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Corporal segments in man
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DOI: http://dx.doi.org/10.20534/ELBLS-17-1-69-73
Gudkov Georgy Vladimirovich, State budgetary educational institution of higher education
"Kuban State Medical University" of Russian Ministry of healthcare, Krasnodar City Doctor of medicine, Professor of Department of clinical immunology, allergology and laboratory diagnostic, Faculty of Vocational Training and Professional Retraining of Specialists E-mail: pol09@mail.ru Filippov Evgeny Fedorovich, State budgetary educational institution of higher education
"Kuban State Medical University" of Russian Ministry of healthcare, Krasnodar City Doctor of medicine, Head of Department of clinical immunology, allergology and laboratory diagnostic, Faculty of Vocational Training and Professional Retraining of Specialists E-mail: filippovef@mail.ru Piven Aleksandr Vladimirovich, Federal state budgetary educational institution of higher education
"Kuban State Medical University", Krasnodar City Student of Master degree program "Biochemistry and molecular biology" E-mail: alexander_piven@mail.ru
Zolotavina Mariya Leonidovna, Federal state budgetary educational institution of higher education "Kuban State Medical University ", Krasnodar City Candidate of biological sciences, associate professor of Biochemistry
and Physiology Department, E-mail: zolotavina-m@mail.ru Kim Natalya Viktorovna, Municipal budgetary healthcare institution "Children's city hospital № 1", Krasnodar City Doctor of clinical laboratory diagnostics E-mail: mea-terra@list.ru
Early diagnostics of fetal aneuploidies by analyzing the polymorphisms of microsatellite DNA of trophoblasts circulating in the maternal bloodstream
Abstract: available risks of invasive prenatal diagnostics in the course of aneuploidy analysis actualize the technological development of non-invasive prenatal diagnostics based on the analysis of short tandem repeats of microsatellite DNA of trophoblasts circulating in the maternal bloodstream.
Keywords: non-invasive prenatal diagnostics, flow cytometry, trophoblasts, trisomy, quantitative fluorescent polymerase chain reaction, whole genome amplification, microcatellite markers.
Methods of invasive prenatal diagnostics allow quiet Material and methods
accurately to detect possible chromosomal abnormalities We have analyzed peripheral blood samples obtained
of the fetus, however, involve the risk of complications: from 15 women with singleton pregnancy in the 8th -13th
threatened miscarriage, development of hemorrhage, etc. [1, 3-9; 2, 113-126]. The alternative approach is a noninvasive prenatal diagnostics, the material for which is considered to be the captured trophoblasts circulating in the maternal blood [3, 5-11; 4, 311-313; 5, 131-139]. Their genetic material can serve as the source of fetal DNA for prenatal genetic screening since the 9th week of gestation [6, 3-9; 7, 308-318; 8, 508-520]. However, extremely low content of trophoblasts in the maternal bloodstream requires improvement of molecular diagnostics methods based on the use of genetic material obtained from isolated cells [9, 218-227; 10, 575-579; 11, 10-20]. First of all, it is necessary to select conditions under which the library of amplified fragments will represent genome of an isolated cell, what will improve efficiency of the fragment analysis of short tandem repeats of microsatellite fetal DNA in the chromosomes, which are the most exposed to numerical aberrations [12, 1009-1019; 13, 65-76; 14, 991-997].
The purpose of the study was to conduct the early diagnostics of fetal aneuploidies based on the analysis of short tandem repeats of microsatellite DNA of tropho-blasts circulating in the maternal bloodstream.
week of gestation from the group with high risk of giving birth to children with congenital pathology. The average age of the surveyed women was 28 years (ranging from 23 to 37 years). In 8 cases (53,3%) the current pregnancy was the first one, and in the rest of the cases — a consecutive one.
Mononuclear cells were isolated from the peripheral heparinized venous blood of pregnant women by density gradient centrifugation "Histopaque-1077" (1.077 g/ml). The obtained sample of mononuclear cells (~ 2x10 7 cells in 800 mcl) was subjected to the negative immunomagnetic separation by markers of mature linearly differentiated cells (kit BD Human Lineage Cell Depletion Set). The negative fraction of cells was dyed with antibodies against HLA-G, Trop-2, CD45 (BD Biosciences, USA), and sorting of tropho-blasts into individual tubes was performed with the flow cytometer sorter BD FACS Arialll (BD Biosciences, USA). Whole genome amplification of DNA of sorted cells was performed with the use of reagent kit PicoPLEX™ WGA Kit (Rubicon Genomics, USA) with subsequent quantitative fluorescent PCR (QF PCR) by markers corresponding to short tandem repeats (STR) of microsatellite DNA of chromosomes
Early diagnostics of fetal aneuploidies by analyzing the polymorphisms of microsatellite DNA.
(reagents Aneufast Multiplex QF-PCR Kit, Genomed, United Kingdom). Fragment analysis of QF PCR products was performed on the genetic analyzer ABI PRISM 3500xL (Applied Biosystems, USA), using the program GeneMapper® Software v5.0.
Results
Density gradient centrifugation with subsequent negative magnetic separation by linear markers of mature hematopoietic cells allowed us to obtain the sample that
was maximally enriched with trophoblasts. The average concentration of the separated cells after the negative separation was ~ 4,72x10 5 cells/ml; depletion degree exceeded 90% (93,1±2,14%).
For subsequent cytometry and sorting within the gate of living nucleated cells, which were negative against the pan-leukocytic marker (CD45), double positive gate HLA-G+Trop-2+ was separated and then was used for further sorting (Figure 1).
Figure 1. Separation of trophoblast sorting gate (Trop-2+HLA-G+) among the population of cells, negative against the marker CD45
The number of cell-candidates, which meet the criteria for sorting CD45-HLA-G+Trop-2+, amounted to 34.4±10.3 cells. These cells after sorting into individual tubes were subjected to whole genome amplification, upon completion of which the electrophoretic control
of individual cells was used for subsequent multiplex QF-PCR to STR-markers of fetal chromosomes (table 1), with subsequent separation of products by capillary electrophoresis. As the greatest density ofDNA fragments fell to the range of400-700 base pairs, the shortest STR-
showed the sufficient concentration of DNA fragments - markers were used (less than 300 base pairs) for 21st, 18th 56,8±10,1 ng/mcl. The library ofamplified DNAfragments and 13th chromosomes (table 1).
Table 1. - STR-markers for detection of polymorphisms in the 21st, 18th and 13th autosomes (maximum ranges of allele lengths are indicated)
chromosome 21 chromosome 18 chromosome 13
D21S1442(136-174 bp) D18S535(126-156 bp) D13S631 (192-218 bp)
D21S1435(142-188 bp) D18S391 (144-168 bp) D13S258(230-281 bp)
D21S1446(200-228 bp) D18S976(164-184 bp) -
Also fetal sex determination was carried out using pseudoautosomal markers of sex chromosomes — X22 (189-253 bp), DXYS267 (330-354 bp) and DXYS218 (266-294 bp), the X-linked HPRT-marker (264-313 bp) and also two markers against non-polymorphic loci — SRY-gene (463 bp) and amelogenin (AMXY: 104 and 109 bp).
Table 2 presents the summarized data about infor-mativity of genotypes of all STR-marker kits for each of the studied fetal chromosomes. Only two (13,3%) out of15 samples had significant contamination of maternal DNA along with a small number of separated tropho-
blasts (observations № 1 and № 4, table 2), what did not allow to conduct an adequate assessment because of significant decrease in the height of allele peaks. Fetal sex was determined in all 15 cases (10 — male and 5 — female). Fetal aneuploidy (trisomy) was detected in 2 pregnant women on the 21st chromosome (observations № 5 and № 14) and in one woman — on 18 th chromosome (observation № 9). The results obtained in retrospective analysis fully corresponded to the data of chorion biopsy both in determination of fetal sex and type of aneuploidy.
Positive correlation between the number of separated trophoblasts and the number of informative peaks of STR-markers for all studied chromosomes amounted to r = + 0,69. Relation between the length of the STR-marker fragment and the number of its successful identifications in the course of capillary electrophoresis was negative and amounted to r = - 0,61.
Thus, size of the fragments, obtained through whole genome amplification of genetic material of individual cells, complicates the reproduction of long alleles of STR-markers. However, this problem can be minimized by increasing the number of separated trophoblast cells.
Table 2. - STR-markers and their genotypes in separated trophoblasts
Gestational age i o Informativity of STR-markers chromosomes
t is « 21st chromosome 18th chromosome 13th chromosome * + ¥
№ is 'i er lab -g ¡25 D21S1442 D21S1435 D21S1446 D18S535 D18S391 D18S976 D13S631 D13S258 -3 1 1 21-th 18-th 13-th >-i T3 9 x
1. 9 10 — — — — — — — — — — — — XY
2. 11 25 + + + + + — + + 7 2n 2n 2n XY
3. 10 26 + — — + + + + — 5 2n 2n 2n XX
4. 8 18 + — — + — — — — 2 2n — — XY
5. 10 45 + + + + + + + + 8 3n 2n 2n XY
6. 12 38 + + — + + + + + 7 2n 2n 2n XY
7. 9 36 + + — + + + + — 6 2n 2n 2n XX
8. 9 53 + + + + + + + + 8 2n 2n 2n XY
9. 11 27 + + — + + + + — 6 2n 3n 2n XY
10. 8 34 + + + + + + + — 7 2n 2n 2n XY
11. 13 44 + + + + + — + + 7 2n 2n 2n XX
12. 10 41 + + — + + + + + 7 2n 2n 2n XY
13. 9 38 + + — + + + + + 7 2n 2n 2n XY
14. 8 39 + + + + + + + — 7 3n 2n 2n XX
15. 11 24 + + — + — + + + 6 2n 2n 2n XX
Number «+» 13 12 6 13 12 11 13 8
Discussion
The current study demonstrated the possibility for technological optimization of enriching and sorting the trophoblast cells that circulate in maternal blood and also use of QF-PCR with subsequent capillary electrophoresis of fluorescent product for analysis of STR-markers of fetal chromosomes, which are the most exposed to numerical aberrations, on the basis of genome of the isolated trophoblasts.
The genomic library of fragments, which represents genome of isolated cells for conduct of microsatellite analysis, is obtained by the method based on MALBAC-technology (Multiple Annealing and Looping-Based Amplification Cycles) [15, 79-102]. The use of this method allowed us to obtain the high concentration of amplified DNA, mostly consisting of fragments about 400-700 bp in length, representing the genome of isolated trophoblast cells.
Microsatellite analysis, carried out in the course ofQF PCR, was performed with account of the limited length of amplified DNA fragments of the matrix, so sequence length of the selected STR-markers of autosomes did not exceed 300 bp. For sex chromosome identification, markers against pseudoautosomal sequences, X-linked marker and two markers against non-polymorphic sequences (SRY-gene and amelogenin) were used. The set of microsatellite markers, which was used in this study, allowed to detect aneuploidies in 3 pregnant women and to determine fetal sex in all cases, as well as to exclude mosaicism and contamination of maternal DNA sample.
Thus, the set of methods implemented on the basis of genome of the trophoblasts isolated from the blood of pregnant woman in early gestation-term, allowed to fulfill the early screening offetal aneuploidy by the chromosomes, which are the most exposed to numerical aberrations, as well as to determine fetal sex.
Early diagnostics of fetal aneuploidies by analyzing the polymorphisms of microsatellite DNA...
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