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Project titleIdentification and assessment of the functional significance of splicing variants in the MYBPC3 gene

AffiliationFederal State Budgetary Scientific Institution “Tomsk National Research Medical Center of the Russian Academy of Sciences”,

KeywordsHypertrophic cardiomyopathy, splicing variants, haploinsufficiency, minigens, variants with unknown significance

Annotation
Most hereditary diseases are caused by mutations that disrupt the structure and function of the proteins. Human genome is characterized by a high degree of genetic polymorphism. The development of modern methods for hereditary diseases diagnostics (primarily methods using NGS technologies) has led to the emergence of a large amount of information about new variants / mutations, but doctors and researchers do not have sufficient data to assess the effect of these variants from the clinical point of view, and so we cannot unambiguously classify new variants as pathogenic or call them as neutral. So, a large number of single nucleotide variants have been identified, which have been assigned the status of “variants with unknown significance (VUS). Thus, the assessment of the effect of DNA variants identified in a patient is one of the most acute problems of modern medical genetics. Hypertrophic cardiomyopathy (HCM) is one of the most common autosomal dominant human diseases with an incidence of 1: 500 (up to 0.2% in the population). Moreover, even full exome sequencing makes it possible to establish the cause of the disease in a little more than 50% of cases, which is the main problem in the diagnostics for this disease. Most of the the MYBPC3 gene variants causing the disease are splicing variants, potentially leading to the formation of a truncated protein, which, however, is not included in the sarcomere, and thus, in this case, the mechanism of the mutation effect is so called "haploinsufficiency", i.e. expression from one “healthy” allele does not provide enough protein. Both exome and "custom" gene panels for NGS usually include only the coding regions of the gene: exons and small adjacent regions of introns containing canonical splice sites. However, in a number of publications in recent years, disease cases have been described where nucleotide substitutions “deep” in an intron can lead to the formation of new splicing sites. This determines the relevance of research in this direction in hypertrophic cardiomyopathy and, in particular, the study of the MYBPC3 gene in this aspect. It is possible that the identification of splicing variants in intron sequences will improve the efficiency of HCM gene diagnostics. In the proposed project, it is planned to evaluate the functional significance of variants in the MYBPC3 gene in patients with HCM, leading to a haploinsufficiency of the myosin-binding protein C gene product using a minigene model in cell culture and assessing the pathogenicity of the identified variants.

Expected results
As a result of the project, new data will be obtained on the functional significance of the variants leading to the disruption of the synthesis of the MYBPC3 gene product in patients with hypertrophic cardiomyopathy. The effect of the putative splicing variants will be verified by in vitro studies using genetically engineered constructs (minigens). The data obtained can be further used to assess the pathogenicity of variants in the MYBPC3 gene detected in Russian patients and will serve as the development of a new area of research in assessing the functional significance of pathogenic variants in hereditary pathology.

Annotation of the results obtained in 2023
During the second year of the project, in silico modeling of complete mutagenesis was carried out on the myosin binding protein C gene sequence. As a result, about a thousand possible single-nucleotide variants were discovered that influenced gain or loss of canonical splice sites within +/- 50 bp with probability >0.5; including 602 possible substitutions for which this probability was >0.8. Further analysis was carried out on evaluation of simultaneous dinucleotide substitutions and the results were compared with available variants n databases. Twelve dinucleotide sites with a potential splicing effect were identified where there is a SNP in one of the two positions, with rare allele frequency >1% in population, including three of them with the same substitution (rs11570058, rs3729953, rs2071304). In one site, there was a dinucleotide substitution previously found in a patient with hypertrophic cardiomyopathy, according to the online resource ClinVar. Comparison of theoretically identified variants significant for splices predicted by SpliceAI with sequencing results of the MYBPC3 gene in 28 patients with hypertrophic cardiomyopathy showed that among all variants found in the experimental patient sample, only variants at canonical splice sites were consistent with the predictions and they could be classified as pathogenic/probably pathogenic. We have created genetically engineered constructs - minigenes, including regions of the MYBPC3 gene with splicing variants and their flanking regions, for variants found in patients and for predicted variants. Site-directed mutagenesis was performed to generate plasmids containing the predicted variants. The chr11:47339649-A-C (hg38) variant affecting the donor splice site in intron 21 (NM_000256.3: c.2067+2T>G), was identified in a 23-year-old patient with an obstructive form of hypertrophic cardiomyopathy. A comparison of the mRNA obtained for minigenes with/without the variant showed that the chr11:47339649-A-C substitution leads to the skipping of exon 21 and exon 22 during the splicing.

Publications

1. Salakhov R.R., Golubenko M.V., Skoblov M.Y., Savchenko R.R., Valiakhmetov N.R., Pavlyukova E.N., Nazarenko M.S. Функциональный анализ новой мутации сплайсинга c.2067+2T>G в гене MYBPC3 при гипертрофической кардиомиопатии Бюллетень сибирской медицины/Bulletin of Siberian Medicine, - (year - 2024)

2. Salakhov R.R., Golubenko M.V., Valiakhmetov N.R., Skoblov M.Y., Nazarenko M.S ОЦЕНКА ФУНКЦИОНАЛЬНОЙ ЗНАЧИМОСТИ ВАРИАНТОВ В ГЕНЕ MYBPC3 У ПАЦИЕНТОВ С ГИПЕРТРОФИЧЕСКОЙ КАРДИОМИОПАТИЕЙ Российский национальный конгресс кардиологов 2023. Сборник тезисов, 2023, С. 742 (year - 2023)

Annotation of the results obtained in 2022
During the first year of the project, NGS data analysis was carried out for several dozen samples of patients with hypertrophic cardiomyopathy. A method for sequencing the whole genes of hypertrophic cardiomyopathy has been used, based on the long PCR products that amplify overlapping fragments of the full target genes sequences, and subsequent sequencing with the monomolecular sequencing technology of Oxford Nanopore Technology. Based on the results of the work, an article was published in the International Journal of Molecular Sciences (doi: 10.3390/ijms232415845). The work was carried out to form a group of DNA samples containing splicing variants and to assess their pathogenicity class according to the accepted criteria. To date, 4 variants have been identified that probably lead to alternative splicing. Three of them were assigned to pathogenic variants according to the criteria and the remaining one was classified as a variant with unknown significance. In addition, the work was carried out to construct genetic vectors based on the pSpl3-Flu2-TKdel plasmid in order to assess the functional significance of variants that likely lead to alternative splicing in the MYBPC3 gene and to find optimal conditions for transformation and transfection of vectors into HEK293 cell culture. As a result, it has been proven that the c.2067+2T>G variant in the MYBPC3 gene leads to the loss of exon 21. During the first year, members of the scientific team took part in three conferences. At the conference "NGS in Medical Genetics - MGNGS'22" (Suzdal), data were presented on the use of monomolecular sequencing technology to search for options by targeted sequencing of the full sequence of genes leading to the development of hypertrophic cardiomyopathy (. In addition, data on the wide variability of the clinical manifestation of the disease with mutations in the genes of hypertrophic cardiomyopathy are presented at the All-Russian Congress of Cardiologists (Kazan). Data on the first experience of using functional analysis to assess the pathogenicity of the variant leading to the disappearance of the donor site of exon 21 of the MYBPC3 gene were presented at the conference “Human Genetics and Pathology” (Tomsk).

Publications

1. Salakhov R.R., Golubenko M.V., Valiakhmetov N.R., Pavlyukova E.N., Zarubin A.A., Babushkina N.P., Kucher A.N., Sleptcov A.A., Nazarenko M.S. Application of Long-Read Nanopore Sequencing to the Search for Mutations in Hypertrophic Cardiomyopathy International Journal of Molecular Sciences, V. 23(24), 15845 (year - 2022) https://doi.org/10.3390/ijms232415845