Sarcomere Mutation-Specific Expression Patterns in Human Hypertrophic Cardiomyopathy

Abstract

Background Heterozygous mutations in sarcomere genes in hypertrophic cardiomyopathy (HCM) are proposed to exert their effect through gain-of-function for missense mutations or loss-of-function for truncating mutations. However, allelic expression from individual mutations has not been sufficiently characterized to support this exclusive distinction in human HCM. Methods and Results Sarcomere transcript and protein levels were analyzed in septal myectomy and transplant specimens from 46 genotyped HCM patients with or without sarcomere gene mutations and 10 control hearts. For truncating mutations in MYBPC3, the average ratio of mutant:wild-type transcripts was ~1:5, in contrast to ~1:1 for all sarcomere missense mutations, confirming that nonsense transcripts are uniquely unstable. However, total MYBPC3 mRNA was significantly increased by ~9 fold in HCM samples with MYBPC3 mutations compared to control hearts and to HCM samples without sarcomere gene mutations. Full-length MYBPC3 protein content was not different between MYBPC3 mutant HCM and control samples and no truncated proteins were detected. By absolute quantification of abundance (AQUA) with multiple reaction monitoring, stoichiometric ratios of mutant sarcomere proteins relative to wild-type were strikingly variable in a mutation-specific manner, with the fraction of mutant protein ranging from 30–84%. Conclusions These results challenge the concept that haploinsufficiency is a unifying mechanism for HCM caused by MYBPC3 truncating mutations. The range of allelic imbalance for several missense sarcomere mutations suggests that certain mutant proteins may be more or less stable, or incorporate more or less efficiently into the sarcomere than wild-type proteins. These mutation-specific properties may distinctly influence disease phenotypes.

Identification of pathogenic gene mutations in LMNA and MYBPC3 that alter RNA splicing

“…First, the minigene splice assay may not identify the precise splice alteration that occurs in vivo. However, of the 53 splice-altering variants identified here, 16 variants were independently characterized by RT-PCR in patient tissues (15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25), alter splicing, and are pathogenic (Table S3). Moreover, the fact that 48 of 53 VUS were identified in patients with an AD cardiomyopathy provides a very strong likelihood that the aberrant splicing observed in the minigene assay occurs in vivo.…”

Impact of Demographic Features, Lifestyle, and Comorbidities on the Clinical Expression of Hypertrophic Cardiomyopathy

“…Overall, however, physiological stimuli and comorbidities seem to exert a modest impact especially on the phenotypic expression of HCM. Future research targeting HCM variability should rather focus on molecular aspects including modifier genes, epigenetic factors, and the role of regulatory systems such as microRNAs, the ubiquitine‐proteasome complex, or nonsense‐mediated RNA decay 78, 79…”

An Investigation of the Molecular Mechanism of Double cMyBP-C Mutation in a Patient with End-Stage Hypertrophic Cardiomyopathy

“…Several studies have shown that truncation mutations in cMyBP-C diminish the total protein level and cause hypertrophic cardiomyopathy through haploinsufficiency [36][37][38]. However, a recent study suggests that consequences of the mutations cannot be predicted solely based on the type or location of the mutation [33]. Our in vitro results showed that full length cMyBP-C protein level was not significantly altered between wild-type, single mutations and double mutation, indicating that both mutant mRNA and protein are stable and UPS is unlikely to be involved in the regulation of mutant protein clearance in H9C2 cells.…”

“…It is important to examine the expression and stability of the single and double point mutations as a recent study showed complexity in the expression of point mutations [33]. To determine the isolated effect of E258K, E441K and their combination (E258K-E441K) on protein stability, we transfected H9C2 cells with WT or single or double mutation of MYBPC3 constructs tagged with GFP.…”