Evaluation of the use of a cardiogenetic gene panel for next generation sequencing in molecular autopsy in sudden cardiac death

"Evaluation of the use of a cardiogenetic gene panel for next generation sequencing in molecular autopsy in sudden cardiac death"

IRN AP09563474

 

Sudden cardiac death (SCD) is an urgent problem of modern health care, which has a high social significance. While in older people, SCD is most commonly caused by coronary artery disease and heart failure, in those under 45 years of age, causes of SCD usually include genetic disorders such as hereditary cardiomyopathies and primary arrhythmogenic diseases. In assessing families in which SCD has occurred, the role of genetic testing has become an important function, both in establishing the primary diagnosis and in screening relatives at risk.

Most of the SCD deceased are young and middle-aged people, some of whom have not previously had any cardiovascular disease, and their health status was assessed as stable and not causing concern. Forensic examination reveals a preexisting structural heart anomaly (positive autopsy) in about two-thirds of sudden deaths, for the remaining one-third of cases, the cause of sudden death remains unexplained after standard forensic examination (negative autopsy), with so-called sudden unexpected death (SUD). In this case, a negative autopsy leaves the relatives of the deceased without explaining the cause of death and with the potential risk for them to die suddenly without clinical symptoms of any cardiovascular disease.

We have developed and tested a panel of 96 genes for identifying genetic predisposition and diagnosing cardiomyopathies and cardiac arrhythmias, based on targeted enrichment of the coding part of genes and subsequent sequencing on NGS platforms [1]. A patent of the Republic of Kazakhstan for a utility model was received [2]. In a subsequent study, this panel was tested in the study of the genetic landscape in arrhythmias of various origins [3-5]. To assess the diagnostic value of new generation sequencing using our developed cardiogenetic panel in molecular autopsy for sudden nonviolent death, we want to test it on DNA isolated from the blood of young people who died from sudden cardiac death (SCD).

In forensic practice, where the ultimate cause of death is not determined by autopsy, such as sudden unexpected death (SUD), molecular autopsy, or molecular autopsy, becomes a key process in the investigation of the cause of death. The combination of clinical and genetic evaluation at autopsy of SCD and SUD-originating families provides a platform for early initiation of therapeutic and preventive strategies with the ultimate goal of reducing sudden death among young people in our community.

 Objective of the project

To evaluate the diagnostic value of next-generation sequencing using our developed cardiogenetic panel in molecular autopsy for sudden nonviolent death.

The research will be carried out by an interdisciplinary group of researchers, including geneticists, molecular biologists, cardiologists, forensic doctors at the Laboratory of Genomic and Personalized Medicine, Center for Life Sciences, PI “National Laboratory Astana”.

 

Table 1 - Composition of the research group for conducting scientific research

№ N

Name, scientific degree

Place of work, position

Role in the project

Ссылки на профили

1

Ainur R. Akilzhanova, MD, PhD, DMSci, associated professor

National Laboratory Astana, Head of the Laboratory of Genomic and Personalized medicine, Leading Researcher

PI

ResearcherID 
N-6931-2015

https://orcid.org/0000-0001-6161-8355

Scopus Author ID 13612246000

 

2

Saule Rakhimova, MD, KBSci

National Laboratory Astana, Laboratory of Genomic and Personalized medicine, Leading Researcher

Leading researcher

Web of Science ResearcherID 
N-4602-2017

https://orcid.org/0000-0002-8245-2400

Scopus Author ID: 16550810200

 

3

Madina Zhalbinova

National Laboratory Astana, Laboratory of Genomic and Personalized medicine, Researcher

Junior Researcher

https://orcid.org/0000-0001-9704-8913

 

4

Ayaulym Chamoieva

Master, Eurasian National University

Assistant Researcher

Web of Science Researcher ID:  ABF-1920-2021

ORCID: 0000-0003-0877-3537

 

 

 

Doctors, forensic medical experts will be involved in the implementation of the project's tasks.

 

 

Name

Working place

Role in the project

Ссылки на профили

1

Zhakupova Tolkyn Zeynakabidenovna, Candidate of Medical Sciences, Associate Professor (Associate Professor)

1/NJSC "Astana Medical University", Professor of the Department of Forensic Medicine

2/Branch of the RGKP Center for Forensic Expertise of the Ministry of Justice of the Republic of Kazakhstan "Research Center for Forensic Expertise", Leading Researcher Senior Researcher,

 

Forensic medical specialist,

physician

ABE-9755-2021

https://orcid.org/0000-0002-5352-2895

Scopus Author ID: 57189262960

2

Ospanova Kulzhami Yesimkhanovna, candidate of medical sciences

1) NJSC "Astana Medical University", Associate Professor of the Department of Forensic Medicine;

2) Branch Kazakhstan "Institute of Forensic Expertise in Nur-Sultan", forensic medical expert Forensic medical expert of the RGKP of the Forensic Science Center of the Ministry of Justice of the Republic of Kazakhstan

 

Medical forensic expert,

physician

 

Scopus ID

57215489748

 

 

3

Polyakova Tatiana Igorevna, Master of Medicine

 

Branch of the RGKP Center for Forensic Examinations of the Ministry of Justice of the Republic of Kazakhstan "Institute of Forensic Examinations in Nur-Sultan", Head of the Department of Complicated Examinations

 

Medical Forensic Expert,

physician

Scopus ID

57189711327

 

 

 

Expected results

For the first time in the Republic of Kazakhstan, using the developed cardiogenetic panel of 96 candidate genes for cardiac arrhythmias, for subsequent targeted NGS sequencing of the entire coding sequence of target genes, a molecular genetic assessment will be carried out and genetic variations will be studied in young people who died sudden nonviolent death.

The genetic variants in the genes for predisposition to cardiomyopathies and cardiac arrhythmias will be studied using the developed target panel for sequencing the next generation among young people who died of sudden nonviolent death.

An assessment of the diagnostic value of the developed gene panel for molecular autopsy in forensic practice will be carried out.

The research results will be published in the form of article publications in foreign peer-reviewed scientific journals:

- at least 1 (one) article published, accepted for publication or submitted to a peer-reviewed scientific publication included in the Science Citation Index Expanded or Social Science Citation Index in the Web of Science database and (or) having a CiteScore percentile in the Scopus database of at least 35 (thirty five).

Achieved results

Genetic variants in genes for predisposition to cardiomyopathies and cardiac arrhythmias were studied using the developed targeted panel for next generation sequencing among young people who died of sudden nonviolent death to assess the diagnostic value of targeted sequencing in molecular autopsy in forensic medical practice.

During the reporting period, a study group was recruited - 9 persons who suddenly died outside of medical institutions (conditionally designated T 1-T 9), aged 25 to 42 years, who died in Nur-Sultan and who were admitted to the study at the general expert department the branch "Center for Forensic Examination of the Ministry of Justice of the Republic of Kazakhstan" of the Institute of Forensic Examination in Nur-Sultan (ISE in Nur-Sultan), 8 men aged 25 to 42 years and one woman 33 years old.

The recruited deceased underwent a full forensic medical examination, forensic histological examination, and biomaterials were taken for molecular research in accordance with the developed protocols.

According to the results of forensic medical studies, the death of persons included in the SCD group was regarded as death of cardiac origin. In a forensic study of 9 recruited patients, the diagnosis of Ischemic heart disease (IHD) was made in four cases, in the rest of the cases it was diagnosed as Secondary cardiomyopathy, since undefined histological characteristics of the heart were revealed.

Depending on the results of the forensic medical examination, the participants included in the study were grouped into 2 groups - SCD (n = 4) CHD postmortem and SCD (n = 5) with nondiagnostic structural heart anomalies, secondary CMB postmortem.

Targeted sequencing of all exon regions and exon-intron junctions of 96 candidate genes associated with the development of hereditary heart syndromes, cardiomyopathies, and cardiac arrhythmias was carried out using a developed panel of genes based on Haloplex technology, Agilent Technologies.

Targeted sequencing and stepwise filtering of annotated variants identified 251 unique variants in 86 genes out of 96 in the whole group of cases studied. The most common genetic mutations are found in the genes TTN, LAMA2, MYBPC3, MYH6, KCNQ1, GAA, SCN5A, RYR2, CACNA1C, KCNJ2 and others. Classification of 251 genetic variants in accordance with the ACMG recommendations showed that 7 (2.9%) variants were classified as pathogenic, 7 (2.9%) - probable pathogenic, 80 (31.9%) - variants of uncertain significance (VUS) , 61 (23.8%) probable benign and 96 (38.4%) benign.

Genetic screening of persons who died from SCD revealed variants with probable pathogenic functional effects with a high frequency in 63% and 35% of SCD cases with nondiagnostic and diagnostic cardiac abnormalities, respectively, showing a high diagnostic value of genetic screening using a gene panel for targeted NGS.

In individuals with SCD with ischemic changes in the heart posthumously, the most frequent mutations were found in the TTN, MYBPC3, LAMA2, MYH6, and GAA genes. In persons with nondiagnostic structural anomalies of the heart, cardiomyopathies, pathogenic variants were revealed in the genes of the ion channels KCNQ1, KCNJ2, SCN5A, RYR2.

Validation of the identified genetic variants with the Sanger direct sequencing method confirmed the accuracy and precision of targeted sequencing using the panel of genes we developed.

Degree of implementation - the next generation targeted sequencing (NGS) method was introduced using the developed panel for targeted enrichment of coding sequences for 96 candidate genes during molecular autopsy of young people who died from SCD. There is an act of implementation.

Published 2 publications in international journals with an impact factor and having Q1 percentile in the Web of Science and Clarivate analytics databases, Scopus percentile of at least 35 - namely 99, 4 theses in materials of international conferences, two presentations at international conferences.

An article was submitted to an international journal with an impact factor and a Scopus percentile of at least 35 in the Web of Science and Clarivate analytics databases. Publications in international journals

  1. Akilzhanova A.R., Abilova Z.M., Rakhimova S.E., Kozhamkulov U.A., Kairov U.E., Akilzhanova G.A., Nuralinov O., Akilzhanova S.K., Abdrakhmanov A.S., and Bekbosynova M.S. Targeted Sequencing Approach to Identifying Rare Genetic Variants Associated with Atrial Fibrillation //The Journal of Heart and Lung Transplantation. – 2021. - Vol 40. - Issue 4S. – P. S238 (IF – 7.865; Q1, процентиль – 99).
  2. Zhalbinova M., Rakhimova S., Bekbosynova M., Andosova S., Abdirova B., and Akilzhanova A. Associations of Gene Polymorphism in Kazakhstani Patients with Implanted LVAD //The Journal of Heart and Lung Transplantation. – 2021. - 40. - Issue 4S. – P.S391 (IF – 7.865; Q1, процентиль – 99).

Abstracts in the materials of the international conferences, presentations

  1. Akilzhanova A.R., Abilova Z.M., Rakhimova S.E., Kozhamkulov U.A., Kairov U.E., Akilzhanova G.A., Nuralinov O., Akilzhanova S.K., Abdrakhmanov A.S., and Bekbosynova M.S. Targeted Sequencing Approach to Identifying Rare Genetic Variants Associated with Atrial Fibrillation //ISHLT2021 41st Annual Meeting & Scientific Sessions – Virtual Experience, April 24-28, 2021 (poster report)
  2. Akilzhanova A. R., Rakhimova S. E., Kozhamkulov U. A., Kairov U.E., Akilzhanova G., Chamoieva A., Zhakupova T. Z. Mutation spectrum in Kazakhstani sudden cardiac death victims revealed by targeted next-generation sequencing of 96 genes associated with cardiac diseases// European Society of Human Genetics ESHG 2021. - the European Human Genetics Conference, Virtual Conference, August, 28–31,2021 (poster report).
  3. Zhakupova T.Z., Ospanova K.E., Akilzhanova A.R. Histological findings during autopsy of sudden death of adolescents // Proceedings of the scholarly abstracts European Academic Science and Research June, 2021. – P.17
  4. Akilzhanova, M.Zhalbinova, A. Chamoieva, D.Samatkyzy, S. Rakhimova, U. Kozhamkulov, U. Kairov, G. Akilzhanova, T. Polyakova, K.Ospanova, T. Zhakupova. Genetic screening of heart genes in Kazakhstani sudden unexplained death victims in a forensic setting by targeted next-generation sequencing// Abstract book of the International Conference “Current Problems of Biological Safety in the Modern Conditions", September 22-23, 2021

Manuscript submitted to international journal OMICS: A Journal of Integrative Biology, Scopus, процентиль 68%, 89/282, Biotechnology

M.Zhalbinova, S. Rakhimova, U. Kozhamkulov, Yu.Pya, J.Lee, Y.Wang, M. Bekbossynova, and A. Akilzhanova. Association of gene polymorphisms with side effects in congestive heart failure patients with implanted mechanical circulatory support device // OMICS: A Journal of Integrative Biology (in press)