Circulating tumour DNA (ctDNA) fragments found in blood represent a critical source of information about the tumour. However, the analysis of ctDNA is challenging due to the overwhelming presence of DNA from non-malignant cells and requires the development of highly sensitive laboratory techniques. This review discusses the major categories of ctDNA biomarkers, their detection methodologies, and explores the potential of emerging technologies within the liquid biopsy field.

Authors discuss the current state and prospects for the development of genetic testing in clinical laboratory diagnostics, recent nucleic acids sequencing technologies, their advantages and applications. Although genome-wide association studies (GWAS) have become a standard practice in identifying SNPs to determine disease susceptibility, this approach has limitations. A novel approach is proposed: integrative genome-wide association analysis (iGWAS), which relies on gene expression information to investigate the associations between SNPs and disease phenotype. Numerous studies have shown that iGWAS can significantly facilitate the search for genetic correlations and is superior to a method that relies only on the search for SNPs. Genetic testing will facilitate the molecular-based reclassification of human diseases. Authors describe the technical aspects of nanopore sequencing, the development of an iPhone app to complement miniature sequencing devices, and the world’s first mobile genomic sequence analyzer, iGenomics.

Cell-free DNA (cfDNA) testing is the core of most liquid biopsy assays. In particular, cfDNA fragmentation features could facilitate non-invasive cancer detection due to their interconnection with tumor-specific epigenetic alterations. However, the final cfDNA fragmentation profile in a purified sample is the result of a complex interplay between informative biological and artificial technical factors. In this work, we use ddPCR to study cfDNA lengths in colorectal cancer patients and observe shorter and more variable cfDNA fragments in accessible chromatin loci compared to the densely packed pericentromeric region. We also report a convenient qPCR system suitable for screening cfDNA samples for artificial high molecular weight DNA contamination.

The review discusses issues related to genetic predisposition and resistance to tuberculosis. Genetic factors largely determine susceptibility to various diseases, including infections. The main focus is on the genes of the major histocompatibility complex and toll-like receptors. A number of genetic polymorphisms responsible for resistance and predisposition to tuberculosis and related clinical consequences are considered. Knowledge of molecular genetic biomarkers is necessary to identify risk groups and carry out predictive measures.

The review discusses issues related to genetic predisposition and resistance to the new coronavirus infection including various factors: epidemiological, gender, ethnic and microbiome. The role of molecular genetic methods in the diagnosis of coronavirus infection is emphasized. The genetic factors largely determine the body’s susceptibility to various diseases, including infectious ones. The focus is on the genes associated with the production of interferons and enzymes that are responsible for anti-viral responses, as well as inflammatory reactions in the lungs. The first group includes genes IFNAR2 and OAS1, which control the production of antiviral proteins, while the second includes DPP9, TYK2 and CCR2, that control the behavior of monocytes. A number of other genetic polymorphisms responsible for resistance and susceptibility to infections and associated clinical consequences are discussed. The knowledge of molecular genetic biomarkers is necessary to identify risk groups, conduct predictive measures, including vaccination against COVID-19.

Nucleic acid fragments found in blood circulation originate mostly from dying cells and carry signs pointing to specific features of the parental cell types. Deciphering these clues may be transformative for numerous research and clinical applications but strongly depends on the development and implementation of robust analytical methods. Remarkable progress has been achieved in the reliable detection of sequence alterations in cell-free DNA while decoding epigenetic information from methylation and fragmentation patterns requires more sophisticated approaches. This review discusses the currently available strategies for detecting and analyzing the epigenetic marks in the liquid biopsies.

Cell-free DNA analysis in cancer patients may reveal tumor-specific alterations, including mutations, aberrant methylation, and chromatin state changes. The latter are connected to shifts in transcription programs and may be considered as epigenetic markers of cancer. Several whole-genome sequencing studies have shown that the chromatin state of parental cells may be partially decoded from cfDNA fragmentation features. In this preliminary study, we aim to test an approach for identifying differentially fragmented regions in cell-free DNA of cancer patients. The study involved cfDNA samples from 24 patients with colon adenocarcinoma, stages I (n=3), II (n=6), III (n=7), and IV (n=8), and 13 healthy individuals. We designed a targeted NGS panel based on a single-primer extension protocol to analyze the lengths of cfDNA fragments. The panel comprised 81 primers covering COAD-specific open-chromatin regions extracted previously from TCGA data. We developed a novel approach for targeted cfDNA fragmentation analysis in multiply regions of interest. We have experimentally identified 10 genomic regions with significant (p<0.01) differences in cfDNA fragmentation in patients with stage IV colorectal cancer and healthy controls. Our evidence supports the concept that targeted analysis of cfDNA fragmentation may facilitate the detection of tumor presence. This strategy may reveal a novel class of cost-effective biomarkers that will serve as analytes themselves or might be incorporated into multidimensional liquid biopsy assays in combination with somatic mutations or aberrant methylation.

The review addresses issues related to genetic predisposition and resistance to sexually transmitted infections (STI). Genetic factors largely determine the susceptibility of the body to various diseases, including infectious ones. The main influence is given to the genes of the main histocompatibility complex and toll-like receptors. Some STI and genetic polymorphisms responsible for resistance and susceptibility to infections and related clinical consequences are considered. Knowledge of molecular genetic biomarkers is necessary to identify risk groups, conduct predictive measures, in particular vaccination.

Epigenetic alterations represent promising therapeutic targets in cancer treatment. Recently it was revealed that small molecules have the potential to act as microRNA silencers. Capacity to bind the discrete stem-looped structure of pre-miR-21 and prevent its maturation opens opportunities to utilize such compounds for the prevention of initiation, progression, and chemoresistance of cancer. Molecular simulations performed earlier identified 3,3′-diindolylmethane (DIM) as a potent microRNA-21 antagonist. However, data on DIM and microRNA-21 interplay is controversial, which may be caused by the limitations of the cell lines.

Fragmentation of DNA is the first and very important step in preparing nucleic acids for NGS. Here we report a novel Fragmentation Through Polymerization (FTP) technique, which is simple, robust and low-cost enzymatic method of fragmentation. This method generates double-stranded DNA fragments that are suitable for the direct use in NGS library construction, and allows to eliminate the need of an additional step of reparation of DNA ends.

The review addresses issues related to genetic predisposition and resistance to infectious diseases. Genetic factors largely determine the susceptibility of the body to various diseases, including infectious ones. A genetic predisposition to tuberculosis, salmonellosis, viral hepatitis, tick-borne encephalitis, Lyme disease, HIV and others is shown. Knowledge of molecular genetic biomarkers is necessary for identifying risk groups, conducting predictive measures, in particular vaccination. The main influence is given to the genes of the main histocompatibility complex; the role of mitochondrial DNA in susceptibility to HIV infection is shown.

As public health systems are being modernized across the world, conventional medicine is undergoing a serious transformation and new medical models are emerging based on personalized, predictive, participatory, precision, mobile, and digital approaches. So far, there is no consensus in the literature and the medical community about the goals, objectives and applications of these models, particularly precision medicine, which is sometimes perceived as merely a fancier term for personalized medicine. The role of laboratory diagnostics in precision medicine is also a matter of intense debate. This review analyzes the currently available information about precision medicine and gives examples of how 5P approaches can be used in clinical practice.

В рамках модернизации системы здравоохранения в настоящее время происходит изменение старых и становление новых направлений современной медицины: персонализированной, предиктивной, превентивной, партисипативной, прецизионной, мобильной и цифровой. Вместе с тем, в научной литературе и медицинском сообществе имеются различные мнения о целях, области применения и задачах указанных направлений здравоохранения и роли в них лабораторной диагностики. Прежде всего это относится к прецизионной медицине, которая воспринимается только как более современное название персонализированной медицины. В обзоре проанализирована и обобщена имеющаяся на сегодняшний день информация и представлены примеры применения подходов медицины 5П в клинической практике.

Total number of deleterious somatic mutations normalized by gene length in 52 CBT samples. (XLSX 132Â kb)

Carotid body tumor (CBT) is a form of head and neck paragangliomas (HNPGLs) arising at the bifurcation of carotid arteries. Paragangliomas are commonly associated with germline and somatic mutations involving at least one of more than thirty causative genes. However, the specific functionality of a number of these genes involved in the formation of paragangliomas has not yet been fully investigated.

Background Colorectal cancer (CRC) is a common cancer worldwide. The main cause of death in CRC includes tumor progression and metastasis. At molecular level, these processes may be triggered by epithelial-mesenchymal transition (EMT) and necessitates specific alterations in cell metabolism. Although several EMT-related metabolic changes have been described in CRC, the mechanism is still poorly understood. Results Using CrossHub software, we analyzed RNA-Seq expression profile data of CRC derived from The Cancer Genome Atlas (TCGA) project. Correlation analysis between the change in the expression of genes involved in glycolysis and EMT was performed. We obtained the set of genes with significant correlation coefficients, which included 21 EMT-related genes and a single glycolytic gene, HK3 . The mRNA level of these genes was measured in 78 paired colorectal cancer samples by quantitative polymerase chain reaction (qPCR). Upregulation of HK3 and deregulation of 11 genes ( COL1A1, TWIST1, NFATC1, GLIPR2, SFPR1, FLNA, GREM1, SFRP2, ZEB2, SPP1, and RARRES1 ) involved in EMT were found. The results of correlation study showed that the expression of HK3 demonstrated a strong correlation with 7 of the 21 examined genes ( ZEB2, GREM1, TGFB3, TGFB1, SNAI2, TWIST1, and COL1A1 ) in CRC. Conclusions Upregulation of HK3 is associated with EMT in CRC and may be a crucial metabolic adaptation for rapid proliferation, survival, and metastases of CRC cells.

The review considers mobile health technologies (mHealth) - a new, rapidly developing field of medicine. Extremely promising are hybrid devices based on microfluidic technologies, mobile devices for monitoring diabetes mellitus, the use of mobile phones in immunoassay, microscopy in the diagnosis of cancer. Amplification technologies of mobile health care are used, in particular, in the diagnosis of infectious and parasitic diseases. All conditions for the emergence of mass open online medicine (IOM) have been created.

Whole-genome amplification (WGA) techniques are used for non-specific amplification of low-copy number DNA, and especially for single-cell genome and transcriptome amplification. There are a number of WGA methods that have been developed over the years. One example is degenerate oligonucleotide-primed PCR (DOP-PCR), which is a very simple, fast and inexpensive WGA technique. Although DOP-PCR has been regarded as one of the pioneering methods for WGA, it only provides low genome coverage and a high allele dropout rate when compared to more modern techniques. Here we describe an improved DOP-PCR (iDOP-PCR). We have modified the classic DOP-PCR by using a new thermostable DNA polymerase (SD polymerase) with a strong strand-displacement activity and by adjustments in primers design. We compared iDOP-PCR, classic DOP-PCR and the well-established PicoPlex technique for whole genome amplification of both high- and low-copy number human genomic DNA. The amplified DNA libraries were evaluated by analysis of short tandem repeat genotypes and NGS data. In summary, iDOP-PCR provided a better quality of the amplified DNA libraries compared to the other WGA methods tested, especially when low amounts of genomic DNA were used as an input material.