[Contents of alpha-fetoprotein in the blood of pregnant women as a criterion of the presence of congenital heart defects in the fetus]. / Issledovanie soderzhaniia al'fa-fetoproteina v syvorotke krovi beremennykh kak kriteriia nalichiia vrozhdennykh porokov razvitii u ploda.

alpha-Fetoprotein (AFP) was measured in the blood of 16 women pregnant with twins at various terms of gestation and 24 pregnant women whose fetuses were found to have anencephaly, patent spina bifida, gastroschisis, renal polycystosis, or Down's disease. In Down's disease AFP level was 7 ng/ml (0.17 multiple of medians, MoM) at 17 weeks gestation and 6 ng/ml (0.12 MoM) at 19 weeks. In the fetal abnormalities studied AFP level was 372 ng/ml on average (6.8 MoM) at 16 to 18 weeks gestation, this being about 10 times higher than the normal level. AFP level in twin pregnancy at the same period was 2.3 MoM. AFP measurements are important for the prenatal diagnosis of fetal status in order to plan follow-up of pregnancy and labor management.

Dystrophin gene analysis and prenatal diagnosis of Duchenne muscular dystrophy in Russia.

Of 126 families referred for counselling of Duchenne muscular dystrophy (DMD), DNA analysis has been suggested to 119 families with at least one affected child or with an affected close male relative of the woman at risk of being a DMD carrier. A large proportion (about 80 per cent) of the families were represented by sporadic cases (only one affected individual). By means of multiplex polymerase chain reactions with different sets of oligoprimers providing amplification of 10-11 different exons, altogether 49 dystrophin gene deletions were identified (41 per cent). Eighteen deletions clustered in the 5' 'hot spot' region of DMD cDNA and 36 in the distal half of the central rod domain around exons 43-53. An unusually high frequency (18 per cent) of deletions involving exons 17-19 was discovered. Large deletions extending through both 'hot spot' regions and thus occupying over 30-40 exons were recorded in five cases (10 per cent). Seventy-six of 94 families were found to be informative by RFLP analysis for intragenic or extragenetic DNA probes. Carrier status was ascertained in 20 and rejected in 32 female relatives in 40 DMD families. Eight DMD-affected fetuses were diagnosed prenatally by direct deletion testing or by RFLP analysis. Feasible interpopulation variations in the dystrophin gene deletion pattern are discussed. The prospects for more effective prenatal diagnosis and carrier detection in high-risk DMD families in Russia are briefly outlined.

Five years' experience of prenatal diagnosis of cystic fibrosis in the former U.S.S.R.

From a total of 490 cystic fibrosis (CF) high-risk families under supervision (mostly Russian Slavs from the European part of the country), DNA data including both direct screening for some CF gene (CFTR) mutations (delF508, G551D and 1677delTA) and allelic polymorphism studies with tightly CF linked DNA markers were collected from 261 families. All full families (129) and 86 CF families with a decreased index child were found to be either fully (42 per cent) or partially (40 per cent) informative for DNA analysis. Prenatal diagnosis (PD) was carried out in 161 CF families. Microvillar enzyme (MVE) assay was applied to all 140 PD at the second trimester either as a single test (88) or in conjunction with DNA analysis (52). The frequency of false-negative results of the MVE assay was 1.3 per cent and that of false-positive results, as judged by the albumin meconium test, was 5.0 per cent. Ambiguous results of MVE analysis were found in 30 cases, 12 of which were verified by DNA analysis. Molecular diagnosis of CF at the first trimester was carried out in 21 cases and four pregnancies were terminated. Altogether, 39 pregnancies with a predicted high risk of CF fetuses were terminated. The low average frequency of delF508 in CF chromosomes of Russian Slavs (50 per cent), its remarkable inter-population variation, and the significant proportion of at-risk families without an affected child determine the necessity of combined molecular and biochemical (MVE assay) approaches for efficient prenatal diagnosis of CF in the former U.S.S.R.

[Analysis of deletions in the dystrophin gene by the multiplex amplification method in patients suffering from Duchenne's muscular dystrophy]. / Analiz deletsii v gene distrofina metodom mul'tipleksnoi amplifikatsii u patsientov, stradaiushchikh miodistrofiei diushenna.

Multiplex polymerase chain reaction was carried out with the material from 68 patients suffering from Duchenne muscular dystrophy in Moscow and Leningrad clinics. Six pairs of oligoprimers were used. Deletions were detected in the material from 22 patients. A new type of deletion was found. Data on deletion frequencies and spectrum were compared with the results published by other authors.

[Analysis of deletion mutations of the dystrophin gene by the multiplex polymerase chain reaction method in the diagnosis of Duchenne muscular dystrophy]. / Analiz deletsionnykh mutatsii gena distrofina metodom mul'tipleksnoi polimeraznoi tsepnoi reaktsii v diagnostike miodistrofii diushenna.

The 33 patients suffering from the Duchenne muscular dystrophy (DMD), 7 healthy donors and a DMD risk family were studied by means of polymerase multiplex chain reaction (MPCR) with 6 oligoprimer pairs for 6 different exons of dystrophin gene. The deletions varying in sizes from 1 to 6 exons were detected in 12 out of 33 DMD patients studied (36.3%). The prenatal diagnosis of DMD was carried out by chorionic villus biopsy on the 1st trimester of pregnancy. Contrary to earlier findings, in elder brother with sever DMD manifestation, no visible deletion was detected in the DNA sample from the male foetus and thus the diagnosis of DMD in foetus was rejected. The perspectives of MPCR in pre and postnatal diagnosis of DMD are discussed.

Frequency of the F508 deletion in cystic fibrosis patients from the European part of the USSR.

The frequency of the F508 deletion (delta F508) has been analyzed in 189 cystic fibrosis (CF) patients from the European part of the USSR, viz. 127 northern Slavonians (Leningrad region), 30 southern Slavonians (the Ukraine), 10 central Slavonians (Moscow region), 14 Moldavians (Kishenev region) and 8 Lithuanians (Vilnius region). The distribution of CF+ chromosomes with and without delta F508 varied significantly in the different ethnic groups studied and correlated with the clinical manifestation of CF. The overall frequency of delta F508 in Slavonian patients is equal to 62.5%, approximately 90% of them being heterozygous or homozygous for this mutation. The frequency of the deletion among 99 Slavonian patients with severe disease manifestation (pancreatic insufficiency, PI) is equal to 67.5%, only 12 patients having pancreatic sufficiency (PS, 17.5%). The highest value of delta F508 (77.4%) is registered in PI/CF patients of the southern Slavonian group; it is much less frequent (about 57%) in relevant groups of Slavonians from the northern and central parts of the country. Unusually low frequencies (24% and 26%) of delta F508 are detected in a few samples of Lithuanian and Moldavian CF patients, respectively. All delta F508+CF-chromosomes of Slavonian origin are associated with haplotypes 2.2.2. defined by the restriction fragment length polymorphism sites KM19/PstI, CS.7/Hin6I and MP6d-9/MspI, although a high proportion (about 25%) of unknown mutations is associated with the same haplotype. Haplotype B (allele 1XV2c/TaqI; allele 2 KM19/PstI) accounts for 91% of delta F508+CF chromosomes. Our data are consistent with the hypothesis of a single origin and subsequent diffusion of this major CF mutation; however, its interpopulational dissemination in Eastern Europe does not follow the suggested south-east to north-west gradient in Western Europe. The significance of these data for prenatal diagnosis and carrier screening of CF mutations is 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.

[Allele polymorphism of the DNA loci MET, D7S8, D7S23, linked to the cystic fibrosis gene in some populations of the USSR, in high risk families and in cystic fibrosis patients]. / Allel'nyi polimorfizm DNK-lokusov MET, D7S8, D7S23, stseplennykh s genom mukovistsidoza, v nekotorykh populiatsiiakh SSSR, v sem'iakh bysokogo riska i u bol'nykh mukovistsidozom.

RELP analysis of DNA loci MET, D7S8 and D7S23 was carried out in Leningrad population and partially in populations of Moscow, Azerbaijan, Ukraine, Buryatia as well as in individuals from high risk families and in cystic fibrosis (CF) patients by means of blot hybridization and polymerase chain reaction. Allelic polymorphism of all loci studied in these three groups was found to be quite similar to that in the North-Western Europe and in whites of the North America. Linkage disequilibrium of the alleles studied with the CF gene was especially pronounced for alleles of the D7S23 locus and gradually decreases from KM-19 through CS-7 to XV-2c DNA probes. The data witness genetic homogeneity of the CF mutation in European populations of the USSR and its similarity to this mutation in Western Europe. The significance of these data for potential diagnosis of CF and for heterozygous carrier detection is 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.

[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.

[The possibility of the incorporation of macromolecules, including exogenous DNA, into the germ cells of male mice. The liposome method and Ca-P coprecipitation method]. / K vozmozhnosti inkorporirovaniia makromolekul, vkluchaia ékzogennuiu DNK, v polovye kletki samtsov myshei. Liposomnyi metod i metod Ca-P kopretsipitatsii.

Liposomes loaded with FITC-labeled albumin in the presence of PEG-1,500 are actively sorbed on the membranes of mature spermatozoa and remain attached even after thorough washing. Immature sperm cells are able to incorporate alien DNA carried by liposomes. In contrast, the mature spermatozoa could not incorporate plasmid DNA loaded with positively charged liposomes. Chlortetracycline in Ca-P coprecipitate crystals is tightly fixed in the postacrosomal region of mature sperms. Intensity of staining of chlortetracycline is stimulated by DNA load in Ca-P coprecipitate as well as by DMSO or EDTA. The method of Ca-P coprecipitation could not provide for plasmid DNA incorporation into taure sperms. Foreign DNA incorporation in postacrosomal regions of sperm heads seems quite possible in experiments with dimethylsulphoxide (DMSO).

[A cytogenetic study of the human chorion for the purpose of the prenatal diagnosis of hereditary diseases]. / Tsitologicheskoe issledovanie khoriona cheloveka s tsel'iu prenatal'noi diagnostiki nasledstvennykh boleznei.

Cytogenetical investigation of 50 diagnostic chorionic villus samples from women with a high risk of giving birth to babies with chromosomal and genic pathology, and of 128 chorionic samples obtained from medical abortions, both on the 8-12th weeks of gestation was performed by means of original direct chromosomal analysis. Chromosomal anomalies were found in 6 cases of diagnostic chorion biopsies (12%) and in 4 cases (3%) of medical abortions. The former group included 5 embryos with autosomal trisomy (4--Ts21 and 1--Ts13) and one embryo with monosomy 18. The latter group contained 2 embryos with X-chromosome monosomy and 2 other with chromosomal mosaicism. A significant prevalence of the female sex was found in the diagnostic group (sex ratio 0.56), but not in the medical abortion one (sex ratio 1.0). Analysis of routine chromosomal preparations and those after in situ hybridization with X-chromosome alfoid-probe YAP 1-10 revealed polyploidy in average in 0.8-1% chorion cells. The feasible causes of sex ratio distortion in embryos of diagnostic group and factors responsible for the rate of polyploidy are discussed. High reliability of originally elaborated direct "shaking-blotting" method of chromosomal preparations from chorionic villus samples is stressed.

[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.

[Activity of enzymes in amniotic fluid in prenatal diagnosis of cystic fibrosis]. / Issledovanie aktivnosti fermentov amnioticheskoi zhidkosti v prenatal'noi diagnostike mukovistsidoza.

The activity of microvillar enzymes--gamma-glutamyltranspeptidase, aminopeptidase, general and intestinal forms of alkalyne phosphotases was studied in amniotic fluid (AF) of 33 women with 25% risk of cystic fibrosis (CF) (mucoviscidoses) in their progeny. The figures obtained in this group were compared with corresponding values of the same enzymes in 100 AF samples from normal pregnancies (negative control) and with 9 AF samples from women which were known to give birth to the children with CF (positive control). CF has been predicted in 5 cases, pregnancies were artificially terminated in 4 women. Biochemical CF prediction was proved by immunochemical assay of albumin contents in meconium of fetal ileum. One woman from the high risk group refused abortion and gave birth to a CF child. Among 26 cases of low CF prediction, 13 resulted in birth of a child without a sign of CF, one - in a child with clear-cut diagnosis of CF and 12 other pregnancies still proceed. The efficiency of complex biochemical, pathomorphological and molecular approaches for verification of intrauterine CF diagnosis in aborted fetuses as well as for detection of heterozygous carriers of CF gene and prenatal diagnosis of CF is discussed.

[Mapping of apolipoprotein A-1 and ceruloplasmin genes on human, rat and mouse chromosomes by hybridization in situ with specific human DNA probes]. / Kartirovanie genov apolipoproteina A-1 i tseruloplazmina na khromosomakh cheloveka, krys i myshei metodom gibridizatsii in situ so spetsificheskimi DNK-zondami cheloveka.

In situ hybridization was carried out on metaphase-prometaphase chromosomes of PGA-stimulated lymphocytes and bone marrow cells obtained from laboratory rats and mice. Plasmid cloned sequences of human apolipoprotein A-1 (Apo A-1) and ceruloplasmin (CP) cDNA fragments have been used as specific probes labelled in nick-translation reaction with 3HdTTP and 3Hd ATP. The data of our study suggest that Apo A-1 is localized in 11q14-22, 9 A2-4 and 5q36 areas in men, mice and rats, respectively. The DNA sequences of human CP cDNA most probably occupy 3q23-25, 13q24-26 and 15q13-20 areas. Heterologous in situ hybridization of other species with DNA probes does not always give reliable results in gene mapping. Thus, the data of heterologous hybridization should be considered with caution.

Chromosomal localization of ceruloplasmin and transferrin genes in laboratory rats, mice and in man by hybridization with specific DNA probes.

Fragments of the natural rat ceruloplasmin (Cp) gene and cDNA copies of rat Cp and transferring (Tf) mRNAs highly labelled by nick translation with 125I-dCTP were used as specific probes for assignment of these genes to the metaphase chromosomes of rat, mouse and man by in situ hybridization. Both Cp and Tf genes were found to be syntenic in rodents, occupying with high probability the regions 9D and 9F1-3 in mice and 7q11-13 and 7q31-34 in rats respectively. The significant increase in silver grain count over chromosome 15 in rats after hybridization with both the Cp and Tf probes suggests the presence of a related pseudogene cluster on this particular chromosome and thus favours its partial homeology to chromosome 7. The localization of silver grains in metaphase chromosome of man indicates subregional assignment of the Tf gene to 3q21. Use of the rat Cp DNA probe does not indicate synteny of the Cp and Tf genes in man and suggests the existence of a related DNA sequence in 15q11-13. The potential and limitations of the in situ hybridization technique with heterologous DNA probes for gene mapping in mammalian species are discussed.

[Characteristics of ceruloplasmin synthesis in mammalian embryogenesis. 2. The yolk sac as the site of the primary expression of the ceruloplasmin gene in rats]. / Osobennosti sinteza tseruloplazmina v émbriogeneze mlekopitaiushchikh. 2. Zheltochnyi meshok kak mesto pervichnoi ékspressii gena tseruloplazmina u krys.

It was shown that the omphaloid placenta and, first of all, visceral wall of yolk sac is the site of primary synthesis of ceruloplasmin (CP), whereas the activation of CP synthesis in the liver cells is secondary and is revealed from the 12th day of embryo-genesis. The CP synthesis in the yolk sac cells proved by selective CP localization in the cells of the yolk sac visceral wall and, first of all, in the cells of visceral endoderm on sections stained by the method of indirect immunofluorescence and using the reaction of soluble peroxidase-antiperoxidase complex. A specific CP-mRNA has been revealed in the yolk sac cells which is actively translated in the polyribosomes isolated from the yolk sac and in the cell-free translation system from the rabbit reticulocytes. on the 14th day of embryogenesis CP amounts to ca. 4% of all polypeptides secreted by the yolk sac cells. As the embryogenesis proceeds, the relative rate of CP synthesis progressively decreases in the yolk sac and increases in the liver cells. CP synthesized by the yolk sac cells has a molecular mass of ca. 122 kD. Possible causes of differences between the "embryonic" and "adult" rat CPs are discussed. A suggestion has been put forward that the time of activation of CP synthesis coincides with the yolk sac formation (8-9th days of embryogenesis) and the cells of visceral endoderm are the site of primary expression of the CP gene.