Unequal allelic expression of wild-type and mutated β-myosin in familial hypertrophic cardiomyopathy

Abstract

Familial hypertrophic cardiomyopathy (FHC) is an autosomal dominant disease, which in about 30% of the patients is caused by missense mutations in one allele of the β-myosin heavy chain (β-MHC) gene (MYH7). To address potential molecular mechanisms underlying the family-specific prognosis, we determined the relative expression of mutant versus wild-type MYH7-mRNA. We found a hitherto unknown mutation-dependent unequal expression of mutant to wild-type MYH7-mRNA, which is paralleled by similar unequal expression of β-MHC at the protein level. Relative abundance of mutated versus wild-type MYH7-mRNA was determined by a specific restriction digest approach and by real-time PCR (RT-qPCR). Fourteen samples from M. soleus and myocardium of 12 genotyped and clinically well-characterized FHC patients were analyzed. The fraction of mutated MYH7-mRNA in five patients with mutation R723G averaged to 66 and 68% of total MYH7-mRNA in soleus and myocardium, respectively. For mutations I736T, R719W and V606M, fractions of mutated MYH7-mRNA in M. soleus were 39, 57 and 29%, respectively. For all mutations, unequal abundance was similar at the protein level. Importantly, fractions of mutated transcripts were comparable among siblings, in younger relatives and unrelated carriers of the same mutation. Hence, the extent of unequal expression of mutated versus wild-type transcript and protein is characteristic for each mutation, implying cis-acting regulatory mechanisms. Bioinformatics suggest mRNA stability or splicing effectors to be affected by certain mutations. Intriguingly, we observed a correlation between disease expression and fraction of mutated mRNA and protein. This strongly suggests that mutation-specific allelic imbalance represents a new pathogenic factor for FHC.Electronic supplementary materialThe online version of this article (doi:10.1007/s00395-011-0205-9) contains supplementary material, which is available to authorized users.

Allele-Specific Silencing of Mutant Myh6 Transcripts in Mice Suppresses Hypertrophic Cardiomyopathy

“…Indeed, the LVWT in 403i-treated MHC 403/+ mice, with or without CsA, was comparable to that in wild-type mice. Based on these findings and evidence for unequal expression of mutated and wildtype MYH7 mRNAs in human HCM hearts (23), we suggest that variable penetrance and severity of HCM may reflect, at least in part, the proportion of wild-type and mutant transcripts and proteins.…”

Sarcomere Mutation-Specific Expression Patterns in Human Hypertrophic Cardiomyopathy

“…A previous study reported variability in the fraction of mutant mRNA for 5 MYH7 variants, 39 but was limited by use of skeletal muscle biopsies, analysis of benign variants, and less sensitive methods than the primer extension technique. While it is possible that differences could be due to specific mutations analyzed, we think this is a less plausible explanation given that non-PTC containing transcripts are not substrates for NMD, and should be intrinsically stable.…”

“…39, 40 An important distinction from previously published methodologies is the recognition of missed cleavages and post-translational modifications which was critical for accurate absolute and relative quantification of wild-type and mutant peptides by AQUA. For one mutation, MYBPC3 Arg495Gln, the mutant peptide was similarly ~2-fold more abundant than wild-type in 2 samples from unrelated HCM patients, implying that preferential expression or stability of the mutant protein is an inherent property of a particular mutant protein rather than a function of other modifying factors.…”

Two Strikes and You’re Out

“…V606 lies in the 50 KDa portion of the myosin S1 head, where it contributes to the actin-binding site 9 . R453C has been associated with more severe human HCM and is located within the γ-phosphate sensing domain of the myosin ATPase 10 . When engineered in vitro into human β myosin, the R453C mutation has reduced actin-activated ATPase and directs slower in vitro sliding of actin filaments 11 .…”

“…However, altered actin binding plus reduced ATPase evoked significantly more hypertrophy, suggesting a disease model wherein the cardiac sarcomeres contain a mixture of myosin heads, some with different actin binding and others with reduced ATPase. These mixed mutations may or may not reflect a situation where each allele produces half the total MHC protein, as V606M and R719W may not be expressed at equal levels to the normal allele 10 . It is also possible that some mutations may alter mRNA stability and splicing, provoking reduced expression of the mutant protein relative to normal MHC, thereby leading to less hypertrophy.…”