzbeks; and 0.80, 0.70, and 0.58 in Russians. The correspondence of genotype frequencies to the Hardy-Weinberg equilibrium was observed with AR STR in Russians and Uzbeks, STRX1 STR in Georgians, and vWF in all three populations. A significant deviation from the equilibrium was found for STRX1 in Russians and Uzbeks and AR in Georgians. The potential of individualization was 0.05 for AR, 0.13 for vWF, and 0.18 for STRX1 in Georgians; 0.04, 0.09, and 0.13, respectively in Uzbeks; and 0.05, 0.14, and 0.07, respectively, in Russians. The allele and genotype frequency distributions of each STR were analyzed in all three populations. Allele frequencies in the populations were compared by the Kolmogorov-Smirnov test. The Russian population significantly differed in allele frequencies of the three STR from Uzbeks and in those of STRX1 and AR from Georgians. Georgians and Uzbeks significantly differed in vWF and STRX1 frequencies. The possibility of using the three STR in molecular diagnosis of the corresponding monogenic diseases, population genetic studies, and personal identification is discussed.

Dating the origin of the CCR5-Delta32 AIDS-resistance allele by the coalescence of haplotypes.

The CCR5-Delta32 deletion obliterates the CCR5 chemokine and the human immunodeficiency virus (HIV)-1 coreceptor on lymphoid cells, leading to strong resistance against HIV-1 infection and AIDS. A genotype survey of 4,166 individuals revealed a cline of CCR5-Delta32 allele frequencies of 0%-14% across Eurasia, whereas the variant is absent among native African, American Indian, and East Asian ethnic groups. Haplotype analysis of 192 Caucasian chromosomes revealed strong linkage disequilibrium between CCR5 and two microsatellite loci. By use of coalescence theory to interpret modern haplotype genealogy, we estimate the origin of the CCR5-Delta32-containing ancestral haplotype to be approximately 700 years ago, with an estimated range of 275-1,875 years. The geographic cline of CCR5-Delta32 frequencies and its recent emergence are consistent with a historic strong selective event (e.g. , an epidemic of a pathogen that, like HIV-1, utilizes CCR5), driving its frequency upward in ancestral Caucasian populations.

[Population features of gene mutation frequency of the CKR-5 receptor, determining sensitivity to the AIDS virus]. / Populiatsionnye osobennosti chastot mutatsii gena khemokinovogo retseptora CKR-5, opredeliaiushchego chuvstvitel'nost' k virusu SPIDa.

Recently, it was shown that a 32-bp deletion in the CKR5 macrophage chemokine receptor gene produced resistance to HIV infection. Frequencies of the CKR5 mutant allele in Russians, Tatars, Uzbeks, Kazakhs, Azerbaijanis, Uigurts, Tuvinians, and Georgians estimated by means of the PCR technique were equal to 0.13, 0.12, 0.85, 0.06, 0.05, 0.04, 0.03, and 0.00, respectively. While the theoretically expected frequency of deletion homozygotes in Russians and Tatars was 1.7%, none of such homozygotes were detected among the 90 persons examined. The data suggested that about 25% of the population in northwestern Russia is highly resistant to HIV infection.

Proportion of the GSTM1 0/0 genotype in some Slavic populations and its correlation with cystic fibrosis and some multifactorial diseases.

A homozygous gene deletion at the glutathione S-transferase M1 (GSTM1) locus of genomic DNA from blood spots was studied by PCR in the group of Slavic populations from the north-western and central-eastern regions of European Russia and in patients with lung cancer (LC), other tumors (OT), endometriosis (E), alcoholic cirrhosis (AC), cystic fibrosis (CF) and chronic bronchitis (CB). The frequencies of the GSTM1 0/0 genotype were 38.8% and 67.5% for both population groups, respectively. The proportion of the GSTM1 gene deletion genotype was estimated as significantly increased in LC (81%), OT (65%), E (81%), AC (77.3%), and in CB (73.6%) patients with symptoms of CB confirmed by X-ray but not in CB patients without X-ray evidence of disease (40.9%). A definite preponderance of GSTM1-0 homozygotes (51.1%) has been registered in CF patients of the pancreatic sufficient group with clear-cut pulmonological manifestations but not in those of the pancreatic insufficient group with predominantly intestinal or mixed clinical symptoms (41.2% and 37.5%, respectively). Earlier clinical manifestations and death before the age of 5 years are typical for GSTM1-deleted CF patients. These data support the notion that GSTM1 deletion should be considered as a convenient genetic marker for the early detection of groups at higher risk of many diseases caused by environmental and genetic factors, where manifestation depends on the lack of detoxification. High levels of GSTM1 0/0 genotypes in E patients favor the substantial contribution of certain environmental toxins in the pathogenesis of this widespread disease.

Allele frequencies and molecular diagnosis in haemophilia A and B patients from Russia and from some Asian republics of the former U.S.S.R.

RFLP analysis of some intra- and extra-genic polymorphic sites of Factor VIII (FVIII) and Factor IX (FIX) genes with relevant DNA probes or by polymerase chain reaction (PCR) was carried out in Slavic populations from the European part of Russia and also in the native ethnic groups of Uzbekistan and Kazahstan. The allele frequencies for the HindIII (intron 19) and XbaI (intron 22) polymorphic sites (PSs) in the FVIII gene were very similar in the two populations studied, but different for the intron 13 (CA)n repeat. Significant variations in the TaqI (intron d) and DdeI (intron a) polymorphisms of the FIX gene were evident between the Russian and Asian populations. Two unusual alleles (4.35 and 4.2 kb) for the extragenic PS St14/TaqI were registered in Slavs and one new allele (380 bp) for the DdeI polymorphic site of FIX was discovered in both Asian populations. Altogether, 210 haemophilia A (HA) and 24 haemophilia B (HB) families were subjected to molecular studies. So far, 160 HA and 12 HB families have been found to be informative for DNA analysis. Carrier status was ascertained in 42 HA and 6 HB female relatives, and rejected in 52 and 10 of them, respectively. The origin of some HA and HB mutations was traced with relevant polymorphic markers in several at-risk families. Prenatal diagnosis was accomplished in 28 HA and three HB families, resulting in the identification of 20 affected male fetuses.

[Molecular diagnosis of fragile X-chromosome syndrome (Martin-Bell syndrome) in patients of native populations]. / Molekuliarnaia diagnostika sindroma fragil'noi X-khromosomy (sindrom Martina-Bella) u bol'nykh otechestvennykh populiatsii.

63 families at-rist of Fragile X-syndrome (FraX) are subjected to Southern blot analysis with the DNA probes Ox1.9 and Ox0.55. Molecular studies have confirmed an initial clinical diagnosis of FraX in 26 families earlier studied cytogenetically and in 11 of 27 families with only some clinical traits of FraX syndrome in proband. Full mutation and premutation condition of FMR-1 gene was ascertained in 34 and rejected in 18 close relatives of probands with the proved FraX syndrome in 37 and families. Four different patterns of pathological alleles are detected at electrophoretograms of DNA samples restricted by endonuclease RcoRI and hybridized to the DNA probe Ox1.9. Prenatal diagnosis of FraX was carried out in two cases at the 1st and 2nd trimester of pregnancy. Perspective of broad application of molecular methods for early diagnostics and prophylactic of FraX syndrome are briefly discussed.

[Complex biochemical and molecular approach to prenatal diagnosis of mucoviscidosis]. / Kompleksnyi biokhimicheskii i molekuliarnyi podkhod v prenatal'noi diagnostike mukovistsidoza.

Prenatal diagnosis of 62 cases of cystic fibrosis was performed in high-risk families at 18-21 gestational weeks using biochemical and molecular tests. The diagnosis was ruled out in 42 and confirmed in 20 cases. The incidence of false-positive results was 8.3% (5 of 62) and that of false-negative results 3.7% (2 of 62). Reliability of prenatal diagnosis in concomitant use of biochemical and molecular tests was over 95%. Advantages of prenatal diagnosis of cystic fibrosis using DNA probes and requirements for its wider adoption in the first trimester are discussed.

[Use of molecular and genetic approaches in prenatal diagnosis and prevention of hemophilia A and Duchenne muscular dystrophy]. / Ispol'zovanie molekuliarno-geneticheskikh podkhodov dlia prenatal'noi diagnostiki i profilaktiki gemofilii A i miodistrofii Diushenna.

Allele polymorphism has been evaluated using blot hybridization and a polymerase cascade of DNA synthesis in 40 families at high risk of hemophilia A (a total of 147 subjects) and in 15 families with Duchenne's myodystrophy, Heterozygous carriage of hemophilia A was identified or confirmed in 18 and ruled out in 4 close female relatives of probands. Prenatal tests for fetal hemophilia A were performed in 5 women from families with hemophilia A (in the 1st trimester in 2 and in the 2nd trimester in 3). Four diagnoses of hemophilia A were confirmed and 1 was ruled out. The DNA methods proved revealing in 34 of 40 families with hemophilia A and in 11 of 15 families with Duchenne's myodystrophy. Three of 9 probands were found to have a deletion of the proximal gene for Duchenne's myodystrophy in the DNA probe area of XY 1.1. Prospects of screening for heterozygous carriage and prenatal identification of hemophilia A and Duchenne's myodystrophy are discussed.

[Detection of carriers of hemophilia A by testing for HindIII polymorphism in the factor VIII gene by PCR]. / Vyiavlenie nositelei gemofilii A pri pomoshchi testirovaniia polimorfnogo saita HindIII v gene faktora VIII metodom PCR.

Representatives of 62 families from Moscow and Leningrad with haemophilia A observed in the pedigree were tested for HindIII polymorphism in the factor VIII gene. The proposed scheme of investigation was based on intron 19 of the FVIII gene amplification by the PCR technique followed by restriction analysis with the inner control of hydrolysis. 207 unrelated X-chromosomes were analysed, the frequency of the incidence of the polymorphic HindIII site in the given population found to be 0.29. The frequency of incidence of the HindIII heterozygotes calculated according to Hardy-Weinberg equation was 0.41. This value evidences for relatively high informativity of this polymorphism for carrier detection and prenatal diagnosis of haemophilia A. 23 families (37%) out of 62 examined in the study were informative for this criteria. The new scheme proved to be effective in testing HindIII polymorphism for haemophilia A carrier detection and prenatal diagnosis. The whole procedure takes one day, the radiolabelled probes are not used. The scheme described was inculcated in the All-Union Research Center for Haematology, Ministry of Health, USSR, Moscow, Research Institute for Obstetrics and Gynecology, Leningrad, Institute of Medical Genetics, Greifswald, DDR.

[Detection of the heterozygote carrier state and prenatal diagnosis of hemophilia A using DNA probes]. / Vyiavlenie geterozigotnogo nositel'stva i prenatal'naia diagnostika gemofilii A pri pomoshchi DNK-zondov.

Southern blot analysis of DNA samples from 110 persons related to 30 high risk hemophilia A families was carried out using intergenic probe St-14 and intergenic probes p 51-61 and p 1.8. Thirty-five hemophilia A patients, 24--their mothers--obligatory carriers and 51 close proband relatives were studied altogether. 16 female proband relatives were diagnosed as hemophilia A carriers, hemophilia A heterozygosity was rejected in three persons. Twenty five families were found to be at risk for prenatal hemophilia A diagnosis. One case of hemophilia A diagnosis in a 10-week fetus has been presented.