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Background: Idiopathic ventricular tachycardia (VT) occurs in structurally normal hearts and accounts for a significant number of all types of VT. The genome-wide association study is the most effective strategy for identifying novel genetic variants for common diseases. However, no genome-wide association study has been reported for idiopathic VT. Methods: We conducted the first genome-wide association study for idiopathic VT in the Chinese Han population using a discovery population with 246 cases and 648 controls and a replication population with 222 cases and >4072 controls. Candidate VT genes were functionally characterized in zebrafish. Real-time RT-PCR analysis was used to determine the effects of candidate genes on expression of ion channels and regulators. Patch-clamping was used to record L-type calcium current from neonatal rat cardiomyocytes with overexpression of candidate genes. Results: We identified 4 significant loci represented by rs78960694 (minor allele frequency [MAF]=5.02% in cases and 1.84% in controls; P =4.30×10 − 12, odds ratio [OR]=3.91) and rs2229095 (MAF=3.25% in cases and 1.63% in controls; P =1.02×10 − 7, OR=3.44) near and in CCR7 , respectively, rs68126098 in NELL1 (MAF=40.98% in cases and 32.07% in controls; P= 2.40×10 − 8, OR=1.53), rs2390325 between PKN2 and LMO4 (MAF=21.19% in cases and 15.12% in controls; P =1.92×10 − 7, OR=1.62), and rs270065 in CSMD1 (MAF=33.63% in cases and 40.25% in controls; P =9.51×10 − 7, OR=0.69). Note that the associations of idiopathic VT for CCR7 variant rs78960694 and NELL1 variant rs68126098 reach genome-wide significance ( P − 8). Overexpression of either PKN2 or CCR7 increased the heart rate in zebrafish, and enhanced expression of CACNA1C, RYR2 , or NOS1AP in zebrafish embryos, HEK293, and AC16 cardiomyocytes. Overexpression of either PKN2 or CCR7 significantly increased L-type Ca2+ current density. Conclusions: The first genome-wide association study identifies 4 novel loci and 2 risk genes ( PKN2 and CCR7 ) for idiopathic VT. These findings identify new molecular determinants for cardiac calcium homeostasis and rhythm maintenance and provide novel targets for diagnosis and treatment for idiopathic VT.

The sodium voltage-gated channel beta subunit 3 (

Liver fibrogenesis is orchestrated by the paracrine signaling interaction between several resident cell types regulating the activation of hepatic stellate cells (HSCs). However, the molecular mechanisms underlying paracrine regulation are largely unknown. The aim of this study is to elucidate the role of Ninjurin2 in the crosstalk between hepatocytes and HSCs and better understand the implications of Ninjurin2 in liver fibrosis.Ninj2 knockout mice (Ninj2We found that hepatic Ninjurin2 expression was significantly increased in fibrotic human liver and MCD diet-induced liver injury mouse models. In the mouse model, hepatocyte-specific overexpression of Ninj2 exacerbates MCD-induced liver fibrosis, while global Ninj2 knockout reverses the phenotype. To mimic hepatocyte-HSC crosstalk during liver fibrosis, we used co-culture systems containing hepatocytes and HSCs and determined that Ninjurin2 overexpression in hepatocytes directly activates HSCs in vitro. Mechanistically, Ninjurin2 directly interacts with insulin-like growth factor 1 receptor (IGF1R) and increases the hepatocyte secretion of the fibrogenic cytokine, platelet-derived growth factor-BB (PDGF-BB) through IGF1R-PI3K-AKT-EGR1 cascade. Inhibition of PDGFRB signaling in HSCs can abolish the profibrogenic effect of Ninjurin2. In addition, we demonstrated that a specific inhibitory Ninjurin2 peptide containing an N-terminal adhesion motif mitigates liver fibrosis and improves hepatic function in the mouse models by negatively regulating the sensitivity of IGF1R to IGF1 in hepatocytes.Hepatic Ninjurin2 plays a key role in liver fibrosis through paracrine regulation of PDGF-BB/PDGFRB signaling in HSCs, and the results suggesting Ninjurin2 may be a potential therapeutic target.

Genetic variants in ninjurin-2 (NINJ2; nerve injury-induced protein 2) confer risk of ischemic strokes and coronary artery disease as well as endothelial activation and inflammation. However, little is known about NINJ2's in vivo functions and underlying mechanisms.The phenotypes of NINJ2 knockout mice were analyzed, and mechanisms of NINJ2 that regulate body weight, insulin resistance, and glucose homeostasis and lipogenesis were investigated in vivo and in vitro.This study found that mice lacking NINJ2 showed impaired adipogenesis, increased insulin resistance, and abnormal glucose homeostasis, all of which are risk factors for strokes and coronary artery disease. Mechanistically, NINJ2 directly interacts with insulin receptor/insulin-like growth factor 1 receptor (INSR/IGF1R), and NINJ2 knockdown can block insulin-induced mitotic clonal expansion during preadipocyte differentiation by inhibiting protein kinase B/extracellular signal-regulated kinase (AKT/ERK) signaling and by decreasing the expression of key adipocyte transcriptional regulators CCAAT/enhancer-binding protein β (C/EBP-β), C/EBP-α, and peroxisome proliferator-activated receptor γ (PPAR-γ). Furthermore, the interaction between NINJ2 and INSR/IGF1R is needed for maintaining insulin sensitivity in adipocytes and muscle via AKT and glucose transporter type 4. Notably, adenovirus-mediated NINJ2 overexpression can ameliorate diet-induced insulin resistance in mice.In conclusion, these findings reveal NINJ2 as an important new facilitator of insulin receptors, and the authors propose a unique regulatory mechanism between insulin signaling, adipogenesis, and insulin resistance.

Brugada syndrome (BrS) is a fatal arrhythmia that causes an estimated 4% of all sudden death in high-incidence areas. SCN5A encodes cardiac sodium channel Na V 1.5 and causes 25 to 30% of BrS cases. Here, we report generation of a knock-in (KI) mouse model of BrS ( Scn5a G1746R/+ ). Heterozygous KI mice recapitulated some of the clinical features of BrS, including an ST segment abnormality (a prominent J wave) on electrocardiograms and development of spontaneous ventricular tachyarrhythmias (VTs), seizures, and sudden death. VTs were caused by shortened cardiac action potential duration and late phase 3 early afterdepolarizations associated with reduced sodium current density ( I Na ) and increased Kcnd3 and Cacna1c expression. We developed a gene therapy using adeno-associated virus serotype 9 (AAV9) vector-mediated MOG1 delivery for up-regulation of MOG1, a chaperone that binds to Na V 1.5 and traffics it to the cell surface. MOG1 was chosen for gene therapy because the large size of the SCN5A coding sequence (6048 base pairs) exceeds the packaging capacity of AAV vectors. AAV9- MOG1 gene therapy increased cell surface expression of Na V 1.5 and ventricular I Na , reversed up-regulation of Kcnd3 and Cacna1c expression, normalized cardiac action potential abnormalities, abolished J waves, and blocked VT in Scn5a G1746R/+ mice. Gene therapy also rescued the phenotypes of cardiac arrhythmias and contractile dysfunction in heterozygous humanized KI mice with SCN5A mutation p.D1275N. Using a small chaperone protein may have broad implications for targeting disease-causing genes exceeding the size capacity of AAV vectors.

AGGF1 is an angiogenic factor with G-Patch and FHA domains 1 described by our group. Gain-of-function mutations in AGGF1 cause Klippel-Trenaunay syndrome, whereas somatic loss-of-function mutations cause cancer. Paraspeckles are small membraneless subnuclear structures with a diameter of 0.5-1 μm, and composed of lncRNA NEAT1 as the scaffold and three core RNA-binding proteins NONO, PSPC1, and PSF. Here, we show that AGGF1 is a key regulatory and structural component of paraspeckles that induces paraspeckle formation, forms an outside rim of paraspeckles, wraps around the NONO/PSF/PSPC1/NEAT1 core, and regulates the size and number of paraspeckles. AGGF1-paraspeckles are larger (1 μm) than conventional paraspeckles. RNA-FISH in combination with immunostaining shows that AGGF1, NONO, and NEAT1_2 co-localize in 20.58% of NEAT1_2-positive paraspeckles. Mechanistically, AGGF1 interacts with NONO, PSF, and HNRNPK, and upregulates NEAT1_2, a longer, 23 kb NEAT1 transcript with a key role in regulation of paraspeckle size and number. RNA-immunoprecipitation shows that AGGF1 interacts with NEAT1, which may be another possible mechanism underlying the formation of AGGF1-paraspeckles. NEAT1_2 knockdown reduces the number and size of AGGF1-paraspeckles. Functionally, AGGF1 regulates alternative RNA splicing as it decreases the exon skipping/inclusion ratio in a CD44 model. AGGF1 is also localized in some nuclear foci without NEAT1 or NONO, suggesting that AGGF1 is an important liquid-liquid phase separation (LLPS) driver for other types of AGGF1-positive nuclear condensates (referred to as AGGF1-bodies). Our results identify a special type of AGGF1-coated paraspeckles and provide important insights into the formation, structure, and function of paraspeckles.

AIM Loss-of-function KCNMA1 variants cause Liang-Wang syndrome (MIM #618729), a newly-identified multiple malformation syndrome with a broad spectrum of developmental and neurological phenotypes. However, the full spectrum of clinical features and underlying pathogenic mechanisms need full elucidation. METHODS Exome sequencing was used to identify pathogenic variants. Patch-clamp recordings were performed to access the effects of KCNMA1 variants on BK channels. Total and membrane protein expression levels of BK channels were characterized using Western blotting. RESULTS We report identification and functional characterization of two new de novo loss-of-function KCNMA1 variants p.(A172T) and p.(A314T) with characteristics of Liang-Wang syndrome. Variant p.(A172T) is associated with developmental delay, cognitive impairment, and ataxia. Mechanistically, p.(A172T) abolishes BK potassium current, inhibits Mg2+ -dependent gating, but shifts conductance-voltage (G-V) curves to more positive potentials when complexed with WT channels. Variant p.(A314T) is associated with developmental delay, intellectual disability, cognitive impairment, mild ataxia, and generalized epilepsy, suppresses BK current amplitude, and shifts G-V curves to more positive potentials when expressed with WT channels. In addition, two new patients with previously reported gain-of-function variants p.(N536H) and p.(N995S) are found to show epilepsy and paroxysmal dyskinesia as reported previously, but also exhibit additional symptoms of cognitive impairment, and dysmorphic features. Furthermore, variants p.(A314T) and p.(N536H) reduced total and membrane levels of BK proteins. CONCLUSION Our findings identified two new loss-of-function mutations of KCNMA1 associated with Liang-Wang syndrome, expanded the spectrum of clinical features associated with gain-of-function KCNMA1 variants, and emphasized the overlapping features shared by gain-of-function and loss-of-function mutations.

Angiogenic factor AGGF1 (AngioGenic factor with G-patch and FHA (Forkhead-Associated) domain 1) blocks neointimal formation (formation of a new or thickened layer of arterial intima) after vascular injury by regulating phenotypic switching of vascular smooth muscle cells (VSMCs). However, the AGGF1 receptor on VSMCs and the underlying molecular mechanisms of its action are unknown. In this study, we used functional analysis of serial AGGF1 deletions to reveal the critical AGGF1 domain involved in VSMC phenotypic switching. This domain was required for VSMC phenotypic switching, proliferation, cell cycle regulation, and migration, as well as the regulation of cell cycle inhibitors cyclin D, p27, and p21. This domain also contains an RDDAPAS motif via which AGGF1 interacts with integrin α7 (ITGA7), but not α8. In addition, we show that AGGF1 enhanced the expression of contractile markers MYH11, α-SMA, and SM22 and inhibited MEK1/2, ERK1/2, and ELK phosphorylation in VSMCs, and that these effects were inhibited by knockdown of ITGA7, but not by knockdown of ITGA8. In vivo, deletion of the VSMC phenotypic switching domain in mice with vascular injury inhibited the functions of AGGF1 in upregulating α-SMA and SM22, inhibiting MEK1/2, ERK1/2, and ELK phosphorylation, in VSMC proliferation, and in blocking neointimal formation. Finally, we show the inhibitory effect of AGGF1 on neointimal formation was blocked by lentivirus-delivered shRNA targeting ITGA7. Our data demonstrate that AGGF1 interacts with its receptor integrin α7 on VSMCs, and this interaction is required for AGGF1 signaling in VSMCs and for attenuation of neointimal formation after vascular injury.

Mutations in cardiac sodium channel NaThe purpose of this study was to identify structural elements involved in MOG1-NaSystematic analyses of large deletions, microdeletions, and point mutations, and glutathione S-transferases pull-down, co-immunoprecipitation, cell surface protein quantification, and patch-clamping of ILarge deletion analysis defined the MOG1-NaOur findings define the MOG1-Na

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Atrial fibrillation (AF) affects30 million individuals worldwide. However, no genetic mutation from human patients with AF has been linked to inflammation. Here, we show that AF-associated human variant p.Ile138Thr in natriuretic peptide A (

Vascular hyperpermeability caused by distorted endothelial cell-cell junctions is associated with the no-reflow phenomenon after opening of the occluded vessels in patients with coronary artery disease (CAD), the leading cause of death worldwide. Coronary no-reflow is observed in ∼30% of CAD patients after percutaneous coronary stenting and is associated with a worse prognosis at follow-up and a higher incidence of death. However, limited tools are available to control vascular hyperpermeability and no-reflow. Losartan, an angiotensin II (Ang II) receptor blocker acting on the Ang II type-1 receptor (AT1R) subtype, is a prescription drug for treating hypertension. Here we show that in a murine model of ischemia and reperfusion (I/R), losartan blocked vascular hyperpermeability and decreased infarct size, hemorrhages, edema, and inflammation. Mechanistically, losartan-mediated inhibition of vascular hyperpermeability is mediated by the inhibition of phosphorylation of Src and vascular endothelial cadherin (VE-cadherin), which increases VEGF receptor 2 (VEGFR2)-Src-VE-cadherin complex formation, resulting in increased cell surface VE-cadherin and inhibition of vascular hyperpermeability. On the other hand, hypoxia and reoxygenation increased the phosphorylation levels of Src and VE-cadherin and reduced the formation of the VEGFR2-Src-VE-cadherin complex, which led to reduced cell surface VE-cadherin and increased vascular hyperpermeability; all were inhibited by losartan. These data suggest that losartan may be used for blocking vascular hyperpermeability associated with I/R.-Li, Y., Yao, Y., Li, J., Chen, Q., Zhang, L., Wang, Q. K. Losartan protects against myocardial ischemia and reperfusion injury

The thymic stromal lymphopoietin (TSLP)/TSLP receptor (TSLPR) axis is involved in multiple inflammatory immune diseases, including coronary artery disease (CAD). To explore the causal relationship between this axis and CAD, we performed a three-stage case-control association analysis with 3,628 CAD cases and 3,776 controls using common variants in the genes TSLP, interleukin 7 receptor (IL7R), and TSLPR. Three common variants in the TSLP/TSLPR axis were significantly associated with CAD in a Chinese Han population [rs3806933T in TSLP, Padj = 4.35 × 10−5, odds ratio (OR) = 1.18; rs6897932T in IL7R, Padj = 1.13 × 10−7, OR = 1.31; g.19646A>GA in TSLPR, Padj = 2.04 × 10−6, OR = 1.20]. Reporter gene analysis demonstrated that rs3806933 and rs6897932 could influence TSLP and IL7R expression, respectively. Furthermore, the “T” allele of rs3806933 might increase plasma TSLP levels (R2 = 0.175, P

Stroke is one of the most common causes of death worldwide. Genetic risk factors have been found to play important roles in the pathology of ischemic stroke. In a previous genome-wide association study, a functional variant (rs9943582, –154G/A) in the 5’ flanking region of the apelin receptor gene (APLNR) was shown to be significantly associated with stroke in the Japanese population. However, the association required validation in other ethnicities. To validate the genetic relationship between APLNR and ischemic stroke in the Chinese Han population, we genotyped rs9943582 in a case–control population containing 1,158 ischemic stroke patients and 1,265 common controls enrolled from the GeneID database, and performed a genetic association study. We detected no allelic or genotypic associations between rs9943582 and ischemic stroke in the Chinese Han GeneID population, although the study population provided sufficient statistical power. This finding indicates that the association between the APLNR variant and ischemic stroke or atherosclerosis may need further validation.

Genome-wide association studies (GWAS) have identified >60 genomic loci for coronary artery disease (CAD), including ADTRP (regulator of TFPI and coagulation) and MIA3 (involved in ER trafficking of collagens). In this study, we hypothesized that some GWAS genes form a molecular regulatory network involved in the pathogenesis of CAD. Global microarray analysis showed that ADTRP regulated expression of MIA3 and PIK3R3 encoding the regulatory subunit 3 of PI3K. ADTRP -mediated up-regulation of PIK3R3 activates AKT, resulting in up-regulation of MIA3 . Knockdown of ADTRP expression promoted oxidized-LDL-mediated monocyte adhesion to endothelial cell (EC) and transendothelial migration of monocytes, inhibited EC proliferation and migration, and increased apoptosis, which was reversed by expression of constitutively active AKT1, while the over-expression of ADTRP in ECs blunted these processes. Knockdown of MIA3 expression also promoted monocyte adhesion to ECs and transendothelial migration of monocytes. However, knockdown of MIA3 increased ADTRP expression, suggesting that MIA3 negatively regulates ADTRP expression. Genetically, no significant interaction between ADTRP SNP rs6903956 and MIA3 SNP rs17465637 was detected in 2,185 CAD patients and 2,156 non-CAD controls by classical two-locus genotypic analysis, relative excess risk due to interaction or INTERSNP programs. We have recently found that one molecular mechanism for gene-gene interaction is positive cyclic cross-regulation of gene expression: Gene A positively regulates gene B, whereas gene B also positively regulates gene A. Here we have identified a different scenario in which gene A positively regulates gene B, but gene B negatively regulates gene A, thereby resulting in lack of gene-gene interaction. In conclusion, we have uncovered a novel molecular signaling pathway involving ADTRP and MIA3 for the pathogenesis of CAD. We show that ADTRP positively regulates PIK3R3 expression, which leads to activation of AKT and up-regulation of MIA3 , thereby regulating endothelial cell functions directly relevant to atherosclerosis. These results further support that positive cyclic cross-regulation of gene expression is a molecular mechanism for gene-gene interaction.

Objective: The endothelium is the initial target that leads to cardiovascular disease. Knowing that the internal mammary arteries (IMA) are resistant to the development of atherosclerosis, which contrasts with coronary arteries (Cor) which are athero-prone, we hypothesize that genes over-expressed in the endothelial cells (ECs) of between these two arteries will identify genes that resist atherosclerosis. Methods and Results: Microarray analysis showed over 1,000 differentially expressed in the ECs of IMA vs Cor. The most statistically significant different gene was the adenosine A 2B receptor. This indicates the A 2B receptor may be involved in a resistance to atherosclerosis. Western blot analysis showed higher A 2B expression in the IMA than in coronary arteries with or without disease from proteins harvested from these human arteries and ECs. Overexpression of A 2B in ECs blunted: monocyte adhesion, cell adhesion molecule expression, migration, and the transendothelial migration of monocytes-- processes directly associated with the development of atherosclerosis. Knockdown of A 2B expression by siRNA promoted these processes. Conclusions: ECs derived from the IMA and Cor are distinctly different in gene expression, which may be responsible for their differential sensitivities for atherosclerosis. This study defined how the A 2B receptor may act as an atherosclerotic-resistance gene, which blunted monocyte adhesion and cell adhesion molecule expression, EC migration and retarded the transendothelial migration of monocytes.

Objectives: This study sought to ascertain the relationship of 9p21 locus with: 1) angiographic coronary artery disease (CAD) burden; and 2) myocardial infarction (MI) in individuals with underlying CAD. Background: Chromosome 9p21 variants have been robustly associated with coronary heart disease, but questions remain on the mechanism of risk, specifically whether the locus contributes to coronary atheroma burden or plaque instability. Methods: We established a collaboration of 21 studies consisting of 33,673 subjects with information on both CAD (clinical or angiographic) and MI status along with 9p21 genotype. Tabular data are provided for each cohort on the presence and burden of angiographic CAD, MI cases with underlying CAD, and the diabetic status of all subjects. Results: We first confirmed an association between 9p21 and CAD with angiographically defined cases and control subjects (pooled odds ratio [OR]: 1.31, 95% confidence interval [CI]: 1.20 to 1.43). Among subjects with angiographic CAD (n = 20,987), random-effects model identified an association with multivessel CAD, compared with those with single-vessel disease (OR: 1.10, 95% CI: 1.04 to 1.17)/copy of risk allele). Genotypic models showed an OR of 1.15, 95% CI: 1.04 to 1.26 for heterozygous carrier and OR: 1.23, 95% CI: 1.08 to 1.39 for homozygous carrier. Finally, there was no significant association between 9p21 and prevalent MI when both cases (n = 17,791) and control subjects (n = 15,882) had underlying CAD (OR: 0.99, 95% CI: 0.95 to 1.03)/risk allele. Conclusions: The 9p21 locus shows convincing association with greater burden of CAD but not with MI in the presence of underlying CAD. This adds further weight to the hypothesis that 9p21 locus primarily mediates an atherosclerotic phenotype.

Aberrant crypt foci (ACF), the earliest neoplastic lesions of the colon, have genetic and epigenetic alterations. Loss of heterozygosity (LOH) of tumor suppressor gene loci is seen in most colon cancers, but it is not known how early in tumorigenesis this takes place. Nine microsatellite markers close to specific genes, that is, APC (5q21), PTPRJ (11p11), p53 (17p13) and DCC (18q21), were analyzed in 32 ACF and samples of normal crypts from the same 28 patients. Six losses of heterozygosity were found in 5 of 32 ACF: 4 losses of heterozygosity were at 11p11, the location of the gene for protein tyrosine phosphatase receptor type J (PTPRJ) and of a second independent region of deletion; the others were at 5q21 and 18q21. Microsatellite instability (MSI) with markers for a single locus was found in 4 of 32 ACF. All the observed allelic alterations (LOH and MSI) were in 8 of 32 ACF. The finding of LOH in ACF with normal expressions of adenomatous polyposis coli (APC) and beta-catenin proteins suggests that LOH can occur very early in colon neoplasia and perhaps even before APC mutations. The finding of 3 of 4 of the losses of heterozygosity at 11p11 for PTPRJ and half of all the losses of heterozygosity in this study at PTPRJ suggest that this gene plays a role early in colon neoplasia.

We previously identified a novel locus for plasma triglyceride (TG) levels on chromosome 1p31-32 by genome-wide linkage analysis in the GeneQuest population with familial and premature coronary artery disease (CAD). Here we tested a hypothesis that variants in LRP8, a gene that is under the 1p31-32 linkage peak and associated with risk of familial and premature CAD and increased platelet activation, are associated with TG levels. Seven tagSNPs that cover the entire LRP8 gene were characterized in 358 GeneQuest Caucasian probands. Only SNP R952Q (rs5174) was associated with TG levels (P-adj = 0.0016), and this finding was replicated in one other independent population of 134 patients with early-onset myocardial infarction (males45; females55; P-adj = 0.0098). TG levels were higher in the group with higher body mass index (BMI ≥ 25) than in the group with lower BMI (BMI25). The association was significant in the overweight group (P-adj = 0.0029) or in the smoking group (P-adj = 0.0004), but not in the group with normal BMI or without smoking history. These results suggest that genetic variant R952Q of LRP8 is associated with increased plasma TG levels in patients who are overweight and have premature CAD/MI and history of smoking.

MEF2A is a member of the myocyte enhancer factor-2 (MEF2) family of transcription factors. We showed that MEF2A was expressed in coronary arteries and mutations or rare variants of MEF2F were associated with risk of coronary artery disease (CAD) in humans. Both positive and negative replication studies were later reported. To further investigate the role of MEF2A in CAD and MI, we have characterized heterozygous MEF2A knockout mice and show here that haploinsufficiency of MEF2A in mice promotes atherosclerosis. Heterozygous MEF2A knockout mice were crossed to apoE-deficient mice, a widely used genetic model for atherosclerosis. The degree of atherogenesis was determined by the aortic root quantitative atherosclerosis assay. In vivo vascular permeability assay was performed using Evans blue dye upon stimulation with mustard oil in ears. Serum total cholesterol, HDL cholesterol, and triglyceride levels were measured with the Stanbio enzymatic kits. No significant differences were detected for total plasma cholesterol, HDL-C, VLDL-Cl and TG levels between MEF2A + ApoE −/− mice and their MEF2A +/+ ApoE −/− littermate controls (P = 0.14 – 0.44). The glucose levels before and after fasting were also similar between the two lines of mice (fasting, 106.25 ± 20.7 vs. 104.8 ± 21.9, P = 0.87; non-fasting, 175.3 ± 82 vs. 179.9 ± 119, P = 0.092). Total aortic lesion area was twofold greater in MEF2A + ApoE −/− mice than in MEF2A +/+ ApoE −/− littermate controls fed on western diet (83408 μm 2 vs. 46621 μm 2 in males, n = 11 vs. 15, P = 0.0003; 137822 μm 2 vs. 61568 μm 2 in females, n = 12 vs. 9, P = 0.00003). Permeability of the vasculature in response to a proinflammatory stimulus was 3.2 fold higher in MEF2A + ApoE −/− mice than in MEF2A + / + ApoE −/− littermate controls (P = 0.000012). MEF2A haploinsufficiency promotes atherosclerotic lesion formation in mice, which may be due to increased vascular permeability. Lipid or glucose levels do not contribute to the formation of atherosclerotic lesions in these mice.

Common atrial fibrillation (AF) is a complex disease, and its pathogenesis involves multiple genetic factors, environmental factors, and interactions among these factors. Genetic factors clearly contribute to the risk of common AF. Parental AF increases by more than threefold the risk of AF under age of 75 years in offspring, and first-degree relatives have almost fivefold more risk of developing AF before the age of 60 years. Candidate gene case—control studies investigated the roles of several genes in common AF and thromboembolism in AF, including KCNE1, KCNE4, KCNE5, GNB3, AGT, CETP, coagulation factor II, α-fibrinogen, factor XIII, and IL6. Genomewide single nucleotide polymorphism association studies is the state-of-the art study design for dissecting the common complex AF trait and have successfully identified two SNPs on chromosome 4q25 that are associ ated with risk of atrial fibrillation. Rare families with AF have been reported, and studies of these families identified mutations in several genes for AF, including KCNQ1, KCNE2, KCNE3, and KCNJ2. Two autosomal dominant AF genes were mapped to chromosome 10 q and 6 q, and one autosomal recessive gene for AF was mapped to 5 p, but these genes have not yet been identified. Also, AF can occur in patients with dilated cardiomyopathy with SCN5A and LMNA mutations, long QT syndrome patients with an ankyrin-B mutation, and short QT syndrome patients with a KCNH2 mutation. Genetic studies of AF will continue to provide insight into molecular mechanisms for the pathogenesis of AF and will facilitate realization of genetic testing and genotype-based therapies (personalized medicine) for AF patients.

To identify the genetic basis of disease in a Chinese family with retinitis pigmentosa (RP).Linkage analysis was performed for 15 family members in the RP family using microsatellite markers flanking candidate genetic loci for known autosomal dominant RP (adRP) and markers covering the entire X chromosome by every 10 cM. To screen for a mutation causing RP, PCR and DNA sequence analyses of the complete coding region (including ORF15) and exon-intron boundaries of the retinitis pigmentosa GTPase regulator (RPGR) gene associated with X-linked RP (xlRP) were carried out for the proband in the RP family. After the mutation was identified, direct DNA sequence analysis was preformed for all 15 family members and 101 controls to determine whether the mutation co-segregated with RP in the family and whether it was present or absent in the controls.Linkage analysis excluded all known adRP loci. However, positive linkage was identified with two markers on the X chromosome, DXS993 and DXS1068, where the RPGR gene is located. Direct DNA sequence analysis revealed a hemizygous mutation, g.ORF15+1166delA (c.2919delA), in affected males. The deletion results in a frameshift leading to early termination of RPGR. The g.ORF15+1166delA mutation arose de novo and co-segregated with all male patients, but was not present in normal family members and 101 controls. The clinical features of the mutation carriers showed intrafamilial variability.The novel g.ORF15+1166delA mutation of RPGR causes X-linked RP in a four generation Chinese family. The deletion arose de novo. An interesting feature of mutation g.ORF15+1166delA is that it was associated with RP in all hemizygous males and four of five heterozygous female carriers in the Chinese family. These results revealed the broader xlRP genotypic and phenotypic spectrum of RPGR mutations.

Transgenic and gene-targeted mice are now frequently used to expand the study of cardiac physiology and pathophysiology owing to the ease with which the mouse genome can be manipulated. There are many measures by which an assessment of the phenotypical expression of the transgenic mouse can be made. In the case of cardiac channelopathies and how they relate to cardiac function, telemetry is a technology that utilizes transmitters that are surgically implanted in animals for the purpose of acquiring biopotentials or physiological parameters. Electrophysiological techniques have also been used to assess cardiac function at the cellular level, by measuring whole-cell ionic currents and/or transmembrane potentials. This chapter will discuss the surgical procedures involved in successfully implanting the transmitter device in a mouse, as well as highlight the recording of and analysis of electrocardiograms. This chapter will also outline the procedures involved in isolating single-ventricular myocytes from a mouse heart. It is a protocol that was developed in our laboratory for which we have routinely and successfully isolated myocytes from both transgenic and nontransgenic mouse hearts. Although no one isolation protocol is alike, we also present our own observations that have assisted in maximizing myocyte bioavailability and yield.

The transgenic mouse technology is a powerful tool that can be used for creating animal models for cardiovascular disease to identify molecular pathogenic mechanisms and for identifying the physiological functions of a novel gene. A transgenic animal can be generated by several methods, which include microinjection of a DNA fragment into the pronucleus, embryonic stem cell manipulation and injection, sperm-mediated transgenesis, and viral infection of preimplanted embryos. The microinjection method is one of the most widely used approaches. This method involves four steps: (1) collection of fertilized eggs from the superovulated female, (2) injection of DNA into the pronucleus of fertilized eggs, (3) transfer of the injected eggs back into the oviduct of a pseudopregnant foster recipient, allowing the eggs to develop into pups, and (4) identification of the transgenic founder and establishment of transgenic lines through further breeding.

Microarray analysis is a powerful technique for high-throughput, global transcriptonomic profiling of gene expression. It holds great promise for analyzing the genetic and molecular bases of cardiovascular diseases and various other complex diseases and permits the analysis of thousands of genes simultaneously, both in diseased and nondiseased tissues and/or cell lines. Microarrays or microchips are made by depositing spots of DNA or oligonucleotides representing thousands of genes on a solid support such as a coated glass surface, and can allow the comparison of gene expression patterns in any two samples. Total RNA is isolated from the tissue or cells of interest, converted to cDNA and then cRNA labeled with biotin, and hybridized to the chips. Hybridization signals are then quantified and compared among different samples. We used oligonucleotide microarrays to obtain an unbiased assessment of expression levels of thousands of genes simultaneously in normal and diseased coronary arteries. Fifty-six genes showed differential expression in atherosclerotic coronary artery tissues, and 49 of them represent new linked genes for coronary artery disease. These studies can generate novel hypotheses relating to the pathologies of disease and further studies with animal models, molecular biology, cell biology, and biochemistry will validate these hypotheses and provide novel insights into the pathogenesis of disease.

A complex disease trait refers to a phenotype that does not follow simple Mendelian segregation attributable to a single gene locus, but instead may be caused by multiple disease loci, their interactions, polygenic inheritance, and environmental effects. Most cardiovascular disorders are thought to have a polygenic basis with complex interactions with environmental factors. A gene that increases or decreases the risk to a complex cardiovascular disease (susceptibility gene) can now be mapped to a specific chromosomal region by model-free linkage analysis, and follow-up molecular genetic studies can identify the specific gene at the locus. This chapter describes a protocol for model-free linkage analysis of a complex trait, as implemented in the popular genetic analysis software-SAGE. In particular, the Haseman-Elston sib-pair regression method is introduced and implemented with examples to demonstrate how to identify susceptibility loci for complex traits.

Many human diseases are associated with cytogenetic abnormalities or chromosomal disorders including translocations, deletions, duplications, inversions, and other complicated chromosomal changes. Fluorescence in situ hybridization (FISH), a technique involving hybridization of labeled probes to chromosomes and detection of hybridization via fluorochromes, has become a popular method for identification and characterization of cytogenetic abnormalities. For FISH analysis, metaphase chromosomes are prepared by mitotic arrest and hypotonic shock, and denatured. Hybridization of digoxigenin- or biotin-labeled probes to these chromosomes is visualized using fluorochromes like fluorescein isothiocyanate and Texas Red. We have successfully applied FISH technology to the characterization of chromosome breakpoints involved in disease-associated cytogenetic abnormalities to identify candidate gene(s) for the disease. FISH is also widely used in clinical diagnosis of chromosomal disorders.

Somatic cell hybrids are generated by fusion of two different parental cells. This technology has been used extensively in the production of monoclonal antibodies and has made significant contributions to the field of human genetics through its applications in gene expression, gene mapping, and positional cloning of human disease genes. In our laboratory, we have employed this technique in the positional cloning of several genes for human diseases associated with cytogenetic abnormalities (chromosomal disorders), including translocations. Somatic cell hybrids are constructed by fusing mouse thymidine kinase-deficient 3T3 fibroblasts with human lymphoblastoid cells, as a result of which specific hybrid cells containing only cytogenetically abnormal human chromosomes involved in a chromosomal disorder can be successfully isolated and cloned. These hybrid cells serve as an excellent tool with which to define the exact chromosomal breakpoints involved in a cytogenetic abnormality and to identify genes at the breakpoints.

Single-nucleotide polymorphisms (SNPs) are DNA sequence variations that occur at a single base in the genome sequence. SNPs are valuable markers for identifying genes responsible for susceptibility to common diseases, and in some cases, they are the causes of human diseases. A genetic study of a complex disease usually involves a case-control association study that requires genotyping of a large number of SNPs in hundreds of patients (cases) and matched controls. A significant difference of the allele frequency or genotypic frequency of a SNP between the two populations is considered to be the evidence for the association between the SNP and disease. A key to a fast and effective case-control association study requires high-throughput genotyping of SNPs. Two assays-the TaqMan SNP genotyping assay and the pyrosequencing assay-have been developed for this purpose and proven to be particularly useful. Here, we present the operative protocol, clarify the key technical issues, and highlight certain cautionary notes for high throughput SNP genotyping using TaqMan and pyrosequencing assays.

Objectives Ventricular tachycardia is a kind of serious arrhythmia. Some recent Genome-wide association studies identified susceptibility locus, among which SNP rs11970286 has a documented role in the regulation of QT interval. However, few researchs foucs on the relevance of common variants and ventricular tachycardia. We hypothesised that this SNP act a role in ventricular tachycardia. Methods 220 patients diagnosed as ventricular tachycardia and 1000 healthy subjects were included in the present study. All patients were genotyped for SNPs using a Rotor-Gene TM 6000 High Resolution Melt system. A case-control analysis were performed by accurate statistical analysis adjusting for potential confounding factors. Results The association of SNP rs11970286 and QT was significant (p=0.018). We also found significant association between SNP rs11970286 and idiopathic ventricular tachycardia, as well as ventricular tachycardia after acute myocardial infarction (p=0.001, OR=2.226; p=0.005, OR=4.010 respectively). Conclusions Our findings suggest that SNP rs11970286 might be risk factors for ventricular tachycardia in Chinese population. And QT interval may confer an intermediate phenotype. The identification of causal genes and mechanisms at the loci remains a major task.

Objectives The prolongation of the PR interval has been considered as an increased risk of atrial fibrillation (AF), pacemaker implantation and mortality. Several recent genome-wide association studies (GWAS) have yielded associations between common variants and echocardiogram (ECG) parametres. SNP rs3825214 in TBX5 gene was shown correlating with PR interval, QRS duration, QT interval and verified significant in diseases such as atrial fibrillation, advanced AV block. The aim of this study is to further assess association between SNP rs3825214 and ECG parametres, AF, ventricular tachycardia, as well as some other arrhythmias associating with sudden cardiac death in mainland Chinese Han population. Methods 692 patients as AF group, 235 patients as VT group, and 856 controls in GeneID population were enrolled for case-control association study. Genotyping was performed using a Rotor-Gene TM 6000 High Resolution Melt system. The associations of both allele and genotype were analysed by rigorous statistical analysis adjusting for potential confounding factors. Results In contrast to previous GWAS results, we did not found PR interval, QRS duration, QT interval significantly associated with SNP rs3825214, but the PR interval shows a tendency of association (p=0.057). QT c was associated with SNP rs3825214 (p=0.047, β=−19.76). A significant association between G allele of SNP rs3825214 and lone atrial fibrillation (LAF) was arresting (p=0.002; P-adj=0.001, OR=0.652).In both AF and LAF group, the distributions were significantly different compared to the control group with p value equals to 0.029 and 0.003 respectively. Assuming a dominant genetic model, the GG shows strong significant association with AF, and especially LAF. The GG genotype could be a profound protective factor under dominant genetic model as the OR was 0.73 after adjusting for sex, age, T2DM, hypertension, stroke, and CAD. Conclusions The study detected the association of allele G of SNP rs3825214 in TBX5 with QT c and lone AF for the first time. The findings expand the GWAS results to other ethnic population and provide new insight into the molecular aetiology involved in the pathogenesis of lone AF.