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.

Detection of SARS-CoV-2 RNA by a Multiplex Reverse-Transcription Loop-Mediated Isothermal Amplification Coupled with Melting Curves Analysis.

Loop-mediated isothermal amplification (LAMP) is a method of nucleic acid amplification that is more stable and resistant to DNA amplification inhibitors than conventional PCR. LAMP multiplexing with reverse transcription allows for the single-tube amplification of several RNA fragments, including an internal control sample, which provides the option of controlling all analytical steps. We developed a method of SARS-CoV-2 viral RNA detection based on multiplex reverse-transcription LAMP, with single-tube qualitative analysis of SARS-CoV-2 RNA and MS2 phage used as a control RNA. The multiplexing is based on the differences in characteristic melting peaks generated during the amplification process. The developed technique detects at least 20 copies of SARS-CoV-2 RNA per reaction on a background of 12,000 MS2 RNA copies. The total time of analysis does not exceed 40 min. The method validation, performed on 125 clinical samples of patients' nasal swabs, showed a 97.6% concordance rate with the results of real-time (RT)-PCR assays. The developed multiplexed LAMP can be employed as an alternative to PCR in diagnostic practice to save personnel and equipment time.

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.