Intrinsic MYH7 expression regulation contributes to tissue level allelic imbalance in hypertrophic cardiomyopathy

Abstract

HCM, the most common inherited cardiac disease, is mainly caused by mutations in sarcomeric genes. More than a third of the patients are heterozygous for mutations in the MYH7 gene encoding for the β-myosin heavy chain. In HCM-patients, expression of the mutant and the wildtype allele can be unequal, thus leading to fractions of mutant and wildtype mRNA and protein which deviate from 1:1. This so-called allelic imbalance was detected in whole tissue samples but also in individual cells. There is evidence that the severity of HCM not only depends on the functional effect of the mutation itself, but also on the fraction of mutant protein in the myocardial tissue. Allelic imbalance has been shown to occur in a broad range of genes. Therefore, we aimed to examine whether the MYH7-alleles are intrinsically expressed imbalanced or whether the allelic imbalance is solely associated with the disease. We compared the expression of MYH7-alleles in non-HCM donors and in HCM-patients with different MYH7-missense mutations. In the HCM-patients, we identified imbalanced as well as equal expression of both alleles. Also at the protein level, allelic imbalance was determined. Most interestingly, we also discovered allelic imbalance and balance in non-HCM donors. Our findings therefore strongly indicate that apart from mutation-specific mechanisms, also non-HCM associated allelic-mRNA expression regulation may account for the allelic imbalance of the MYH7 gene in HCM-patients. Since the relative amount of mutant mRNA and protein or the extent of allelic imbalance has been associated with the severity of HCM, individual analysis of the MYH7-allelic expression may provide valuable information for the prognosis of each patient.

Keywords: Allelic imbalance; Beta-myosin; Hypertrophic cardiomyopathy; MYH7.

Fig. 1

Relative quantification of mutant and wildtype MYH7-mRNA in HCM-patients. a Defined mixtures of synthetic plasmids encoding for the wildtype and the respective mutated sequences were amplified by quantitative PCR and quantified densitometrically from allele specific restriction digests. Quantifications were performed in at least 4 independent experiments. The expected ratios are indicated by the grey line. b Relative quantification of mutant and wildtype MYH7-mRNA in M. soleus or myocardial tissue from HCM-patients. Quantification was performed on RNA extracted from at least two pieces of tissue at least in duplicate analysis. Each dot represents a single quantification analysis. The asterisks indicate significant deviation from 50% (one way ANOVA; p < 0.0001)

Fig. 2

Relative quantification of the MYH7 alleles in myocardium of non-HCM control individuals using heterozygous single nucleotide polymorphisms. a Defined mixtures of synthetic plasmids encoding for the wildtype and T63T (triangle) I989I (cross) or A1702A (open circle) variant, respectively were amplified by quantitative PCR and quantified densitometrically from allele specific restriction digests. Quantifications were performed in at least four independent experiments. The expected ratios are indicated by the grey line. b Relative quantification of MYH7-mRNA in myocardial tissue from 11 non-HCM control individuals carrying the variants T636T, I989I, and A1702A, respectively. Quantification was performed on RNA extracted from at least two pieces of tissue at least in duplicate analysis. Each dot represents a single quantification analysis. The asterisks indicate significant deviation from 50% (one way ANOVA; p < 0.0001)