Identification of differentially methylated genes in first-trimester placentas with trisomy 16.

The presence of an extra chromosome in the embryo karyotype often dramatically affects the fate of pregnancy. Trisomy 16 is the most common aneuploidy in first-trimester miscarriages. The present study identified changes in DNA methylation in chorionic villi of miscarriages with trisomy 16. Ninety-seven differentially methylated sites in 91 genes were identified (false discovery rate (FDR) < 0.05 and Δß > 0.15) using DNA methylation arrays. Most of the differentially methylated genes encoded secreted proteins, signaling peptides, and receptors with disulfide bonds. Subsequent analysis using targeted bisulfite massive parallel sequencing showed hypermethylation of the promoters of specific genes in miscarriages with trisomy 16 but not miscarriages with other aneuploidies. Some of the genes were responsible for the development of the placenta and embryo (GATA3-AS1, TRPV6, SCL13A4, and CALCB) and the formation of the mitotic spindle (ANKRD53). Hypermethylation of GATA3-AS1 was associated with reduced expression of GATA3 protein in chorionic villi of miscarriages with trisomy 16. Aberrant hypermethylation of genes may lead to a decrease in expression, impaired trophoblast differentiation and invasion, mitotic disorders, chromosomal mosaicism and karyotype self-correction via trisomy rescue mechanisms.

ABCA1 and ABCG1 DNA methylation in epicardial adipose tissue of patients with coronary artery disease.

BACKGROUND: Recent studies have focused on the potential role of epicardial adipose tissue (EAT) in the development of coronary artery disease (CAD). ABCA1 and ABCG1 transporters regulate cell cholesterol content and reverse cholesterol transport. We aimed to determine whether DNA methylation and mRNA levels of the ABCA1 and ABCG1 genes in EAT and subcutaneous adipose tissue (SAT) were associated with CAD. METHODS: Paired EAT and SAT samples were collected from 82 patients undergoing elective cardiac surgery either for coronary artery bypass grafting (CAD group, N = 66) or valve surgery (NCAD group, N = 16). ABCA1 and ABCG1 mRNA levels in EAT and SAT samples were analyzed using real time polymerase chain reaction, ABCA1 protein levels in EAT samples were assessed by western blotting. ABCA1 and ABCG1 DNA methylation analysis was performed in 24 samples from the CAD group and 9 samples from the NCAD group via pyrosequencing. RESULTS: DNA methylation levels in the ABCA1 promoter and ABCG1 cg27243685 and cg06500161 CpG sites were higher in EAT samples from patients with CAD compared with NCAD (21.92% vs 10.81%, p = 0.003; 71.51% vs 68.42%, p = 0.024; 46.11% vs 37.79%, p = 0.016, respectively). In patients with CAD, ABCA1 and ABCG1 DNA methylation levels were higher in EAT than in SAT samples (p < 0.05). ABCA1 mRNA levels in EAT samples were reduced in the subgroup of patients with CAD and concomitant carotid artery disease or peripheral artery disease compared with the NCAD group (p = 0.024). ABCA1 protein levels in EAT samples tended to be lower in CAD patients than in the NCAD group (p = 0.053). DNA methylation levels at the ABCG1 cg27243685 site positively correlated with plasma triglyceride concentration (r = 0.510, p = 0.008), body mass index (r = 0.556, p = 0.013) and waist-to-hip ratio (r = 0.504, p = 0.012) in SAT samples. CONCLUSION: CAD is associated with ABCA1 and ABCG1 DNA hypermethylation in EAT. CAD with concomitant carotid artery disease or peripheral artery disease is accompanied by decreased ABCA1 gene expression in EAT. DNA methylation levels at the ABCG1 cg27243685 locus in SAT are associated with hypertriglyceridemia and obesity.

NLRP7 variants in spontaneous abortions with multilocus imprinting disturbances from women with recurrent pregnancy loss.

PURPOSE: Comparative analysis of multilocus imprinting disturbances (MLIDs) in miscarriages from women with sporadic (SPL) and recurrent pregnancy loss (RPL) and identification of variants in the imprinting control gene NLRP7 that may lead to MLIDs. METHODS: Chorionic cytotrophoblast and extraembryonic mesoderm samples from first-trimester miscarriages were evaluated in 120 women with RPL and 134 women with SPL; 100 induced abortions were analyzed as a control group. All miscarriages had a normal karyotype. Epimutations in 7 imprinted genes were detected using methyl-specific PCR and confirmed with DNA pyrosequencing. Sequencing of all 13 exons and adjusted intron regions of the NLRP7 gene was performed. RESULTS: Epimutations in imprinted genes were more frequently detected (p < 0.01) in the placental tissues of miscarriages from women with RPL (7.1%) than in those of women with SPL (2.7%). The predominant epimutation was postzygotic hypomethylation of maternal alleles of imprinted genes (RPL, 5.0%; SPL, 2.1%; p < 0.01). The frequency of MLID was higher among miscarriages from women with RPL than among miscarriages from women with SPL (1.7% and 0.4%, respectively, p < 0.01). Variants in NLRP7 were detected only in miscarriages from women with RPL. An analysis of the parental origin of NLRP7 variants revealed heterozygous carriers in families with RPL who exhibited spontaneous abortions with MLIDs and compound heterozygosity for NLRP7 variants. CONCLUSION: RPL is associated with NLRP7 variants that lead to germinal and postzygotic MLIDs that are incompatible with normal embryo development. TRIAL REGISTRATION: Not applicable.

Method of targeted bisulfite massive parallel sequencing of the human LINE-1 retrotransposon promoter.

The methylation index of the LINE-1 promoter is one of the most commonly used markers for assessing the global level of genome methylation in various human cells and tissues. We developed an NGS-based protocol for DNA methylation analysis of the LINE-1 retrotransposon promoter. This approach allows assessment of the DNA methylation index of 19 CpG sites in the LINE-1 promoter that have the highest tissue- or tumor-specific variability. The method provides a DNA methylation profile for analyzing either the methylation index of each CpG site independently or the mean DNA methylation index across the LINE-1 promoter. The results obtained using the developed method corresponded well to the level of methylation assessed using a commercially available kit for DNA pyrosequencing. In addition, our method provides much more information: 1) the DNA methylation profile of a significant part of the LINE-1 promoter and 2) the level of DNA methylation at individual LINE-1 loci in the genome. The method of targeted bisulfite massive parallel sequencing of the human LINE-1 retrotransposon promoter can be used in large-scale studies of the global level of genome methylation in normal human cells or tumors. To accomplish this, we modified the targeted massive parallel sequencing method based on 16S Metagenomic Sequencing Library Preparation protocol (Illumina, USA) by:•Introduction of the stage of bisulfite conversion of DNA.•Development of specific primers for the LINE-1 sequence.

DNA Hypomethylation of the MPO Gene in Peripheral Blood Leukocytes Is Associated with Cerebral Stroke in the Acute Phase.

Dysregulation of the oxidant-antioxidant system contributes to the pathogenesis of cerebral stroke (CS). Epigenetic changes of redox homeostasis genes, such as glutamate-cysteine ligase (GCLM), glutathione-S-transferase-P1 (GSTP1), thioredoxin reductase 1 (TXNRD1), and myeloperoxidase (MPO), may be biomarkers of CS. In this study, we assessed the association of DNA methylation levels of these genes with CS and clinical features of CS. We quantitatively analyzed DNA methylation patterns in the promoter or regulatory regions of 4 genes (GCLM, GSTP1, TXNRD1, and MPO) in peripheral blood leukocytes of 59 patients with CS in the acute phase and in 83 relatively healthy individuals (controls) without cardiovascular and cerebrovascular diseases. We found that in both groups, the methylation level of CpG sites in genes TXNRD1 and GSTP1 was ≤ 5%. Lower methylation levels were registered at a CpG site (chr1:94,374,293, GRCh37 [hg19]) in GCLM in patients with ischemic stroke compared with the control group (9% [7%; 11.6%] (median and interquartile range) versus 14.7% [10.4%; 23%], respectively, p < 0.05). In the leukocytes of patients with CS, the methylation level of CpG sites in the analyzed region of MPO (chr17:56,356,470, GRCh3 [hg19]) on average was significantly lower (23.5% [19.3%; 26.7%]) than that in the control group (35.6% [30.4%; 42.6%], p < 0.05). We also found increased methylation of MPO in smokers with CS (27.2% [23.5%; 31.1%]) compared with nonsmokers with CS (21.7% [18.1%; 24.8%]). Thus, hypomethylation of CpG sites in GCLM and MPO in blood leukocytes is associated with CS in the acute phase.

Identification of a candidate genetic variant for the Himalayan color pattern in dogs.

Acromelanism is a temperature-dependent hypopigmentation pattern commonly manifested as the Himalayan coat color found in rabbits, rats, mice, minks, and gerbils, wherein the extreme "points" are dark and the torso is pale. It is known as the Siamese pattern in cats. Himalayan color is genetically determined by the allelic variant ch of the locus C, later identified as the tyrosinase gene TYR. The tyrosinase functions at the initial steps of melanin production, and alteration of its activity by sequence changes results in pigmentation defects in vertebrates. The presence of acromelanism in dogs has not been described until now. We analyzed a DNA sample of a dachshund with a unique coat color resembling the Himalayan type. Sequencing of the coding part of the TYR gene from the proband revealed a homozygous variant (c.230G > A) in exon 1, leading to an amino acid substitution (p.R77Q) in a conserved region of the protein. The proband's mother, which is black-and-tan, is a heterozygous carrier of the c.230A allele, while none of the 210 dogs of different breeds, unrelated to the proband, carried the c.230A allele. These results suggest that the identified sequence variant is likely the cause of the Himalayan coloration of the proband.

LINE-1 retrotransposon methylation in chorionic villi of first trimester miscarriages with aneuploidy.

PURPOSE: High frequency of aneuploidy in meiosis and cleavage stage coincides with waves of epigenetic genome reprogramming that may indicate a possible association between epigenetic mechanisms and aneuploidy occurrence. This study aimed to assess the methylation level of the long interspersed repeat element 1 (LINE-1) retrotransposon in chorionic villi of first trimester miscarriages with a normal karyotype and aneuploidy. METHODS: The methylation level was assessed at 19 LINE-1 promoter CpG sites in chorionic villi of 141 miscarriages with trisomy of chromosomes 2, 6, 8-10, 13-15, 16, 18, 20-22, and monosomy X using massive parallel sequencing. RESULTS: The LINE-1 methylation level was elevated statistically significant in chorionic villi of miscarriages with both trisomy (45.2 ± 4.3%) and monosomy X (46.9 ± 4.2%) compared with that in induced abortions (40.0 ± 2.4%) (p < 0.00001). The LINE-1 methylation levels were specific for miscarriages with different aneuploidies and significantly increased in miscarriages with trisomies 8, 14, and 18 and monosomy X (p < 0.05). The LINE-1 methylation level increased with gestational age both for group of miscarriages regardless of karyotype (R = 0.21, p = 0.012) and specifically for miscarriages with trisomy 16 (R = 0.48, p = 0.007). LINE-1 methylation decreased with maternal age in miscarriages with a normal karyotype (R = - 0.31, p = 0.029) and with trisomy 21 (R = - 0.64, p = 0.024) and increased with paternal age for miscarriages with trisomy 16 (R = 0.38, p = 0.048) and monosomy X (R = 0.73, p = 0.003). CONCLUSION: Our results indicate that the pathogenic effects of aneuploidy in human embryogenesis can be supplemented with significant epigenetic changes in the repetitive sequences.

Structural Variability, Expression Profile, and Pharmacogenetic Properties of TMPRSS2 Gene as a Potential Target for COVID-19 Therapy.

The human serine protease serine 2 TMPRSS2 is involved in the priming of proteins of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and represents a possible target for COVID-19 therapy. The TMPRSS2 gene may be co-expressed with SARS-CoV-2 cell receptor genes angiotensin-converting enzyme 2 (ACE2) and Basigin (BSG), but only TMPRSS2 demonstrates tissue-specific expression in alveolar cells according to single-cell RNA sequencing data. Our analysis of the structural variability of the TMPRSS2 gene based on genome-wide data from 76 human populations demonstrates that a functionally significant missense mutation in exon 6/7 in the TMPRSS2 gene is found in many human populations at relatively high frequencies, with region-specific distribution patterns. The frequency of the missense mutation encoded by rs12329760, which has previously been found to be associated with prostate cancer, ranged between 10% and 63% and was significantly higher in populations of Asian origin compared with European populations. In addition to single-nucleotide polymorphisms, two copy number variants were detected in the TMPRSS2 gene. A number of microRNAs have been predicted to regulate TMPRSS2 and BSG expression levels, but none of them is enriched in lung or respiratory tract cells. Several well-studied drugs can downregulate the expression of TMPRSS2 in human cells, including acetaminophen (paracetamol) and curcumin. Thus, the interactions of TMPRSS2 with SARS-CoV-2, together with its structural variability, gene-gene interactions, expression regulation profiles, and pharmacogenomic properties, characterize this gene as a potential target for COVID-19 therapy.

Genomic structural variations for cardiovascular and metabolic comorbidity.

The objective of this study was to identify genes targeted by both copy number and copy-neutral changes in the right coronary arteries in the area of advanced atherosclerotic plaques and intact internal mammary arteries derived from the same individuals with comorbid coronary artery disease and metabolic syndrome. The artery samples from 10 patients were screened for genomic imbalances using array comparative genomic hybridization. Ninety high-confidence, identical copy number variations (CNVs) were detected. We also identified eight copy-neutral changes (cn-LOHs) > 1.5 Mb in paired arterial samples in 4 of 10 individuals. The frequencies of the two gains located in the 10q24.31 (ERLIN1) and 12q24.11 (UNG, ACACB) genomic regions were evaluated in 33 paired arteries and blood samples. Two patients contained the gain in 10q24.31 (ERLIN1) and one patient contained the gain in 12q24.11 (UNG, ACACB) that affected only the blood DNA. An additional two patients harboured these CNVs in both the arteries and blood. In conclusion, we discovered and confirmed a gain of the 10q24.31 (ERLIN1) and 12q24.11 (UNG, ACACB) genomic regions in patients with coronary artery disease and metabolic comorbidity. Analysis of DNA extracted from blood indicated a possible somatic origin for these CNVs.

A comparison of genome-wide DNA methylation patterns between different vascular tissues from patients with coronary heart disease.

Epigenetic mechanisms of gene regulation in context of cardiovascular diseases are of considerable interest. So far, our current knowledge of the DNA methylation profiles for atherosclerosis affected and healthy human vascular tissues is still limited. Using the Illumina Infinium Human Methylation27 BeadChip, we performed a genome-wide analysis of DNA methylation in right coronary artery in the area of advanced atherosclerotic plaques, atherosclerotic-resistant internal mammary arteries, and great saphenous veins obtained from same patients with coronary heart disease. The resulting DNA methylation patterns were markedly different between all the vascular tissues. The genes hypomethylated in athero-prone arteries to compare with atherosclerotic-resistant arteries were predominately involved in regulation of inflammation and immune processes, as well as development. The great saphenous veins exhibited an increase of the DNA methylation age in comparison to the internal mammary arteries. Gene ontology analysis for genes harboring hypermethylated CpG-sites in veins revealed the enrichment for biological processes associated with the development. Four CpG-sites located within the MIR10B gene sequence and about 1 kb upstream of the HOXD4 gene were also confirmed as hypomethylated in the independent dataset of the right coronary arteries in the area of advanced atherosclerotic plaques in comparison with the other vascular tissues. The DNA methylation differences observed in vascular tissues of patients with coronary heart disease can provide new insights into the mechanisms underlying the development of pathology and explanation for the difference in graft patency after coronary artery bypass grafting surgery.

Dm nxf1/sbr gene affects the formation of meiotic spindle in female Drosophila melanogaster.

The small bristles (sbr) gene of Drosophila melanogaster belongs to the family of nuclear export factor (NXF) genes that participate in mRNA nuclear export. During meiosis, females of Drosophila melanogaster that carry various combinations of mutant alleles of the Dm nxf1/sbr gene exhibit disruption of the division spindle and misalignment of chromosomes at the metaphase plate. Meiosis of sbr ( 5 ) /+ females is characterized by the formation of tripolar spindles during the first cell division. According to the sequencing results, the sbr ( 5 ) (l(1)K4) lethal allele is a deletion of 492 nucleotides. In SBR(5) protein, 57 of the 146 amino acids that have been lost by deletion belong to the NTF2-like domain.