Molecular genetic methods in biomedical research. Part III: human gene diagnostics in clinical practice

Application of molecular genetic methods in the diagnosis and treatment of human diseases is extremely wide due to a huge amount of hereditary information contained in the human genome. Gene diagnostics allows establishing predisposition to diseases, identification of genetic abnormalities and prediction of pathological outcomes. In addition, gene diagnostics also enables prediction of the individual response to treatment in order to achieve the maximum therapeutic effect. Among all molecular genetic methods, polymerase chain reaction (PCR) diagnostics is a leading approach. Technical simplicity, low cost, high sensitivity and reliability of the method have made PCR diagnostics a routine modality for the risk assessment, diagnostics, and monitoring of the treatment efficiency. Here, we consider the application of PCR diagnostics for the abovementioned tasks and talk about the real-life examples of detecting mutations and chromosomal aberrations which may cause a disease. Further, we discuss the prospects of using a semi-quantitative PCR in medical practice and focus on pharmacogenetics as a key component of a personalised therapy. The lecture is aimed primarily at biomedical students and physicians and represents a continuation of the previous lectures published in Fundamental and Clinical Medicin.

TECHNOLOGY OF BOVINE LEUKEMIA VIRUS GENODIAGNOSTICS IN CATTLE, IN PRODUCED RAW MATERIALS AND PRODUCTS

The most important task of the dairy cattle industry is to obtain high quality raw milk. To achieve it, a set of measures is required, including aimed at increasing the biological safety of produced raw materials. The aim of the study was to create a scientific and methodological basis for the Bovine leukemia virus (BLV) gene diagnostics in a combined format of pathogen indication and identification. This required updating the strategy of BLV PCR-RFLP genotyping, consistent with its phylogenetic classification, taking into account the growing knowledge about the genetic diversity of 11 genotypes of the studied viral pathogen. When staging nested PCR, oligonucleotide primers were used, which initiate at the final stage of the reaction the production of a 444 bp env-gene fragment of the pathogen. Five restriction endonucleases were used in PCR-RFLP BLV genotyping of: PvuII, SspI, AsuHPI, HaeIII, and BstX2I. As a result of verification of the developed Bovine leukemia virus method for gene identification with an updated genotyping strategy, a technical result was obtained, expressed in the ability to identify all 11 BLV genotypes discovered to date by interpreting the generated 58 genotype-associated combinations of PCR-RFLP profiles.

Potential of whole-genome sequencing-based pharmacogenetic profiling

Pharmacogenetics represents a major driver of precision medicine, promising individualized drug selection and dosing. Traditionally, pharmacogenetic profiling has been performed using targeted genotyping that focuses on common/known variants. Recently, whole-genome sequencing (WGS) is emerging as a more comprehensive short-read next-generation sequencing approach, enabling both gene diagnostics and pharmacogenetic profiling, including rare/novel variants, in a single assay. Using the example of the pharmacogene CYP2D6, we demonstrate the potential of WGS-based pharmacogenetic profiling as well as emphasize the limitations of short-read next-generation sequencing. In the near future, we envision a shift toward long-read sequencing as the predominant method for gene diagnostics and pharmacogenetic profiling, providing unprecedented data quality and improving patient care.

Development of accelerated genodiagnosis method of pertussis and pertussis-like diseases on the basis of mPCR-RT

The aim of the work was to develop an accelerated genodiagnosis method based on mPCR-RT for the detection DNA of B. pertussis, B. parapertussis, B. holmesii. Materials and methods. The study used 104 strains of microorganisms, of which: 50 strains of B. pertussis, 37 - B. parapertussis, 17 - heterologous species of microorganisms. Assessment of analytical specificity was carried out using DNA strains of various microorganisms with a concentration at least 109 GE / ml. To check the analytical sensitivity we studied a series of serial dilutions of bacterial cultures of the control strains B. pertussis № 143, B. parapertussis № 38b, B. holmesii DSM 13416 with a concentration of 5x109 - 5 μm/ml. Results. Insertion sequences were chosen as diagnostic targets: for B. parapertussis - a specific fragment IS1001, for B. holmesii - a specific fragment hlIS1001, for B.pertussis - a fragment IS481. To develop a genodiagnosis method specific primers were designed and combined into a single multi-primer mixture, the composition of the reaction mixture and the amplification conditions were selected. The analytical sensitivity of the developed method for detecting pertussis and pertussis-like pathogens was 5×101 GE / ml. Verification of the developed methodology of gene diagnostics showed 100% analytical specificity. Conclusion. An accelerated genodiagnosis method based on mPCR-RT has been developed, it allows you to identify DNA of B. pertussis, B. parapertussis, B. holmesii, which expands the possibilities of examining patients with suspected pertussis and pertussis-like diseases in order to increase laboratory confirmation of the diagnosis.

Typing and classification of non-tuberculous mycobacteria isolates

Background: There are a large and growing number of non-tuberculous mycobacteria (NTM) species that have been isolated, identified, and described in the literature, yet there are many clinical isolates which are not assignable to known species even when the genome has been sequenced. Additionally, a recent manuscript has proposed the reclassification of the Mycobacterium genus into five distinct genera. Methods: We describe using a community standard fast average nucleotide identity (ANI) approximation method, MASH, for classifying NTM genomes by comparison to a resource of type strain genomes and proxy genomes. We evaluate the genus reclassification proposal in light of our ANI, MLST, and pan-genome work. Results: We describe here a sequencing study of hundreds of clinical NTM isolates. To aid in characterizing these isolates we defined a multi-locus sequence typing (MLST) schema for NTMs which can differentiate strains at the species and subspecies level using eight ribosomal protein genes. We determined and deposited the allele profiles for 2,802 NTM and Mycobacterium tuberculosis complex strains in PubMLST. Conclusions: The MLST schema and our pan-genome analysis of Mycobacteria can help inform the design of marker-gene diagnostics. The ANI comparisons likewise can assist in the classification of unknown genomes, even from previously unknown species.

Congenital nephrotic syndrome: is early aggressive treatment needed? Yes

Congenital nephrotic syndrome (CNS) was primarily considered one disease entity. Hence, one treatment protocol was proposed in the beginning to all CNS patients. Today, with the help of gene diagnostics, we know that CNS is a heterogeneous group of disorders and therefore, different treatment protocols are needed. The most important gene defects causing CNS are NPHS1, NPHS2, WT1, LAMB2, and PLCE1. Before active treatment, all infants with CNS died. It was stated already in the mid-1980s that intensive medical therapy followed by kidney transplantation (KTx) should be the choice of treatment for infants with severe CNS. In Finland, early aggressive treatment protocol was adopted from the USA and further developed for treatment of children with the Finnish type of CNS. The aim of this review is to state reasons for “early aggressive treatment” including daily albumin infusions, intensified nutrition, and timely bilateral nephrectomy followed by KTx at the age of 1–2 years.

Hypotheses of genetic aspects of the pathogenesis of type 1 Chiari malformation

Relevance. The study of the genetic mechanisms of the Chiari malformation is based on the study of genes of a possible predisposition to this pathology in combination with environmental factors that form the pathogenetic chain of the disease. Objectives of the research — to analyze foreign and Russian publications. Based on the literature, study the hypothesis of the genetic aspects of the pathogenesis of Chiari malformation. Findings. Based on the data of domestic and foreign literature, one can judge a breakthrough in the study of the genetic nature of the Chiari malformation, however, there is still no consensus on the pathogenesis of this disease, the responsible gene causing the pathology also remains unidentified. Further study of the genetically determined mechanism of the malformation will help in an interdisciplinary approach for gene diagnostics and personalized prevention of the craniovertebral region pathology.

Typing and classification of non-tuberculous mycobacteria isolates

Background: There are a large and growing number of non-tuberculous mycobacteria (NTM) species that have been isolated, identified, and described in the literature, yet there are many clinical isolates which are not assignable to known species even when the genome has been sequenced. Additionally, a recent manuscript has proposed the reclassification of the Mycobacterium genus into five distinct genera. Methods: We describe using a fast average nucleotide identity (ANI) approximation method, MASH, for classifying NTM genomes by comparison to a resource of type strain genomes and proxy genomes. We evaluate the genus reclassification proposal in light of our ANI, MLST, and pan-genome work. Results: We describe here a sequencing study of hundreds of clinical NTM isolates. To aid in characterizing these isolates we defined a multi-locus sequence typing (MLST) schema for NTMs which can differentiate strains at the species and subspecies level using eight ribosomal protein genes. We determined and deposited the allele profiles for 2,802 NTM and Mycobacterium tuberculosis complex strains in PubMLST. Conclusions: The MLST schema and our pan-genome analysis of Mycobacteria can help inform the design of marker-gene diagnostics. The ANI comparisons likewise can assist in the classification of unknown genomes, even from previously unknown species.

The acantamoeba lesions of the cornea (diagnosis) (Review of literature)

The Aсanthamoeba keratitis is a rare, but very dangerous disease of cornea; its development is connected with contact lens wearing, and corneal microtraumas with contamination of particles of the soil or water. The disease has no pronounced distinguishing clinical signs therefore it is difficult to distinguish them from herpetic or fungal keratitis. Nevertheless, it can be suspected on the basis of anamnestic data (use of contact lenses, corneal injury with contamination by the soil or water), uneven strong pain syndrome, ring-shaped infiltration of cornea, absence of effect of traditional therapy. Isolation of Acanthamoeba by bioculture method, morphological research of corneal bioptat (by light and luminescent microscopy), gene diagnostics, in – vivo confocal microscopy of the cornea can give additional help in the diagnosis. Also perspective diagnostic methods of Acanthamoeba keratitis are given. Nevertheless, so far there is no conventional technique of diagnosis of this disease.