An Efficient 2D Protocol for Differentiation of iPSCs into Mature Postmitotic Dopaminergic Neurons: Application for Modeling Parkinson's Disease.

About 15% of patients with parkinsonism have a hereditary form of Parkinson's disease (PD). Studies on the early stages of PD pathogenesis are challenging due to the lack of relevant models. The most promising ones are models based on dopaminergic neurons (DAns) differentiated from induced pluripotent stem cells (iPSCs) of patients with hereditary forms of PD. This work describes a highly efficient 2D protocol for obtaining DAns from iPSCs. The protocol is rather simple, comparable in efficiency with previously published protocols, and does not require viral vectors. The resulting neurons have a similar transcriptome profile to previously published data for neurons, and have a high level of maturity marker expression. The proportion of sensitive (SOX6+) DAns in the population calculated from the level of gene expression is higher than resistant (CALB+) DAns. Electrophysiological studies of the DAns confirmed their voltage sensitivity and showed that a mutation in the PARK8 gene is associated with enhanced store-operated calcium entry. The study of high-purity DAns differentiated from the iPSCs of patients with hereditary PD using this differentiation protocol will allow for investigators to combine various research methods, from patch clamp to omics technologies, and maximize information about cell function in normal and pathological conditions.

Diversity and Differential Expression of MicroRNAs in the Human Skeletal Muscle with Distinct Fiber Type Composition.

The ratio of fast- and slow-twitch fibers in human skeletal muscle is variable and largely determined by genetic factors. In this study, we investigated the contribution of microRNA (miRNA) in skeletal muscle fiber type composition. The study involved biopsy samples of the vastus lateralis muscle from 24 male participants with distinct fiber type ratios. The miRNA study included samples from five endurance athletes and five power athletes with the predominance of slow-twitch (61.6-72.8%) and fast-twitch (69.3-80.7%) fibers, respectively. Total and small RNA were extracted from tissue samples. Total RNA sequencing (N = 24) revealed 352 differentially expressed genes between the groups with the predominance of fast- and slow-twitch muscle fibers. Small RNA sequencing showed upregulation of miR-206, miR-501-3p and miR-185-5p, and downregulation of miR-499a-5p and miR-208-5p in the group of power athletes with fast-twitch fiber predominance. Two miRtronic miRNAs, miR-208b-3p and miR-499a-5p, had strong correlations in expression with their host genes (MYH7 and MYH7B, respectively). Correlations between the expression of miRNAs and their experimentally validated messenger RNA (mRNA) targets were calculated, and 11 miRNA-mRNA interactions with strong negative correlations were identified. Two of them belonged to miR-208b-3p and miR-499a-5p, indicating their regulatory links with the expression of CDKN1A and FOXO4, respectively.

The Impact of the Anticoagulant Type in Blood Collection Tubes on Circulating Extracellular Plasma MicroRNA Profiles Revealed by Small RNA Sequencing.

Pre-analytical factors have a significant influence on circulating microRNA (miRNA) profiling. The aim of this study was a comprehensive assessment of the impact of the anticoagulant type in blood collection tubes on circulating plasma miRNA profiles using small RNA sequencing. Blood from ten healthy participants (five males and five females from 25 to 40 years old) was taken in collection tubes with four different anticoagulants: acid citrate dextrose (ACD-B), sodium citrate, citrate-theophylline-adenosine-dipyridamole (CTAD) and dipotassium-ethylenediaminetetraacetic acid (K2 EDTA). Platelet-free plasma samples were obtained by double centrifugation. EDTA plasma samples had elevated levels of hemolysis compared to samples obtained using other anticoagulants. Small RNA was extracted from plasma samples and small RNA sequencing was performed on the Illumina NextSeq 500 system. A total of 30 samples had been successfully sequenced starting from ~1 M reads mapped to miRNAs, allowing us to analyze their diversity and isoform content. The principal component analysis showed that the EDTA samples have distinct circulating plasma miRNA profiles compared to samples obtained using other anticoagulants. We selected 50 miRNA species that were differentially expressed between the sample groups based on the type of anticoagulant. We found that the EDTA samples had elevated levels of miRNAs which are abundant in red blood cells (RBC) and associated with hemolysis, while the levels of some platelet-specific miRNAs in these samples were lowered. The ratio between RBC-derived and platelet-derived miRNAs differed between the EDTA samples and other sample groups, which was validated by quantitative PCR. This study provides full plasma miRNA profiles of 10 healthy adults, compares them with previous studies and shows that the profile of circulating miRNAs in the EDTA plasma samples is altered primarily due to an increased level of hemolysis.

Rare single nucleotide variants in COL5A1 promoter do not play a major role in keratoconus susceptibility associated with rs1536482.

BACKGROUND: Keratoconus is a chronic degenerative disorder of the cornea characterized by thinning and cone-shaped protrusions. Although genetic factors play a key role in keratoconus development, the etiology is still under investigation. The occurrence of single-nucleotide polymorphisms (SNPs) associated with keratoconus in Russian patients is poorly studied. The purpose of this study was to validate whether three reported keratoconus-associated SNPs (rs1536482 near the COL5A1 gene, rs2721051 near the FOXO1 gene, rs1324183 near the MPDZ gene) are also actual for a Russian cohort of patients. Additionally, we investigated the COL5A1 promoter sequence for single-nucleotide variants (SNVs) in a subgroup of keratoconus patients with at least one rs1536482 minor allele (rs1536482+) to assess the role of these SNVs in keratoconus susceptibility associated with rs1536482. METHODS: This case-control study included 150 keratoconus patients and two control groups (main and additional, 205 and 474 participants, respectively). We performed PCR targeting regions flanking SNVs and the COL5A1 promoter, followed by Sanger sequencing of amplicons. The additional control group was genotyped using an SNP array. RESULTS: The minor allele frequency was significantly different between the keratoconus and control cohorts (main and combined) for rs1536482, rs2721051, and rs1324183 (p-value < 0.05). The rare variants rs1043208782 and rs569248712 were found in the COL5A1 promoter in two out of 94 rs1536482+ keratoconus patients. CONCLUSION: rs1536482, rs2721051, and rs1324183 were associated with keratoconus in a Russian cohort. SNVs in the COL5A1 promoter do not play a major role in keratoconus susceptibility associated with rs1536482.

Circulating Extracellular miRNA Analysis in Patients with Stable CAD and Acute Coronary Syndromes.

Extracellular circulating microRNAs (miRNAs) are currently a focus of interest as non-invasive biomarkers of cardiovascular pathologies, including coronary artery disease (CAD) and acute coronary syndromes (ACS): myocardial infarction with and without ST-segment elevation (STEMI and NSTEMI) and unstable angina (UA). However, the current data for some miRNAs are controversial and inconsistent, probably due to pre-analytical and methodological variances in different studies. In this work, we fulfilled the basic pre-analytical requirements provided for circulating miRNA studies for application to stable CAD and ACS research. We used quantitative PCR to determine the relative plasma levels of eight circulating miRNAs that are potentially associated with atherosclerosis. In a cohort of 136 adult clinic CAD patients and outpatient controls, we found that the plasma levels of miR-21-5p and miR-146a-5p were significantly elevated in ACS patients, and the level of miR-17-5p was decreased in ACS and stable CAD patients compared to both healthy controls and hypertensive patients without CAD. Within the ACS patient group, no differences were found in the plasma levels of these miRNAs between patients with positive and negative troponin, nor were any differences found between STEMI and NSTEMI. Our results indicate that increased plasma levels of miR-146a-5p and miR-21-5p can be considered general ACS circulating biomarkers and that lowered miR-17-5p can be considered a general biomarker of CAD.

Identification of Clinically Significant Prostate Cancer by Combined PCA3 and AMACR mRNA Detection in Urine Samples.

PURPOSE: Preclinical evaluation of PCA3 and AMACR transcript simultaneous detection in urine to diagnose clinical significant prostate cancer (prostate cancer with Gleason score ≥7) in a Russian cohort. PATIENTS AND METHODS: We analyzed urine samples of patients with a total serum PSA ≥2 ng/mL: 31 men with prostate cancer scheduled for radical prostatectomy, 128 men scheduled for first diagnostic biopsy (prebiopsy cohort). PCA3, AMACR, PSA and GPI transcripts were detected by multiplex reverse transcription quantitative polymerase chain reaction, and the results were used for scores for calculation and statistical analysis. RESULTS: There was no significant difference between clinically significant and nonsignificant prostate cancer PCA3 scores. However, there was a significant difference in the AMACR score (patients scheduled for radical prostatectomy p=0.0088, prebiopsy cohort p=0.029). We estimated AUCs, optimal cutoffs, sensitivities and specificities for PCa and csPCa detection in the prebiopsy cohort by tPSA, PCA3 score, PCPT Risk Calculator and classification models based on tPSA, PCA3 score and AMACR score. In the clinically significant prostate cancer ROC analysis, the PCA3 score AUC was 0.632 (95%CI: 0.511-0.752), the AMACR score AUC was 0.711 (95%CI: 0.617-0.806) and AUC of classification model based on the PCA3 score, the AMACR score and total PSA was 0.72 (95%CI: 0.58-0.83). In addition, the correlation of the AMACR score with the ratio of total RNA and RNA of prostate cells in urine was shown (tau=0.347, p=6.542e-09). Significant amounts of nonprostate RNA in urine may be a limitation for the AMACR score use. CONCLUSION: The AMACR score is a good predictor of clinically significant prostate cancer. Significant amounts of nonprostate RNA in urine may be a limitation for the AMACR score use. Evaluation of the AMACR score and classification models based on it for clinically significant prostate cancer detection with larger samples and a follow-up analysis is promising.

Elevated Plasma Levels of Circulating Extracellular miR-320a-3p in Patients with Paroxysmal Atrial Fibrillation.

The potential of extracellular circulating microRNAs (miRNAs) as non-invasive biomarkers of atrial fibrillation (AF) has been confirmed by a number of recent studies. However, the current data for some miRNAs are controversial and inconsistent, probably due to pre-analytical and methodological differences. In this work, we attempted to fulfill the basic pre-analytical requirements provided for circulating miRNA studies for application to paroxysmal atrial fibrillation (PAF) research. We used quantitative PCR (qPCR) to determine the relative plasma levels of circulating miRNAs expressed in the heart or associated with atrial remodeling or fibrillation with reported altered plasma/serum levels in AF: miR-146a-5p, miR-150-5p, miR-19a-3p, miR-21-5p, miR-29b-3p, miR-320a-3p, miR-328-3p, miR-375-3p, and miR-409-3p. First, in a cohort of 90 adult outpatient clinic patients, we found that the plasma level of miR-320a-3p was elevated in PAF patients compared to healthy controls and hypertensive patients without AF. We further analyzed the impact of medication therapies on miRNA relative levels and found elevated miR-320a-3p levels in patients receiving angiotensin-converting-enzyme inhibitors (ACEI) therapy. Additionally, we found that miR-320a-3p, miR-21-5p, and miR-146a-5p plasma levels positively correlated with the CHA2DS2-Vasc score and were elevated in subjects with CHA2DS2-Vasc ≥ 2. Our results indicate that, amongst the analyzed miRNAs, miR-320a-3p may be considered as a potential PAF circulating plasma biomarker, leading to speculation as to whether this miRNA is a marker of platelet state change due to ACEI therapy.

Dataset on transcriptome profiling of corneal endothelium from patients with Fuchs endothelial corneal dystrophy.

Fuchs endothelial corneal dystrophy (FECD) is a bilateral inherited eye disease with advanced forms only treatable by corneal transplantation. The pathogenesis of FECD has not been worked out yet, however, trinucleotide repeat polymorphism CTG18.1 in the TCF4 gene has recently been associated with late-onset FECD. Gene expression profiling of corneal endothelium with and without this expansion can help elucidate molecular mechanisms of the disease development. Current data article represents whole transcriptome profiles of corneal endothelium obtained from 12 patients with FECD and 6 control tissues from eye bank donors. RNA sequencing data is available at NCBI Sequence Read Archive under Accession No. PRJNA524323. In addition, each patient and donor were genotyped for CTG18.1 expansion and the corresponding numbers of CTG repeats in the TCF4 gene are provided within this article. The dataset includes samples from FECD patients both with and without CTG18.1 expansion.

CTG18.1 Expansion is the Best Classifier of Late-Onset Fuchs' Corneal Dystrophy Among 10 Biomarkers in a Cohort From the European Part of Russia.

Purpose: To assess the occurrence and diagnostic performance of nine single-nucleotide variants (SNVs) in the TCF4, SLC4A11, LOXHD1, and AGBL1 genes and the CTG18.1 trinucleotide repeat expansion in a Russian cohort of Fuchs' endothelial corneal dystrophy (FECD) patients. Methods: This retrospective case-control study included 100 patients diagnosed with FECD (cases) and 100 patients with cataracts (controls). Blood DNA was used to perform PCR and subsequent Sanger sequencing of rs613872 and rs17595731 in TCF4, c.99-100delTC, rs267607065, rs267607064, and rs267607066 in SLC4A11, rs113444922 in LOXHD1, and rs181958589 and rs185919705 in AGBL1. The number of CTG18.1 trinucleotide repeats was determined by a combination of conventional PCR or triplet primed PCR with fragment analysis. Results: At least one rs613872 marker allele was found in 78% of FECD patients and 21% of controls, and at least one rs17595731 marker allele was found in 14% and 2%, respectively. CTG18.1 trinucleotide expansion (>40 repeats) was detected in 72% of FECD patients and 5% of controls. Marker alleles of the tested SNVs in SLC4A11, LOXHD1, and rs185919705 in AGBL1 were not found in our FECD cohort. One FECD patient carried the marker allele of the rs181958589 SNV. Analysis of the diagnostic performance of individual markers in TCF4 and their combinations showed that the CTG18.1 repeat expansion was the best classifier for FECD (AUC = 0.84). Conclusions: Patients carrying CTG18.1 repeat expansion constituted a high proportion of the Russian FECD cohort; therefore, this marker is suitable for development of diagnostic and therapeutic approaches.

Novel RNA biomarkers of prostate cancer revealed by RNA-seq analysis of formalin-fixed samples obtained from Russian patients.

Due to heterogeneous multifocal nature of prostate cancer (PCa), there is currently a lack of biomarkers that stably distinguish it from benign prostatic hyperplasia (BPH), predict clinical outcome and guide the choice of optimal treatment. In this study RNA-seq analysis was applied to formalin-fixed paraffin-embedded (FFPE) tumor and matched normal tissue samples collected from Russian patients with PCa and BPH. We identified 3384 genes differentially expressed (DE) (FDR < 0.05) between tumor tissue of PCa patients and adjacent normal tissue as well as both tissue types from BPH patients. Overexpression of four of the discovered genes (ANKRD34B, NEK5, KCNG3, and PTPRT) was validated by RT-qPCR. Furthermore, the enrichment analysis of overrepresented microRNA and transcription factor (TF) recognition sites within DE genes revealed common regulatory elements of which 13 microRNAs and 53 TFs were thus linked to PCa for the first time. Moreover, 8 of these TFs (FOXJ2, GATA6, NFE2L1, NFIL3, PRRX2, TEF, EBF2 and ZBTB18) were found to be differentially expressed in this study making them not only candidate biomarkers of prostate cancer but also potential therapeutic targets.