Andreas Perrot

Genetic counselling and testing in cardiomyopathies: a position statement of the European Society of Cardiology Working Group on Myocardial and Pericardial Diseases

Advances in molecular genetics present new opportunities and challenges for cardiologists who manage patients and families with cardiomyopathies. The aims of this position statement of the European Society of Cardiology Working Group on Myocardial and Pericardial Diseases are to review the general issues related to genetic counselling, family screening and genetic testing in families with a cardiomyopathy, and to provide key messages and suggestions for clinicians involved in their management.

Risk Factors for Malignant Ventricular Arrhythmias in Lamin A/C Mutation Carriers A European Cohort Study

OBJECTIVES The purpose of this study was to determine risk factors that predict malignant ventricular arrhythmias (MVA) in Lamin A/C (LMNA) mutation carriers. BACKGROUND LMNA mutations cause a variety of clinical phenotypes, including dilated cardiomyopathy and conduction disease. Many LMNA mutation carriers have a poor prognosis, because of a high frequency of MVA and progression to end-stage heart failure. However, it is unclear how to identify mutation carriers that are at risk for MVA. METHODS In this multicenter cohort of 269 LMNA mutation carriers, we evaluated risk factors for MVA, defined as sudden cardiac death, resuscitation, and appropriate implantable cardioverter-defibrillator (ICD) treatment. RESULTS In a median follow-up period of 43 months (interquartile range: 17 to 101 months), 48 (18%) persons experienced a first episode of MVA: 11 persons received successful cardiopulmonary resuscitation, 25 received appropriate ICD treatment, and 12 persons died suddenly. Independent risk factors for MVA were nonsustained ventricular tachycardia, left ventricular ejection fraction <45% at the first clinical contact, male sex, and non-missense mutations (ins-del/truncating or mutations affecting splicing). MVA occurred only in persons with at least 2 of these risk factors. There was a cumulative risk for MVA per additional risk factor. CONCLUSIONS Carriers of LMNA mutations with a high risk of MVA can be identified using these risk factors. This facilitates selection of LMNA mutation carriers who are most likely to benefit from an ICD.

RBM20 , a gene for hereditary cardiomyopathy, regulates titin splicing

Alternative splicing has a major role in cardiac adaptive responses, as exemplified by the isoform switch of the sarcomeric protein titin, which adjusts ventricular filling. By positional cloning using a previously characterized rat strain with altered titin mRNA splicing, we identified a loss-of-function mutation in the gene encoding RNA binding motif protein 20 (Rbm20) as the underlying cause of pathological titin isoform expression. The phenotype of Rbm20-deficient rats resembled the pathology seen in individuals with dilated cardiomyopathy caused by RBM20 mutations. Deep sequencing of the human and rat cardiac transcriptome revealed an RBM20-dependent regulation of alternative splicing. In addition to titin (TTN), we identified a set of 30 genes with conserved splicing regulation between humans and rats. This network is enriched for genes that have previously been linked to cardiomyopathy, ion homeostasis and sarcomere biology. Our studies emphasize the key role of post-transcriptional regulation in cardiac function and provide mechanistic insights into the pathogenesis of human heart failure.

Mutations in the Human Muscle LIM Protein Gene in Families With Hypertrophic Cardiomyopathy

Background—Muscle LIM protein (MLP) is an essential nuclear regulator of myogenic differentiation. Additionally, it may act as an integrator of protein assembly of the actin-based cytoskeleton. MLP-knockout mice develop a marked cardiac hypertrophy reaction and dilated cardiomyopathy (DCM). MLP is therefore a candidate gene for heritable forms of hypertrophic cardiomyopathy (HCM) and DCM in humans. Methods and Results—We analyzed 1100 unrelated individuals (400 patients with DCM, 200 patients with HCM, and 500 controls) for mutations in the human CRP3 gene that encodes MLP. We found 3 different missense mutations in 3 unrelated patients with familial HCM but detected no mutation in the DCM group or the controls. All mutations predicted an amino acid exchange at highly conserved residues in the functionally important LIM1 domain, which is responsible for interaction with &agr;-actinin and with certain muscle-specific transcription factors. Protein-binding studies indicate that mutations in the CRP3 gene lead to a decreased binding activity of MLP to &agr;-actinin. All 3 index patients were characterized by typical asymmetrical septal hypertrophy. Family studies revealed cosegregation of clinically affected individuals with the respective mutations in MLP. Conclusion—Here, we present evidence that mutations in the CRP3/MLP gene can cause HCM.

Clinical ResearchGenetic DisordersRisk Factors for Malignant Ventricular Arrhythmias in Lamin A/C Mutation Carriers: A European Cohort Study

OBJECTIVES The purpose of this study was to determine risk factors that predict malignant ventricular arrhythmias (MVA) in Lamin A/C (LMNA) mutation carriers. BACKGROUND LMNA mutations cause a variety of clinical phenotypes, including dilated cardiomyopathy and conduction disease. Many LMNA mutation carriers have a poor prognosis, because of a high frequency of MVA and progression to end-stage heart failure. However, it is unclear how to identify mutation carriers that are at risk for MVA. METHODS In this multicenter cohort of 269 LMNA mutation carriers, we evaluated risk factors for MVA, defined as sudden cardiac death, resuscitation, and appropriate implantable cardioverter-defibrillator (ICD) treatment. RESULTS In a median follow-up period of 43 months (interquartile range: 17 to 101 months), 48 (18%) persons experienced a first episode of MVA: 11 persons received successful cardiopulmonary resuscitation, 25 received appropriate ICD treatment, and 12 persons died suddenly. Independent risk factors for MVA were nonsustained ventricular tachycardia, left ventricular ejection fraction <45% at the first clinical contact, male sex, and non-missense mutations (ins-del/truncating or mutations affecting splicing). MVA occurred only in persons with at least 2 of these risk factors. There was a cumulative risk for MVA per additional risk factor. CONCLUSIONS Carriers of LMNA mutations with a high risk of MVA can be identified using these risk factors. This facilitates selection of LMNA mutation carriers who are most likely to benefit from an ICD.

Connective tissue growth factor overexpression in cardiomyocytes promotes cardiac hypertrophy and protection against pressure overload.

Connective tissue growth factor (CTGF) is a secreted protein that is strongly induced in human and experimental heart failure. CTGF is said to be profibrotic; however, the precise function of CTGF is unclear. We generated transgenic mice and rats with cardiomyocyte-specific CTGF overexpression (CTGF-TG). To investigate CTGF as a fibrosis inducer, we performed morphological and gene expression analyses of CTGF-TG mice and rat hearts under basal conditions and after stimulation with angiotensin II (Ang II) or isoproterenol, respectively. Surprisingly, cardiac tissues of both models did not show increased fibrosis or enhanced gene expression of fibrotic markers. In contrast to controls, Ang II treated CTGF-TG mice displayed preserved cardiac function. However, CTGF-TG mice developed age-dependent cardiac dysfunction at the age of 7 months. CTGF related heart failure was associated with Akt and JNK activation, but not with the induction of natriuretic peptides. Furthermore, cardiomyocytes from CTGF-TG mice showed unaffected cellular contractility and an increased Ca2+ reuptake from sarcoplasmatic reticulum. In an ischemia/reperfusion model CTGF-TG hearts did not differ from controls. Our data suggest that CTGF itself does not induce cardiac fibrosis. Moreover, it is involved in hypertrophy induction and cellular remodeling depending on the cardiac stress stimulus. Our new transgenic animals are valuable models for reconsideration of CTGFs profibrotic function in the heart.

Expression profiling of human idiopathic dilated cardiomyopathy

OBJECTIVE To investigate the global changes accompanying human dilated cardiomyopathy (DCM) we performed a large-scale expression screen using myocardial biopsies from a group of DCM patients with moderate heart failure. By hierarchical clustering and functional annotation of the deregulated genes we examined extensive changes in the cellular and molecular processes associated to DCM. METHODS The expression profiles were obtained using a whole genome covering library (UniGene RZPD1) comprising 30336 cDNA clones and amplified RNA from myocardiac biopsies from 10 DCM patients in comparison to tissue samples from four non-failing, healthy donors. RESULTS By setting stringent selection criteria 364 differentially expressed, sequence-verified non-redundant transcripts were identified with a false discovery rate of <0.001. Numerous genes and ESTs were identified representing previously recognised, as well as novel DCM-associated transcripts. Many of them were found to be upregulated and involved in cardiomyocyte energetics, muscle contraction or signalling. Two hundred and twenty-two deregulated transcripts were functionally annotated and hierarchically clustered providing an insight into the pathophysiology of DCM. Data was validated using the MLP-deficient mouse, in which several differentially expressed transcripts identified in the human DCM biopsies could be confirmed. CONCLUSIONS We report the first genome-wide expression profile analysis using cardiac biopsies from DCM patients at various stages of the disease. Although there is a diversity of links between the cytoskeleton and the initiation of DCM, we speculate that genes implicated in intracellular signalling and in muscle contraction are associated with early stages of the disease. Altogether this study represents the most comprehensive and inclusive molecular portrait of human cardiomyopathy to date.

Beyond the sarcomere: CSRP3 mutations cause hypertrophic cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is a frequent genetic cardiac disease and the most common cause of sudden cardiac death in young individuals. Most of the currently known HCM disease genes encode sarcomeric proteins. Previous studies have shown an association between CSRP3 missense mutations and either dilated cardiomyopathy (DCM) or HCM, but all these studies were unable to provide comprehensive genetic evidence for a causative role of CSRP3 mutations. We used linkage analysis and identified a CSRP3 missense mutation in a large German family affected by HCM. We confirmed CSRP3 as an HCM disease gene. Furthermore, CSRP3 missense mutations segregating with HCM were identified in four other families. We used a newly designed monoclonal antibody to show that muscle LIM protein (MLP), the protein encoded by CSRP3, is mainly a cytosolic component of cardiomyocytes and not tightly anchored to sarcomeric structures. Our functional data from both in vitro and in vivo analyses suggest that at least one of MLPs mutated forms seems to be destabilized in the heart of HCM patients harbouring a CSRP3 missense mutation. We also present evidence for mild skeletal muscle disease in affected persons. Our results support the view that HCM is not exclusively a sarcomeric disease and also suggest that impaired mechano-sensory stress signalling might be involved in the pathogenesis of HCM.

A gain-of-function TBX20 mutation causes congenital atrial septal defects, patent foramen ovale and cardiac valve defects

Background Ostium secundum atrial septal defects (ASDII) account for approximately 10% of all congenital heart defects (CHD), and mutations in cardiac transcription factors, including TBX20, were identified as an underlying cause for ASDII. However, very little is known about disease penetrance in families and functional consequences of inherited TBX20 mutations. Methods The coding region of TBX20 was directly sequenced in 170 ASDII patients. Functional consequences of one novel mutation were investigated by surface plasmon resonance, CD spectropolarymetry, fluorescence spectrophotometry, luciferase assay and chromatin immunoprecipitation. Results We found a novel mutation in a highly conserved residue in the T-box DNA binding domain (I121M) segregating with CHD in a three generation kindred. Four mutation carriers revealed cardiac phenotypes in terms of cribriform ASDII, large patent foramen ovale or cardiac valve defects. Interestingly, tertiary hydrophobic interactions within the mutant TBX20 T-box were significantly altered leading to a more dynamic structure of the protein. Moreover, Tbx20-I121M resulted in a significantly enhanced transcriptional activity, which was further increased in the presence of co-transcription factors GATA4/5 and NKX2-5. Occupancy of DNA binding sites on target genes was also increased. Conclusions We suggest that TBX20-I121M adopts a more fluid tertiary structure leading to enhanced interactions with cofactors and more stable transcriptional complexes on target DNA sequences. Our data, combined with that of others, suggest that human ASDII may be related to loss-of-function as well as gain-of-function TBX20 mutations.

Mutations in GATA4, NKX2.5, CRELD1, and BMP4 Are Infrequently Found in Patients With Congenital Cardiac Septal Defects

Cardiac septal defects constitute the majority ofcongenitalheartdisease(CHD)inhumansandfamilialrecurrenceisreportedtoexceed5%[Burnetal.,1998].Previously, mutations in GATA4 and NKX2.5 havebeendescribedtobepathogenicforostiumsecundumatrial septal defects (ASDII) and ventricular septaldefects(VSD)[Schottetal.,1998;Gargetal.,2003].Incontrast, CRELD1 and BMP4 constitute functionalcandidatesforregulardevelopmentoftheendocardialcushionandmutationsinthesegenescause atrioven-tricular septal defects (AVSD) in animal models andhumans [Jiao et al., 2003; Robinson et al., 2003]. Wehypothesizedthatmutationsin GATA4(NM_002052),NKX2.5 (NM_004387), CRELD1 (NM_015513), andBMP4(NM_001202)canbeidentifiedinalargecohortof patients withcongenital septal defects with a focusonASDII.Weanalyzedthecodingregionofthesefourgenesin205patientswithcongenitalseptaldefectsbysinglestrandedconformationalpolymorphism(SSCP)and sequencing. The patient cohort was assembledout of 110 patients with isolated ASDII. Of these,four subjects (3.6%) mentioned a familial history andformal segregation analysis of pedigrees suggestedan autosomal dominant inheritance. However, familyrelatives were not studied systematically. To thishomogenous ASDII patient cohort we added agroupof95individualswithdifferentcongenitalseptaldefects (60 ASDII, 22 perimembranous VSD, and13 AVSD) and concomitant minor cardiac malforma-tions (Aortic coarctation ¼CoA, persistent ductusarteriosus¼PDA or partial anomalous venousreturn¼PAPVR). These patients were included as asubgroup in a candidate gene approach reportedpreviously [Ozcelik et al., 2006]. All patients wereattending the Department for Congenital Heart Dis-ease, German Heart Institute Berlin (GHIB). Patientswith syndromic appearance and/or limb malforma-tions were excluded from the genetic study andcontrolsubjectswerematchedforethnicity.Thestudyprotocol was approved by the Institutional ReviewBoard of the GHIB and Charite´.A heterozygous c.1750C>T mutation of GATA4,which predicts p.A411V, was identified in a cauca-sian patient with multiperforated ASDII and PAPVR.After exclusion in 600 control chromosomes weconsideredthevarianttobeanovelASDIIassociatedmutation representing the fifth GATA4 mutationidentified in a patient with ASDII. The carrier was a73-year-old female with ASDII and sustained atrial