Christian Geier

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.

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.

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.

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

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. Hum Mutat 29(6), 879–885, 2008.

A missense variant in desmoglein-2 predisposes to dilated cardiomyopathy.

Familial Dilated Cardiomyopathy (FDCM) is caused by mutations in genes encoding myocardial force transduction proteins. Desmoglein-2 (DSG2) and Desmocollin-2 (DSC2) provide cellular adhesion and force transduction by cell-to-cell anchorage. To test whether perturbations of DSG2 or DSC2 exhibit a pathogenic impact on DCM pathogenesis, we sequenced both genes in 73 patients with FDCM and assessed prevalence of missense variations in matched control cohorts. We detected two missense variations in DSG2 (V55M and V919G) which were absent in 360 control alleles. Surprisingly, both variants were previously reported in patients with arrhythmogenic right ventricular cardiomyopathy. Yet, in the present study only the DSG2-V55M variant showed segregation with DCM in a family pedigree. Subsequent, analysis of 538 patients with idiopathic DCM and 617 consecutive control individuals resulted in identification of thirteen DSG2-V55M carriers with DCM, whereas only three control subjects harbored the variant. DSG2 immunostaining revealed pale structures of the intercalated disc in myocardium of one unique homozygous DSG2-V55M carrier. Furthermore, myocardial desmosomal structures were significantly shortened when compared to DCM myocardium negative for DSG2-V55M. Thus, our study identified the DSG2-V55M polymorphism as a novel risk variant for DCM associated with shortened desmosomes of the cardiac intercalated disc.

Contractures and hypertrophic cardiomyopathy in a novel FHL1 mutation

We investigated a large German family (n = 37) with male members who had contractures, rigid spine syndrome, and hypertrophic cardiomyopathy. Muscle weakness or atrophy was not prominent in affected individuals. Muscle biopsy disclosed a myopathic pattern with cytoplasmic bodies. We used microsatellite markers and found linkage to a locus at Xq26‐28, a region harboring the FHL1 gene. We sequenced FHL1 and identified a new missense mutation within the third LIM domain that replaces a highly conserved cysteine by an arginine (c.625T>C; p.C209R). Our finding expands the phenotypic spectrum of the recently identified FHL1‐associated myopathies and widens the differential diagnosis of Emery–Dreifuss–like syndromes. ANN NEUROL 2010;67:136–140

Genetic deletion of arginine 14 in phospholamban causes dilated cardiomyopathy with attenuated electrocardiographic R amplitudes

BACKGROUND Familial dilated cardiomyopathy is a highly heterogeneous genetic disease. Thus, identification of disease-causing mutations is a challenging and time-consuming task. Genotype-phenotype associations may alleviate identification of the underlying mutation. OBJECTIVE The purpose of this study was to investigate cardiac phenotypes within a family harboring a familial dilated cardiomyopathy-related mutation in the gene encoding phospholamban. METHODS Complete genetic and clinical analyses were performed in a family with familial dilated cardiomyopathy due to the PLN-R14Del mutation. Family relatives were studied by ECG, Holter ECG, echocardiography, ECG body surface potential mapping, and cardiac magnetic resonance imaging. RESULTS A candidate gene approach resulted in identification of a heterozygous deletion of arginine 14 in the gene encoding phospholamban (PLN-R14Del) segregating with dilated cardiomyopathy in the family pedigree. Mutation carriers suffered from familial dilated cardiomyopathy associated with cardiac death between the ages of 26 and 50 years. Interestingly, all adult mutation carriers revealed strikingly attenuated R amplitudes on standard ECG, regardless of the absence or presence of echocardiographic abnormalities. Gadolinium-enhanced cardiac magnetic resonance imaging showed late enhancement in PLN-R14Del carriers with preserved ejection fraction. Late enhancement was regionally related to areas of most pronounced R-amplitude attenuation as assessed by body surface potential mapping. CONCLUSION Attenuated R amplitudes were identified as an early ECG phenotype in a family with familial dilated cardiomyopathy due to the PLN-R14Del mutation. All adults harboring PLN-R14Del had attenuated R waves irrespective of echocardiographic abnormalities. The study findings suggest a mutation-related remodeling process preceding ventricular dysfunction.

Genetic and phenotypic analysis of dilated cardiomyopathy with conduction system disease: Demand for strategies in the management of presymptomatic lamin A/C mutant carriers

One‐third of cases of dilated cardiomyopathy (DCM) is of familial aetiology. Several genes have been reported to cause the autosomal dominant form of DCM.

Back to square one: what do we know about the functions of Muscle LIM Protein in the heart?

Muscle LIM Protein (MLP) is small, just 198 amino acid long protein, which is specifically expressed in slow skeletal muscle and cardiac tissues. This article will focus on the cardiac functions of MLP: the current knowledge about localisation data, binding partners and animal models for the protein will be summarised, and the role of MLP in maintaining a healthy heart be discussed. This review will furthermore attempt to identify gaps in our knowledge—and hence future research potential—with a special focus on MLP’s role in cardiac mechano-signalling.