Generation of the ICGi019-B-1 and ICGi019-B-2 lines via correction of the p.Met659Ile (c.1977G>A) variant in MYH7 of patient-specific induced pluripotent stem cells using CRISPR/Cas9

The problem of interpretation of the genetic data from patients with inherited cardiovascular diseases still remains relevant. To date, the clinical significance of approximately 40 % of variants in genes associated with in herited cardiovascular diseases is uncertain, which requires new approaches to the assessment of their pathogenetic contribution. A combination of the induced pluripotent stem cell (iPSC) technology and editing the iPSC genome with CRISPR/Cas9 is thought to be the most promising tool for clarifying variant pathogenicity. A variant of unknown significance in MYH7, p.Met659Ile (c.1977G>A), was previously identified in several genetic screenings of hypertrophic cardiomyopathy patients. In this study, the single nucleotide substitution was corrected with CRISPR/Cas9 in iPSCs generated from a carrier of the variant. As a result, two iPSC lines (ICGi019-B-1 and ICGi019-B-2) were generated and characterized using a standard set of methods. The iPSC lines with the corrected p.Met659Ile (c.1977G>A) variant in MYH7 possessed a morphology characteristic of human pluripotent cells, expressed markers of the pluripotent state (the OCT4, SOX2, NANOG transcription factors and SSEA-4 surface antigen), were able to give rise to derivatives of three germ layers during spontaneous differentiation, and retained a normal karyotype (46,XY). No CRISPR/Cas9 off-target activity was found in the ICGi019-B-1 and ICGi019-B-2 iPSC lines. The maintenance of the pluripotent state and normal karyotype and the absence of CRISPR/Cas9 off-target activity in the iPSC lines with the corrected p.Met659Ile (c.1977G>A) variant in MYH7 allow using the iPSC lines as an isogenic control for further studies of the variant pathogenicity and its impact on the hypertrophic cardiomyopathy development.

1. Akhtar M., Elliott P. The genetics of hypertrophic cardiomyopathy. Glob Cardiol Sci Pract. 2018;2018(3):36. doi 10.21542/gcsp.2018.36

2. Bashyam M.D., Purushotham G., Chaudhary A.K., Rao K.M., Acharya V., Mohammad T.A., Nagarajaram H.A., Hariram V., Narasimhan C. A low prevalence of MYH7/MYBPC3 mutations among Familial Hypertrophic Cardiomyopathy patients in India. Mol Cell Biochem. 2012;360(1-2):373-382. doi 10.1007/s11010-011-1077-x

3. Bhagwan J.R., Mosqueira D., Chairez-Cantu K., Mannhardt I., Bodbin S.E., Bakar M., Smith J.G.W., Denning C. Isogenic models of hypertrophic cardiomyopathy unveil differential phenotypes and mechanism-driven therapeutics. J Mol Cell Cardiol. 2020;145: 43-53. doi 10.1016/j.yjmcc.2020.06.003

4. Chai A.C., Cui M., Chemello F., Li H., Chen K., Tan W., Atmanli A., McAnally J.R., Zhang Y., Xu L., Liu N., Bassel-Duby R., Olson E.N. Base editing correction of hypertrophic cardiomyopathy in human cardiomyocytes and humanized mice. Nat Med. 2023;29(2):401-411. doi 10.1038/s41591-022-02176-5

5. Cheng J., Novati G., Pan J., Bycroft C., Žemgulyte A., Applebaum T., Pritzel A., … Senior A.W., Jumper J., Hassabis D., Kohli P., Avsec Ž. Accurate proteome-wide missense variant effect prediction with AlphaMissense. Science. 2023;381(6664):eadg7492. doi 10.1126/science.adg7492

6. Cohn R., Thakar K., Lowe A., Ladha F.A., Pettinato A.M., Romano R., Meredith E., Chen Y.S., Atamanuk K., Huey B.D., Hinson J.T. A contraction stress model of hypertrophic cardiomyopathy due to sarcomere mutations. Stem Cell Rep. 2019;12(1):71-83. doi 10.1016/j.stemcr.2018.11.015

7. Dementyeva E.V., Vyatkin Y.V., Kretov E.I., Elisaphenko E.A., Medvedev S.P., Zakian S.M. Genetic analysis of patients with hypertrophic cardiomyopathy. Genes Cells. 2020a;15(3):68-73. doi 10.23868/202011011 (in Russian)

8. Dementyeva E.V., Kovalenko V.R., Zhiven M.K., Ustyantseva E.I., Kretov E.I., Vyatkin Y.V., Zakian S.M. Generation of two clonal iPSC lines, ICGi019-A and ICGi019-B, by reprogramming peripheral blood mononuclear cells of a patient suffering from hypertrophic cardiomyopathy and carrying a heterozygous p.M659I mutation in MYH7. Stem Cell Res. 2020b;46:101840. doi 10.1016/j.scr.2020.101840

9. Escribá R., Larrañaga-Moreira J.M., Richaud-Patin Y., Pourchet L., Lazis I., Jiménez-Delgado S., Morillas-García A., … de la Pompa J.L., Brugada R., Monserrat L., Barriales-Villa R., Raya A. iPSC-based modeling of variable clinical presentation in hypertrophic cardiomyopathy. Circ Res. 2023;133(2):108-119. doi 10.1161/circresaha.122.321951

10. Funakoshi S., Yoshida Y. Recent progress of iPSC technology in cardiac diseases. Arch Toxicol. 2021;95(12):3633-3650. doi 10.1007/s00204-021-03172-3

11. Gähwiler E.K.N., Motta S.E., Martin M., Nugraha B., Hoerstrup S.P., Emmert M.Y. Human iPSCs and genome editing technologies for precision cardiovascular tissue engineering. Front Cell Dev Biol. 2021;9:639699. doi 10.3389/fcell.2021.639699

12. Geske J.B., Ommen S.R., Gersh B.J. Hypertrophic cardiomyopathy: clinical update. JACC Heart Fail. 2018;6(5):364-375. doi 10.1016/j.jchf.2018.02.010

13. Guo G., Wang L., Li X., Fu W., Cao J., Zhang J., Liu Y., … Liu G., Zhang Y., Dong J., Tao H., Zhao X. Enhanced myofilament calcium sensitivity aggravates abnormal calcium handling and diastolic dysfunction in patient-specific induced pluripotent stem cell-derived cardiomyocytes with MYH7 mutation. Cell Calcium. 2024;117: 102822. doi 10.1016/j.ceca.2023.102822

14. Guo H., Liu L., Nishiga M., Cong L., Wu J.C. Deciphering pathogenicity of variants of uncertain significance with CRISPR-edited iPSCs. Trends Genet. 2021;37(12):1109-1123. doi 10.1016/j.tig.2021.08.009

15. Hendel A., Bak R.O., Clark J.T., Kennedy A.B., Ryan D.E., Roy S., Steinfeld I., … Bacchetta R., Tsalenko A., Dellinger D., Bruhn L., Porteus M.H. Chemically modified guide RNAs enhance CRISPRCas genome editing in human primary cells. Nat Biotechnol. 2015; 33(9):985-989. doi 10.1038/nbt.3290

16. Hesaraki M., Bora U., Pahlavan S., Salehi N., Mousavi S.A., Barekat M., Rasouli S.J., Baharvand H., Ozhan G., Totonchi M. A novel missense variant in actin binding domain of MYH7 is associated with left ventricular noncompaction. Front Cardiovasc Med. 2022;9:839862. doi 10.3389/fcvm.2022.839862

17. Liang X., Potter J., Kumar S., Zou Y., Quintanilla R., Sridharan M., Carte J., Chen W., Roark N., Ranganathan S., Ravinder N., Chesnut J.D. Rapid and highly efficient mammalian cell engineering via Cas9 protein transfection. J Biotechnol. 2015;208:44-53. doi 10.1016/j.jbiotec.2015.04.024

18. Livak K.J., Schmittgen T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2–ΔΔCT method. Methods. 2001;25(4):402-408. doi 10.1006/meth.2001.1262

19. Ma N., Zhang J.Z., Itzhaki I., Zhang S.L., Chen H., Haddad F., Kitani T., Wilson K.D., Tian L., Shrestha R., Wu H., Lam C.K., Sayed N., Wu J.C. Determining the pathogenicity of a genomic variant of uncertain significance using CRISPR/Cas9 and human-induced pluripotent stem cells. Circulation. 2018;138(23):2666-2681. doi 10.1161/circulationaha.117.032273

20. Malakhova A.A., Grigor’eva E.V., Pavlova S.V., Malankhanova T.B., Valetdinova K.R., Vyatkin Y.V., Khabarova E.A., Rzaev J.A., Zakian S.M., Medvedev S.P. Generation of induced pluripotent stem cell lines ICGi021-A and ICGi022-A from peripheral blood mononuclear cells of two healthy individuals from Siberian population. Stem Cell Res. 2020;48:101952. doi 10.1016/j.scr.2020.101952

21. Mosqueira D., Mannhardt I., Bhagwan J.R., Lis-Slimak K., Katili P., Scott E., Hassan M., … Williams P.M., Gaffney D., Eschenhagen T., Hansen A., Denning C. CRISPR/Cas9 editing in human pluripotent stem cell-cardiomyocytes highlights arrhythmias, hypocontractility, and energy depletion as potential therapeutic targets for hypertrophic cardiomyopathy. Eur Heart J. 2018;39(43):3879-3892. doi 10.1093/eurheartj/ehy249

22. Parrotta E.I., Lucchino V., Scaramuzzino L., Scalise S., Cuda G. Modeling cardiac disease mechanisms using induced pluripotent stem cell-derived cardiomyocytes: progress, promises and challenges. Int J Mol Sci. 2020;21(12):4354. doi 10.3390/ijms21124354

23. Pasipoularides A. Challenges and controversies in hypertrophic cardiomyopathy: clinical, genomic and basic science perspectives. Rev Esp Cardiol (Engl Ed). 2018;71(3):132-138. doi 10.1016/j.rec.2017.07.003

24. Pavlova S.V., Shulgina A.E., Zakian S.M., Dementyeva E.V. Studying pathogenetic contribution of a variant of unknown significance, p.M659I (c.1977G>A) in MYH7, to the development of hypertrophic cardiomyopathy using CRISPR/Cas9-engineered isogenic induced pluripotent stem cells. Int J Mol Sci. 2024;25(16):8695. doi 10.3390/ijms25168695

25. Richard P., Charron P., Carrier L., Ledeuil C., Cheav T., Pichereau C., Benaiche A., … Desnos M., Schwartz K., Hainque B., Komajda M., EUROGENE Heart Failure Project. Hypertrophic cardiomyopathy: distribution of disease genes, spectrum of mutations, and implications for a molecular diagnosis strategy. Circulation. 2003;107(17): 2227-2232. doi 10.1161/01.CIR.0000066323.15244.54

26. Shafaattalab S., Li A.Y., Gunawan M.G., Kim B., Jayousi F., Maaref Y., Song Z., Weiss J.N., Solaro R.J., Qu Z., Tibbits G.F. Mechanisms of arrhythmogenicity of hypertrophic cardiomyopathy-associated troponin T (TNNT2) variant I79N. Front Cell Dev Biol. 2021;9: 787581. doi 10.3389/fcell.2021.787581

27. Smith J.G.W., Owen T., Bhagwan J.R., Mosqueira D., Scott E., Mannhardt I., Patel A., Barriales-Villa R., Monserrat L., Hansen A., Eschenhagen T., Harding S.E., Marston S., Denning C. Isogenic pairs of hiPSC-CMs with hypertrophic cardiomyopathy/LVNC-associated ACTC1 E99K mutation unveil differential functional deficits. Stem Cell Rep. 2018;11(5):1226-1243. doi 10.1016/j.stemcr.2018.10.006

28. Sorogina D.A., Grigor’eva E.V., Malakhova A.A., Pavlova S.V., Medvedev S.P., Vyatkin Y.V., Khabarova E.A., Rzaev J.A., Zakian S.M. Creation of induced pluripotent stem cells ICGi044-B and ICGi044-C using reprogramming of peripheral blood mononuclear cells of a patient with Parkinson’s disease associated with с.1492T>G mutation in the GLUD2 gene. Russ J Dev Biol. 2023;54(1):104-111. doi 10.1134/S1062360423010125

29. Wang L., Kim K., Parikh S., Cadar A.G., Bersell K.R., He H., Pinto J.R., Kryshtal D.O., Knollmann B.C. Hypertrophic cardiomyopathy-linked mutation in troponin T causes myofibrillar disarray and pro-arrhythmic action potential changes in human iPSC cardiomyocytes. J Mol Cell Cardiol. 2018;114:320-327. doi 10.1016/j.yjmcc.2017.12.002