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

Abstract

Hypertrophic cardiomyopathy (HCM) is a cardiovascular pathology that is caused by variants in genes encoding sarcomere-associated proteins. However, the clinical significance of numerous variants in HCM-associated genes is still unknown. CRISPR/Cas9 is a tool of nucleotide sequence editing that allows for the unraveling of different biological tasks. In this study, introducing a mutation with CRISPR/Cas9 into induced pluripotent stem cells (iPSCs) of a healthy donor and the directed differentiation of the isogenic iPSC lines into cardiomyocytes were used to assess the pathogenicity of a variant of unknown significance, p.M659I (c.1977G > A) in MYH7, which was found previously in an HCM patient. Using two single-stranded donor oligonucleotides with and without the p.M659I (c.1977G > A) mutation, together with CRISPR/Cas9, an iPSC line heterozygous at the p.M659I (c.1977G > A) variant in MYH7 was generated. No CRISPR/Cas9 off-target activity was observed. The iPSC line with the introduced p.M659I (c.1977G > A) mutation in MYH7 retained its pluripotent state and normal karyotype. Compared to the isogenic control, cardiomyocytes derived from the iPSCs with the introduced p.M659I (c.1977G > A) mutation in MYH7 recapitulated known HCM features: enlarged size, elevated diastolic calcium level, changes in the expression of HCM-related genes, and disrupted energy metabolism. These findings indicate the pathogenicity of the variant.

Keywords: CRISPR/Cas9; cardiomyocyte; hypertrophic cardiomyopathy; induced pluripotent stem cells; variants of unknown significance.

Figure 1

Introducing p.M659I (c.1977G > A) variant in MYH7. (a) Design of single-guide RNA and single-stranded donor oligonucleotides for MYH7 editing. Nucleotide sequence of a part of MYH7 Intron 17 and Exon 18 is provided. The localization of protospacer for the single-guide RNA, PAM, and the single-stranded donor oligonucleotides is shown in grey, magenta, and yellow, respectively. Positions of the target c.1977G > A substitution and synonymous substitution in PAM are indicated with red rectangles. (b) One iPSC clone heterozygous at p.M659I (c.1977G > A) mutation in MYH7 was generated (ICGi022-A + MYH7 p.M659I). Nucleotide sequence of the same region in the ICGi022-A iPSC line used for MYH7 editing is provided for comparison. Positions of the target c.1977G > A substitution and synonymous substitution in PAM are indicated with red rectangles.

Figure 2

Characteristics of the iPSC line with introduced p.M659I (c.1977G > A) mutation in MYH7. (a) Positive expression of pluripotent state markers: the OCT4, NANOG, and SOX2 transcription factors and TRA-1-60 surface antigen in the iPSC line. Scale bar—100 µm. (b) The iPSC line can be spontaneously differentiated into derivatives of three germ layers: ectoderm (TUBB3, βIII-tubulin), mesoderm (αSMA, smooth muscle α-actin), and endoderm (CK18, cytokeratin 18). Scale bar—100 µm. (c) Expression level of OCT4, NANOG, and SOX2 in the iPSC line with introduced p.M659I (c.1977G > A) mutation in MYH7 (ICGi022-A + MYH7 p.M659I) is comparable to that in the ICGi022-A iPSC line used for MYH7 editing. (d) The iPSC line retains normal karyotype, 46,XX. (e) The iPSC line with introduced p.M659I (c.1977G > A) mutation in MYH7 (+ MYH7 p.M659I) is not contaminated with mycoplasma. Control +, positive control for mycoplasma contamination.

Figure 3

Cardiomyocytes derived from the iPSC line with introduced p.M659I (c.1977G > A) mutation in MYH7 have enlarged size and elevated diastolic calcium level. (a) Examples of immunofluorescence staining of cardiomyocytes derived from two iPSC lines of healthy donors (ICGi021-A and ICGi022-A), the iPSC line with introduced p.M659I (c.1977G > A) mutation in MYH7 (ICGi022-A + MYH7 p.M659I), and iPSC line from the patient with the variant (ICGi019-B) with antibodies to sarcomeric α-actinin. Scale bar—50 µm. (b) Comparison of cardiomyocyte areas for the iPSC lines from healthy donors and with p.M659I (c.1977G > A) mutation in MYH7 (introduced with CRISPR/Cas9 or patient-specific). (c) Comparison of diastolic calcium level in cardiomyocytes derived from the iPSC lines of healthy donors and with p.M659I (c.1977G > A) mutation in MYH7 (introduced with CRISPR/Cas9 or patient-specific).

Figure 4

Cardiomyocytes derived from the iPSC line with introduced p.M659I (c.1977G > A) mutation in MYH7 demonstrate disrupted energy metabolism and changes in expression pattern of HCM-related genes. (a) Comparison of oxygen consumption rate (OCR) in cardiomyocytes derived from the iPSC line with introduced p.M659I (c.1977G > A) mutation in MYH7 (ICGi022-A + MYH7 p.M659I) and the ICGi022-A iPSC line used for MYH7 editing. (b) Comparison of extracellular acidification rate (ECAR) in cardiomyocytes derived from the iPSC line with introduced p.M659I (c.1977G > A) mutation in MYH7 (ICGi022-A + MYH7 p.M659I) and the ICGi022-A iPSC line used for MYH7 editing. (c) Comparison of gene expression level in cardiomyocytes derived from the iPSC line with introduced p.M659I (c.1977G > A) mutation in MYH7 (ICGi022-A + MYH7 p.M659I) and the ICGi022-A iPSC line used for MYH7 editing.