Nexilin mutations destabilize cardiac Z-disks and lead to dilated cardiomyopathy.

Z-disks, the mechanical integration sites of heart and skeletal muscle cells, link anchorage of myofilaments to force reception and processing. The key molecules that enable the Z-disk to persistently withstand the extreme mechanical forces during muscle contraction have not yet been identified. Here we isolated nexilin (encoded by NEXN) as a novel Z-disk protein. Loss of nexilin in zebrafish led to perturbed Z-disk stability and heart failure. To evaluate the role of nexilin in human heart failure, we performed a genetic association study on individuals with dilated cardiomyopathy and found several mutations in NEXN associated with the disease. Nexilin mutation carriers showed the same cardiac Z-disk pathology as observed in nexilin-deficient zebrafish. Expression in zebrafish of nexilin proteins encoded by NEXN mutant alleles induced Z-disk damage and heart failure, demonstrating a dominant-negative effect and confirming the disease-causing nature of these mutations. Increasing mechanical strain aggravated Z-disk damage in nexilin-deficient skeletal muscle, implying a unique role of nexilin in protecting Z-disks from mechanical trauma.

Repeated replication and a prospective meta-analysis of the association between chromosome 9p21.3 and coronary artery disease.

BACKGROUND: Recently, genome-wide association studies identified variants on chromosome 9p21.3 as affecting the risk of coronary artery disease (CAD). We investigated the association of this locus with CAD in 7 case-control studies and undertook a meta-analysis. METHODS AND RESULTS: A single-nucleotide polymorphism (SNP), rs1333049, representing the 9p21.3 locus, was genotyped in 7 case-control studies involving a total of 4645 patients with myocardial infarction or CAD and 5177 controls. The mode of inheritance was determined. In addition, in 5 of the 7 studies, we genotyped 3 additional SNPs to assess a risk-associated haplotype (ACAC). Finally, a meta-analysis of the present data and previously published samples was conducted. A limited fine mapping of the locus was performed. The risk allele (C) of the lead SNP, rs1333049, was uniformly associated with CAD in each study (P<0.05). In a pooled analysis, the odds ratio per copy of the risk allele was 1.29 (95% confidence interval, 1.22 to 1.37; P=0.0001). Haplotype analysis further suggested that this effect was not homogeneous across the haplotypic background (test for interaction, P=0.0079). An autosomal-additive mode of inheritance best explained the underlying association. The meta-analysis of the rs1333049 SNP in 12,004 cases and 28,949 controls increased the overall level of evidence for association with CAD to P=6.04x10(-10) (odds ratio, 1.24; 95% confidence interval, 1.20 to 1.29). Genotyping of 31 additional SNPs in the region identified several with a highly significant association with CAD, but none had predictive information beyond that of the rs1333049 SNP. CONCLUSIONS: This broad replication provides unprecedented evidence for association between genetic variants at chromosome 9p21.3 and risk of CAD.

An alternative splice variant in Abcc6, the gene causing dystrophic calcification, leads to protein deficiency in C3H/He mice.

Dystrophic cardiac calcification (DCC) is an autosomal recessive trait characterized by calcium phosphate deposits in myocardial tissue. The Abcc6 gene locus was recently found to mediate DCC; however, at the molecular level the causative variants remain to be determined. Examining the sequences of Abcc6 cDNA in DCC-resistant C57BL/6 and DCC-susceptible C3H/He mice, we identified a missense mutation (Cys to Thr at codon 619, rs32756904) at the 3'-border of exon 14 that creates an additional donor splice site (GT). Accordingly, an alternative transcript variant was detected, lacking the last 5 bp of exon 14 (-AGG(C/T)GCTgtga-) in DCC-susceptible C3H/He mice that carry the Thr allele. The 5-bp deletion was found to result in premature termination at codon 684, in turn leading to protein deficiency in DCC-susceptible mouse tissue as well as in cells transfected with Abcc6 cDNA lacking the last 5 bp of exon 14. All mouse strains that were found to carry the Thr allele, including C3H/He, DBA/2J, and 129S1/SvJ, were also found to be positive for DCC. In summary, we identified a splice variant leading to a 5-bp deletion in the Abcc6 transcript that gives rise to protein deficiency both in vivo and in vitro. The fact that all mouse strains that carry the deletion also develop dystrophic calcifications further suggests that the underlying splice variant affects the biological function of MRP6 protein and is a cause of DCC in mice.

Association between arterial pressure and coronary artery calcification.

OBJECTIVES: Coronary artery calcification (CAC) determined by electron beam computed tomography is a predictor of future cardiovascular events. This study investigates conditions affecting CAC severity in patients with coronary artery disease (CAD) undergoing coronary angiography. METHODS: Presence and degree of CAC were assessed angiographically in 877 CAD patients grouped into no visible CAC (n = 333), mild to moderate CAC (n = 321), and severe CAC (n = 223). Regression analyses investigated relationships between CAC and demographic data, cardiovascular risk factors, and coronary anatomy. RESULTS: Prevalences of hypertension and systolic blood pressure (SBP) values were higher in individuals with CAC (moderate CAC: 49.5%, 137.5 +/- 18.6 mmHg; severe CAC: 58.3%, 142.1 +/- 20.4 mmHg) compared to individuals with CAD but no CAC (42.0%, 134.0 +/- 18.4 mmHg; both P < 0.001). Likewise, pulse pressure was significantly elevated with increasing degree of CAC (no CAC, 52.3 +/- 13.6 mmHg vs moderate CAC, 55.7 +/- 14.4 mmHg vs severe CAC, 59.1 +/- 15.4 mmHg; P < 0.001). Further determinants of CAC were age, positive family history for CAC and severity of CAD. No differences in CAC severity were found in relation to body mass index, low-density lipoprotein-cholesterol, diabetes, and smoking habits. In multivariate analysis, CAC was independently related to age, SBP or pulse pressure, respectively, positive family history for CAC, and the severity of CAD. CONCLUSIONS: Of the cardiovascular risk factors, SBP and pulse pressure display the strongest relationship with angiographic detection of CAC. Mechanistic studies need to clarify whether hypertension causes CAC, or whether coronary calcium deposition serves as a marker for a higher degree of vascular calcification and, thus, impaired vascular compliance and higher blood pressure levels.

Ultrafine mapping of Dyscalc1 to an 80-kb chromosomal segment on chromosome 7 in mice susceptible for dystrophic calcification.

In mice, dystrophic cardiovascular calcification (DCC) is controlled by a major locus on proximal mouse chromosome 7 named Dyscalc1. Here we present a strategy that combines in silico analysis, expression analysis, and extensive sequencing for ultrafine mapping of the Dyscalc1 locus. We subjected 15 laboratory mouse strains to freeze-thaw injury of the heart, and association with respective genotypes allowed condensation of the Dyscalc1 locus to 1 Mb. Within this region, 51 known and predicted genes were studied in DCC-susceptible C3H/He and DCC-resistant C57BL/6 mice with respect to mRNA expression in response to injury. Five genes displayed differential expression. Genotyping of seven novel single nucleotide polymorphisms (SNPs) within these genes revealed an 80-Kb region in NZB mice that were found positive for calcification though carrying otherwise alleles from DCC-resistant mice. This microheterogeneity in NZB mice was evolutionary conserved in all DCC-susceptible mouse strains and contains the genes EMP-3, BC013491, and Abcc6 (partially). The flanking SNPs are rs3703247 and NT_039420.5_2757991. mRNA levels of EMP-3 were found to be upregulated in response to injury in both C57BL/6 and C3H/He mice. Sequencing of EMP-3 revealed an SNP leading to an amino acid substitution (p.T153I) that was found in all mouse strains susceptible for DCC but not in resistant strains such as C57BL/6 mice. Thus, the p.T153I changes might affect the biological function of EMP-3 gene product after injury. Using this combined approach, we ultrafine-mapped the Dyscalc1 locus to an 80-Kb region and identified EMP-3 as a new candidate gene for DCC.

Novel missense mutations (p.T596M and p.P1797H) in NOTCH1 in patients with bicuspid aortic valve.

The bicuspid aortic valve (BAV) is the most common congenital cardiac malformation, occurring in 1-2% of the population. In a recent report, mutations in NOTCH1 a signaling and transcriptional regulator have been shown to cause BAV in two families. This study provides data on systematic sequencing in search for novel mutations in NOTCH1 gene in a large sample BAV. For the first time, we report results of a systematic mutation-analysis based on DNA-sequencing of all coding exons and adjacent splice consensus sequences of NOTCH1 gene. Our analyses revealed 57 NOTCH1 sequence variants. Twenty-one variants are located within exons and 36 within intronic or 5'-UTR sequences. Thirty-five variants were described previously as polymorphisms. The remaining 22, however, were neither listed in public SNP databases nor in the literature and were therefore considered novel. Seventeen variants were found only once (MAF = 1%), of these 15 were novel. Two sequence variants led to amino acid substitutions (p.T596M and p.P1797H) and are located in highly conserved regions of the NOTCH1 protein. In addition, these two mutations could not be detected in at least 327 healthy controls by using RFLP-analysis. The functional relevance of the other 13 novel and rare variants could not be proven without further functional examination. In this study, we provide a new evidence that the mutations in the NOTCH1 gene may trigger the underlying mechanism causing the valve calcification, especially in BAV. In conclusion, NOTCH1 gene mutations do not only play a role in familiar BAV, but can also be observed in approximately 4% of sporadic cases.