[Genetic and clinical characteristics of 22q11.2 deletion syndrome].

In a group of 140 patients with typical phenotype, the 22q11.2 microdeletion was detected in 43 patients (32%) using FISH and MLPA methods. There were no deletions of other chromosomal loci leading to phenotypes similar to the 22q11.2 deletion syndrome (22q11.2DS). Sequencing of the TBX1 gene did not detect any mutations, except for some common neutral polymorphisms. For the first time in the Russian Federation, the diagnostic efficiency of 22q11.2DS appeared to be 32%, as a result of the application of a combination of genetic approaches for a large group of patients with suspected 22q11.2DS.

[Generation and application of dynamic standard reference intervals for analyzing results of comparative genomic hybridization].

The present work was aimed at generating the dynamic standard reference intervals (DSRI) and their application for chromosomal-aberration (CA) analysis. The evaluation of the generated DSRI was performed using the DNA samples from four patients with already known CA. High-resolution comparative genomic hybridization analysis (HR-CGH) allowed us to not only identify all of the CAs, that were not revealed by CGH, but also to detect the breakpoints and to determine the size of chromosomal imbalance.

Hidden X chromosomal mosaicism in a 46,XX male.

We report on a 37-year-old XX male with complex hidden X chromosomal mosaicism. The patient had fully mature male genitalia with hypoplastic testes descended in the scrotum and no sign of undervirilization. Hormonal examination demonstrated hypergonadotropic hypogonadism, semen analysis showed severe oligoasthenoteratozoospermia. In situ hybridization revealed the presence of 3 SRY-positive cell lines bearing 1, 2 or 3 X chromosomes. Skewed inactivation of the paternal SRY-bearing X chromosome was detected by molecular analysis of the androgen receptor gene.

Unique mosaic X/Y translocation/insertion in infant 45,X male.

We report on a 45,X male with hydrocephaly, lobar holoprosencephaly and ichthyosis. In situ hybridization and molecular analysis have demonstrated the presence of a mosaic SRY-bearing derivative X chromosome that included Yp and heterochromatic Yq fragments.

Analysis of cell-free fetal DNA in plasma and serum of pregnant women.

Sixty blood samples from pregnant women during gestational weeks 9-28 were investigated. Cell-free fetal DNA was extracted from maternal plasma or serum to be detected by nested PCR for determination of fetal gender. The SRY gene as a marker for fetal Y chromosome was detected in 34/36 women carrying a male fetus. In 3/24 women carrying female fetuses, the SRY sequence was also detected. Overall, fetal sex was correctly predicted in 91.7% of the cases. Therefore, the new, non-invasive method of prenatal diagnosis of fetal gender for women at risk of producing children with X-linked disorders is reliable, secure, and can substantially reduce invasive prenatal tests.

[Investigation of fetal cells isolated from maternal blood by different methods]. / Issledovanie kletok ploda, vydelennykh iz krovi materi razlichnymi sposobami.

This paper presents the results of investigation of the isolated from maternal blood by 4 different methods according to the optimized protocols. All women had male fetuses. The mononuclear cells with fetal erythroblasts were preisolated by using density-gradient centrifugation of the maternal blood in the Ficoll solution. Fetal cells were detected by FISH for Y-chromosomal sequences. The fetal cells were 2.1% by fluorescence activated cell sorting (FACS); 3.8% by magnetic field sorting, and 2.6% by two-stage density gradient precipitation. The fetal lymphocytes were investigated through air-cultivation of peripheral maternal lymphocytes. Their proportion was 3.9% in the culture samples. The findings lead to the conclusion that the new non-invasive approach is useful for prenatal diagnosis of chromosomal aneuploidies.

[A noninvasive approach to studying fetal cells for prenatal diagnosis of chromosomal aneuploidies]. / Neinvazivnyi podkhod k issledovaniiu kletok ploda v tseliakh prenatal'noi diagnostiki khromosomnykh aneuploidii.

Fetal cells isolated from maternal peripheral blood during the second trimester of pregnancy were analyzed. Blood samples were centrifuged in a Ficoll-Paque gradient, the mononuclear cell fraction was isolated and stained with fluorescent monoclonal antibodies against glycophorine A (GPA + PE), transferrin (CD71 + FITC), and Hoechst 33342. Fluorescence-activated cell sorting (FACS) was conducted on a Vantage flow cytofluorimeter (Becton Dickinson). Fluorescence in situ hybridization (FISH) with Y chromosome-specific DNA probe revealed fetal cells that exhibited Y signal in all 20 blood samples obtained from women pregnant with healthy male fetuses. The concentration of these fetal cells averaged about 1.34% and ranged from 0.1 to 4.2% in different blood samples. In six cases, blood samples were obtained from pregnant women, in which prenatal cytogenetic analysis revealed various fetal aneuploidies. Using FISH with DNA probes specific for chromosomes X, 18, and 13/21, Fetal cells with chromosomal aberrations were detected in these six maternal blood samples at a concentration from 1.5 to 5.6% (on average 3.7%). These results indicate the possibility of a new noninvasive approach, which is safe for both mother and fetus when used for isolation of fetal cells from pregnant women's blood samples and prenatal diagnosis of a broad spectrum of fetal cell chromosomal aberrations.

[Maternal blood fetal cells: new noninvasive approach in prenatal diagnosis of hereditary diseases]. / Kletki ploda v krovi materi: novyi neinvazivnyi podkhod v prenatal'noi diagnostike nasledstvennykh boleznei.

A new noninvasive approach to prenatal diagnosis of hereditary diseases is being actively developed, which is based on the use of different fetal cells contained in pregnant females. Due to the fact that the native concentration of fetal cells is extremely low, their isolation requires the application of different know-how enrichment and sorting techniques. Either the FISH method with chromosome-specific probes or PCR is used to examine the cell fraction isolated, which detects fetal sex, Mendelian disorders such as beta-globin mutations. Promising results in the prenatal diagnosis of chromosomal aneuploidies were achieved in isolating and examining fetal erythroblasts. The results of numerous studies on the optimization of a protocol for isolating and reliably examining fetal cells from the blood of pregnant females allow the new noninvasive approach to the prenatal diagnosis of hereditary diseases to be considered to be highly promising.

[Dependence of the concentration of maternal serum markers on the haptoglobin gene]. / Zavisimost' kontsentratsii materinskikh syvorotochnykh markerov ot gena gaptoglobina.

This paper is the first in a series devoted to the investigation of the possible effect of maternal genotype on the levels of fetal alpha-fetoprotein (AFP) and human chorionic gonadotrophin (HCG) entering maternal blood. We studied the possible association between the maternal haptoglobin system and levels of maternal AFP and HCG, which are markers of fetal pathology. Haptoglobin types were determined in groups of pregnant women with different levels of serum markers. Distribution of haptoglobin types differed from the theoretically expected in one out of five groups tested. In the group with low AFP level, a significant decrease in frequency of the Hp*2 allele was found. A statistically significant decrease of the mean haptoglobin concentration in the group with low levels of AFP and elevated levels of HCG was observed. A reduction in serum haptoglobin concentration was shown to be accompanied by a decrease of serum iron concentration. The possible mechanism underlying the influence of maternal genotype on the levels of AFP and HCG in maternal blood are discussed.

[Chorionic gonadotropin concentration dependence on the phenotype of the Rhesus system]. / Zavisimost' kontsentratsii khorionicheskogo gonadotropina ot fenotipa sistemy rezus.

The phenotypes of the rhesus system in pregnant females were studied for impact on maternal blood chorionic gonadotropin (CGT) levels in the second trimester. The polymorphism of the rhesus system was determined in the groups of pregnant females (n = 1591) having different blood CGT levels. There was a significant increase in the frequency of the Rh(d) phenotype (p < 0.01) among the pregnant women with lower chorionic gonadotropin levels. There was also an evident bimodality in the distribution among RH(d) pregnant females.

[Chromosomal mosaicism of extraembryonic cell membrane, detected during prenatal diagnosis]. / Khromosomnyi mozaitsizm kletok vnezarodyshevykh obolochek, vyiavlennyi pri provedenii prenatal'noi diagnostiki.

Data on detection of chromosomal mosaicism in amnionic cells and chorionic villi obtained by prenatal cytogenic diagnosis are presented. The frequency of chromosomal mosaicism in preparations of amniotic fluid cell culture was 2.6% (6 out of 226), and that in "direct" villus preparations was 1.6% (13 out of 774). The necessity perform an additional analysis of other fetal cells or neonatal lymphocytes to specify the diagnosis was shown. The analysis of the outcome of pregnancies during which chromosomal mosaicism in the extraembryonic cells was detected indicates that these women from a high-risk group of at, both genetically and obstetrically: in only 8 out of 19 cases did pregnancies end in normal deliveries at term; in three cases, spontaneous abortions occurred at 16-31 weeks of gestation; in three cases, the pregnancies were terminated due to fetal chromosomal aberrations in nonmosaic form; the outcome of pregnancy in five cases was preterm delivery of an underweight newborn.

[Various biochemical parameters in serum of pregnant women carrying fetuses with various congenital defects of development]. / Nekotorye biokhimicheskie pokazateli syvorotki krovi beremennykh plodom s riadom vrozhdennykh porokov razvitiia.

The number of changes in some maternal serum biochemical parameters for some congenital malformations are presented. In all pregnancies we observed low levels of LAT, AST, CPK, HBD, AP and high levels of GGTP, THY, amylase. These data demonstrate total depression in metabolism of women with an affected fetus. The biological basis of altered levels of enzymes in pregnancies with affected fetus is unclear.

[Translocation 11q;22q: a clinico-cytogenetic study]. / Translokatsii 11q; 22q: kliniko-tsitogeneticheskoe issledovanie.

Seven families with translocations t(11; 22) identified at our Institute and analysis of the literature showed that the imbalance resulted from such translocations is always due to nondisjunction 3:1. Nondisjunction occurs more often in the 1st meiotic division, and is more rare in the second one. Expressed prezygotic selection against spermia with an additional chromosome greatly increases the risk of having an imbalanced child for the women-carriers as compared to men-carriers. The phenotype of the patients with +der(22)t(11; 22) is composed of the features characteristic for trisomy 22q (cleft lip and palate, preauricular papillomas and fistulas, rectal atresia or stenosis) and trisomy 11q (long philtrum with the upper lip hanging over, renal al; asia and hypoplasia). Diaphragmatic hernias are found to be common for the patients with +der(22)t(11; 22).

[Significance of the maternal levels of serum alpha-fetoprotein, human chorionic gonadotropin and unconjugated estriol in various congenital developmental defects in pregnancy trimester II]. / Znacheniia urovnei materinskogo syvorotochnogo al'fa-fetoproteina, khorionicheskogo gonadotropina cheloveka i nekon"iugirovannogo estriola pri riade vrozhdennykh porokov razvitiia vo II trimestre beremennosti.

Maternal serum alpha-fetoprotein (MSAFP), human chorionic gonadotropin (hCG) and unconjugated oestriol (uE3) concentrations were measured in maternal serum samples from 21 pregnancies with neural-tube defects, 4 pregnancies with ventral wall defects (VWD) and 1662 unaffected pregnancies in women. These congenital malformations were confirmed by ultrasound scanning. The mean multiplate of the median (MoM) for AFP and uE3 was significantly different from the control values in cases of open NTD (AFP median MoM = 5.95, p < 0.001, uE3 median MoM = 0.2, p < 0.001), while hCG values did not differ from those of matched controls (hCG median MoM = 0.9). The biological basis of altered levels of uE3 in pregnancies with fetal NTDs is unclear.

[Prenatal diagnosis as a method of prevention of congenital and hereditary diseases]. / Prenatal'naia diagnostika kak put' profilaktiki vrozhdennykh i nasledstvennykh zabolevanii.

The program of the prevention of congenital and hereditary diseases with the aid of prenatal diagnosis includes a complex of different methods: ultrasonography, invasive procedures made at different times of pregnancy, obstetrical monitoring, immunochemical blood tests, fetal cytogenetic analysis, pathological, anatomical, and syndromological studies in abortuses. Emphasis is laid on the use of the data on ultrasound screening of the pregnant and screening of the mother's blood for some factors that form a group of women at a greater genetic risk, who require prenatal diagnosis. The efficacy of the preventive measures can be enhanced with combined use of instrumental, obstetrical and laboratory research methods. The establishment of the correct and early diagnosis may, on the one hand, remove the tension and concern in the family; on the other hand, it may prevent bearing a sick child and provide the married couple with a based genetic counselling about progeny.

[Detection of chromosomal abnormalities using cordocentesis]. / Vyiavlenie khromosomnoi patologii u plodov pri ispol'zovanii kordotsenteza.

Four cases of cytogenetic prenatal diagnosis of fetuses with chromosomal aberrations are presented: (1) the Patau syndrome; (2) and (4) the Down syndrome; (3) the Klinefelter syndrome. Cordocentesis has been shown to be expedient for rapid and accurate determination of fetus karyotype. Indicative for cytogenetic examination were ultrasonic data, maternal age, the values of AFP, HGG and nonconjugated estreol in maternal serum. Comparison of ultrasonic examination of fetuses with the data on abortus autotopsia was undertaken. The results demonstrate importance of ultrasonic, cytogenetic, biochemical and morphological research in prenatal malformation diagnosis.

[Results of the cytogenetic prenatal diagnosis in the first and second trimesters of pregnancy]. / Rezul'taty tsitogeneticheskoi prenatal'noi diagnostiki v I i II trimestrakh beremennosti.

The authors analyze the results of prenatal diagnosis carried out in women during the first and second pregnancy trimesters. Cytogenetic screening was carried out in risk-group pregnant women, i.e. in women aged over 36, in cases with family history of chromosome mutation or a previous child with chromosome aberration, or a sex-linked disease in one of family members, etc. Chromosome preparations from amniotic fluid cell cultures and 'direct' chromosome preparations from chorionic villi were analyzed. The authors discuss the efficacy of prenatal diagnosis in various pregnancy periods, fetal tissue collection methods and quantities necessary for the examination.

Use of 4-trifluoromethylumbelliferyl-alpha-L-iduronide as a new substrate for detection of alpha-L-iduronidase deficiency in human tissues and for rapid prenatal diagnosis of Hurler disease.

Results are presented of alpha-L-iduronidase assays in the leukocytes of normal individuals, patients with Hurler disease and heterozygous carriers. The assays were carried out using 4-methylumbelliferyl-alpha-L-iduronide and 4-trifluoromethylumbelliferyl-alpha-L-iduronide as substrates. It was shown that 4-trifluoromethylumbelliferyl-alpha-L-iduronide, along with the commonly used 4-methylumbelliferyl-alpha-L-iduronide, can serve as a specific substrate for alpha-L-iduronidase and is therefore suitable for demonstrating the enzyme deficiency in patients with Hurler disease, as well as the decrease of enzyme activity in heterozygous disease carriers. Using the two substrates a prenatal diagnosis of Hurler disease in a fetus was made on the basis of the lack of enzyme activity in amniotic fluid cell cultures. The diagnosis was confirmed by the results of alpha-L-iduronidase activity assay in fetal liver and kidney. It was found that 4-trifluoromethylumbelliferyl-alpha-L-iduronide is highly efficient for the rapid detection of alpha-L-iduronidase deficiency directly in pieces of tissues and in placenta, which is important for the prenatal diagnosis of Hurler disease.

[Use of 4-methylumbelliferryl-alpha-L-iduronide and 4-trifluoromethylumbelliferryl-alpha-L-iduronide for detecting alpha-L-iduronidase deficiencies in human tissue and for rapid prenatal diagnosis of Hurler disease]. / Ispol'zovanie 4-metilumbelliferil-alpha-L-iduronida i 4-triftormetil- umbelliferil-alpha-L-iduronida dlia vyiavleniia nedostatochnosti alpha-L-iduronidazy v tkaniakh cheloveka i dlia bystroi prenatalnoi diagnostiki bolezni gurler.

Activity of alpha-L-iduronidase was studied in leukocytes of healthy persons, of patients with Hurler disease and of heterozygous carriers of the disease where 4-methylumbelliferyl-alpha-L-iduronide and 4-trifluoromethylumbelliferyl-alpha-L-iduronide were used as substrates. 4-Trifluoromethylumbelliferyl-alpha-L-iduronide proved to be also a specific substrate of alpha-L-iduronidase and enabled to detect the enzyme deficiency in patients with Hurler disease as well as a decrease of the enzymatic activity in heterozygous carriers of the disease. Using these two substrates prenatal diagnosis of Hurler disease was carried out in fetus which exhibited absence of the enzymatic activity in cell culture from amniotic fluid. The diagnosis was corroborated after analysis of alpha-L-iduronidase activity in liver and kidney tissues of the fetus. 4-Trifluoromethylumbelliferyl-alpha-L-iduronide was very effective in express detection of alpha-L-iduronidase deficiency immediately in tissue slices as well as in placenta which is of importance in prenatal diagnosis of Hurler disease.

[Prenatal diagnosis of triploidy by transabdominal aspiration of chorionic villi]. / Prenatal'naia diagnostika triploidii pri transabdominal'noi aspiratsii vorsin khoriona.

A case of triploidy identified in second trimester fetal diagnosis is presented. Cytogenetic study was undertaken in chorionic willi obtained by transabdominal placentocentesis. The diagnosis was confirmed by cytogenetic analysis of cultured amniotic fluid cells. The observation was revealed within a programme of combined ultrasound and cytogenetic prenatal monitoring, in association with maternal age. The fetus aborted at 23 weeks of pregnancy was abnormal, including congenital malformations and hypoplasia of internal organs and placenta.