Functional characterisation of AI-prioritised sarcomeric variants in hypertrophic cardiomyopathy using iPSC-derived cardiomyocytes
I Balakrishnan, S Cong, V Ho, Y H Lin, A Murugiah, J P Chee, A Lee, C Y Khoo, H K Teo, C T Ng, D Sim, W K Lim, S S Jamuar, D Hausenloy, C RamachandraAbstract
Background
Hypertrophic cardiomyopathy (HCM), the most common inherited cardiac disease, is predominantly caused by pathogenic sarcomeric gene variants. However, the high prevalence of variants of uncertain significance (VUS), particularly among individuals of Asian ancestry, remains a major barrier to accurate genetic interpretation and clinical decision-making.
Purpose
We aimed to evaluate whether human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) can provide functional evidence to support pathogenicity assessment of two sarcomeric VUS (MYH7, MYBPC3) identified in patients with HCM.
Methods
Two VUS, MYH7 c.1956G>C (p.R625S) and MYBPC3 c.3814G>C (p.V1272L) were identified from a local HCM biobank and prioritised for functional characterisation based on high in silico pathogenicity scores. To assess VUS pathogenicity, two CRISPR-Cas9 gene editing approaches were undertaken. First, each VUS was introduced into a healthy control iPSC line. Second, the corresponding VUS was reverted to wild type in a patient-derived iPSC line. All lines were differentiated into cardiomyocytes to enable isogenic comparison of structural and functional abnormalities (Figure 1).
Results
Isogenic iPSC-CMs carrying MYH7 p.R625S and MYBPC3 p.V1272L exhibited shared pathological HCM phenotypes compared to their isogenic healthy controls, including cellular hypertrophy, impaired relaxation, and abnormal myofibril mechanics across independent clones (Figure 2). Consistently, patient-derived iPSC-CMs showed an attenuated phenotype when MYH7 p.R625S was reverted to wild-type. Mechanistically, both variants appeared to result in slower linear-phase relaxation kinetics, whereas MYBPC3 p.V1272L had an additional effect of haploinsufficiency, as evidenced by reduced protein levels.
Conclusion
Our findings demonstrate that human iPSC-CM models provide reproducible functional evidence supporting pathogenicity of two VUS in sarcomeric HCM genes. Both variants induced cellular hypertrophy and impaired relaxation, highlighting the utility of iPSC platforms for elucidating functional consequences and potentially improving interpretation of high-priority VUS in HCM. Beyond variant classification, this platform may enable patient specific therapeutic testing and support precision cardiovascular care.Isogenic iPSC-CM Editing StrategyFunctional Impact of HCM VUS