Evaluation of Epigenetic Age Based on DNA Methylation Analysis of Several CpG Sites in Ukrainian Population

Epigenetic clocks are the models, which use CpG methylation levels for the age prediction of an organism. Although there were several epigenetic clocks developed there is a demand for development and evaluation of the relatively accurate and sensitive epigenetic clocks that can be used for routine research purposes. In this study, we evaluated two epigenetic clock models based on the 4 CpG sites and 2 CpG sites in the human genome using the pyrosequencing method for their methylation level estimation. The study sample included 153 people from the Ukrainian population with the age from 0 to 101. Both models showed a high correlation with the chronological age in our study sample (R2 = 0.85 for the 2 CpG model and R2 = 0.92 for the 4 CpG model). We also estimated the accuracy metrics of the age prediction in our study sample. For the age group from 18 to 80 MAD was 5.1 years for the 2 CpG model and 4.1 years for the 4 CpG model. In this regard, we can conclude, that the models evaluated in the study have good age predictive accuracy, and can be used for the epigenetic age evaluation due to the relative simplicity and time-effectiveness.

Rapid Pyrosequencing Method for FMO3 Non-Synonymous Genetic Variant Evaluation in A Korean Population

Background: The aim of this study was to develop a feasible pyrosequencing method to detect non-synonymous single nucleotide polymorphisms (SNPs) of the flavin-containing monooxygenase 3 (FMO3) gene and compare the ethnic differences in the frequencies of these alleles. Methods and Results: This pyrosequencing method was used to identify four non-synonymous FMO3 SNPs, including c.855C>T (rs909530), c.441C>T (rs1800822), c.923A>G (rs2266782), and c.472G>A (rs2266782). The allele frequencies of these SNPs in 122 unrelated Korean subjects were analyzed, and were as follows: 44.7% for c.855C>T, 23.4% for c.441C>T, 23.0% for c.923A>G, and 27.1% for c.472G>A. Linkage disequilibrium (LD) analysis showed that c.923A>G and c.472G>A were in strong LD (D′ = 0.8289, r2 = 0.5332). Conclusions: The designed pyrosequencing method was successfully applied to identify the c.855C>T, c.441C>T, c.923A>G, and c.472G>A SNPs. The frequencies were similar to those reported previously in a Japanese population. However, in general, large differences between ethnicities were found.

Combined RT-qPCR and Pyrosequencing of a SARS-CoV-2 Spike Glycoprotein Polybasic Cleavage Motif Uncovers Rare Pediatric COVID-19 Spectrum Diseases of Unusual Presentation

BackgroundSurveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections is essential for the global containment measures with regard to the ongoing pandemic. Diagnostic gold standard is currently reverse transcription of the (+)RNA genome and subgenomic RNAs and subsequent quantitative polymerase chain reaction (RT-qPCR) from nasopharyngeal swabs or bronchoalveolar lavages. In order to further improve the diagnostic accuracy, particularly for the reliable discrimination between negative and false-negative specimens, we propose the combination of the RT-qPCR workflow with subsequent pyrosequencing of a S-gene amplicon. This extension might add important value mainly in cases with low SARS-CoV-2 load, where RT-qPCR alone can deliver conflicting results.sResultsWe successfully established a combined RT-qPCR and S-gene pyrosequencing method which can be optionally exploited after routine diagnostics or for epidemiologic studies. This allows a more reliable interpretation of conflicting RT-qPCR results in specimens with relatively low viral loads and close to the detection limits of qPCR (CT values >30). After laboratory implementation and characterization of a best practice protocol we tested the combined method in a large pediatric cohort from two German medical centers (n=769). Pyrosequencing after RT-qPCR enabled us to uncover 6 previously unrecognized cases of pediatric SARS-CoV-2 associated diseases, partially exhibiting unusual and heterogeneous presentation. Moreover, it is notable that in the course of RT-qPCR/pyrosequencing method establishment we did not observe any case of false-positive diagnosis when confirmed SARS-CoV-2-positive specimens were used from foregoing routine testing.ConclusionsThe proposed protocol allows a specific and sensitive detection of SARS-CoV-2 close to the detection limits of RT-qPCR. Combined RT-qPCR/pyrosequencing does not negatively affect preceding RT-qPCR pipeline in SARS-CoV-2 diagnostics and can be optionally applied in routine to inspect conflicting RT-qPCR results.

Pyrosequencing method in the diagnosis of Gilbert syndrome

Метод пиросеквенирования позволяет выявить различные варианты полиморфизма (ТА)5/6/7/8 в гомо- и гетерозиготном состоянии. Частота выявление синдрома Жильбера у амбулаторных пациентов составила 24%. Применение набора «АмплиСенс®Пироскрин UGT1A1» в клинической практике может быть использовано для диагностики синдрома Жильбера и оценки побочных эффектов при назначении лекарств. The pyrosequencing is a reliable and robust technique for different variants of polymorphism (TA)5/6/7/8 verification in homo- and heterozygous state. The frequency of GS in outpatient patients was 24%. The application of «AmpliSens® Pyroscreen UGT1A1» kit in clinical practice.