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

TL;DR

Researchers corrected a genetic variant in patient-derived stem cells using CRISPR/Cas9 to study its role in heart disease.

Contribution

Generated and characterized isogenic iPSC lines with corrected MYH7 variant for studying hypertrophic cardiomyopathy.

Findings

Corrected iPSC lines showed normal pluripotency markers and morphology.

No CRISPR/Cas9 off-target effects were detected in the corrected lines.

Lines retained normal karyotype and can serve as isogenic controls for pathogenicity studies.

Abstract

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 inherited 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.

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