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.

Fig. 1

Experimental approach to minimize heteroduplex formation. a Schematic representation of restriction fragments produced by Nde II digestion of wild-type and mutated homoduplexes for mutation R723G (upper panels). Formation of heteroduplexes (lower two panels) generates one refractory restriction site (gray arrows), thus producing the 125-bp fragment from both strands of the heteroduplexes. b For reconditioning PCR, after 35 PCR cycles 1:100 dilutions of the product were subjected to another PCR and quantified after each cycle. The reaction was exponential until the seventh cycle. IOD, integrated optical density (SD from densitometric analysis at different exposure times). c 3.5% agarose gel with restriction digests of PCR products of a sample from the left ventricular lateral wall of a patient heterozygous for mutation R723G. The 35-bp fragment is not seen due to its small size. Cleavage of the reconditioning PCR products of three, four, five or six cycles (lanes 3C–6C) yielded mutated MYH7-mRNA fractions of 73, 82, 77 and 75%, respectively (on average 74 ± 8% in this muscle sample). There was no indication for heteroduplex-induced increase of mutated MYH7-mRNA at increasing cycle numbers. Lanes 1–3 12, 15 and 18 μl of equimolar DNA standard; B blank; U undigested PCR product

Fig. 2

Relative quantification of R723G mutated and wild-type sequences in defined plasmid mixtures and biopsies. a Restriction fragments of the 281-bp PCR product generated from defined mixtures of R723G-mutant and wild-type plasmid (Lanes 2–8). Lane L, equimolar DNA standard; lane B, blank; lane 1, undigested product. The increasing ratio (indicated above lanes) of mutant versus wild-type sequence resulted in brighter bands of 125 bp and weaker bands of 90 bp. b Input versus experimentally determined mutation-specific DNA in plasmid mixtures (n = 3 assays). The dashed line indicates the expected values. c Representative gel with the undigested 281-bp PCR product (lane 1) and restriction fragments after reconditioning PCR for three, four, five and six cycles (lanes 2–5) from a M. soleus sample of patient H27. L, equimolar DNA standard; B, blank. d Fraction of mutated mRNA in the left ventricular wall (light columns) and M. soleus (dark columns) of patients from three unrelated families with mutation R723G. The age (years) of patients at M. soleus biopsy is indicated; *patient H27 was transplanted ≈ 1 year after soleus biopsy)

Fig. 3

Allele-specific RT-qPCR for quantification of mutated versus wild-type MYH7-mRNA in myocardium. For RT-qPCR, mRNA from cardiac tissue samples was reverse transcribed using a MYH7-specific RT-primer. Subsequent comparative quantitative PCR was accomplished using identical primers and differently labeled mutation or wild-type specific probes in one reaction tube (Online Resource). RT-qPCR data are shown in comparison to data from restriction digest approach performed on the same cardiac samples from left ventricular (LV) anterior wall and right ventricular (RV) wall of H27

Fig. 4

MS spectra of native and synthetic β-MHC wild-type peptides and of peptides with mutation R723G. The Lys-C digest fraction of β-myosin containing native wild-type and R723G-mutated peptides was analyzed by nanoLC–ESI-MS after adding equimolar amounts of stable isotope-labeled synthetic wild-type and mutant internal standard peptides. The traces represent ion signals of the fivefold charged synthetic mutant (a; m/z 764.1), synthetic wild-type (b; m/z 783.9), native mutant (c; m/z 763.1) and native wild-type (d; m/z 782.8) peptides. Note the quite large difference in peak areas between native wild-type and mutated peptides mainly representing the difference in abundance of the two peptides and thus of the two parent proteins in the muscle sample

Fig. 5

Fractions of mutated MYH7-mRNA and β-myosin for different FHC mutations. The fractions of mutated mRNA (gray bars) and protein (black bars) in M. soleus are summarized for mutations V606M, I736T, R719W and R723G. Additionally, the R723G-mutated mRNA fractions in myocardium and previously published protein levels of mutations G584R and V606M [26] are depicted. mRNA data shown here are from the restriction digest approach. All data are also listed in Table 1. All mutations significantly deviate from equal abundance of wild-type and mutated transcript with similar deviations also at the protein level. Similar deviations of mRNA and protein levels were found in M. soleus and myocardium for mutation R723G (LV, left ventricle; RV, right ventricle). Note the intra- and/or inter-familial similarity in mutated MYH7-mRNA/protein expression for I736T, R723G and V606M

Fig. 6

Secondary structure of MYH7 pre-mRNA and mature mRNA. The RNAfold program (minimum free energy) was used to compare the secondary structure of the MYH7 mRNA of mutated and wild-type isoforms. Mutations R719W and I736T generate identical structures as the wild-type sequence and are therefore not depicted. a Secondary mRNA structures of wild-type (NM_000257), mutation V606M (c.1922G>A) and mutation R723G (c.2273C>G). b Secondary structure of the pre-mRNA of wild-type exon 15 to exon 21 (position 6,992–10,380, NG_007884). Enlarged boxes: sites of structural alterations caused by mutations V606M (c.1922G>A, green arrow) and R723G (c.2273C>G, red arrow). Blue arrow in the wild-type sequence: location of mutation R719W that does not alter the pre-mRNA structure