Aberrant methylation of placental development genes in chorionic villi of spontaneous abortions with trisomy 16.

In humans, aneuploidy is incompatible with the birth of healthy children and mainly leads to the death of embryos in the early stages of development in the first trimester of pregnancy. Trisomy 16 is the most common aneuploidy among spontaneous abortions of the first trimester of pregnancy. However, the mechanisms leading to the death of embryos with trisomy 16 remain insufficiently investigated. One of these potential mechanisms is abnormal placental development, including aberrant remodeling of spiral arteries. Spiral artery remodeling involves the migration of trophoblast cells into the maternal spiral arteries, replacing their endothelium and remodeling to ensure a stable embryonic nutrition and oxygen supply. This is a complex process which depends on many factors from both the embryo and the mother. We analyzed the methylation level of seven genes (ADORA2B, NPR3, PRDM1, PSG2, PHTLH, SV2C, and TICAM2) involved in placental development in the chorionic villi of spontaneous abortions with trisomy 16 (n = 14), compared with spontaneous abortions with a normal karyotype (n = 31) and the control group of induced abortions (n = 10). To obtain sequencing libraries, targeted amplification of individual gene regions using designed oligonucleotide primers for bisulfite-converted DNA was used. The analysis was carried out using targeted bisulfite massive parallel sequencing. In the group of spontaneous abortions with trisomy 16, the level of methylation of the PRDM1 and PSG2 genes was significantly increased compared to induced abortions (p = 0.0004 and p = 0.0015, respectively). In the group of spontaneous abortions, there was no increase in the level of methylation of the PRDM1 and PSG2 genes, but the level of methylation of the ADORA2B gene was significantly increased compared to the induced abortions (p = 0.032). The results obtained indicate the potential mechanisms of the pathogenetic effect of trisomy 16 on the placental development with the participation of the studied genes.

A case report of Pallister-Killian syndrome with an unusual mosaic supernumerary marker chromosome 12 with interstitial 12p13.1-p12.1 duplication.

Pallister-Killian syndrome (PKS) is a rare inherited disease with multiple congenital anomalies, profound intellectual disability, and the presence in the karyotype of sSMC - i(12)(p10). The frequency of PKS may be underestimated due to problems with cytogenetic diagnosis caused by tissue-specific mosaicism and usually a low percentage of peripheral blood cells containing sSMC. Such tissue-specific mosaicism also complicates a detailed analysis of the sSMC, which, along with the assessment of mosaicism in different tissues, is an important part of cytogenetic diagnosis in PKS. Unfortunately, a full-fledged diagnosis in PKS is either practically impossible or complicated. On the one hand, this is due to problems with the biopsy of various tissues (skin biopsy with fibroblast culture is most often used in practice); on the other - a low percentage of dividing peripheral blood cells containing sSMC, which often significantly complicates the analysis of its composition and organization. In the present study, a detailed analysis of sSMC was carried out in a patient with a characteristic clinical picture of PKS. A relatively high percentage of peripheral blood cells with sSMC (50%) made it possible to perform a detailed molecular cytogenetic analysis of de novo sSMC using chromosomal in situ suppression hybridization (CISS-hybridization), multicolor FISH (mFISH), multicolor chromosome banding (MCB), array CGH (aCGH), and quantitative real-time PCR (qPCR), and short tandem repeat (STR) - analysis. As a result, it was found that the sSMC is not a typical PKS derivative of chromosome 12. In contrast to the classical i(12)(p10) for PKS, the patient's cells contained an acrocentric chromosome consisting of 12p material. Clusters of telomeric repeats were found at the both ends of the sSMC. Furthemore, the results of aCGH and qPCR indicate the presence of interstitial 8.9 Mb duplication at 12p13.1-p12.1 within the sSMC, which leads to different representations of DNA from different segments of 12p within cells containing sSMC. The obtained data raise the question of the instability of the sSMC and, as a consequence, the possible presence of additional rearrangements, which, in traditional cytogenetic analysis of patients with PKS, are usually described as i(12)(p10).

Expression of the NUP153 and YWHAB genes from their canonical promoters and alternative promoters of the LINE-1 retrotransposon in the placenta of the first trimester of pregnancy.

The placenta has a unique hypomethylated genome. Due to this feature of the placenta, there is a potential possibility of using regulatory elements derived from retroviruses and retrotransposons, which are suppressed by DNA methylation in the adult body. In addition, there is an abnormal increase in the level of methylation of the LINE-1 retrotransposon in the chorionic trophoblast in spontaneous abortions with both normal karyotype and aneuploidy on different chromosomes, which may be associated with impaired gene transcription using LINE-1 regulatory elements. To date, 988 genes that can be expressed from alternative LINE-1 promoters have been identified. Using the STRING tool, genes (NUP153 and YWHAB) were selected, the products of which have significant functional relationships with proteins highly expressed in the placenta and involved in trophoblast differentiation. This study aimed to analyze the expression of the NUP153 and YWHAB genes, highly active in the placenta, from canonical and alternative LINE-1 promoters in the germinal part of the placenta of spontaneous and induced abortions. Gene expression analysis was performed using real-time PCR in chorionic villi and extraembryonic mesoderm of induced abortions (n = 10), adult lymphocytes (n = 10), spontaneous abortions with normal karyotype (n = 10), and with the most frequent aneuploidies in the first trimester of pregnancy (trisomy 16 (n = 8) and monosomy X (n = 6)). The LINE-1 methylation index was assessed in the chorionic villi of spontaneous abortions using targeted bisulfite massive parallel sequencing. The level of expression of both genes from canonical promoters was higher in blood lymphocytes than in placental tissues (p < 0.05). However, the expression level of the NUP153 gene from the alternative LINE-1 promoter was 17 times higher in chorionic villi and 23 times higher in extraembryonic mesoderm than in lymphocytes (p < 0.05). The expression level of NUP153 and YWHAB from canonical promoters was higher in the group of spontaneous abortions with monosomy X compared to all other groups (p <0.05). The LINE-1 methylation index negatively correlated with the level of gene expression from both canonical (NUP153 - R = -0.59, YWHAB - R = -0.52, p < 0.05) and alternative LINE-1 promoters (NUP153 - R = -0.46, YWHAB - R = -0.66, p < 0.05). Thus, the observed increase in the LINE-1 methylation index in the placenta of spontaneous abortions is associated with the level of expression of the NUP153 and YWHAB genes not only from alternative but also from canonical promoters, which can subsequently lead to negative consequences for normal embryogenesis.

Influence of human peripheral blood samples preprocessing on the quality of Hi-C libraries.

The genome-wide variant of the chromatin conformation capture technique (Hi-C) is a powerful tool for revealing patterns of genome spatial organization, as well as for understanding the effects of their disturbance on disease development. In addition, Hi-C can be used to detect chromosomal rearrangements, including balanced translocations and inversions. The use of the Hi-C method for the detection of chromosomal rearrangements is becoming more widespread. Modern high-throughput methods of genome analysis can effectively reveal point mutations and unbalanced chromosomal rearrangements. However, their sensitivity for determining translocations and inversions remains rather low. The storage of whole blood samples can affect the amount and integrity of genomic DNA, and it can distort the results of subsequent analyses if the storage was not under proper conditions. The Hi-C method is extremely demanding on the input material. The necessary condition for successfully applying Hi-C and obtaining high-quality data is the preservation of the spatial chromatin organization within the nucleus. The purpose of this study was to determine the optimal storage conditions of blood samples for subsequent Hi-C analysis. We selected 10 different conditions for blood storage and sample processing. For each condition, we prepared and sequenced Hi-C libraries. The quality of the obtained data was compared. As a result of the work, we formulated the requirements for the storage and processing of samples to obtain high-quality Hi-C data. We have established the minimum volume of blood sufficient for conducting Hi-C analysis. In addition, we have identified the most suitable methods for isolation of peripheral blood mononuclear cells and their long-term storage. The main requirement we have formulated is not to freeze whole blood.

Comparative cytogenetics of anembryonic pregnancies and missed abortions in human.

Miscarriage is an important problem in human reproduction, affecting 10-15 % of clinically recognized pregnancies. The cases of embryonic death can be divided into missed abortion (MA), for which the ultrasound sign of the embryo death is the absence of cardiac activity, and anembryonic pregnancy (AP) without an embryo in the gestational sac. The aim of this study was to compare the frequency of chromosomal abnormalities in extraembryonic tissues detected by conventional cytogenetic analysis of spontaneous abortions depending on the presence or absence of an embryo. This is a retrospective study of 1551 spontaneous abortions analyzed using GTG-banding from 1990 to 2022 (266 cases of AP and 1285 cases of MA). A comparative analysis of the frequency of chromosomal abnormalities and the distribution of karyotype frequencies depending on the presence of an embryo in the gestational sac was carried out. Statistical analysis was performed using a chi-square test with a p <0.05 significance level. The total frequency of chromosomal abnormalities in the study was 53.6 % (832/1551). The proportion of abnormal karyotypes in the AP and MA groups did not differ significantly and amounted to 57.1 % (152/266) and 52.9 % (680/1285) for AP and MA, respectively (p = 0.209). Sex chromosome aneuploidies and triploidies were significantly less common in the AP group than in the MA group (2.3 % (6/266) vs 6.8 % (88/1285), p = 0.005 and 4.9 % (13/266) vs 8.9 % (114/1285), p = 0.031, respectively). Tetraploidies were registered more frequently in AP compared to MA (12.4 % (33/266) vs. 8.2 % (106/1285), p = 0.031). The sex ratio among abortions with a normal karyotype was 0.54 and 0.74 for AP and MA, respectively. Thus, although the frequencies of some types of chromosomal pathology differ between AP and MA, the total frequency of chromosomal abnormalities in AP is not increased compared to MA, which indicates the need to search for the causes of AP at other levels of the genome organization, including microstructural chromosomal rearrangements, monogenic mutations, imprinting disorders, and epigenetic abnormalities.

Generation of iPS cell line (ICGi040-A) from skin fibroblasts of a patient with ring small supernumerary marker chromosome 4.

Human induced pluripotent stem cell (iPSC) line, ICGi040-A, was obtained from skin fibroblasts derived from a male patient with mosaic ring small supernumerary marker chromosome 4 (sSMS(4)) and infertility. ICGi040-A cells have karyotype 47,XY,+r(4) in 97% of cells and express a set of pluripotent markers, as well as are able to differentiate in vitro into derivatives of all three embryonic germ layers.

Complex biology of constitutional ring chromosomes structure and (in)stability revealed by somatic cell reprogramming.

Human ring chromosomes are often unstable during mitosis, and daughter cells can be partially or completely aneuploid. We studied the mitotic stability of four ring chromosomes, 8, 13, 18, and 22, in long-term cultures of skin fibroblasts and induced pluripotent stem cells (iPSCs) by GTG karyotyping and aCGH. Ring chromosome loss and secondary aberrations were observed in all fibroblast cultures except for r(18). We found monosomy, fragmentation, and translocation of indexed chromosomes. In iPSCs, aCGH revealed striking differences in mitotic stability both between iPSC lines with different rings and, in some cases, between cell lines with the same ring chromosome. We registered the spontaneous rescue of karyotype 46,XY,r(8) to 46,XY in all six iPSC lines through ring chromosome loss and intact homologue duplication with isoUPD(8)pat occurrence, as proven by SNP genotype distribution analysis. In iPSCs with other ring chromosomes, karyotype correction was not observed. Our results suggest that spontaneous correction of the karyotype with ring chromosomes in iPSCs is not universal and that pluripotency is compatible with a wide range of derivative karyotypes. We conclude that marked variability in the frequency of secondary rearrangements exists in both fibroblast and iPSC cultures, expanding the clinical significance of the constitutional ring chromosome.

[Evolutionary Aspects of Genomic Imprinting].

Genomic imprinting is an epigenetic phenomenon that differentiates maternal and paternal copies of genes in the genome and causes monoallelic expression depending on parental origin. Imprinting is an evolutionary puzzle, as it bears the costs of diploidization without its advantages, namely, protection from recessive mutations. The aim of this review is to answer the question of why genomic imprinting arose and became fixed in the evolution of angiosperms, insects, marsupials, and placental mammals.

Generation of iPSC line ICGi024-A from human skin fibroblasts of a patient with ring chromosome 18.

Ring chromosome 18 is a rare chromosomal disorders that usually originate de novo and correlate with clinical manifestation: developmental delay as well as microcephaly, brain and ocular malformations, hypotonia and skeletal abnormalities. We generate iPSC clonal cell line ICGi024-A with pluripotency properties which were demonstrated in vitro by three germ layer differentiation capacity. ICGi024-A can be used for disease modeling and fundamental investigation of ring chromosome instability.

Establishment of an induced pluripotent stem cell line (ICGi025-A) from fibroblasts of a patient with 46,XY,r(8)/45,XY,-8 mosaicism.

Ring chromosomes are structural aberrations commonly associated with disease phenotype. We consider necessary to create the iPSCs with a ring chromosome 8, which can be used for disease modeling and related research. The ICGi025-A iPSCs line was obtained by the reprogramming of the skin fibroblasts from a 1-year-old boy with 46,XY,r(8)/45,XY,-8 mosaicism, developmental delay, microcephaly, dysmorphic features, diffuse muscle hypotonia, moderate proximal muscle weakness, feeding problems, and motor alalia. The iPSCs had expression of the pluripotency-associated markers. In vitro differentiated cells expressed the markers of the cells of three germ layers. That data allowed us to conclude that ICGi025-A cells were pluripotent.

Generation of two induced pluripotent stem cell lines from peripheral blood mononuclear cells of a patient with Wilson's disease.

Wilson's disease is an inherited disorder associated with copper accumulation in the liver, brain and other vital organs. Wilson's disease is caused by mutations in the ATP7B gene. Over 300 mutations of ATP7B have been described. Despite the disease is autosomal recessive, the patient whose PBMCs were reprogrammed in the study harbours heterozygous mutation c.3207C > A (p.H1069Q). Detailed analysis of the ATP7B complete gene sequencing data has not revealed other known disease associated mutation. The generated iPSC lines maintained the original genotype, expressed pluripotency markers, had normal karyotype and demonstrated the ability to differentiate into derivatives of the three germ layers.

Karyotype evaluation of repeated abortions in primary and secondary recurrent pregnancy loss.

PURPOSE: To study the contribution of embryo chromosomal abnormalities in primary and secondary recurrent pregnancy loss (RPL) and to analyze the recurrence of chromosomal constitution in miscarriages from the same couple. METHODS: Retrospective study of abortion karyotypes in RPL families based on the mother's primary or secondary RPL status (563 embryo specimens, 335 samples from primary, and 228 samples from secondary RPL). RPL was defined as two or more consecutive miscarriages. One hundred eight cases of recurrent embryo/fetal loss in 51 families were analyzed to assess the probability of having the same karyotype pattern (recurrent normal or recurrent abnormal) in both previous and subsequent pregnancy loss. The karyotypes of abortions were established using standard cytogenetic analysis, as well as interphase fluorescence in situ hybridization (FISH) and comparative genomic hybridization (CGH). RESULTS: The frequency of aberrations was 43.9% in abortions from primary RPL versus 52.6% in secondary RPL (p = 0.041). Women 35 years of age or older were the main contributors to this difference. The odds ratio of a subsequent abortion having the same karyotype pattern (normal or abnormal) as the previous one was 6.98 (p = 0.0013). CONCLUSION: The frequency of abnormalities is higher in abortions from the secondary RPL versus primary RPL group, and this difference is due to the relative deficiency of miscarriages with abnormal karyotypes in older women with primary RPL. The probability of having the same karyotype pattern (recurrent normal or recurrent abnormal) in the previous and subsequent abortion is increased significantly compared with chance.

Generation of four iPSC lines from two siblings with a microdeletion at the CNTN6 gene and intellectual disability.

The human induced pluripotent stem cell (iPSC) lines, ICGi009-A, ICGi009-B, ICGi013-A and ICGi013-B, were generated from skin fibroblasts of two siblings with intellectual disability. Both patients were carriers of CNTN6 gene microdeletion (Kashevarova et al., 2014). iPSC lines have normal karyotype, express pluripotency markers, are able to differentiate in vitro into derivatives of all three germ layers and represent a unique tool to study neurodevelopmental disorders.

Generation of the induced pluripotent stem cell line, ICAGi002-A, from unaffected carrier megabase scaled duplication involving the CNTN6 gene.

The 3p26.3 microduplication involving the CNTN6 gene cause developmental delay and the intellectual disability. However, the incomplete penetrance is described for this copy number variation (CNV). Here we describe ICAGi002-A line, which is supposed to use as a model for studying of the penetrance of the CNV in 3p26.3. The ICAGi002-A iPSCs line was obtained by the reprogramming of the skin fibroblasts from a healthy donor with 3p26.3 microduplication involving the CNTN6 gene. The ICAGi002-A cells was pluripotent as it was shown by the expression of the pluripotency-associated markers and in vitro differentiation into the cells of three germ layers.

Induced pluripotent stem cell line, ICAGi001-A, derived from human skin fibroblasts of a patient with 2p25.3 deletion and 2p25.3-p23.3 inverted duplication.

Skin fibroblasts from a patient with developmental delay and chromosome 2p25.3 deletion syndrome were reprogrammed into induced pluripotent stem cells (iPSCs) and the clonal stem cell line ICAGi001-A (iTAF9-11) was established. ICAGi001-A pluripotency was demonstrated in vitro by three germ layer differentiation capacity. This line is a good model for studying of the developmental delay and brain disorder.

Induced pluripotent stem cell line, IMGTi003-A, derived from skin fibroblasts of an intellectually disabled patient with ring chromosome 13.

Skin fibroblasts from a patient with neurodevelopmental and speech delay, anxiety disorder, macrocephaly, microorchidism, multiple anomalies of internal organs and ring chromosome 13 were reprogrammed into induced pluripotent stem cells (iPSCs) to generate a clonal stem cell line IMGTi003-A (iTAF6-6). IMGTi003-A pluripotency was demonstrated by three germ layer differentiation capacity in vitro, and this cell line had a mosaic karyotype with 46,XY,r(13) as a predominant cell subpopulation. IMGTi003-A line is a good model for studying of the mitotic instability of the ring chromosome 13.

Generation of two iPSC lines (IMGTi001-A and IMGTi001-B) from human skin fibroblasts with ring chromosome 22.

Skin fibroblasts from a patient with intellectual disability and ring chromosome 22 were reprogrammed into induced pluripotent stem cells (iPSCs) to establish a clonal stem cell lines, IMGTi001-A (iTAF5-29) and IMGTi001-B (iTAF5-32). Because of ring chromosome mitotic instability these cell lines show mosaic karyotypes with 46,XX,r(22) in >83% cells, 45,XX,-22 as minor class and sporadically cells with other karyotypes. Differentiation in derivatives of all three germ layers was shown in teratoma assay for IMGTi001-A, and in embryoid bodies for both cell lines. To our knowledge, human iPSC lines with ring chromosome are described for the first time.

[Preimplantation Genetic Diagnosis by Blastocentesis: Problems and Perspectives].

The discovery of cell-free DNA in blastocoele fluid opens new perspectives for the development of preimplantation genetic diagnosis of human chromosomal and genetic diseases. In this review we analyzed the results of the first studies, which made it possible to evaluate the effectiveness of the application of a new source of biological material and showed a high degree of agreement between the results of molecular karyotyping with cell-free DNA and blastocyst cells. The results suggest the possibility of developing a noninvasive method of preimplantation genetic diagnosis, which may open a new round of progress in the field of assisted reproductive technologies and the genetics of early stages of human ontogenesis.

MOLECULAR KARYOTYPING BY USING CELL-FREE DNA FROM HUMAN BLASTOCOELE FLUID, EMBRYOBLAST AND TROPHOBLAST CELLS.

In the present study, we have carried out a comparative analysis of molecular karyotypes of cell free DNA from blastocoele fluid, trophectoderm and inner cell mass of human blastocysts at the preimplantation stages of development, using the comparative genomic hybridization. Different types of chromosomal abnormalities distribution between trophectoderm, inner cell mass and blastocoele fluid were identified. It was first shown that the source of cell free DNA in the blastocoele fluid may arise from the cells of inner cell mass in addition to the trophectoderm cells, which are traditionally used in preimplantation genetic diagnosis of chromosomal aberrations. These results support the promising of blastocentesis as a new technology of preimplantation genetic diagnosis, which provides the possibility of integral analysis of chromosome abnormalities in different cells of the blastocyst.

[Estimation of association of CNTN6 copy number variation with idiopathic intellectual disability].

Analysis of the prevalence of copy number variations of the CNTN6 gene, recently selected as a new candidate gene for intellectual disorders, was performed. Real-time PCR did not detect any change in the number of CNTN6 gene copies in a group of 200 patients with impaired intellectual development. However, taking into account our data from the previous aCGH analysis and published data, the overall frequency of microdeletions and microduplications of CNTN6 was estimated as 1: 265 (0.4%). The common phenotypic features of 40 patients with microdeletions and microduplications of CNTN6 appeared to be the autism spectrum disorders, developmental delay, intellectual disability, seizures, cognitive impairment, cardiological defects, and behavioral problems.