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

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 ß-myosin heavy chain (ß-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 ß-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.

Novel correlations between the genotype and the phenotype of hypertrophic and dilated cardiomyopathy: results from the German Competence Network Heart Failure.

AIMS: Hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) can both be due to mutations in the genes encoding ß-myosin heavy chain (MYH7) or cardiac myosin-binding protein C (MYBPC3). The aim of the present study was to determine the prevalence and spectrum of mutations in both genes in German HCM and DCM patients and to establish novel genotype-to-phenotype correlations. METHODS AND RESULTS: Coding exons and intron flanks of the two genes MYH7 and MYBPC3 of 236 patients with HCM and 652 patients with DCM were sequenced by conventional and array-based means. Clinical records were established following standard protocols. Mutations were detected in 41 and 11% of the patients with HCM and DCM, respectively. Differences were observed in the frequency of splice site and frame-shift mutations in the gene MYBPC3, which occurred more frequently (P< 0.02, P< 0.001, respectively) in HCM than in DCM, suggesting that cardiac myosin-binding protein C haploinsufficiency predisposes to hypertrophy rather than to dilation. Additional novel genotype-to-phenotype correlations were found in HCM, among these a link between MYBPC3 mutations and a particularly large thickness of the interventricular septum (P= 0.04 vs. carriers of a mutation in MYH7). Interestingly, this correlation and a link between MYH7 mutations and a higher degree of mitral valve regurgitation held true for both HCM and DCM, indicating that the gene affected by a mutation may determine the magnitude of structural and functional alterations in both HCM and DCM. CONCLUSION: A large clinical-genetic study has unravelled novel genotype-to-phenotype correlations in HCM and DCM which warrant future investigation of both the underlying mechanisms and the prognostic use.

A network against failing hearts--introducing the German "Competence Network Heart Failure".

Heart failure (HF) has been identified as one of the most threatening diseases for the western civilisation, posing a risk to health for a rising number of patients. Acknowledging the medical problem of HF to be both economically and socially threatening the German Federal Ministry of Research and Education (BMBF) initiated a nationwide research network aiming to find new ways in prevention, alleviation and treatment of the widespread disease. The "Competence Network Heart Failure" (CNHF), initiated in 2003, bundles the scientific expertise in a large-scale research network; its aims are the coordination of basic and applied clinical research as well as dissemination of findings into clinical practice in order to consolidate and perpetuate the achieved improvements. The scope of this paper is to introduce the CNHF and to provide an overview of the tasks and hitherto attained achievements to a broad spectrum of health care providers.

Bisoprolol vs. carvedilol in elderly patients with heart failure: rationale and design of the CIBIS-ELD trial.

BACKGROUND: Chronic heart failure (CHF) is a widespread disease with severe quality of life impairment and a poor prognosis. Beta-blockers are the mainstay of CHF therapy; yet they are under-prescribed and under-dosed in clinical practice. This is particularly evident in elderly patients, which may be due to a fear of side-effects or intolerance. Beta-blockers have further not been adequately tested in patients with diastolic CHF, which is particularly common in elderly patients. Finally, comparative data on the use of different beta-blockers in patients with CHF is scarce. AIM: To compare the tolerance of bisoprolol and carvedilol in elderly patients with CHF. METHODS: CIBIS-ELD is an investigator-initiated, multi-centre, 1:1 randomised, double-blind, phase III trial comparing bisoprolol and carvedilol in patients >or=65 years with systolic or diastolic CHF. Recruitment started in April 2005 and is anticipated to be completed by April 2008 with at least 800 patients enrolled. PERSPECTIVE: This is the first large scale head to head beta-blockers trial in an elderly population with CHF. Besides determining which of two standard beta-blockers is best tolerated in elderly patients with systolic or diastolic CHF, we expect to gain further insight into the treatment of the particular population of patients with diastolic CHF.

A DNA resequencing array for pathogenic mutation detection in hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is a heterogeneous autosomal dominant cardiac disorder with a prevalence of 1 in 500. Over 450 different pathogenic mutations in at least 16 genes have been identified so far. The large allelic and genetic heterogeneity of HCM requires high-throughput, rapid, and affordable mutation detection technologies to efficiently integrate molecular screening into clinical practice. We developed a custom DNA resequencing array that contains both strands of all coding exons (160), splice-site junctions, and 5'UTR regions of 12 genes that have been clearly implicated in HCM (MYH7, MYBPC3, TNNT2, TPM1, TNNI3, MYL3, MYL2, CSRP3, PLN, ACTC, TNNC1, and PRKAG2). We analyzed a first series of 38 unrelated patients with HCM (17 familial, 21 sporadic). A total of 953,306 bp across the 38 patients were sequenced with a mean nucleotide call rate of 96.92% (range: 93-99.9%). Pathogenic mutations (single nucleotide substitutions) in MYH7, MYBPC3, TNNI3, and MYL3 (six known and six novel) were identified in 60% (10/17) of familial HCM and 10% of sporadic cases (2/21). The high-throughput HCM resequencing array is the most rapid and cost-effective tool for molecular testing of HCM to date; it thus has considerable potential in diagnostic and predictive testing, and prognostic stratification.

Dysfunction of dysferlin-deficient hearts.

Mutations in the gene encoding dysferlin cause limb-girdle muscular dystrophy 2B (LGMD2B), a disorder that is believed to spare the heart. We observed dilated cardiomyopathy in two out of seven LGMD2B patients and cardiac abnormalities in three others. Cardiac biopsies showed that dysferlin was completely absent from the sarcolemma and appeared to be trapped within the cardiomyocytes. SJL/J mice (33-week-old) had diminished end-systolic pressure and reduced dP/dt; however, the hearts were histologically normal. Gene expression profiles of cardiac tissue were obtained and later confirmed by quantitative RT-PCR. Dysferlin-deficient and control mice had different gene expression patterns in terms of cardiomyocyte Z-disc and signal transduction proteins. CapZ, LIM-domain-binding protein 3 (LDB3, MLP), cypher (ZASP), desmin, and the cardiac ankyrin-repeated protein (CARP) were differentially expressed, compared to controls. Mechanical stress induced by the nonselective beta-adrenergic agonist isoproterenol (5 mg/kg body weight) given daily for 10 days resulted in reduced fractional shortening and increased cardiac fibrosis in SJL/J mice as compared to controls. Isoproterenol also caused metalloproteinase-2 upregulation in SJL/J mice. In A/J mice, the effect of isoproterenol injection was even more dramatic and lead to premature death as well as marked sarcolemmal injury as demonstrated by Evans blue dye penetration. Our data suggest that disturbances in dysferlin as well as Z-line proteins and transcription factors particularly under mechanical stress cause cardiomyopathy.

The G-231A polymorphism in the endothelin-A receptor gene is associated with lower aortic pressure in patients with dilated cardiomyopathy.

BACKGROUND: The endothelin system (ES) plays an important role in blood pressure (BP) regulation and also in the pathophysiology of idiopathic dilated cardiomyopathy (DCM). Recently, we demonstrated that a genetic polymorphism in the endothelin A (ET(A)) receptor gene was associated with survival in DCM patients. The aim of this study was to determine whether polymorphisms in the ET(A) receptor gene might be associated with the severity of DCM. METHODS: One hundred twenty-four consecutively recruited unrelated patients with DCM, who underwent a detailed phenotyping protocol, were genotyped for the ET(A) receptor G-231A polymorphism using a hybridization technique with allele-specific oligonucleotides. RESULTS: The exon 1 G-231A polymorphism of the ET(A) receptor gene, upstream of the translation start site, was significantly associated with directly measured intra-aortic pressure in that -231A allele carriers had significantly lower systolic (P = .0043), as well as mean (P = .0016) and diastolic (P = .0041) aortic pressure compared to noncarriers. The association of ET(A) G-231A with aortic pressure was independent from other factors such as prior medication, left ventricular end-diastolic diameter, age, gender, and New York Heart Association (NYHA) functional classification. However, no such association was seen for cuff BP and survival rates were not significantly different between -231A allele carriers and -231G homozygotes (log rank test, P = .66). No significant association with any other parameter investigated in the present study could be observed, even when men and women were analyzed separately. CONCLUSIONS: Our results suggest an association of genetic variation in the ET(A) receptor gene with aortic pressure in patients with DCM.

A systematic meta-analysis of the efficacy and heterogeneity of disease management programs in congestive heart failure.

BACKGROUND: We sought to systematically combine the evidence on efficacy of disease management programs (DMPs) in the treatment of congestive heart failure (CHF), to identify and explain heterogeneity of results from prior studies of DMPs, and to assess potential publication bias from these studies. METHODS AND RESULTS: We conducted a systematic literature search on randomized clinical trials investigating the effect of DMPs on CHF outcomes and performed meta-analyses and meta-regressions comparing DMPs and standard care for mortality and rehospitalization. We included 36 studies from 13 different countries (with data from 8341 patients). Our meta-analysis yielded a pooled risk difference of 3% (95% confidence interval [CI] 1-6%, P < .01) for mortality and of 8% (95% CI 5-11%, P < .0001) for rehospitalization, both favoring DMP. Factors explaining heterogeneity between studies included severity of disease, proportion of beta-blocker at baseline, country, duration of follow-up, and mode of postdischarge contact. No statistically significant publication bias was detected. CONCLUSION: DMPs have the potential to reduce morbidity and mortality for patients with CHF. The benefit of the intervention depends on age, severity of disease, guideline-based treatment at baseline, and DMP modalities. Future studies should directly compare the effect of different aspects of disease management programs for different populations.

Composite polymorphisms in the ryanodine receptor 2 gene associated with arrhythmogenic right ventricular cardiomyopathy.

OBJECTIVE: Mutations in the cardiac ryanodine receptor (RYR2) gene have been reported to cause arrhythmogenic right ventricular cardiomyopathy (ARVC). The molecular mechanisms by which genetic modifications lead to ARVC are still not well understood. METHODS: ARVC patients were screened for mutations in the RYR2 gene by denaturing HPLC and DNA sequencing. Single channel measurements were carried out with RyR2 channels purified from explanted hearts of ARVC patients. RESULTS: None of the published RYR2 mutations were found in our ARVC-cohort. However, we identified two single nucleotide polymorphisms (SNPs) in exon 37 of the human RYR2 gene which lead to the amino acid exchanges G1885E and G1886S, respectively. Both SNPs together were found exclusively in 3 out of 85 ARVC patients in a composite heterozygous fashion (genotype T4). This genotype was associated with ARVC (p<0.05) but not with dilated cardiomyopathy (DCM, 79 patients) or none-failing controls (463 blood donors). However, either one of the two SNPs were identified in further 7 ARVC patients, in 11 DCM patients, and in 64 blood donors. The SNP leading to G1886S may create a protein kinase C phosphorylation site in the human RyR2. Single channel recordings at pCa4.3 revealed four conductance states for the RyR2 of genotype T4 and a single open state for the wild type RyR2. At pCa7.7, the lowest subconductance state of the RyR2 channel of genotype T4 persisted with a greatly enhanced open probability indicating a leaky channel. CONCLUSION: The RyR2 channel leak under diastolic conditions could cause SR-Ca2+ depletion, concomitantly arrhythmogenesis and heart failure in a subgroup of ARVC patients of genotype T4. A change in the RyR2 subunit composition due to the combined expression of both SNPs alters the behaviour of the tetrameric channel complex.

Novel sequence variants in dysferlin-deficient muscular dystrophy leading to mRNA decay and possible C2-domain misfolding.

Mutations in the gene encoding dysferlin (DYSF) cause the allelic autosomal recessive disorders limb girdle muscular dystrophy 2B and Miyoshi myopathy. It encompasses 55 exons spanning 150 kb of genomic DNA. Dysferlin is involved in membrane repair in skeletal muscle. We identified three families with novel sequence variants in DYSF. All affected family members showed limb girdle weakness and had reduced or absent dysferlin protein on immunohistochemistry. All exons of DYSF were screened by genomic sequencing. Five novel variants in DYSF were found: two missense mutations (c.895G>A and c.4022T>C), one 5' donor splice-site variant (c.855+1delG), one nonsense mutation (c.1448C>A), and a variant in the 3'UTR of DYSF (c.*107T>A). All alterations were confirmed by restriction enzyme analysis and not found in 400 control alleles. Nonsense mediated RNA decay or changes in the three-dimensional protein structure resulting in intracellular dysferlin aggregates and finally the lack of dysferlin protein were identified as consequences of the novel DYSF variants.

Gene expression analysis of human tissue from patients with cardiomyopathies: a new tool for guiding therapies in the future?

The complete sequencing of the human genome led to the development of a number of new molecular technologies. DNA microarrays represent an exciting new tool for gene expression analysis in human tissue. Measurements of the expressions of many thousands of genes in parallel is possible now. Microarrays may be used for various applications in medicine. They can be used to find novel prognostic and predictive markers as well as new disease classifications into clinically relevant subgroups. While there has been great progress in cancer research in this field, there are significantly less expression data available concerning the heart. In order to identify genes and pathways involved in the pathogenesis of cardiomyopathies, we have looked recently for alterations at cellular and molecular levels in heart tissue from cardiomyopathy patients. We showed that a special group of genes is differentially regulated in dilated cardiomyopathy. The first results in hypertrophic cardiomyopathy also showed similar findings. The surgeon's role in the clinical application of gene expression profiling is crucial. He provides a link between the patient and the laboratory scientists playing a significant role in focusing research on the clinically relevant problems. Gene expression profiles may help to better characterize the course and prognosis of the individual patient in the future. The long-term goal is to find a tool that will help to guide medical and surgical therapies in cardiomyopathies and other heart diseases.

Increased susceptibility to complement attack due to down-regulation of decay-accelerating factor/CD55 in dysferlin-deficient muscular dystrophy.

Dysferlin is expressed in skeletal and cardiac muscles. However, dysferlin deficiency results in skeletal muscle weakness, but spares the heart. We compared intraindividual mRNA expression profiles of cardiac and skeletal muscle in dysferlin-deficient SJL/J mice and found down-regulation of the complement inhibitor, decay-accelerating factor/CD55, in skeletal muscle only. This finding was confirmed on mRNA and protein levels in two additional dysferlin-deficient mouse strains, A/J mice and Dysf-/- mice, as well as in patients with dysferlin-deficient muscular dystrophy. In vitro, the absence of CD55 led to an increased susceptibility of human myotubes to complement attack. Evidence is provided that decay-accelerating factor/CD55 is regulated via the myostatin-SMAD pathway. In conclusion, a novel mechanism of muscle fiber injury in dysferlin-deficient muscular dystrophy is demonstrated, possibly opening therapeutic avenues in this to date untreatable disorder.

[Familial dilated cardiomyopathy]. / Familiäre dilatative kardiomyopathie.

Dilated cardiomyopathy (DCM) is the most frequent form of primary myocardial diseases and the third most common cause of heart failure. Clinically, DCM is characterized by a progressive course of ventricular dilatation and systolic dysfunction. The life expectancy is limited and varies according to the underlying etiology with a median survival time of about 5 years after diagnosis. Myocarditis, immunologic abnormalities, toxic myocardial damage, and genetic factors are all assumed to be causes. Familial occurrence of DCM, mostly as an autosomal dominant trait, is more common than generally believed and is responsible for 20-30% of all cases of DCM. Candidate gene screening and linkage analyses in large families were successful in identifying 24 disease genes. There is a wide variability in the onset, course and severity of the disease even within the same family. In addition, genotype-phenotype correlations included only small numbers of affected. This implies that in most cases no conclusion can be drawn from the clinical manifestation of DCM to the responsible disease gene. Mutations in the beta-myosin heavy chain and in cardiac troponin T are common causes of pure familial DCM. DCM associated with conduction disease is mainly due to mutations in lamin A/C and X-linked DCM is often caused by mutations in dystrophin. All other disease genes are rare causes of familial DCM. Genetic screening in all known disease genes is not possible, but more efficient screening methods are awaited in the near future. Until then, clinical examination of family members and, in case of familial DCM, genetic counseling are recommended in the work-up of patients with idiopathic DCM.

[Arrhythmogenic right ventricular cardiomyopathy]. / Die arrhythmogene rechtsventrikuläre kardiomyopathie.

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a primary myocardial disorder that is characterized by localized or diffuse atrophy of predominantly right ventricular myocardium with subsequent replacement by fatty and fibrous tissue. Arrhythmias of right ventricular origin are the main clinical manifestation. Affected patients present with ventricular premature beats and nonsustained or sustained ventricular tachycardia demonstrating a left bundle branch block pattern. However, since ventricular tachycardia may also degenerate into ventricular fibrillation, sudden death may be the first manifestation of ARVC.In recent years, ARVC has been more and more recognized as an important and frequent cause of ventricular tachyarrhythmias and sudden cardiac death, particularly in young patients and athletes, with apparently normal hearts. Evidence of the disease is found in 30-50% of family members. ARVC is a genetically heterogeneous disease. The diagnosis is based on electrocardiographic abnormalities and the identification of regional or global right ventricular dysfunction and fibrolipomatosis. Although several potentially causative genes have been identified, currently, genetic testing is not part of the routine diagnostic work-up.An implantable cardioverter-defibrillator is indicated in selected high-risk patients with ARVC (i. e., patients with life-threatening ventricular tachycardia or survivors of sudden cardiac death). The clinical course of the disease is often characterized by progression. In individual patients heart transplantation may become necessary.

Returning hypertrophy after surgery in a patient with hypertrophic cardiomyopathy caused by a myosin-binding protein C mutation.

We report a 13-year follow-up of a patient who underwent both myectomy and septal ablation due to hypertrophic cardiomyopathy caused by a cardiac myosin-binding protein C gene mutation. After myectomy the patient again developed significant septal hypertrophy at the operated septal area with a need for a second interventional therapy. This exceptional case underscores the remarkable ability of the heart muscle to show a continuous hypertrophic process over many years.

Prevalence of cardiac beta-myosin heavy chain gene mutations in patients with hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is a frequent, autosomal-dominant cardiac disease and manifests predominantly as left ventricular hypertrophy. Mutations in the cardiac beta-myosin heavy chain gene (MYH7) are responsible for the disease in about 30% of cases where mutations were identified. We clinically evaluated a large group of 147 consecutive HCM patients from three cardiology centers in Germany, Poland, and Kyrgyzstan according to the same protocol. The DNA of the patients was systematically analyzed in the whole coding region of the MYH7 gene using PCR, single-strand conformation polymorphism analysis, and automated sequencing. Eleven different missense mutations (including seven novel ones) in 11 unrelated patients were identified, showing a mutation frequency of 7.5% in the study population. We further examined the families of five patients (three of German, one of Polish, and one of Kyrgyz origin) with 32 individuals in total. We observed a clear, age-dependent penetrance with onset of disease symptoms in the fourth decade of life. Genotype-phenotype correlations were different for each mutation, whereas the majority was associated with an intermediate/malign phenotype. In conclusion, we report a systematic molecular screening of the complete MYH7 gene in a large group of consecutive HCM patients, leading to a genetic diagnosis in 38 individuals. Information about the genotype in an individual from one family could be very useful for the clinician, especially when dealing with healthy relatives in doubt of their risk about developing HCM. The increasing application of genetic screening and the increasing knowledge about genotype-phenotype correlations will hopefully lead to an improved clinical management of HCM patients.