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Unequal Allelic Expression of Wild-Type and Mutated B-Myosin in Familial Hypertrophic Cardiomyopathy

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Unequal Allelic Expression of Wild-Type and Mutated B-Myosin in Familial Hypertrophic Cardiomyopathy Basic Res Cardiol (2011) 106:1041–1055 DOI 10.1007/s00395-011-0205-9 ORIGINAL CONTRIBUTION Unequal allelic expression of wild-type and mutated b-myosin in familial hypertrophic cardiomyopathy Snigdha Tripathi • Imke Schultz • Edgar Becker • Judith Montag • Bianca Borchert • Antonio Francino • Francisco Navarro-Lopez • Andreas Perrot • Cemil O¨ zcelik • Karl-Josef Osterziel • William J. McKenna • Bernhard Brenner • Theresia Kraft Received: 26 May 2011 / Revised: 29 June 2011 / Accepted: 7 July 2011 / Published online: 19 July 2011 Ó The Author(s) 2011. This article is published with open access at Springerlink.com Abstract Familial hypertrophic cardiomyopathy (FHC) genotyped and clinically well-characterized FHC patients is an autosomal dominant disease, which in about 30% of were analyzed. The fraction of mutated MYH7-mRNA in the patients is caused by missense mutations in one allele five patients with mutation R723G averaged to 66 and 68% of the b-myosin heavy chain (b-MHC) gene (MYH7). To of total MYH7-mRNA in soleus and myocardium, respec- address potential molecular mechanisms underlying the tively. For mutations I736T, R719W and V606M, fractions family-specific prognosis, we determined the relative of mutated MYH7-mRNA in M. soleus were 39, 57 and expression of mutant versus wild-type MYH7-mRNA. We 29%, respectively. For all mutations, unequal abundance found a hitherto unknown mutation-dependent unequal was similar at the protein level. Importantly, fractions of expression of mutant to wild-type MYH7-mRNA, which is mutated transcripts were comparable among siblings, in paralleled by similar unequal expression of b-MHC at the younger relatives and unrelated carriers of the same protein level. Relative abundance of mutated versus wild- mutation. Hence, the extent of unequal expression of type MYH7-mRNA was determined by a specific restriction mutated versus wild-type transcript and protein is charac- digest approach and by real-time PCR (RT-qPCR). Four- teristic for each mutation, implying cis-acting regulatory teen samples from M. soleus and myocardium of 12 mechanisms. Bioinformatics suggest mRNA stability or splicing effectors to be affected by certain mutations. Intriguingly, we observed a correlation between disease Electronic supplementary material The online version of this expression and fraction of mutated mRNA and protein. article (doi:10.1007/s00395-011-0205-9) contains supplementary material, which is available to authorized users. S. Tripathi Á I. Schultz Á E. Becker Á J. Montag Á B. Borchert Á A. Perrot Á C. O¨ zcelik B. Brenner Á T. Kraft (&) Charite´-Unversita¨tsmedizin Berlin, Experimental and Clinical Institute of Molecular and Cell Physiology, Research Center (ECRC) am Max-Delbru¨ck-Centrum fu¨r Hannover Medical School, Carl Neuberg Str. 1, Molekulare Medizin, Kardio-Genetisches Labor, 30625 Hannover, Germany 13125 Berlin, Germany e-mail: [email protected] C. O¨ zcelik Á K.-J. Osterziel Present Address: Charite´-Universita¨tsmedizin Berlin, Kardiologie am Campus I. Schultz Virchow-Klinikum, 13353 Berlin, Germany Niederwiesenring 4, 63110 Rodgau, Germany Present Address: Present Address: K.-J. Osterziel B. Borchert Kardiologische Gemeinschaftspraxis, Marienstraße 9, Department of Cardiac, Thoracic, Transplantation and Vascular 92224 Amberg, Germany Surgery, Hannover Medical School, Carl-Neuberg Str. 1, 30625 Hannover, Germany W. J. McKenna Institute of Cardiovascular Science, University College London, A. Francino Á F. Navarro-Lopez London WC1E 6BT, United Kingdom Hospital Clinic/IDIBAPS, University of Barcelona, 08036 Barcelona, Spain 123 1042 Basic Res Cardiol (2011) 106:1041–1055 This strongly suggests that mutation-specific allelic expression of different genes in humans [8, 13, 48]. Thus, imbalance represents a new pathogenic factor for FHC. allelic imbalance could well be quite significant for disease expression in genetic disorders [29], as has been shown, Keywords Hypertrophic cardiomyopathy Á Allelic e.g., for hyperkalemic periodic paralysis in horses [50]. imbalance Á Cardiac b-myosin heavy chain Á Myosin Evidence for allelic imbalance in FHC arises from our missense mutation Á mRNA quantification previous work on functional effects of b-MHC missense mutations. Since model systems of FHC often do not reveal the same mutation effect as it may occur in humans [19, 25], Introduction we study these mutations in M. soleus fibers and ventricular biopsies of FHC patients. In muscle fibers from individuals Familial hypertrophic cardiomyopathy (FHC), the most who were essentially asymptomatic but carried b-MHC common genetic heart disease, is characterized by hyper- mutations G584R or V606M, respectively, we did not detect trophy of the left ventricle and the inter-ventricular septum any effects on sarcomere function. The fraction of mutated (IVS), in the absence of overt etiological factors [23]. The b-MHC for mutations V606M and G584R was only 12 and hypertrophy is often asymmetric and variable in severity 23% of the total b-MHC in the sarcomeres, respectively [16]. The prevalence of hypertrophic cardiomyopathy is 1 [26]. This raised several questions: (1) Is the deviation from in 500; it affects individuals at every age [22]. FHC is often the expected 50-to-50 ratio of wild-type versus mutated associated with arrhythmias, syncope and progression to myosin at the protein level paralleled by similar changes at heart failure or sudden cardiac death even at young age [23]. the mRNA level due to allelic imbalance? (2) Is an unequal The genetically heterogeneous disease is transmitted as ratio of mutant versus wild-type protein and mRNA a an autosomal dominant trait [38]. Mutations in more than common feature in FHC caused by myosin mutations and 18 genes, mostly encoding sarcomeric proteins, have been how is it correlated to the severity of the disease? linked to this disease in humans (Human Gene Mutation In the present study, we addressed these questions by Database at http://www.hgmd.cf.ac.uk/ac/index.php). To measuring the relative abundance of mutated and wild-type date, approximately 30% of all genotyped cases were found MYH7-mRNA (RefSeq accession NM_000257) as well as to be associated with missense mutations in the b-myosin of the encoded protein for several different mutations in M. heavy chain (b-MHC) gene (MYH7) with chromosomal soleus and cardiac muscle biopsies of FHC patients with localization 14q12 [34]. Notably, the human b-MHC iso- different disease severity. Wild-type and mutated MYH7- form (UniProt ID P12883), besides being the principal mRNAs are co-expressed in the same cells/muscle fibers ventricular myosin isoform, is also expressed in the slow and therefore are subjected to identical conditions. Thus, skeletal muscle fibers, e.g., of musculus soleus [35]. variations due to confounding factors such as environ- Several mutations in the b-MHC are associated with early mental influences and drugs, of hormones, or differences in onset and high incidence of sudden cardiac death, while tissue sampling or experimental procedures could be min- others have a less severe disease expression and outcome imized. For all five MYH7 missense mutations studied, we [3]. This is usually explained by the type and location of the found a deviation from the usually expected equimolar mutations within the molecule and their different effects on ratio of wild-type versus mutated b-MHC. The deviation structure and function of the encoded protein [32]. Fur- was very similar at the protein- and mRNA level and was thermore, even among affected members of the same family, identical in myocardial tissue and M. soleus. Most inter- variability in the clinical manifestations of FHC has been estingly, the unequal abundance appears to be directly observed, which points to additional risk factors [23]. related to the particular missense mutation, because it is A further mechanism for different clinical prognosis of found to be essentially the same in all carriers of a given FHC-related mutations, however, could be the relative level mutation, including relatives of different generations and of mutated versus wild-type protein in patients with dif- unrelated individuals. Our results suggest that unequal ferent mutations or even in patients with the same mutation. allelic expression of b-MHC contributes to the complex Nearly all FHC patients are heterozygous for the respective phenotype of FHC. mutation, and both mutated and wild-type proteins are assumed to be co-dominantly expressed from the corre- sponding alleles. Equal proportions of the allelic messages Materials and methods of the MYH7 gene and thus of the encoded protein are assumed to be present in every muscle cell. Yet, a higher or Patients and muscle biopsies lower expression of the two allelic transcripts and proteins resulting in a deviation from an expected 50-to-50 ratio, The present study was performed on muscle biopsies from known as allelic imbalance, has been shown previously for individuals previously characterized clinically as FHC 123 Basic Res Cardiol (2011) 106:1041–1055 1043 patients and genetically as carriers of point mutations in the RNA isolation, synthesis of sscDNA and PCR b-MHC head domain. All patients were heterozygous for the mutations they carried. In Table 1, clinical details For mutations I736T, R719W and V606M, total RNA was available to us and the age of all patients at the time of isolated from approximately 3 mg pieces of frozen muscle biopsy are given. using Trizol (GIBCO, Karlsruhe, Germany). Isolated RNA For all patients,
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