Brain-heart interactions and cardiac ventricular arrhythmias

A wide range of evidence implicates the brain as playing a significant role in ventricular arrhythmias and sudden cardiac death. The mechanism is thought to involve the intermediary of the autonomic nervous system. Here we briefly consider possible mechanisms by which central neural processing may modulate the myocardial electrophysiology and hence the arrhythmia substrate.     

Cardiac disease modeling using induced pluripotent stem cell-derived human cardiomyocytes

Causative mutations and variants associated with cardiac diseases have been found in genes encoding cardiac ion channels, accessory proteins, cytoskeletal components, junctional proteins, and signaling molecules. In most cases the functional evaluation of the genetic alterationhas been carried out by expressing the mutated proteins in in-vitro heterologous systems. While these studies have provided a wealth of functional details that have greatly enhanced the understanding of the pathological mechanisms, it has always been clear that heterologous expression of the mutant protein bears the intrinsic limitation of the lack of a proper intracellular environment and the lack of pathological remodeling. The results obtained from the application of the next generation sequencing technique to patients suffering from cardiac diseases have identified several loci, mostly in non-coding DNA regions, which still await functional analysis. The isolation and culture of human embryonic stem cells has initially provided a constant source of cells from which cardiomyocytes(CMs) can be obtained by differentiation. Furthermore, the possibility to reprogram cellular fate to a pluripotent state, has opened this process to the study of genetic diseases. Thus induced pluripotent stem cells(i PSCs) represent a completely new cellular model that overcomes the limitations of heterologous studies. Importantly, due to the possibility to keep spontaneously beating CMs in culture for several months, during which they show a certain degree of maturation/aging, this approach will also provide a system in which to address the effect of long-term expression of the mutated proteins or any other DNA mutation, in terms of electrophysiological remodeling. Moreover, since i PSC preserve the entire patients’ genetic context, the system will help the physicians in identifying the most appropriate pharmacological intervention to correct the functional alteration. This article summarizes the current knowledge of cardiac genetic diseases modelled with i PSC.     

Calcium overload and cardiac function

The changes in cardiac function caused by calcium overload are reviewed. Intracellular Ca²⁺ may increase in different structures [e.g. sarcoplasmic reticulum (SR), cytoplasm and mitochondria] to an excessive level which induces electrical and mechanical abnormalities in cardiac tissues. The electrical manifestations of Ca²⁺ overload include arrhythmias caused by oscillatory (V_os) and non-oscillatory (V_ex) potentials. The mechanical manifestations include a decrease in force of contraction, contracture and aftercontractions. The underlying mechanisms involve a role of Na⁺ in electrical abnormalities as a charge carrier in the Na⁺-Ca²⁺ exchange and a role of Ca²⁺ in mechanical toxicity. Ca²⁺ overload may be induced by an increase in [Na⁺]_i through the inhibition of the Na⁺-K⁺ pump (e.g. toxic concentrations of digitalis) or by an increase in Ca²⁺ load (e.g. catecholamines). The Ca²⁺ overload is enhanced by fast rates. Purkinje fibers are more susceptible to Ca²⁺ overload than myocardial fibers, possibly because of their greater Na⁺ load. If the SR is predominantly Ca²⁺ overloaded, V_os and fast discharge are induced through an oscillatory release of Ca²⁺ in diastole from the SR; if the cytoplasm is Ca²⁺ overloaded, the non-oscillatory V_ex tail is induced at negative potentials. The decrease in contractile force by Ca²⁺ overload appears to be associated with a decrease in high energy phosphates, since it is enhanced by metabolic inhibitors and reduced by metabolic substrates. The ionic currents I_os and I_ex underlie V_os and V_ex, respectively, both being due to an electrogenic extrusion of Ca²⁺ through the Na⁺-Ca²⁺ exchange. I_os is an oscillatory current due to an oscillatory release of Ca²⁺ in early diastole from the Ca²⁺-overloaded SR, and I_ex is a non-oscillatory current due to the extrusion of Ca²⁺ from the Ca²⁺-overloaded cytoplasm. I_os and I_ex can be present singly or simultaneously. An increase in [Ca²⁺]_i appears to be involved in the short- and long-term compensatory mechanisms that tend to maintain cardiac output in physiological and pathological conditions. Eventually, [Ca²⁺]_i may increase to overload levels and contribute to cardiac failure. Experimental evidence suggests that clinical concentrations of digitalis increase force in Ca²⁺-overloaded cardiac cells by decreasing the inhibition of the Na⁺-K⁺ pump by Ca²⁺, thereby leading to a reduction in Ca²⁺ overload and to an increase in force of contraction.     

单核苷酸多态性检测方法的研究进展

单核苷酸多态性（single nucleotide polymorphism, SNP）的研究已成为人类后基因组时代的主要内容之一。因此建立高度自动化和高通量的SNP检测分析技术十分重要。文章系统地介绍了最新发展的几种SNP检测技术的原理和检测平台,详细阐述了等位基因特异性杂交、内切酶酶切技术、引物延伸法、寡核苷酸连接反应等SNP检测原理,以及平板读数仪、基因芯片、微球阵列技术和质谱仪等检测平台,并对SNP高通量检测技术的发展进行了展望。     

The risk of ventricular arrhythmias in a Dutch CRT population: CRT-defibrillator versus CRT-pacemaker

Background

Patients eligible for cardiac resynchronisation therapy (CRT) have an indication for primary prophylactic implantable cardioverter defibrillator (ICD) therapy. However, response to CRT might influence processes involved in arrhythmogenesis and therefore change the necessity of ICD therapy in certain patients.

Method

In 202 CRT-defibrillator patients, the association between baseline variables, 6-month echocardiographic outcome (volume response: left ventricular end-systolic volume decrease < ≥15 % and left ventricular ejection fraction (LVEF) ≤ >35 %) and the risk of first appropriate ICD therapy was analysed retrospectively.

Results

Fifty (25 %) patients received appropriate ICD therapy during a median follow-up of 37 (23–52) months. At baseline ischaemic cardiomyopathy (hazard ratio (HR) 2.0, p = 0.019) and a B-type natriuretic peptide level > 163 pmol/l (HR 3.8, p < 0.001) were significantly associated with the risk of appropriate ICD therapy. After 6 months, 105 (52 %) patients showed volume response and 51 (25 %) reached an LVEF > 35 %. Three (6 %) patients with an LVEF > 35 % received appropriate ICD therapy following echocardiography at ± 6 months compared with 43 patients (29 %) with an LVEF ≤ 35 % (p = 0.001). LVEF post-CRT was more strongly associated to the risk of ventricular arrhythmias than volume response (LVEF > 35 %, HR 0.23, p = 0.020).

Conclusion

Assessing the necessity of an ICD in patients eligible for CRT remains a challenge. Six months post-CRT an LVEF > 35 % identified patients at low risk of ventricular arrhythmias. LVEF might be used at the time of generator replacement to identify patients suitable for downgrading to a CRT-pacemaker.     

Susceptibility genes for age-related maculopathy on chromosome 10q26

On the basis of genomewide linkage studies of families affected with age-related maculopathy (ARM), we previously identified a significant linkage peak on 10q26, which has been independently replicated by several groups. We performed a focused SNP genotyping study of our families and an additional control cohort. We identified a strong association signal overlying three genes, PLEKHA1, LOC387715, and PRSS11. All nonsynonymous SNPs in this critical region were genotyped, yielding a highly significant association (P < .00001) between PLEKHA1/LOC387715 and ARM. Although it is difficult to determine statistically which of these two genes is most important, SNPs in PLEKHA1 are more likely to account for the linkage signal in this region than are SNPs in LOC387715; thus, this gene and its alleles are implicated as an important risk factor for ARM. We also found weaker evidence supporting the possible involvement of the GRK5/RGS10 locus in ARM. These associations appear to be independent of the association of ARM with the Y402H allele of complement factor H, which has previously been reported as a major susceptibility factor for ARM. The combination of our analyses strongly implicates PLEKHA1/LOC387715 as primarily responsible for the evidence of linkage of ARM to the 10q26 locus and as a major contributor to ARM susceptibility. The association of either a single or a double copy of the high-risk allele within the PLEKHA1/LOC387715 locus accounts for an odds ratio of 5.0 (95% confidence interval 3.2-7.9) for ARM and a population attributable risk as high as 57%.     

Utility of cardiac imaging in patients with ventricular tachycardia

Ventricular tachycardia (VT) is a life-threatening arrhythmia that may be idiopathic or result from structural heart disease. Cardiac imaging is critical in the diagnostic workup and risk stratification of patients with VT. Data gained from cardiac imaging provides information on likely mechanisms and sites of origin, as well as risk of intervention. Pre-procedural imaging can be used to plan access route(s) and identify patients where post-procedural intensive care may be required. Integration of cardiac imaging into electroanatomical mapping systems during catheter ablation procedures can facilitate the optimal approach, reduce radiation dose, and may improve clinical outcomes. Intraprocedural imaging helps guide catheter position, target substrate, and identify complications early. This review summarises the contemporary imaging modalities used in patients with VT, and their uses both pre-procedurally and intra-procedurally.     

Single‐nucleotide polymorphism discovery and validation in high‐density SNP array for genetic analysis in European white oaks

An Illumina Infinium SNP genotyping array was constructed for European white oaks. Six individuals of Quercus petraea and Q. robur were considered for SNP discovery using both previously obtained Sanger sequences across 676 gene regions (1371 in vitro SNPs) and Roche 454 technology sequences from 5112 contigs (6542 putative in silico SNPs). The 7913 SNPs were genotyped across the six parental individuals, full‐sib progenies (one within each species and two interspecific crosses between Q. petraea and Q. robur) and three natural populations from south‐western France that included two additional interfertile white oak species (Q. pubescens and Q. pyrenaica). The genotyping success rate in mapping populations was 80.4% overall and 72.4% for polymorphic SNPs. In natural populations, these figures were lower (54.8% and 51.9%, respectively). Illumina genotype clusters with compression (shift of clusters on the normalized x‐axis) were detected in ~25% of the successfully genotyped SNPs and may be due to the presence of paralogues. Compressed clusters were significantly more frequent for SNPs showing a priori incorrect Illumina genotypes, suggesting that they should be considered with caution or discarded. Altogether, these results show a high experimental error rate for the Infinium array (between 15% and 20% of SNPs potentially unreliable and 10% when excluding all compressed clusters), and recommendations are proposed when applying this type of high‐throughput technique. Finally, results on diversity levels and shared polymorphisms across targeted white oaks and more distant species of the Quercus genus are discussed, and perspectives for future comparative studies are proposed.     

SNP genotyping and population genomics from expressed sequences – current advances and future possibilities

With the rapid increase in production of genetic data from new sequencing technologies, a myriad of new ways to study genomic patterns in nonmodel organisms are currently possible. Because genome assembly still remains a complicated procedure, and because the functional role of much of the genome is unclear, focusing on SNP genotyping from expressed sequences provides a cost‐effective way to reduce complexity while still retaining functionally relevant information. This review summarizes current methods, identifies ways that using expressed sequence data benefits population genomic inference and explores how current practitioners evaluate and overcome challenges that are commonly encountered. We focus particularly on the additional power of functional analysis provided by expressed sequence data and how these analyses push beyond allele pattern data available from nonfunction genomic approaches. The massive data sets generated by these approaches create opportunities and problems as well – especially false positives. We discuss methods available to validate results from expressed SNP genotyping assays, new approaches that sidestep use of mRNA and review follow‐up experiments that can focus on evolutionary mechanisms acting across the genome.     

Development and evaluation of high‐density Axiom®CicerSNP Array for high‐resolution genetic mapping and breeding applications in chickpea

To accelerate genomics research and molecular breeding applications in chickpea, a high‐throughput SNP genotyping platform ‘Axiom^®CicerSNP Array’ has been designed, developed and validated. Screening of whole‐genome resequencing data from 429 chickpea lines identified 4.9 million SNPs, from which a subset of 70 463 high‐quality nonredundant SNPs was selected using different stringent filter criteria. This was further narrowed down to 61 174 SNPs based on p‐convert score ≥0.3, of which 50 590 SNPs could be tiled on array. Among these tiled SNPs, a total of 11 245 SNPs (22.23%) were from the coding regions of 3673 different genes. The developed Axiom^®CicerSNP Array was used for genotyping two recombinant inbred line populations, namely ICCRIL03 (ICC 4958 × ICC 1882) and ICCRIL04 (ICC 283 × ICC 8261). Genotyping data reflected high success and polymorphic rate, with 15 140 (29.93%; ICCRIL03) and 20 018 (39.57%; ICCRIL04) polymorphic SNPs. High‐density genetic maps comprising 13 679 SNPs spanning 1033.67 cM and 7769 SNPs spanning 1076.35 cM were developed for ICCRIL03 and ICCRIL04 populations, respectively. QTL analysis using multilocation, multiseason phenotyping data on these RILs identified 70 (ICCRIL03) and 120 (ICCRIL04) main‐effect QTLs on genetic map. Higher precision and potential of this array is expected to advance chickpea genetics and breeding applications.     

Characterization of a Wheat Breeders’ Array suitable for high‐throughput SNP genotyping of global accessions of hexaploid bread wheat (Triticum aestivum)

Targeted selection and inbreeding have resulted in a lack of genetic diversity in elite hexaploid bread wheat accessions. Reduced diversity can be a limiting factor in the breeding of high yielding varieties and crucially can mean reduced resilience in the face of changing climate and resource pressures. Recent technological advances have enabled the development of molecular markers for use in the assessment and utilization of genetic diversity in hexaploid wheat. Starting with a large collection of 819 571 previously characterized wheat markers, here we describe the identification of 35 143 single nucleotide polymorphism‐based markers, which are highly suited to the genotyping of elite hexaploid wheat accessions. To assess their suitability, the markers have been validated using a commercial high‐density Affymetrix Axiom^® genotyping array (the Wheat Breeders’ Array), in a high‐throughput 384 microplate configuration, to characterize a diverse global collection of wheat accessions including landraces and elite lines derived from commercial breeding communities. We demonstrate that the Wheat Breeders’ Array is also suitable for generating high‐density genetic maps of previously uncharacterized populations and for characterizing novel genetic diversity produced by mutagenesis. To facilitate the use of the array by the wheat community, the markers, the associated sequence and the genotype information have been made available through the interactive web site ‘CerealsDB’.     

应用创造酶切位点法检测单碱基突变

应用引物错配技术结合单碱基突变位点而配合成一个酶切位点,使之成为可用PCR-RFLP方法分析的突变位点,是对单碱基突变位点进行基因型鉴定的有效而简捷的手段。本文以鸡胞外脂肪酸结合蛋白(Extracelluar fatty acid binding protein,EX-FABP)基因单碱基突变的基因型检测为例,探讨了应用创造酶切位点PCR（Created Restriction Site PCR,CRS-PCR）检测单碱基突变基因型的思路、方法和策略。 Abstract:Created Restriction Site PCR (CRS-PCR) is a simple and efficient method to identify SNP genotypes.One or more mismatch bases are used in a primer to create a restriction site by combining SNP site after PCR.The CRS-PCR products can be genotyped with a way the same as PCR-RFLP.In the study,Extracelluar fatty acid binding protein (EX-FABP)　gene was served as an example for establishing the CRS-PCR method.Strategy of CRS-PCR was also discussed.     

Identification of Polymorphic Markers by High-Resolution Melting (HRM) Assay for High-Throughput SNP Genotyping in Maize

The development of next generation sequencing (NGS) and high throughput genotyping are important techniques for the QTL mapping and genetic analysis of different crops. High-resolution melting (HRM) is an emerging technology used for detecting single-nucleotide polymorphisms (SNPs) in various species. However, its use is still limited in maize. The HRM analysis was integrated with SNPs to identify three types of populations (NIL population, RIL population and natural population), and the useful tags were screened. The patterns of temperature-shifted melting curves were investigated from the HRM analysis, and compared these with the kit. Among all 48 pairs of primers, 10 pairs of them were selected: six pairs of primers for the NIL population, three pairs of primers for the RIL population, and one pair of primer for the natural population. The marker for the natural population was developed with a matching rate of 80% for the plant height trait, based on the data of the phenotypic characteristics measured in the field. This study provides an effective method for maize genotyping in the classification of maize germplasm resources, which can be applied to other plants for high-throughput SNP genotyping or further mapping.     

Spatiotemporal monitoring of the rare northern dragonhead (Dracocephalum ruyschiana,Lamiaceae) — SNP genotyping and environmental niche modeling herbarium specimens

The species we have studied the spatiotemporal genetic change in the northern dragonhead, a plant species that has experienced a drastic population decline and habitat loss in Europe. We have added a temporal perspective to the monitoring of northern dragonhead in Norway by genotyping herbarium specimens up to 200 years old. We have also assessed whether northern dragonhead has achieved its potential distribution in Norway. To obtain the genotype data from 130 herbarium specimens collected from 1820 to 2008, mainly from Norway (83) but also beyond (47), we applied a microfluidic array consisting of 96 SNP markers. To assess temporal genetic change, we compared our new genotype data with existing data from modern samples. We used sample metadata and observational records to model the species'' environmental niche and potential distribution in Norway. Our results show that the SNP array successfully genotyped all included herbarium specimens. Hence, with the appropriate design procedures, the SNP array technology appears highly promising for genotyping old herbarium specimens. The captured genetic diversity correlates negatively with distance from Norway. The historical‐modern comparisons reveal similar genetic structure and diversity across space and limited genetic change through time in Norway, providing no signs of any regional bottleneck (i.e., spatiotemporal stasis). The regional areas in Norway have remained genetically divergent, however, both from each other and more so from populations outside of Norway, rendering continued protection of the species in Norway relevant. The ENM results suggest that northern dragonhead has not fully achieved its potential distribution in Norway and corroborate that the species is anchored in warmer and drier habitats.