PMC

MYBPC3 protein level. A. Representative western blot and B. Graph summary of densitometric analysis for MYBPC3 protein normalized to GAPDH and expressed as a % of the average of controls run on each gel. Results are shown for control, non-failing hearts (N=10), and HCM hearts without sarcomere mutations (N=14), with MYBPC3 mutations (N=22), and with sarcomere mutations other than MYBPC3 (N=10). *P<0.001 for HCM with MYBPC3 mutations vs HCM with or without other sarcomere mutations, †P<0.05 for HCM without sarcomere mutations vs controls and for HCM with non-MYBPC3 sarcomere mutations vs controls.

MYBPC3 localization. A. Representative western blot for MYBPC3 of samples containing various MYBPC3 mutations shows that all MYBPC3 protein is detected in the myofilament fraction, and none in the supernatant. B. Representative immunofluorescent images of formalin-fixed heart tissue from human hearts, reacted with antibodies to MYBPC3 (green) and α-actinin (red) showing correct localization of MYBPC3 to the A-b and of the sarcomere in a doublet pattern. This localization pattern was observed in all truncating and missense mutant samples examined.

Sarcomere gene expression in human HCM samples. A. (Left) Allele-specific expression from wild-type and MYBPC3 splice-error PTC-containing transcripts quantified by RT-qPCR with custom designed primers (). (Right) Allele-specific expression from wild-type and MYBPC3 PTC-containing transcripts (without splice errors) quantified by custom-designed single base extension reactions. These mutations are either single nucleotide substitutions that encode for a termination codon, or an insertion that causes a frameshift. B. Quantification of wild-type and single nucleotide substitution mutant sarcomere gene transcripts without PTCs by custom-designed single base extension reactions. For the MYBPC3 exon splice site mutations, the mutant fraction is the proportion of detectable missense transcript. The remainder of the samples contain true missense mutations. In instances where 2 samples carried the same mutation, these are designated as (a) and (b) respectively in this figure, and maintained throughout the manuscript. C. Total MYBPC3 transcript abundance (left panel) and total MYH7 transcript abundance (right panel) in carriers of MYBPC3 (n=18) or MYH7 (n=6) mutations, compared to HCM samples in which no sarcomere mutation (n=7) was identified and control (non-HCM donor, n=7) hearts. *P=0.02 vs. control hearts, †P=0.003 vs. non-sarcomere HCM. D. RT-PCR analysis showing uniformly increased intensity of amplified MYBPC3 sequence in samples containing MYBPC3 mutations compared to the other 3 groups.

Stoichiometry of wild-type and mutant sarcomere protein in human HCM determined by absolute quantification of abundance (AQUA). (A) Representative chromatogram from HCM sample MYBPC3 Arg495Gln (a) showing peptide abundance of wild-type and mutant peptides relative to their corresponding AQUA peptides. (B) Graph summary of relative peptide abundance of wild-type and mutant peptides from individual samples. The abundance of each peptide(s) (fmol) was calculated, summed in cases of missed cleavages or post-translational modifications (), and expressed as a relative percentage of the total (abundance of wild-type + mutant peptides for each sample). Three regions (septum, LV lateral wall, and right ventricle) were analyzed in 2 samples with MYH7 mutations obtained from explanted hearts at the time of transplantation. For both B and C, color coding is used to designate genes harboring mutations: MYH7 (blue), MYBPC3 (green), TNNT2 (red), TPM1 (yellow), MYL2 (purple). (C) Relationship of patient age at time of surgery (left panel) and age at time of diagnosis of HCM (right panel) to mutant allele fraction. There was a significantly greater degree of allelic imbalance (in either direction) in patients who had surgery ≤ 40 y/o, compared to those > 40 y/o (P<0.01) and in patients who were diagnosed ≤ 30 y/o, compared to those diagnosed > 30 y/o (P<0.05).