Abstract
Up to 60% of hypertrophic cardiomyopathy cases are due to mutations responsible for sarcomere functioning. However, clinical significance is known not for all variants found in hypertrophic cardiomyopathy-associated genes. Using methods of nucleotide sequence editing opens new prospects for clarifying clinical significance of the variants. A trinucleotide deletion c.1543_1545delAAC (p.Asn515del) with uncertain significance was introduced in MYBPC3 of induced pluripotent stem cells (iPSCs) of a healthy donor by CRISPR/Cas9. Three iPSC lines (ICGi022-A-3, ICGi022-A-4, and ICGi022-A-5) homozygous at the mutation were generated. The iPSC lines with the introduced deletion demonstrated morphology characteristic of human pluripotent cells, normal karyotype (46,XX), expressed markers of the pluripotent state (OCT4, NANOG, TRA-1-60, SSEA4), and were able to give rise to derivatives of three germ layers during spontaneous differentiation. Studying properties of cardiomyocytes obtained under directed differentiation of the ICGi022-A-3, ICGi022-A-4, and ICGi022-A-5 iPSC lines allows establishing pathogenetic contribution of the p.Asn515del variant in MYBPC3 to hypertrophic cardiomyopathy development.
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ACKNOWLEDGMENTS
Analysis of immunofluorescent staining was performed using the Joint Access Center for Microscopy of Biological Objects supported by the Budget Project of ICG SB RAS FWNR-2022-0015.
Funding
This study was supported by the Russian Science Foundation (grant no. 22-15-00271).
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S.V. Pavlova, E.V. Dementyeva, and S.M. Zakian planned the experiment, analyzed the data obtained, and wrote the manuscript. S.V. Pavlova and L.Sh. Shayakhmetova designed single-guide RNA and donor oligonucleotide to introduce mutation using CRISPR/Cas9. S.V. Pavlova performed experiments on iPSC genome editing. E.V. Dementyeva, K.A. Pronyaeva, and A.E. Shulgina characterized the edited iPSC lines.
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Statement of compliance with standards of research involving humans as subjects. The study was approved by the Ethics Committee of FSBI Federal Neurosurgical Center, Novosibirsk, protocol no. 1 from March 14, 2017. Voluntary informed consent was obtained from the patient.
Statement on the welfare of animals. All applicable international, national and/or institutional principles for the care and use of animals were complied with for feeder generation.
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Pavlova, S.V., Shayakhmetova, L.S., Pronyaeva, K.A. et al. Generation of Induced Pluripotent Stem Cell Lines ICGi022-A-3, ICGi022-A-4, and ICGi022-A-5 with p.Asn515del Mutation Introduced in MYBPC3 Using CRISPR/Cas9. Russ J Dev Biol 54, 96–103 (2023). https://doi.org/10.1134/S1062360423010113
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DOI: https://doi.org/10.1134/S1062360423010113