Methods and Results—We have, consequently, expressed, in COS7 cells, GFP-prelamin A (GFPLaA) fusion constructs incorporating the six variants in pedigrees with nonsegregation (R101P, A318T, R388H, R399C, S437Hfsx1, and R654X), the four variants in pedigrees with unknown segregation [R89L, R166P (in 2 families), I210S, R471H], and three additional missense variants (R190Q, E203K, L215P) that segregated with disease. Confocal immunofluorescence microscopy was used to characterize GFP-lamin A localization and nuclear morphology. Abnormal phenotypes were observed for 10/13 (77%) variants (R89L, R101P, R166P, R190Q, E203K, I210S, L215P, R388H, S437Hfsx1, R654X), including 4/6 demonstrating nonsegregation and 3/4 with uncertain segregation. All seven variants affecting coil 1B, and the lamin A-only mutation, R654X, exhibited membrane-bound GFP-lamin A aggregates and nuclear shape abnormalities. Unexpectedly, R388H largely restricted GFP-lamin A to the cytoplasm. Equally unexpected were unique streaked aggregates with S437Hfsx1, and giant aggregates with both S437Hfsx1 and R654X.

Conclusions—This work expands the recognized spectrum of lamin A localization abnormalities in DCM. It also provides evidence supporting pathogenicity of 10 of 13 tested LMNA variants, including some with uncertain or nonsegregation.

Methods and Results—Mitochondria, isolated from the left ventricle in a canine model of dyssynchronous (DHF) or resynchronized (CRT) heart failure, were analyzed by a classical, gel-based, proteomic approach. Two-dimensional gel electrophoresis (2DE) revealed that 31 mitochondrial proteins where changed when controlling the false discovery rate at 30%. Key enzymes in anaplerotic pathways, such as pyruvate carboxylation and branched chain amino acid oxidation , were increased. These concerted changes, along with others, suggested that CRT may increase the pool of Krebs cycle intermediates, and fuel oxidative phosphorylation. Nearly 50% of observed changes pertained to subunits of the respiratory chain. ATP synthase beta subunit of complex V was less degraded and its phosphorylation modulated by CRT, associated with increased formation (2-fold, p=0.004) and specific activity (+20%, p=0.05) of the mature complex. The importance of these modifications was supported by coordinated changes in mitochondrial chaperones and proteases. CRT increased the mitochondrial respiratory control index with tightened coupling when isolated mitochondria were re-exposed to substrates for both complexes I (glutamate and malate) or II (succinate), an effect likely related to ATP synthase subunit modifications and complex quantity and activity.

Conclusions—CRT potently impacts both the mitochondrial proteome, and performance associated with improved cardiac function.

Methods and Results—After permanent occlusion of the distal middle cerebral artery (MCA), infarct volume was determined for 16 inbred strains of mice, chromosome substitution strains, and for two intercross cohorts, F2(B6xBALB/c) and F2(B6xSWR/J). Genome-wide linkage analysis was performed for infarct volume as a quantitative trait. Infarct volume varied up to 30-fold between strains, with heritability estimated at 0.88. Overall, three QTL were identified that modulate infarct volume, with a major locus (Civq1) on chromosome 7 accounting for more than 50% of the variation, with a combined LOD score of 21.7. Interval-specific SNP haplotype analysis for Civq1 results in 12 candidate genes.

Conclusions—The extent of ischemic tissue damage after distal MCA occlusion in inbred strains of mice is modulated by genetic variation mapping to at least 3 different loci. A single locus on chromosome 7 determines the majority of the observed variation in the trait. This locus appears to be identical to LSq1, a locus conferring limb salvage and reperfusion in a mouse model of hindlimb ischemia. The identification of the genes underlying these loci may uncover novel genetic and physiological pathways that modulate cerebral infarction, and provide new targets for therapeutic intervention in ischemic stroke, and possibly other ischemic diseases.

Methods and Results—We now report evidence of shared genetic basis for disruption of the elastic lamina (medial disruption) and medial calcification in an F₂ mouse intercross between C57BL/6J and C3H/HeJ on a hyperlipidemic apolipoprotein E (ApoE-/-) null background. We identified three quantitative trait loci (QTLs), on chromosomes 6, 13 and 18, that are common to both traits, and two additional QTLs for medial calcification on chromosomes 3 and 7. Medial disruption, including severe disruptions leading to aneurysm formation, and medial calcification were highly correlated and occurred concomitantly in the cross. The chromosome 18 locus showed a striking male sex-specificity for both traits. To identify candidate genes, we integrated data from microarray analysis, genetic segregation, and clinical traits. The chromosome 7 locus contains the Abcc6 gene, known to mediate myocardial calcification. Using transgenic complementation, we show that Abcc6 also contributes to aortic medial calcification.

Conclusions—Our data indicate that calcification, though possibly contributory, does not always lead to medial disruption, and that in addition to aneurysm formation, medial disruption may be the precursor to calcification.

Methods and Results—We analyzed the up- and downregulated gene sets with Whole Genome rVISTA to determine the transcription factor binding sites (TFBSs) overrepresented in the 5 kb promoter regions of the 3,274 genes. The downregulated gene set yielded 144 TFBSs that were overrepresented in the subset when compared to the entire genome. In contrast, the upregulated gene set yielded only 13 distinct overrepresented TFBSs. Interestingly, as classified by TRANSFAC®, 8 of the 13 transcription factors (TFs) binding to these regions belong to the ETS family. Additionally, NFKB and its subunits p50 and p65 showed enrichment. Immunohistochemical analyses in 10 of the TFs from the upregulated analysis showed 9 to be present in AAA tissue. Based on Gene Ontology analysis of biological process categories of the upregulated target genes of enriched TFs, 10 TFs had enrichment in immune system process among their target genes.

Conclusions—Our genome-wide analysis provides further evidence of ETS and NFKB involvement in AAA. Additionally, our results provide novel insight for future studies aiming to dissect the pathogenesis of AAA and have uncovered potential therapeutic targets for AAA prevention.

Methods and Results—We genotyped 20 polymorphisms in 360 male twins (mean age: 54) from the Vietnam Era Twin Registry. Current depressive symptoms were measured with the Beck Depression Inventory-II (BDI-II). IL-6 was assessed using a commercially available ELISA kit. Genotype associations were analyzed using generalized estimating equations. To study how SLC6A4 genetic vulnerability influences the relationship between depressive symptoms and IL-6, bivariate models were constructed using structural equation modeling. Of the 20 polymorphisms examined, the effective number of independent tests was 6 and the threshold of significance after Bonferroni correction was 0.008. There were 6 SNPs significantly associated with BDI (P≤0.008), including rs8071667, rs2020936, rs25528, rs6354, rs11080122 and rs8076005, and 1 SNP borderline associated (rs12150214, P=0.017). Of these 7 SNPs, 3 were also significantly associated with IL-6 (P<0.008), including rs25528, rs6354 and rs8076005, and the other 4 were borderline associated (P=0.009~0.025). The subjects with one copy of the minor allele of these 7 SNPs had higher BDI scores and IL-6 levels. Further bivariate modeling revealed that approximately 10% of the correlation between BDI and IL-6 could be explained by the SLC6A4 gene.

Conclusions—Genetic vulnerability involving the SLC6A4 gene is significantly associated with both increased depressive symptoms and elevated IL-6 plasma levels. Common pathophysiological processes may link depression and inflammation, and implicate the serotonin pathway in neural-immune interactions.

Methods and Results—Detailed cardiac information was collected for BMD and XLDCM patients with defined dystrophin gene deletion mutations. Patients were grouped based on the dystrophin protein domain affected by the deletion. Deletions affecting the amino-terminal domain are associated with early-onset DCM (mid-20's), while deletions removing part of the rod domain and hinge 3 have a later onset DCM (mid-40's). Further, we modeled the effects of the most common mutations occurring in the rod domain on the overall structure of the dystrophin protein. By combining genetic and protein information, this analysis revealed a strong correlation between specific protein structural modifications and DCM age of onset.

Conclusions—We identified specific regions of the dystrophin gene that when mutated predispose BMD patients to early-onset DCM. In addition, we propose that some mutations lead to early-onset DCM via specific alterations in protein folding. These findings have potential implications for early intervention in the cardiac care of BMD patients and for therapeutic approaches that target the heart in dystrophinopathies.

Methods and Results—Families were included if at least 5 family members were carriers of the SCN5A mutation identified in the proband. Thirteen large families composed of 115 mutation carriers were studied. The signature type I ECG was present in 54 mutation carriers (BrS-ECG+) (47%). In 5 families, we found 8 individuals affected by BrS, but with a negative genotype (mutation-negative BrS-ECG+). Among these 8 mutation-negative BrS-ECG+ individuals, 3, belonging to 3 different families, had a spontaneous type I ECG, while 5 had a type I ECG only after administration of sodium channel blockers. One of these 8 individuals had also experienced syncope. Mutation carriers had, on average, longer PR and QRS intervals than non-carriers, demonstrating that these mutations exerted functional effects.

Conclusions—Our results suggest that SCN5A mutations are not directly causal to the occurrence of a BrS-ECG+ and that genetic background may play a powerful role in the pathophysiology of BrS. These findings add further complexity to concepts regarding the causes of BrS, and are consistent with the emerging notion that the pathophysiology of BrS includes various elements beyond mutant sodium channels.

Methods and Results—Genetic inactivation of NaPi2a activity from klotho^-/-mice reversed the severe hyperphosphatemia to mild hypophosphatemia or normophosphatemia. Importantly, despite significantly higher serum calcium and 1,25-dihydroxyvitamin D levels in klotho^-/-/NaPi2a^-/- mice, the vascular and soft tissue calcifications were reduced. Extensive soft tissue anomalies and cardiovascular calcification were consistently noted in klotho^-/-mice by 6 weeks of age; however, these vascular and soft tissue abnormalities were absent even in 12-week-old double knockout mice. Klotho^-/-/NaPi2a^-/- mice also regained body weight and did not develop the generalized tissue atrophy often noted in klotho^-/-single knockout mice.

Conclusion—Our in vivo genetic manipulation studies have provided compelling evidence for a pathologic role of increased NaPi2a activities in regulating abnormal mineral ion metabolism and soft tissue anomalies in klotho^-/- mice. Notably, our results suggest that serum phosphate levels are the important in vivo determinant of calcification, and that lowering serum phosphate levels can reduce or eliminate soft tissue and vascular calcification, even in presence of extremely high serum calcium and 1,25-dihydroxyvitamin D levels. These in vivo observations have significant clinical importance and therapeutic implications for chronic kidney disease patients with cardiovascular calcification.

Methods and Results—These associations were investigated in 712 African-American and 705 Hispanic participants from the MESA candidate gene sub-study. Individual ancestry was estimated from 199 genetic markers using STRUCTURE. Associations of ancestry and coronary artery calcium (CAC) and common and internal carotid intima media thickness (cIMT) were evaluated using log-binomial and linear regression models. Splines indicated linear associations of ancestry with subclinical CVD measures in African-Americans, but presence of threshold effects in Hispanics. Among African Americans, each standard deviation (SD) increase in European ancestry was associated with an 8% (95% CI (1.02, 1.15), p=0.01) greater CAC prevalence. Each SD increase in European ancestry was also associated with a 2% (95% CI (-3.4%, -0.5%), p=0.008) lower common cIMT in African Americans. Among Hispanics, the highest tertile of European ancestry was associated with a 34% greater CAC prevalence, p=0.02 as compared to lowest tertile.

Conclusions—The linear association of ancestry and subclinical CVD suggests that genetic effects may be important in determining CAC and cIMT among African-Americans. Our results also suggest that CAC and common cIMT may be important phenotypes for further study with admixture mapping.

Methods and Results—We validated the contribution of 42 SNPs (33 identified in GWAS and 9 previously reported SNPs not included in GWAS chips) and of longitudinally measured key non-genetic variables (ART, underlying conditions, sex, age, ethnicity and HIV disease parameters) to dyslipidemia in 745 HIV-infected study participants (n=34565 lipid measurements; median follow-up, 7.6 years). The relative impact of SNPs and ART to lipid variation in the study population, and their cumulative influence on sustained dyslipidemia at the level of the individual were calculated. SNPs were associated with lipid changes consistent with GWAS estimates. SNPs explained up to 7.6% (non-HDL cholesterol), 6.2% (HDL cholesterol), and 6.8% (triglycerides) of lipid variation; ART explained 3.9% (non-HDL cholesterol), 1.5% (HDL cholesterol), and 6.2% (triglycerides). An individual with the most dyslipidemic antiretroviral and genetic background had an approximately 3-5 fold increased risk of sustained dyslipidemia, compared to an individual with the least dyslipidemic therapy and genetic background.

Conclusions—In the HIV-infected population treated with ART, the weight of the contribution of common SNPs and ART to dyslipidemia was similar. When selecting an ART regimen, genetic information should be considered in addition to the dyslipidemic effects of ART agents.

Methods and Results—We identified 80 mutation-carriers in 15 index families segregating the Y111C-KCNQ1 mutation during a national inventory of mutations causing the long QT syndrome. Twenty-four mutation-carriers <40 years of age experienced syncope (30%). One mutation-carrier had an aborted cardiac arrest (1.25%). No case of sudden cardiac death was reported during a mean non-medicated follow-up of 25±20 years. This corresponds to a low incidence rate of life-threatening cardiac events (0.05%/yr vs. 0.3%/yr, p=0.025). In 8 Y111C families connected by a common ancestor, the natural history of the mutation was assessed investigating the survival over the age of 40 for 107 non-medicated ascertained mutation-carriers (n=24) and family members (n=83) born between 1873 and 1968. In total, 4 deaths in individuals below the age of 40 were noted: one case of non-cardiac death and 3 infant deaths, between 1873 and 1915.

Conclusions—The dominant-negative Y111C-KCNQ1 mutation, associated with a severe phenotype in vitro, presents with a low incidence of life-threatening cardiac events in a Swedish population. This finding of discrepancy emphasizes the importance of clinical observations in the risk stratification of the long QT syndrome.

Methods and Results—Five tagging single nucleotide polymorphisms (tagSNPs) (rs2248690, rs2070633, rs2070635, rs4917, rs6787344) capturing the common genetic variation of the fetuin-A coding gene alpha2- Heremans-Schmid glycoprotein (AHSG) were genotyped in a case-cohort comprising 214 MI cases, 154 IS cases and 2,152 persons who remained free of cardiovascular disease events of the EPIC-Potsdam study. One SNP (rs6787344) was discarded due to Hardy-Weinberg disequilibrium. All AHSG tagSNPs were associated with fetuin-A plasma levels (P-values<0.0001). AHSG rs4917 C>T showed the strongest association explaining 21.2% of the phenotypic variance independent of potential confounding factors (+35.5 µg/mL increase per C-allele, P=2x10^-121). Furthermore, the rs4917 C-allele showed a significant association with MI (adjusted hazard rate ratio (RR) 1.34, 95% CI 1.05-1.70, P=0.02). Based on this association, the expected RR for MI corresponding to 1 standard deviation in fetuin-A was 1.54 and, thus, strikingly matches the previously observed association between fetuin-A plasma levels and MI risk (RR 1.59).

Conclusions—The present data provide evidence for the causal nature of the recently reported association between fetuin-A plasma levels and MI risk, thereby suggesting an involvement of fetuin-A in the pathogenesis of cardiovascular disease.

Methods and Results—We genotyped 44 non-synonymous single nucleotide polymorphisms (nsSNPs) from 11 genes involved in homocysteine metabolism and found only 14 to be polymorphic. These 14 nsSNPs were genotyped in 546 individuals recruited from a tertiary care centre in New Delhi, India and it was found that choline dehydrogenase (CHDH A119C) and methylenetetrahydrofolate reductase (MTHFR C677T) were significantly associated with plasma total homocysteine levels (p=0.009 and p=0.001 respectively). These 2 SNPs were further genotyped in 330 individuals recruited from the same centre and the association remained significant even after increasing the sample size. Further, we found the possibility of a significant interaction between vegetarian diet and the two polymorphisms that could explain the variation of homocysteine levels. We also genotyped all the polymorphic nsSNPs in apparently healthy individuals recruited from 24 different sub-populations (based on their linguistic lineage) spread across the country to determine their basal frequencies. The frequencies of these SNPs varied significantly between linguistic groups.

Conclusion—Vegetarian diet along with CHDH A119C and MTHFR C677T play an important role in modulating the homocysteine levels in Indian population.