Cardiogenetics of the Future: Sequencing Helps Treat Heart Disease

© iStock

Future cardiologists, geneticists, and IT specialists gathered at HSE University to learn how to 'read' DNA for diagnosing cardiovascular diseases. They explored modern sequencing methods and the complexities of working with digital twins of cardiac patients.

The International Laboratory of Bioinformatics of the HSE Faculty of Computer Science hosted a summer school titled 'Cardiogenetics: From Sequencing to Constructing a Cardio Panel.' The event brought together 18 participants, including physicians, biologists, and IT professionals.

Galina Okhrimenko, Junior Research Fellow at the International Laboratory of Bioinformatics, began her introductory lecture by explaining that sequencing—a method for determining the nucleotide sequence of DNA and RNA— can be applied in a wide range of contexts. Genetic testing helps identify disease risks based on family history, guide treatment selection in neonatal screening and pregnancy monitoring, and ultimately assist in diagnosing existing diseases, including hereditary ones.

Typically, a physician may refer a patient for genetic testing. The biological material is then sequenced, and the resulting data is analysed to identify potential disorders. Based on these findings, a clinician or a medical data interpreter determines the likely cause of the disease. The physician evaluates the patient’s clinical picture and examines early signs of the condition. Taking the patient’s medical history into account, they may decide to prescribe further genetic testing.

Individual genes, gene panels, or all coding regions of the genome—which contain information on about 85% of hereditary diseases—can be sequenced. Whole-genome sequencing enables the detection of abnormalities in both coding and regulatory regions. A patient visits a laboratory, where DNA is isolated from venous blood (the preferred option), histological samples (in oncology), or other biological materials. After extraction, a library of DNA fragments with attached adapters is prepared. This library is then loaded into a sequencer, which produces sequences of individual genes or the entire genome, represented in a four-letter nucleotide code.

Galina Okhrimenko

Galina Okhrimenko reminded participants that DNA is composed of nucleotides and interacts with proteins to form chromosomes. The human genome contains about 3.2 billion nucleotides, while the coding regions account for only a small fraction of it.

The speaker highlighted several genome sequencing technologies. Sanger sequencing, the first-generation method introduced in 1977, remains the gold standard due to its high accuracy. Next-generation sequencing (NGS), a second-generation technology, enables parallel sequencing of multiple samples and the processing of large volumes of data. Third-generation systems can read up to two million nucleotides without fragmentation, significantly improving the accuracy of sequencing complex genomic regions.

Okhrimenko further noted that equipment from Illumina and Nanopore provides the highest throughput for sequencing and genotyping. While Sanger sequencing remains the most precise method, other approaches have inherent error rates. NGS technologies, in contrast, allow researchers to efficiently analyse large genomic datasets.

The choice of sequencing technology depends on the objective. Sanger sequencing is preferred for targeted analysis of a specific genomic region. Illumina sequencing works best for examining multiple genes, whereas third-generation methods are most efficient for whole-genome studies.

For each analysis, a test tube is prepared with DNA, a primer, DNA polymerase, and other reagents. A genome fragment is then loaded into the sequencing instrument. Experiments indicate that per 1,000 normal nucleotides, approximately one modified nucleotide occurs, preventing the addition of the next base and causing the DNA chain to terminate.

The speaker described a sequencer as a cabinet-like instrument that incorporates an electron microscope. For genome-wide sequencing, DNA is isolated and then fragmented, with adapters added to enable sequencing and enrich target sites. Fragmentation is performed using ultrasound or enzymatic cleavage. During the sequencing process, index and unique sequences are selected, and primers are attached to guide replication. Next, denaturation occurs, producing single-stranded DNA that can be loaded into a sequencing cell. This cell contains multiple lanes with specialised wells holding nucleotides. The loaded DNA fragments bind to oligonucleotides, primers are added, and a complementary DNA strand is synthesised. The original fragment is then washed away, leaving a new fragment that complements the original sequence. Afterward, hybridisation takes place, and the signal from external radiation is monitored. This process results in the formation of a final cluster that mirrors the original DNA or genome fragments, which can then be recorded and sequenced.

As a result of sequencing, millions of DNA fragments are read and subsequently processed by bioinformatics specialists to investigate the underlying causes of genetic disorders.

Okhrimenko explained that students would learn how to assess the quality of sequencing data, identify single-nucleotide substitutions, and perform data filtering. In practical sessions, they would gain experience searching for mutations in provided samples and analysing the data in the context of the patient’s clinical profile, including potential pathologies.

Linux to Assist Researchers

German Ashniev, a visiting lecturer at the HSE FCS Big Data and Information Retrieval School, explained to the audience the specifics of using the Linux operating system and its Ubuntu distribution in bioinformatics. These open-source operating systems have been updated with specialised packages for bioinformatics applications.

German Ashniev

He noted that while Ubuntu operates differently from the typical Windows environment, it allows users to write commands, view their history and execution, and access files through system links. Users can also manage folder permissions using both the graphical interface and keyboard shortcuts.

Next, Ashniev explained the principles of working with files and data from Illumina and other sequencing systems in Linux and Ubuntu, highlighting the differences in processing files from various types of equipment. Users can perform tasks such as viewing, editing, moving, renaming, copying, deleting, and searching for files and directories, using the autocomplete feature to simplify navigation. Thus, the introductory lecture covered the basic Linux tools and utilities for file system management and data processing, including: pwd, cd, ls, cat, zcat/gunzip/gzip, cp, mv, rm, mkdir, nano, awk, grep, find, sort, uniq, and less. Effective file organisation—whether on a server, a personal computer, or conceptually in one’s workflow—requires creating a structured system with multiple directories and a home directory to store essential documents.

He explained the specifics of file operations in Linux, including copying, editing, and moving documents between directories, as well as the proper procedure for deleting files to avoid losing important data. He also cautioned against common mistakes, such as deleting files in root directories. Additionally, he noted that Linux allows users to create multiple environments for different tasks, helping prevent conflicts between programs.

Biotechnology Campus Tour

As part of the program, the summer school participants visited the Biotechnology Campus and were introduced to the '100,000+ Me' Russian Genome Project.

Ivan Antonov, a bioinformatics specialist at the Biotechnology Campus, explained that most DNA and genome analyses were conducted with samples from healthy adult volunteers, although patients with cardiovascular and oncological diseases also participated. A separate collection of ethnic genomes was assembled. Most donors provided blood samples. Recently, the programme for collecting genetic material has been expanded to include one million participants.

The speaker explained that studying mutations allows researchers to assess their pathogenicity. In particular, mutations that occur frequently in different individuals are unlikely to be the cause of disease.

Antonov added that the geographic distribution of genetic differences among Russia’s ethnic groups closely corresponds to the map of their historical settlement in the country, despite the large number of Russians with mixed ethnic backgrounds.

The speaker also explained that diseases can be classified as monogenic, caused by a single mutation, or multifactorial, such as complex immune system disorders, which require expert evaluation of multiple contributing factors. In oncology, effective treatment relies on DNA analysis of both tumour tissue and healthy cells collected through biopsy.

He emphasised the importance of research in pharmacogenetics, which, among other uses, helps identify how genes influence severe side effects from specific drugs or groups of medications.

Antonov highlighted the study of ancient genomes as a fascinating area of research, enabling scientists to investigate the evolution of DNA and genomes and to gain insights into the diseases and likely causes of death among prehistoric humans.

Yegor Gotsmanov, Research Fellow at the Biotechnology Campus, demonstrated the operation of gene information processing equipment to the students of the School of Cardiogenetics. He explained that blood collection tubes are first sterilised in a specialised airlock. Next, cells are isolated from the collected blood samples. The size of the DNA fragments is then assessed, and if sufficient, the DNA is extracted from the cell nucleus, and its purity is evaluated using spectrometry. Once verified, the DNA fragments are sent to the library. Gotsmanov noted that around 2% of samples are rejected in typical projects, while more complex studies can reject up to 10% of samples.

The fragments are then allocated to double-stranded or single-stranded sequencers, and copies are prepared for the gene library. Special reagents are added, and the nucleotides are repeatedly read using substrates and chips. Gotsmanov noted that about 60 complete genomes are processed daily.

Student Projects

On the final day of the school, students presented their projects on applying sequencing techniques to the diagnosis and treatment of cardiovascular diseases.

Maxim Nikulin, a sixth-year student at the ICM of Sechenov University, presented his project, 'Analysis of Whole-Exome Sequencing Data Using a Virtual Cardiogenetic Panel.' He aimed to assess the presence of pathogenic or likely pathogenic variants associated with cardiac diseases in a patient with a family history of cardiac conduction disorders.

The analysis was based on whole-exome sequencing data obtained using the Illumina Genome Analyzer IIx and the Illumina TruSeq Enrichment Kit, followed by NGS bioinformatic processing. Variant interpretation was performed in accordance with ACMG pathogenicity guidelines.

A total of 76 databases were examined, including 1,074 variants in the GenCC cardiomyopathy panel and 10 variants from the ClinGen dilated cardiomyopathy panel. The study identified a pathogenic variant in the LMNA gene, which encodes a key structural protein of the nuclear envelope. Such pathogenic variants are linked to a high risk of fatal arrhythmias, requiring substantial adjustments in patient care, including the implantation of a cardioverter-defibrillator and cascade family screening.

According to the speaker, retraining the source data improves the quality of bioinformatic analysis by filtering out low-quality variants. The variant identified requires confirmation through Sanger sequencing. Understanding the patient’s genotype informs a significantly altered approach to clinical management.

Alexander Milek presented his project, 'Cardiogenetic Testing of a Digital Twin of a Patient with an Inherited Cardiac Conduction Disorder.' The project focused on developing a cardiogenetic panel for inherited cardiac conduction disorders, identifying gene variants from the patient’s DNA sequencing, filtering and classifying them by frequency and clinical relevance, and comparing the findings with clinical databases to evaluate pathogenicity.

Among the 118 substitutions detected in the panel’s genes, two were previously known, one was highly probable, and one was rare. Milek stressed that no pathogenic variants were identified in the main genes linked to cardiac conduction disorders, underscoring the interpretive challenges in these inherited conditions. Milek stressed that no pathogenic variants were identified in the main genes linked to cardiac conduction disorders, underscoring the interpretive challenges in these inherited conditions.

A non-pathogenic variant in the PRKAG2 gene is associated with PRKAG2 syndrome, which can lead to hypertrophic cardiomyopathy, cardiac conduction disorders, and Wolff-Parkinson-White syndrome. The identified variants of uncertain significance (VUS) and unclassified variants should be further investigated in future studies and functional assays.

Maria Poptsova

Wrapping up the school’s programme, Maria Poptsova, Head of the HSE FCS International Laboratory of Bioinformatics, noted that the students had acquired valuable new knowledge, successfully completed the programme, and presented projects demonstrating the use of advanced genetic analysis techniques in patient diagnosis and treatment. She further highlighted the interdisciplinary scope of bioinformatics and expressed hope that the new knowledge would help specialists from various fields better understand the methods used by experts in related professions, fostering collaboration and improving work outcomes.

The school was supported by Yandex Cloud. Materials on the interpretation of genetic variants were partially based on resources developed by the Institute of Bioinformatics.