Aim. To present an approach to collection of biosamples of patients with rare and scientifically interesting clinical situations for solving the problems of personalized medicine, as well as to analyze related 8-year experience.Material and methods. The approach and a collection of biosamples of blood and its derivatives is developed at the National Medical Research Center for Therapy and Preventive Medicine within the project "Interesting Cases at the National Medical Research Center for Therapy and Preventive Medicine". The collection of biomaterial from project patients was carried out on a planned basis with the inpatient department, as well as upon referral from outpatient doctors. All included patients signed informed consent. Each biosample is accompanied by an extensive annotation, including socio-demographic, clinical, genetic and other types of data. The article presents the project results as of August 12, 2024.Results. An expert group developed 15 disease groups and related inclusion criteria. At the time of analysis, 4525 inpatients and outpatients were included in the project. Positive changes in the number of people included annually is noted. Genetic testing was performed on >2500 patients. The proposed approach allows solving a wide range of clinical and research problems in personalized medicine as follows: timely diagnosis or clarification; formation of patient cohorts to study the genetic aspects of diseases; identification of new genetic variants of hereditary diseases; development of genetic diagnostic panels; study of rare diseases; reduction of sample creation time in case of novel scientific ideas.Conclusion. The proposed approach to the collection and preservation of biosamples and related clinical, socio-demographic, genetic and other types of data in patients with rare clinical cases of scientific interest is important and effective for solving practical and research problems of personalized medicine. The algorithm is well developed, standardized and easily implemented within the clinics, regardless of their size. Preanalytical phase standardization creates the prerequisites for multicenter national and international cooperation.

Over the past decade, circulating small non-coding ribonucleic acid molecules (microRNAs) have demonstrated their potential as minimally invasive diagnostic and prognostic biomarkers of various diseases. Standardization of preanalytical and analytical factors, including collection, processing and storage of biosamples, plays a significant role in the reliability and reproducibility of circulating microRNA quantification. To date, there is no consensus regarding the data normalization used in the analysis of circulating microRNA expression. The review aim is to consider modern original papers on various storage conditions of biobanked plasma and serum samples with subsequent isolation of circulating microRNAs for analysis.

To implement a modern personalized approach in practical healthcare, the latest biomedical technologies should be developed and genetic research should be performed. The analysis of a substantial quantity of data is essential for the investigation of the prevalence of genetic risk factors for various diseases, drug resistance genes, the development of genetic panels to determine the individual risk of pathologies, as well as the creation of genetic risk scores. The review demonstrates through the use of illustrative examples that contemporary biobanks have become a vital component in the field of genetics research, both in Russia and globally. These specialized institutions are capable of accumulating, storing, and utilizing a substantial quantity of biological samples and related data, which is essential for advancing genetic research. The data collected in biobanks and associated clinical information form the basis for large-scale genetic studies conducted in different countries. The efficacy of genetic advancements, such as the early diagnosis of diseases, is contingent upon the number of biobanks, the establishment of collaborative networks among them, and the capacity to leverage digital platforms uniting diverse databases. Biobanks and biobanking have emerged as the foundation for the advancement of personalized medicine.

Circulating microribonucleic acids (microRNAs) are promising biomarkers of various diseases, but their clinical laboratory use requires highly sensitive, reproducible, reliable and sustainable methods for their accurate plasma and serum quantification. The preanalytical phase of studies conducted using biospecimens consists of their collection, processing, storage and transportation. Preanalytical conditions remain the main distorting factors in microRNA studies, and standardization of these conditions, carried out in biobanks, can improve the reproducibility of results and their comparison. The review aim is to consider the main contemporary original studies on preanalytical factors, which are an important source of variability in studies on circulating microRNAs at the stages from blood collection to plasma or serum production.

High-throughput sequencing of small ribonucleic acid (RNA) molecules is widely used to search for markers of various diseases, as well as to study the regulation of gene expression. The data processing protocol consists of many stages, including the stages of analyzing the initial data quality and sequencing results, mapping and studying the expression profile of the detected small RNA molecules. A whole arsenal of programs and specific packages has already been developed to implement each study step. The instrumental composition of the final bioinformatics protocol is critically important for the correct data processing and study reproduction. This review describes the most universal protocol for processing the results of high-throughput sequencing of small RNA molecules, including all the main stages and the most widely used programs.

Coronary collateral circulation (CCC) above the coronary chronic total occlusion is an alternative blood supply to the ischemic myocardium and increases survival among patients with coronary artery disease. In this regard, identification of novel markers associated with the CCC severity has diagnostic potential for patient stratification. It has been shown that circulating microRNAs play an important role in almost all cardiovascular aspects, including the association of some microRNAs with the CCC severity. The aim of this review is to consider the main modern studies on association of circulating microRNAs and CCC severity in coronary chronic total occlusion in patients with coronary artery disease, followed by a functional analysis of the identified microRNAs.

Aim. To validate and evaluate the accuracy of 4 genetic risk scores (GRSs) for hypertension (HTN), previously created on European samples, on a population sample of the Ivanovo Oblast.Material and methods. For genetic analysis, targeted next-generation sequencing was used on a sample of the Central Russia (n=1682) based on the biobank collection. Four GRSs associated with HTN, previously developed for the European population, were selected for validation. The coefficient of determination and the area under the ROC curve were used as quality metrics for regression models. Additional validation was carried out to include all nucleotide sequence variants, regardless of linkage disequilibrium level. A combined GRS was compiled based on coefficients from individual GRSs using the clumping + thresholding (C+T) method.Results. The study demonstrated that the predictive value of previously developed GRSs when used for Central Russian population is lower than in the original studies. The proportion of explained variance was 0,5-0,8%. The best predictive ability (proportion of explained variance — 2,5%) was demonstrated using previously developed GRSs (Evangelou E, et al., 2018), which includes the largest number of nucleotide sequence variants (n=852).Conclusion. GRSs for HTN, developed on European samples, is not recommended for Russian population without preliminary validation. To create original GRSs, combining statistical parameters (β-coefficients and p-value) from different GRS is not recommended.

Background: The present study investigates the feasibility of using three previously published genome-wide association studies (GWAS) results on blood lipids to develop polygenic risk scores (PRS) for population samples from the European part of the Russian Federation. Methods: Two population samples were used in the study – one from the Ivanovo region (n = 1673) and one from the Vologda region (n = 817). We investigated three distinct approaches to PRS development: using the straightforward PRS approach with original effect sizes and fine-tuning with PRSice-2 and LDpred2. Results: In total, we constructed 56 PRS scales related to four lipid phenotypes: low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, total cholesterol, and triglyceride levels. Compared with previous results for the Russian population, we achieved an additional R2 increase of 2–4%, depending on the approach and lipid phenotype studied. Overall, the R2 PRS estimates approached those described for other populations. We also evaluated the clinical utility of blood lipid PRS for predicting carotid and femoral artery atherosclerosis. Specifically, we found that PRS for total cholesterol, low-density lipoprotein cholesterol, and triglycerides were positively correlated with ultrasound parameters of carotid and femoral artery atherosclerosis (ρ = 0.09–0.13, p < 0.001), whereas PRS for high-density lipoprotein cholesterol were inversely correlated with the number of plaques in the femoral arteries (ρ = −0.08, p = 8.71 × 10−3). Conclusions: PRS fine-tuning using PRSice-2 add LDpred2 improves the performance of blood lipid PRS. Our study demonstrates the potential for further use of blood lipid PRS for prediction of atherosclerosis risk.

Familial dysbetalipoproteinemia (FD) is a highly atherogenic, prevalent genetically based lipid disorder. About 10% of FD patients have rare APOE variants associated with autosomal dominant FD. However, there are insufficient data on the relationship between rare APOE variants and FD. Genetic data from 4720 subjects were used to identify rare APOE variants and investigate their pathogenicity for autosomal dominant FD. We observed 24 variants in 86 unrelated probands. Most variants were unique (66.7%). Five identified APOE variants (p.Glu63ArgfsTer15, p.Gly145AlafsTer97, p.Lys164SerfsTer87, p.Arg154Cys, and p.Glu230Lys) are causal for autosomal dominant FD. One of them (p.Lys164SerfsTer87) was described for the first time. When we compared clinical data, it was found that carriers of pathogenic or likely pathogenic APOE variants had significantly higher triglyceride levels (median 5.01 mmol/L) than carriers of benign or likely benign variants (median 1.70 mmol/L, p = 0.034) and variants of uncertain significance (median 1.38 mmol/L, p = 0.036). For the first time, we estimated the expected prevalence of causal variants for autosomal dominant FD in the population sample: 0.27% (one in 619). Investigating the spectrum of APOE variants may advance our understanding of the genetic basis of FD and underscore the importance of APOE gene sequencing in patients with lipid metabolism disorders.

Aim. To investigate the association of 10 circulating plasma microRNAs with collateral flow degree in chronic total occlusion (CTO) patients with coronary artery disease (CAD).Materials and methods. Plasma expression levels of 10 circulating miRNAs were measured by real-time PCR using Taqman technology in a sample of 43 subjects. The study included patients with CAD and CTO with good (n=13) or poor (n=10) coronary collateral circulation (CCC) based on Rentrop classification and a control group of patients without significant coronary stenosis (n=20).Results. Significant differences in expression levels were found for 7 circulating miRNAs in patients with CTO and good CCC and for 5 microRNAs in the combined group of patients with CTO compared to the control group. Among the 7 microRNAs, decreased expression of hsa-miR-126-5p, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-miR-15b-5p, hsa-miR-21-5p, hsa-miR-23a-3p and increased expression of hsa-miR-451a were detected. For the first time, we showed that the level of 2 microRNAs (hsa-miR-23a-3p, hsa-miR-21-5p) is significantly reduced and the level of hsa-miR-451a is increased in patients with CAD with good CCC.Conclusion. Plasma microRNAs with significant differences obtained can be used for further studies on a larger sample size as candidate biomarkers for assessing the severity of ССС in the presence of CTO.

Patients with genetically-based hyperlipidemias exhibit a wide phenotypic variability. Investigation of clinical and biochemical features is important for identifying genetically-based hyperlipidemias, determining disease prognosis, and initiating timely treatment. We analyzed genetic data from 3374 samples and compared clinical data, lipid levels (low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol, triglycerides, and lipoprotein (a)), frequency, age at onset of coronary heart disease (CHD), and the severity of carotid and femoral atherosclerosis (plaque number, maximum stenosis, total stenosis, maximum plaque height, and plaque score) among patients with familial hypercholesterolemia (FH), familial dysbetalipoproteinemia (FD), polygenic hypercholesterolemia (HCL), severe HCL, and those without lipid disorders (n = 324). FH patients exhibited the highest LDL-C (median 8.03 mmol/L, p < 0.001). FD patients had elevated triglyceride levels (median 4.10 mmol/L), lower LDL-C (median 3.57 mmol/L), and high-density lipoprotein cholesterol (median 1.03 mmol/L) compared to FH, polygenic HCL, and severe HCL, p < 0.05. FH and FD patients had similar early onset of CHD, with a median age of 44 and 40 years and comparable frequencies of 29.5% and 31.0%, respectively. They were more likely to develop CHD than subjects without lipid disorders (p = 0.042 and p < 0.001, respectively). Additionally, FH patients had higher a carotid plaque number, total carotid stenosis, and carotid plaque score. This study presents the first simultaneous comparison of clinical and biochemical features among FD, FH, polygenic, and severe HCL, along with the first comprehensive evaluation of carotid and femoral atherosclerosis ultrasound parameters in FD patients. The results highlight distinct phenotypic features unique to each hyperlipidemia analyzed and underscore FH and FD as the most atherogenic hyperlipidemias.

A genetic diagnosis of primary cardiomyopathies can be a long-unmet need in patients with complex phenotypes. We investigated a three-generation family with cardiomyopathy and various extracardiac abnormalities that had long sought a precise diagnosis. The 41-year-old proband had hypertrophic cardiomyopathy (HCM), left ventricular noncompaction, myocardial fibrosis, arrhythmias, and a short stature. His sister showed HCM, myocardial hypertrabeculation and fibrosis, sensorineural deafness, and congenital genitourinary malformations. Their father had left ventricular hypertrophy (LVH). The proband’s eldest daughter demonstrated developmental delay and seizures. We performed a clinical examination and whole-exome sequencing for all available family members. All patients with HCM/LVH shared a c.4411-2A>C variant in ALPK3, a recently known HCM-causative gene. Functional studies confirmed that this variant alters ALPK3 canonical splicing. Due to extracardiac symptoms in the female patients, we continued the search and found two additional single-gene disorders. The proband’s sister had a p.Trp329Gly missense in GATA3, linked to hypoparathyroidism, sensorineural deafness, and renal dysplasia; his daughter had a p.Ser251del in WDR45, associated with beta-propeller protein-associated neurodegeneration. This unique case of three monogenic disorders in one family shows how a comprehensive approach with thorough phenotyping and extensive genetic testing of all symptomatic individuals provides precise diagnoses and appropriate follow-up, embodying the concept of personalized medicine. We also present the first example of a splicing functional study for ALPK3 and describe the genotype–phenotype correlations in cardiomyopathy.

Aim. To analyze contrast-enhanced cardiac magnetic resonance imaging (MRI) in patients with phenotypic manifestations of left ventricular non-compaction (LVNC) and related genetic mutations, as well as to determine the relationship between mutations and types of left ventricular (LV) remodeling and with a number of other morphological and functional cardiac parameters.Material and methods. From the registry of patients with LVNC and their relatives, patients with morphological signs of LVNC and 4 related mutations (MYH7, MYBPC3, TTN, and desmin genes (DES, DSG2, DSP and DSC2)). All patients underwent contrast-enhanced cardiac MRI, based on which the type of LV remodeling was determined.Results. The study included 44 patients who, according to genetic analysis, had mutations in sarcomeric genes responsible for LVNC development. In each patient, the type of LV remodeling was determined based on cardiac MRI results. We found that if patients with LVNC have mutations in the MYBPC3 and TTN genes, the chance of LV dilatation remodeling is significantly higher. On the contrary, in the presence of a DES gene mutation, the probability of this LV remodeling is lower, and milder morphological manifestations of LVNC are noted.Conclusion. The combination of cardiac MRI data and genetic analysis improves the morphological and functional stratification of patients with LVNC.

Aim. To study genetic causes of decreased low-density lipoprotein cholesterol (LDL-C) in Russian patients.Material and methods. The study included the following Epidemiology of Cardiovascular Diseases and their Risk Factors in Regions of Russian Federation (ESSE-RF) participants: individuals with LDL-C<5th percentile, taking into account sex and age (n=52), who underwent targeted sequencing of protein-coding regions of 6 genes (APOB, PCSK9, MTTP, ANGPTL3, SAR1B, APOC3) and determination of the genetic risk score (GRS) for hypercholesterolemia; and a representative sample of the Ivanovo region population (ESSEIvanovo, n=1667), for which only GRS was determined. Genetic testing was performed using next generation sequencing.Results. In 10 (19,2%) of 52 participants with decreased LDL-C levels, the following rare variants potentially associated with hypocholesterolemia were identified: 8 — leading to a premature termination codon in the APOB gene, 1 — leading to a premature termination codon in the APOC3 gene and 1 missense variant in the PCSK9 gene. Of the 10 identified variants, 6 are described by us for the first time. GRS in the LDL-C group (0,27±0,25) was significantly lower than in the ESSE-Ivanovo population sample (0,43±0,27) (p=4,7×10-06).Conclusion. Genetic reasons explain decreased LDL-C levels (<5th percentile) in 32,7% of patients, of which only monogenic variants were identified in 13,5%, a combination of monogenic and polygenic hypocholesterolemia — in 5,7%, and polygenic hypocholesterolemia — in 13,5%.

Genetic overdiagnosis of long QT syndrome (LQTS) becomes a critical concern due to the high clinical significance of DNA diagnosis. Current guidelines for LQTS genetic testing recommend a limited scope and strict referral based on the Schwartz score. Nevertheless, LQTS may be underdiagnosed in patients with borderline phenotypes. We aimed to evaluate the total yield of rare variants in cardiac genes in LQTS patients. The cohort of 82 patients with LQTS referral diagnosis underwent phenotyping, Schwartz score counting, and exome sequencing. We assessed known LQTS genes for diagnostics, as per guidelines, and a broader set of genes for research. Diagnostic testing yield reached 75% in index patients; all causal variants were found in KCNQ1, KCNH2, and SCN5A genes. Research testing of 248 heart-related genes achieved a 50% yield of molecular diagnosis in patients with a low Schwartz score (<3.5). In patients with LQTS-causing variants, each additional rare variant in heart-related genes added 0.94 points to the Schwartz score (p value = 0.04), reflecting the more severe disease in such patients than in those with causal variants but without additional findings. We conclude that the current LQTS genetic diagnosis framework is highly specific but may lack sensitivity for patients with a Schwartz score <3.5. Improving referral criteria for these patients could enhance DNA diagnosis. Also, our results suggest that additional variants in cardiac genes may affect the severity of the disease in the carriers of LQTS-causing variants, which may aid in identifying new modifier genes.