Loci cg06256735 and cg15815843 in the MFAP5 gene regulatory regions are hypomethylated in varicose veins apparently due to active demethylation.

Varicose vein disease (VVD) is a common health problem worldwide. Microfibril Associated Protein 5 (MFAP5) is one of the potential key players in its pathogenesis. Our previous microarray analysis revealed the cg06256735 and cg15815843 loci in the regulatory regions of the MFAP5 gene as hypomethylated in varicose veins which correlated with its up-regulation. The aim of this work was to validate preliminary microarray data, estimate the level of 5-hydroxymethylcytosine (5hmC) at these loci, and determine the methylation status of one of them in different layers of the venous wall. For this, methyl- and hydroxymethyl-sensitive restriction techniques were used followed by real-time PCR and droplet digital PCR, correspondingly, as well as bisulfite pyrosequencing of +/- oxidized DNA. Our microarray data on hypomethylation at the cg06256735 and cg15815843 loci in whole varicose vein segments were confirmed and it was also demonstrated that the level of 5hmC at these loci is increased in VVD. Specifically, among other layers of the venous wall, t. intima is the main contributor to hypomethylation at the cg06256735 locus in varicose veins. Thus, it was shown that hypomethylation at the cg06256735 and cg15815843 loci takes place in VVD, with evidence to suggest that it happens through their active demethylation leading to up-regulation of the MFAP5 gene, and t. intima is most involved in this biochemical process.

Phosphoramidate Azole Oligonucleotides for Single Nucleotide Polymorphism Detection by PCR.

Detection of the Kirsten rat sarcoma gene (KRAS) mutational status is an important factor for the treatment of various malignancies. The most common KRAS-activating mutations are caused by single-nucleotide mutations, which are usually determined by using PCR, using allele-specific DNA primers. Oligonucleotide primers with uncharged or partially charged internucleotide phosphate modification have proved their ability to increase the sensitivity and specificity of various single nucleotide mutation detection. To enhance the specificity of single nucleotide mutation detection, the novel oligonucleotides with four types of uncharged and partially charged internucleotide phosphates modification, phosphoramide benzoazole (PABA) oligonucleotides (PABAO), was used to prove the concept on the KRAS mutation model. The molecular effects of different types of site-specific PABA modification in a primer or a template on a synthesis of full-length elongation product and PCR efficiency were evaluated. The allele-specific PCR (AS-PCR) on plasmid templates showed a significant increase in analysis specificity without changes in Cq values compared with unmodified primer. PABA modification is a universal mismatch-like disturbance, which can be used for single nucleotide polymorphism discrimination for various applications. The molecular insights of the PABA site-specific modification in a primer and a template affect PCR, structural features of four types of PABAO in connection with AS-PCR results, and improvements of AS-PCR specificity support the further design of novel PCR platforms for various biological targets testing.

Epigenome-Wide Changes in the Cell Layers of the Vein Wall When Exposing the Venous Endothelium to Oscillatory Shear Stress.

Epigenomic changes in the venous cells exerted by oscillatory shear stress towards the endothelium may result in consolidation of gene expression alterations upon vein wall remodeling during varicose transformation. We aimed to reveal such epigenome-wide methylation changes. Primary culture cells were obtained from non-varicose vein segments left after surgery of 3 patients by growing the cells in selective media after magnetic immunosorting. Endothelial cells were either exposed to oscillatory shear stress or left at the static condition. Then, other cell types were treated with preconditioned media from the adjacent layer's cells. DNA isolated from the harvested cells was subjected to epigenome-wide study using Illumina microarrays followed by data analysis with GenomeStudio (Illumina), Excel (Microsoft), and Genome Enhancer (geneXplain) software packages. Differential (hypo-/hyper-) methylation was revealed for each cell layer's DNA. The most targetable master regulators controlling the activity of certain transcription factors regulating the genes near the differentially methylated sites appeared to be the following: (1) HGS, PDGFB, and AR for endothelial cells; (2) HGS, CDH2, SPRY2, SMAD2, ZFYVE9, and P2RY1 for smooth muscle cells; and (3) WWOX, F8, IGF2R, NFKB1, RELA, SOCS1, and FXN for fibroblasts. Some of the identified master regulators may serve as promising druggable targets for treating varicose veins in the future.

Allele-Specific PCR for PIK3CA Mutation Detection Using Phosphoryl Guanidine Modified Primers.

Phosphoryl guanidine (PG) is the novel uncharged modification of internucleotide phosphates of oligonucleotides. Incorporating PG modification into PCR primers leads to increased discrimination between wild-type and mutated DNA, providing extraordinary detection limits in an allele-specific real-time polymerase chain reaction (AS-PCR). Herein, we used PG-modification to improve the specificity of AS primers with unfavorable Pyr/Pur primer's 3'-end mismatch in the template/primer complex. Two mutations of the PIK3CA gene (E542K, E545K) were chosen to validate the advantages of the PG modification. Several primers with PG modifications were synthesized for each mutation and assessed using AS-PCR with the plasmid controls and DNA obtained from formalin-fixed paraffin-embedded (FFPE) tissues. The assay allows the detection of 0.5% of mutated DNA on the wild-type DNA plasmid template's background with good specificity. Compared with ddPCR, the primers with PG-modification demonstrated 100% specificity and 100% sensitivity on the DNA from FFPE with mutation presence higher than 0.5%. Our results indicate the high potential of PG-modified primers for point mutation detection. The main principle of the developed methodology can be used to improve the specificity of primers regardless of sequences.

A Novel Thermostable and Processive Reverse Transcriptase from a Group II Intron of Anoxybacillus flavithermus.

Reverse transcriptases (RTs) are a family of enzymes that synthesize DNA using an RNA template and are involved in retrovirus propagation and telomere lengthening. In vitro, RTs are widely applied in various methods, including RNA-seq, RT-PCR, and RT-LAMP. Thermostable RTs from bacterial group II introns are promising tools for biotechnology due to their higher thermostability, fidelity, and processivity compared to commonly used M-MuLV RT and its mutants. However, the diversity of group II intron-encoded RTs is still understudied. In this work, we biochemically characterized a novel RT from a thermophilic bacterium, Anoxybacillus flavithermus, which was isolated from a hot spring in New Zealand and has an optimal growth temperature of around 60 °C. The cloned RT, named Afl RT, retained approximately 40% of the specific activity after a 45 min incubation at 50 °C. The optimal pH was 8.5, the optimal temperature was between 45 and 50 °C, and Mn2+ ions were found to be an optimal cofactor. The processivity analysis with MS2 phage gRNA (3569 b) demonstrated that Afl RT elongated fully up to 36% of the template molecules. In reverse transcription and RT-qLAMP, the enzyme allowed up to 10 copies of MS2 phage genomic RNA to be detected per reaction. Thus, Afl RT holds great potential for a variety of practical applications that require the use of thermostable and processive RTs.

Comparison of Reverse Transcriptase (RT) Activities of Various M-MuLV RTs for RT-LAMP Assays.

Reverse transcriptases (RTs) are a family of enzymes synthesizing DNA using RNA as a template and serving as indispensable tools in studies related to RNA. M-MuLV RT and its analogs are the most commonly used RTs. RTs are widely applied in various diagnostics methods, including reverse-transcription loop-mediated isothermal amplification (RT-LAMP). However, the performance of different RTs in LAMP remains relatively unknown. Here, we report on the first direct comparison of various M-MuLV RTs in RT-LAMP, including enzymes with a different number of mutations and fusions with Sto7d. Several parameters were assessed, namely: optimal reaction temperature, enzyme concentration, reverse transcription time, a minimal amount of RNA template, and tolerance to inhibitors. Mutations increased the optimal reaction temperature from 55 °C to 60-65 °C. All of the RTs were suitable for RT-LAMP with RNA templates in the range of 101-106 copies per reaction. Highly mutated enzymes were 1.5-3-fold more tolerant to whole blood, blood plasma, and guanidinium, but they were two-fold more sensitive to high concentrations of NaCl. The comparison of different RTs presented here could be helpful for selecting the optimal enzyme when developing novel LAMP-based diagnostic tests.

Development of a multiplex allele-specific qPCR approach for testing PIK3CA mutations in patients with colorectal cancer.

Phosphatidylinositol 3-kinases (PI3Ks) are lipid kinases involved in cellular growth and division. Somatic mutations in one of the PI3K catalytic subunit genes, PIK3CA, are frequently found in numerous malignancies, including colorectal cancer (CRC). Several PIK3CA inhibitors are approved for the treatment of breast cancer and lymphoma. Activating mutations in PIK3CA tend to occur in exons 9 and 20, with mutations in other exons 1, 4, and 7 being less common. Most test systems for PIK3CA mutation screening are designed to detect mutations in exons 9 and 20, leaving exons 1-7 overlooked. We have developed a multiplex AS-PCR to screen for PIK3CA mutations in exons 1, 4, 7, 9, and 20. Validation was performed on 515 CRC samples of patients from Siberia and the Far East of Russia. The assay sensitivity was 0.05-0.5% of mutant DNA, and the overall PIK3CA mutation frequency was 13.01%, with 9.32% of mutations in exon 9, 1.94% in exon 20, and 1.74% in exons 1-7. The assay designed is suitable for the analysis of activating PIK3CA mutations in formalin-fixed paraffin-embedded tissue samples. The present work is the first study characterizing the PIK3CA mutation frequency in CRC patients from the eastern part of Russia.

Quantitative and structural characteristics of mitochondrial DNA in varicose veins.

OBJECTIVE: We examined quantitative (in terms of mtDNA/nuclear DNA) and structural (in terms of common deletions in the MT-ND4 gene region) characteristics of mitochondrial DNA (mtDNA) in varicose veins (VVs) and venous wall layers by comparing mitochondrial genome parameters, as well as mitochondrial function (in terms of mitochondrial membrane potential (MtMP)), in varicose vein (VV) vs. non-varicose vein (NV) tissue samples. METHODS: We analyzed paired great saphenous vein samples (VV vs. NV segments from each patient left after venous surgery) harvested from patients with VVs. Relative mtDNA level and the proportion of no-deletion mtDNA were determined by a multiplex quantitative PCR (qPCR), confirming the latter with a more sensitive method - droplet digital PCR (ddPCR). Mitochondria's functional state in VVs was assessed using fluorescent (dependent on MtMP) live-staining of mitochondria in venous tissues. RESULTS: Total mtDNA level was lower in VV than in NV samples (predominantly in the t. media layer). ddPCR analysis showed lower proportion of no-deletion mtDNA in VVs. Because of the decrease in relative MtMP in VVs, our results suggest a possible reduction of mitochondrial function in VVs. CONCLUSION: Quantitative and structural changes (copy number and integrity) of mtDNA are plausibly involved in VV pathogenesis. Future clinical studies implementing the mitochondrial targeting may be eventually fostered after auxiliary mechanistic studies.

Multiplex Droplet Digital PCR Assay for Detection of MET and HER2 Genes Amplification in Non-Small Cell Lung Cancer.

Non-small-cell lung cancer (NSCLC), a subtype of lung cancer, remains one of the most common tumors with a high mortality and morbidity rate. Numerous targeted drugs were implemented or are now developed for the treatment of NSCLC. Two genes, HER2 and MET, are among targets for these specific therapeutic agents. Alterations in HER2 and MET could lead to primary or acquired resistance to commonly used anti-EGFR drugs. Using current methods for detecting HER2 and MET amplifications is time and labor-consuming; alternative methods are required for HER2 and MET testing. We developed the first multiplex droplet digital PCR assay for the simultaneous detection of MET and HER2 amplification in NSCLC samples. The suitability of qPCR was assessed for the optimization of multiplex ddPCR. The optimal elongation temperature, reference genes for DNA quantification, and amplicon length were selected. The developed ddPCR was validated on control samples with various DNA concentrations and ratios of MET and HER2 genes. Using ddPCR, 436 EGFR-negative NSCLC samples were analyzed. Among the tested samples, five specimens (1.15%) showed a higher ratio of MET, and six samples (1.38%) showed a higher ratio of HER2. The reported multiplex ddPCR assay could be used for the routine screening of MET and HER2 amplification in NSCLC samples.

M-MuLV reverse transcriptase: Selected properties and improved mutants.

Reverse transcriptases (RTs) are enzymes synthesizing DNA using RNA as the template and serving as the standard tools in modern biotechnology and molecular diagnostics. To date, the most commonly used reverse transcriptase is the enzyme from Moloney murine leukemia virus, M-MuLV RT. Since its discovery, M-MuLV RT has become indispensable for modern RNA studies; the range of M-MuLV RT applications is vast, from scientific tasks to clinical testing of human pathogens. This review will give a brief description of the structure, thermal stability, processivity, and fidelity, focusing on improving M-MuLV RT for practical usage.

Allele-Specific PCR for KRAS Mutation Detection Using Phosphoryl Guanidine Modified Primers.

Establishing the Kirsten rat sarcoma (KRAS) mutational status is essential in terms of managing patients with various types of cancer. Allele-specific real-time polymerase chain reaction (AS-PCR) is a widely used method for somatic mutations detection. To improve the limited sensitivity and specificity, several blocking methods have been introduced in AS-PCR to block the amplification of wild-type templates. Herein, we used a novel modified oligonucleotide with internucleotide phosphates reshaped 1,3-dimethyl-2-imino-imidazolidine moieties (phosphoryl guanidine (PG) groups) as primers and blockers in the AS-PCR method. Four common KRAS mutations were chosen as a model to demonstrate the advantages of the PG primers and blockers utilizing a customized PCR protocol. The methods were evaluated on plasmid model systems providing a KRAS mutation detection limit of 20 copies of mutant DNA in a proportion as low as 0.1% of the total DNA, with excellent specificity. PG-modification can serve as the universal additional mismatch-like disturbance to increase the discrimination between wild-type and mutated DNA. Moreover, PG can serve to increase primer specificity by a synergetic effect with additional mismatch and would greatly facilitate medical research.

Absence of EGFR C797S Mutation in Tyrosine Kinase Inhibitor-Naïve Non-Small Cell Lung Cancer Tissues.

EGFR tyrosine-kinase inhibitors (TKIs) are used as targeted therapeutics for the treatment of advanced non-small cell lung cancer (NSCLC) with EGFR-activating mutations. EGFR C797S is common causes of acquired resistance to third-generation TKIs. There is wide-spread opinion that resistance-conferring mutation present even in a small proportion of cancer cells before the start of therapy could potentially predict poor response to a targeted drug. In our study, we tested whether C797S can be found in previously untreated NSCLCs. We analyzed DNA samples extracted from formalin-fixed paraffin-embedded (FFPE) tumor tissue sections of 470 lung adenocarcinoma patients, including 235 samples with activating EGFR mutations. Screening was performed using highly sensitive droplet digital PCR assay. No tumor samples with baseline C797S were identified. C797S does not occur in TKI-naïve NSCLCs and provide evidence that screening for this mutation before TKIs administration may not be necessary.

Molecular characterization of Mycoplasma pneumoniae infections in Moscow from 2015 to 2018.

The aim of this study was to assess which Mycoplasma pneumoniae genotypes were present in Moscow during the years 2015-2018 and whether the proportion between detected genotypes changed over time. We were also interested in the presence of macrolide resistance (MR)Mycoplasma pneumoniae. We performed multilocus variable-number tandem-repeat (VNTR) analysis (MLVA), SNP typing, and mutation typing in the 23S rRNA gene from 117 M. pneumoniae clinical isolates. Our analysis suggests two major MLVA types: 4572 and 3562. In 2017-2018, MLVA type 4572 gradually became predominant. In general, the SNP type range is the same as described earlier for European countries. The analysis of MR mutations showed that 7% of the isolates had an A2063G mutation in the 23S rRNA gene with no isolates carrying an A2064G mutation. In 2017-2018, MLVA type 4572 (SNP type 1) begins to spread in Moscow, which was widespread globally, especially in Asian countries. SNP typing of our sample showed higher discriminatory power than MLVA typing.

DNA methylation and gene expression profiling reveal MFAP5 as a regulatory driver of extracellular matrix remodeling in varicose vein disease.

AIM: To integrate transcriptomic and DNA-methylomic measurements on varicose versus normal veins using a systems biological analysis to shed light on the interplay between genetic and epigenetic factors. MATERIALS & METHODS: Differential expression and methylation were measured using microarrays, supported by real-time quantitative PCR and immunohistochemistry confirmation for relevant gene products. A systems biological 'upstream analysis' was further applied. RESULTS: We identified several potential key players contributing to extracellular matrix remodeling in varicose veins. Specifically, our analysis suggests MFAP5 acting as a master regulator, upstream of integrins, of the cellular network affecting the varicose vein condition. Possible mechanism and pathogenic model were outlined. CONCLUSION: A coherent model proposed incorporates the relevant signaling networks and will hopefully aid further studies on varicose vein pathogenesis.

Mycoplasma hyorhinis reduces sensitivity of human lung carcinoma cells to Nutlin-3 and promotes their malignant phenotype.

PURPOSE: MDM2 inhibitors are promising anticancer agents that induce cell cycle arrest and tumor cells death via p53 reactivation. We examined the influence of Mycoplasma hyorhinis infection on sensitivity of human lung carcinoma cells NCI-H292 to MDM2 inhibitor Nutlin-3. In order to unveil possible mechanisms underlying the revealed effect, we investigated gene expression changes and signal transduction networks activated in NCI-H292 cells in response to mycoplasma infection. METHODS: Sensitivity of NCI-Н292 cells to Nutlin-3 was estimated by resazurin-based cell viability assay. Genome-wide transcriptional profiles of NCI-H292 and NCI-Н292Myc.h cell lines were determined using Illumina Human HT-12 v3 Expression BeadChip. Search for key transcription factors and key node molecules was performed using the geneXplain platform. Ability for anchorage-independent growth was tested by soft agar colony formation assay. RESULTS: NCI-Н292Myc.h cells were shown to be 1.5- and 5.2-fold more resistant to killing by Nutlin-3 at concentrations of 15 and 30 µM than uninfected NCI-Н292 cells (P < 0.05 and P < 0.001, respectively). Transcriptome analysis revealed differential expression of multiple genes involved in cancer progression and metastasis as well as epithelial-mesenchymal transition (EMT). Moreover, we have shown experimentally that NCI-Н292Myc.h cells were more capable of growing and dividing without binding to a substrate. The most likely mechanism explaining the observed changes was found to be TLR4- and IL-1b-mediated activation of NF-κB pathway. CONCLUSIONS: Our results provide evidence that mycoplasma infection is an important factor modulating the effect of MDM2 inhibitors on cancer cells and is able to induce EMT-related changes.

Allele-Specific Biased Expression of the CNTN6 Gene in iPS Cell-Derived Neurons from a Patient with Intellectual Disability and 3p26.3 Microduplication Involving the CNTN6 Gene.

Copy number variations (CNVs) of the human CNTN6 gene caused by megabase-scale microdeletions or microduplications in the 3p26.3 region are often the cause of neurodevelopmental disorders, including intellectual disability and developmental delay. Surprisingly, patients with different copy numbers of this gene display notable overlapping of neuropsychiatric symptoms. The complexity of the study of human neuropathologies is associated with the inaccessibility of brain material. This problem can be overcome through the use of reprogramming technologies that permit the generation of induced pluripotent stem (iPS) cells from fibroblasts and their subsequent in vitro differentiation into neurons. We obtained a set of iPS cell lines derived from a patient carrier of the CNTN6 gene duplication and from two healthy donors. All iPS cell lines displayed the characteristics of pluripotent cells. Some iPS cell lines derived from the patient and from healthy donors were differentiated in vitro by exogenous expression of the Ngn2 transcription factor or by spontaneous neural differentiation of iPS cells through the neural rosette stage. The obtained neurons showed the characteristics of mature neurons as judged by the presence of neuronal markers and by their electrophysiological characteristics. Analysis of allele-specific expression of the CNTN6 gene in these neuronal cells by droplet digital PCR demonstrated that the level of expression of the duplicated allele was significantly reduced compared to that of the wild-type allele. Importantly, according to the sequencing data, both copies of the CNTN6 gene, which were approximately 1 Mb in size, showed no any additional structural rearrangements.

Allele rs2010963 C of the VEGFA gene is associated with the decreased risk of primary varicose veins in ethnic Russians.

Objective To study the association of polymorphisms rs699947, rs2010963, rs3025039 in the VEGFA gene region and rs1870377, rs2305949, rs2071559 in the VEGFR2 gene region with the risk of primary varicose veins in ethnic Russians. Methods Genotypes were determined by real-time PCR allelic discrimination. The case group consisted of 448 patients with primary varicose veins and the control group comprised 609 individuals without a history of chronic venous disease. Association was studied by logistic regression analysis. Results Allele rs2010963 C was associated with the decreased risk of varicose veins (additive model of inheritance: odds ratio = 0.73, 95% confidence interval = 0.59-0.91, P = 0.004). Conclusions Our results provide evidence that polymorphism rs2010963 located in the 5' untranslated region of the VEGFA gene can influence genetic susceptibility to primary varicose veins in Russians. Otherwise, it can be in linkage disequilibrium with another functional single nucleotide polymorphism that can alter the level of vascular endothelial growth factor A protein.

Angiopoietin-Like Proteins 4 (ANGPTL4) Gene Polymorphisms and Risk of Brain Arteriovenous Malformation.

BACKGROUND: Brain arteriovenous malformations (BAVMs) are formed by hypertrophied arterial vessels (afferents, feeders), a large number of arteriovenous shunts which become tangled to form a body (nidus) of malformation, which then expands draining proximal veins. The aim of this study was a replication of single nucleotide polymorphism (SNP) rs11672433 association with BAVM development with the subsequent meta-analysis of published data. METHODS: A total of 252 Russian patients with brain BAVMs and 480 control subjects were included in the present study. Genotyping was performed using real-time polymerase chain reaction with competitive hydrolysis probes. RESULTS: In our case-control study, we found no significant association with brain arteriovenous malformation for the SNP rs11672433 of ANGPTL4 gene (odds ratio .82, 95% confidence interval = .57-1.17 P value = .27) as well as in meta-analysis (odds ratio 1.18, 95% confidence interval = .81-1.73, P value = .39). CONCLUSIONS: Our data showed that SNP rs11672433 was not associated with the BAVM Russian population and the following meta-analysis did not detect an association in total. Thus, in spite of the fact that ANGPTL4 (protein) participates in the angiogenesis regulation processes, we consider that SNP rs11672433, a high-frequency locus in the ANGPTL4 gene, does not influence the predisposition to BAVM or its effect is too small to be detected in the present size sample set.

Functional polymorphism rs1024611 in the MCP1 gene is associated with the risk of varicose veins of lower extremities.

OBJECTIVE: Monocyte chemoattractant protein 1 (MCP-1) is a chemokine responsible for monocyte, basophil, and T-lymphocyte attraction. Polymorphism rs1024611 located in the regulatory region of the MCP1 gene has previously been shown to be associated with increased MCP-1 production. In our study, we aimed to examine the association of rs1024611 with the risk of primary varicose veins (PVVs) of lower extremities. METHODS: The case group comprised 470 patients with PVVs, and the control group included 269 individuals without a history of chronic venous disease. All cases and controls were ethnic Russians. Genotypes were determined by real-time polymerase chain reaction allelic discrimination. Association was studied by logistic regression analysis. RESULTS: We revealed the association of genotype G/G with the increased risk of PVVs (odds ratio [OR], 1.87; 95% confidence interval [CI], 1.02-3.44; P = .04). In the subgroup analysis, association was revealed only in patients with C2 Clinical, Etiology, Anatomy, and Pathophysiology class (allele G: OR, 1.62 [95% CI, 1.13-2.33; P = .008]; genotype G/G: OR, 3.22 [95% CI, 1.43-7.27; P = .005]), in patients with age at onset of PVVs before 30 years (allele G: OR, 1.41 [95% CI, 1.08-1.85; P = .01]; genotype G/G: OR, 2.35 [95% CI, 1.22-4.55; P = .01]), and in patients who declared no family history (allele G: OR, 1.46 [95% CI, 1.02-2.09; P = .04]; genotype G/G: OR, 2.50 [95% CI, 1.11-5.63; P = .03]). CONCLUSIONS: Our results provide evidence for MCP-1 involvement in the development of PVVs and indicate that inflammation could be implicated in the pathogenesis of this condition.