Decreased RNA-degradation underlies allelic imbalance of MYH7 in cardiac tissue of Hypertrophic Cardiomyopathy patients with mutation R723G

Mutation p.R723G (c.2167C>G) in the MYH7-gene, which encodes for β-myosin heavy chain, causes Hypertrophic Cardiomyopathy (HCM). Patients with this mutation are heterozygous, they encode for a mutant (R723G) and a wildtype (WT) allele. These patients are severely affected from the disease. We have shown previously that high fractions of mutant mRNA can be associated with severe disease courses. In line with this assumption, patients with mutation R723G exhibit a so-called allelic imbalance of on average 67% R723G- and 33% WT MYH7-mRNA and protein in cardiac tissue. In this study, we identified mechanisms that could cause this allelic imbalance.

Since mRNA and protein allelic imbalance are comparable in all R723G-patients, we can exclude post-transcriptional mechanism as primary cause of the increased fraction of R723G-protein. Thus, increased fractions of R723G-mRNA must underlie the imbalance that either could be caused by enhanced mRNA-production or reduced mRNA-degradation rates.

To test mRNA production, we examined transcriptional activity of R723G and WT alleles and performed relative quantification of WT- and R723G mRNA transcription rates. Mutation-specific RNA-FISH analysis on patient-derived hiPSC-CMs with mutation R723G indicated that 46%±9% of transcribed alleles encoded for the R723G-allele. In addition, comparable fractions of nuclei transcribed WT- or R723G-alleles, respectively. Allele-specific RT-PCR on HeLa cells co-transfected with expression-vectors for R723G- and WT-mRNA showed that both mRNAs were produced in comparable amounts. Thus, altered production rate cannot underlie R723G-allelic imbalance.

In contrast, we show that mutation R723G alters mRNA-degradation. We performed a degradation assay by inhibiting transcription using Actinomycin D and subsequently analyzed whether the ratio of R723G- vs. WT-mRNA changes over time. Indeed, we detected a significant increase in R723G-mRNA relative to WT-mRNA, suggesting a prolongation of R723G-mRNA half-life. A potential modifier of mRNA half-life is its secondary structure. To test for alterations in RNA-structure caused by mutation R723G, we performed Selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) analysis. In line with our assumption, we show a significantly altered SHAPE pattern for the mutant mRNA. Therefore, our results suggest that mutation R723G alters MYH7 mRNA-secondary structure, which enhances its stability and thereby causes allelic imbalance in HCM-patients at tissue level. Specific increase of R723G-mRNA degradation could therefore provide a potential approach to improve HCM-phenotypes.