Abstract Body: Introduction: Hypertrophic cardiomyopathy (HCM) is a monogenic disorder caused by heterozygous (HET) mutations in sarcomere genes such as MYH7. Recently, human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) carrying HET HCM mutations have shed new insights into the molecular mechanisms of HCM. However, the cause of phenotype variability and the role of allelic imbalance in phenotype penetrance of HET missense mutations such as MYH7 G256E remain unclear.
Hypothesis: We hypothesize that hiPSC CMs carrying clinically variable penetrant HCM missense mutations such as MYH7 G256E show a gene dose-dependent disease phenotype indicating their susceptibility for allelic imbalance as a cause for variable disease phenotype penetrance.
Methods: We generated HET and homozygous (HOM) hiPSC-lines for variable penetrant HCM mutation MYH7 G256E. We performed multiplexed single-cell RNA sequencing and quantitative proteomics analyses on wildtype (WT), HET, and HOM iPSC-CMs at day 30 of cardiac differentiation. Additional Digital Droplet PCR studies were performed to investigate the MYH7 mutant allele expression. The functional contractile phenotype was assessed with Sony SI8000 Cell motion imaging system.
Results: We detected a large clone-to-clone and batch-to-batch variability in HET iPSC-CMs on a transcriptomic and proteomic level that obscured the identification of a consistent mutant phenotype in HET MYH7 G256E vs WT CMs. These phenotypes correlated with a 1:1 bulk ratio of mutant vs WT mRNA and protein expression, while overall MYH7 gene and protein expression remained comparable. Transcriptomic and proteomic analyses of WT, HET, and HOM CMs show a gene dose-dependent regulation of known hypertrophy response markers such as NPPB (mRNA: HET vs WT: +1.76x p .adj=7.0*10^-7, HOM vs WT: +5.42x p.adj=1.5*10^-48; protein: HET vs WT: +1.15x, 95% CI [0.86, 1.58]; HOM vs WT: +1.51x, 95% CI [1.12, 2.01]) which functionally correlated with enhanced contractility (contraction velocity in μm/s: WT: 5.84 +/-2.24, HET: 6.04 +/-2.89, HOM: 7.77 +/-3.352, p=0.3*10^-2 ANOVA) and the more frequent occurrence of arrhythmias (WT: 1/46, HET: 0/55, HOM: 5/41).
Conclusions: Hypertrophic disease phenotype in MYH7 G256E mutation iPSC-CMs shows a gene dose-dependent penetrance on a transcriptomic, proteomic, and functional level. This indicates susceptibility to allelic imbalance as a potential cause for variable disease penetrance.