Generation of patient-specific induced pluripotent stem cells (iPSCs) and their subsequent differentiation into cardiomyocytes opened new opportunities for studying pathogenesis of inherited...

Hypertrophic cardiomyopathy is one of the most common cardiovascular pathologies. In most cases, the disease is caused by mutations in genes encoding for sarcomeric proteins. However, high genetic heterogeneity of hypertrophic cardiomyopathy makes it difficult to interpret results of patients' genetic studies. The aim of this study is to check if hypertrophic cardiomyopathy in 15 patients suffering from the disease is due to genetic causes. In the course of genetic analysis, a known pathogenic mutation p.Gln1233Ter in MYBPC3 causing hypertrophic cardiomyopathy was found only in one patient. In six patients, mutations with uncertain clinical significance were identified in hypertrophic cardiomyopathy-associated genes LDB3, MYBPC3, MyH7, MYL2, and MyPn. Three of the mutations, p.Ile730Asn in LDB3, p.Asn515del in MYBPC3, p.Arg955Trp in MYPN were found for the first time in association with hypertrophic cardiomyopathy. In two patients, novel mutations, p.Ser478Trp in MyBpC3 and p.Asn989Ile in MYPN, were identified. Thus, hypertrophic cardiomyopathy may be accounted for by genetic causes in 8 patients more but the role of these mutations in the disease development needs to be clarified.

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.

Hypertrophic cardiomyopathy (HCM) is a common cardiovascular disease. However, effective methods of its therapy have not been developed so far. To date patient-specific induced pluripotent stem cell-derived cardiomyocytes are supposed to be a useful tool for studying HCM molecular mechanisms and to help find new approaches to HCM therapy. Using non-integrating episomal vectors, we generated an iPSC line from peripheral blood mononuclear cells of an HCM patient carrying a heterozygous p.N515del mutation in MYBPC3. The iPSC line expressed pluripotency markers, gave rise to derivatives of three germ layers during spontaneous differentiation, had normal karyotype, and retained the patient-specific mutation.

Hypertrophic cardiomyopathy (HCM) is one of the most frequent cardiovascular diseases but no methods to prevent its progression have been developed. Cardiomyocytes derived from patient-specific induced pluripotent stem cells can become a platform to study pathogenesis of the disease and to search for more effective therapy methods. We generated two iPSC lines from peripheral blood mononuclear cells of an HCM patient with heterozygous p.M659I mutation in MYH7 using episomal vectors. The iPSC lines expressed pluripotency markers, demonstrated ability to spontaneously differentiate into derivatives of three germ layers, and retained the mutation.

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.