Screening assays for heart function mutants in Drosophila

The rapid life cycle and genetic tractability of Drosophila make it an ideal organism for large-scale genetic screens. Here we describe a novel assay for pupal heart rate and rhythmicity as well as techniques to measure adult cardiac stress response. These assays can be powerfully combined to concurrently screen for both mutations affecting cardiac function and mutations affecting the age-dependent decline in adult cardiac stress response. Mutations identified in such screens have the potential to contribute greatly to the understanding of both congenital heart disease and the regulation of age-dependent decline in cardiac function in the human population.     

Molecular insights into cardiomyopathies associated with desmin (<Emphasis Type="Italic">DES</Emphasis>) mutations

Increasing usage of next-generation sequencing techniques pushed during the last decade cardiogenetic diagnostics leading to the identification of a huge number of genetic variants in about 170 genes associated with cardiomyopathies, channelopathies, or syndromes with cardiac involvement. Because of the biochemical and cellular complexity, it is challenging to understand the clinical meaning or even the relevant pathomechanisms of the majority of genetic sequence variants. However, detailed knowledge about the associated molecular pathomechanism is essential for the development of efficient therapeutic strategies in future and genetic counseling. Mutations in DES, encoding the muscle-specific intermediate filament protein desmin, have been identified in different kinds of cardiac and skeletal myopathies. Here, we review the functions of desmin in health and disease with a focus on cardiomyopathies. In addition, we will summarize the genetic and clinical literature about DES mutations and will explain relevant cell and animal models. Moreover, we discuss upcoming perspectives and consequences of novel experimental approaches like genome editing technology, which might open a novel research field contributing to the development of efficient and mutation-specific treatment options.     

Identification of Genetic Alterations,as Causative Genetic Defects in Long QT Syndrome,Using Next Generation Sequencing Technology

Background

Long QT Syndrome is an inherited channelopathy leading to sudden cardiac death due to ventricular arrhythmias. Despite that several genes have been associated with the disease, nearly 20% of cases remain without an identified genetic cause. Other genetic alterations such as copy number variations have been recently related to Long QT Syndrome. Our aim was to take advantage of current genetic technologies in a family affected by Long QT Syndrome in order to identify the cause of the disease.

Methods

Complete clinical evaluation was performed in all family members. In the index case, a Next Generation Sequencing custom-built panel, including 55 sudden cardiac death-related genes, was used both for detection of sequence and copy number variants. Next Generation Sequencing variants were confirmed by Sanger method. Copy number variations variants were confirmed by Multiplex Ligation dependent Probe Amplification method and at the mRNA level. Confirmed variants and copy number variations identified in the index case were also analyzed in relatives.

Results

In the index case, Next Generation Sequencing revealed a novel variant in TTN and a large deletion in KCNQ1, involving exons 7 and 8. Both variants were confirmed by alternative techniques. The mother and the brother of the index case were also affected by Long QT Syndrome, and family cosegregation was observed for the KCNQ1 deletion, but not for the TTN variant.

Conclusions

Next Generation Sequencing technology allows a comprehensive genetic analysis of arrhythmogenic diseases. We report a copy number variation identified using Next Generation Sequencing analysis in Long QT Syndrome. Clinical and familiar correlation is crucial to elucidate the role of genetic variants identified to distinguish the pathogenic ones from genetic noise.     

Human genetic susceptibility to infectious disease

Recent genome-wide studies have reported novel associations between common polymorphisms and susceptibility to many major infectious diseases in humans. In parallel, an increasing number of rare mutations underlying susceptibility to specific phenotypes of infectious disease have been described. Together, these developments have highlighted a key role for host genetic variation in determining the susceptibility to infectious disease. They have also provided insights into the genetic architecture of infectious disease susceptibility and identified immune molecules and pathways that are directly relevant to the human host defence.     

Prevention and control of emerging infections: a challenge for the 3rd millennium

In the last 30 years, several emerging infections due to novel viruses have been identified, from haemorrhagic fever viruses to HIV, from the SARS-Coronavirus to Avian influenza viruses. Ecological and genetic changes are important determinants of the emergence of new viral infections, driving to an increase of R0 (the basic reproductive number) through increasing the probability of transmission. The current H5N1 epidemic may be considered a prepandemic paradigm that needs thorough investigation.     

A novel mutation in the SCN5A gene is associated with Brugada syndrome

Brugada syndrome (BS) is an inherited cardiac disorder associated with a high risk of sudden cardiac death and is caused by mutations in the SCN5A gene encoding the cardiac sodium channel alpha-subunit (Na(v)1.5). The aim of this study was to identify the genetic cause of familial BS and characterize the electrophysiological properties of a novel SCN5A mutation (W1191X). Four families and one patient with BS were screened for SCN5A mutations by PCR and direct sequencing. Wild-type (WT) and mutant Na(v)1.5 channels were expressed in tsA201 cells, and the sodium currents (I(Na)) were analyzed using the whole-cell patch-clamp technique. A novel mutation, W1191X, was identified in a family with BS. Expression of the WT or the mutant channel (Na(v)1.5/W1191X) co-transfected with the beta(1)-subunit in tsA201 cells resulted in a loss of function of Na(v)1.5 channels. While voltage-clamp recordings of the WT channel showed a distinct acceleration of Na(v)1.5 activation and fast inactivation kinetics, the Na(v)1.5/W1191X mutant failed to generate any currents. Co-expression of the WT channel and the mutant channel resulted in a 50% reduction in I(Na). No effect on activation and inactivation were observed with this heterozygous expression. The W1191X mutation is associated with BS and resulted in the loss of function of the cardiac sodium channel.     

A novel technique for ligation of the cephalic vein reduces hemorrhaging during a two-in-one insertion of dual cardiac device leads

The cutdown technique for the cephalic vein is a common access route for transvenous cardiac device leads (TVLs), and sometimes one cephalic vein can accomodate two TVLs. We examined a novel ligation technique to balance the hemostasis and lead maneuverability for this two-in-one insertion. A total of 22 patients scheduled for cardiac device implantations with two or more leads were enrolled. The ipsilateral cephalic vein was identified for inserting the TVLs with a cutdown. If two TVLs could be introduced into one cephalic vein, hemostasis was established by ligating the venous wall between the TVLs. We measured the amount of hemorrhaging per minute and the operators assessed the lead maneuverability before and after the ligation. We successfully implanted cardiac devices in 15 patients (68%) with this novel method, whereas only one TVL could be introduced via the cephalic vein in 7 patients. As for the successful patients, hemorrhaging from the gap was significantly reduced (5.6?±?7.3 to 0.41?±?0.36g/min, p?=?0.016) after the novel ligation. The lead maneuverability was well maintained so there was no difficulty placing the leads into the cardiac chambers in all cases. No major complications were observed. In the present study, the novel ligation method provided significant hemostasis as well as a preserved maneuverability. It could be an optional choice for insertion of multiple TVLs.     

A novel,major, and validated QTL for the effective tiller number located on chromosome arm 1BL in bread wheat

Plant Molecular Biology - A novel and major QTL for the effective tiller number was identified on chromosomal arm 1BL and validated in two genetic backgrounds The effective tiller number (ETN)...     

Molecular genetics of atherosclerosis

Atherosclerosis is a complex multifocal arterial disease involving interactions of multiple genetic and environmental factors. Advances in techniques of molecular genetics have revealed that genetic polymorphisms significantly influence susceptibility to atherosclerotic vascular diseases. A large number of candidate genes, genetic polymorphisms and susceptibility loci associated with atherosclerotic diseases have been identified in recent years and their number is rapidly increasing. In this review we focus on some of the major candidate genes and genetic polymorphisms associated with human atherosclerotic vascular diseases.     

Cardiac desmosomal (dys)function and myocyte viability

Cardiac desmosomes form intercellular junctions at the boundaries or intercalated discs between neighboring cardiomyocytes and are essential for proper cell-to-cell coupling between cardiomyocytes and normal mechanical and electrical function of myocardial tissue. Genetic mutations in desmosomal proteins have been associated with arrhythmogenic right ventricular cardiomyopathy (ARVC), a clinically and genetically heterogeneous cardiac inherited disorder. The disease is characterized by progressive replacement of cardiomyocytes by fibrofatty tissue, ultimately resulting in ventricular dilation, cardiac dysfunction, and the occurrence of life-threatening arrhythmias. Despite increasing knowledge on the genetic basis of ARVC, the etiopathogenesis of the disease remains less understood. The recent development of a number of transgenic mouse models with either heterozygous knock-out of desmosomal proteins or over-expression of aberrant desmosomal components has provided novel insight into the potential mechanisms and final common pathways involved in ARVC. The various disease mechanisms are likely not mutually exclusive and may each contribute to the pathogenesis of ARVC. However, their applicability in the diverse genetic forms of the disease remains uncertain, and may only be ascertained by careful investigation of sequential histopathological and (sub)cellular changes occurring over time during the disease process. Insight into the etiopathogenesis of ARVC will be crucial for the future development of new therapies aimed at delaying onset or progression of this disease.     

Beyond the cardiac myofilament: hypertrophic cardiomyopathy- associated mutations in genes that encode calcium-handling proteins

Traditionally regarded as a genetic disease of the cardiac sarcomere, hypertrophic cardiomyopathy (HCM) is the most common inherited cardiovascular disease and a significant cause of sudden cardiac death. While the most common etiologies of this phenotypically diverse disease lie in a handful of genes encoding critical contractile myofilament proteins, approximately 50% of patients diagnosed with HCM worldwide do not host sarcomeric gene mutations. Recently, mutations in genes encoding calcium-sensitive and calcium-handling proteins have been implicated in the pathogenesis of HCM. Among these are mutations in TNNC1- encoded cardiac troponin C, PLN-encoded phospholamban, and JPH2-encoded junctophilin 2 which have each been associated with HCM in multiple studies. In addition, mutations in RYR2-encoded ryanodine receptor 2, CASQ2-encoded calsequestrin 2, CALR3-encoded calreticulin 3, and SRI-encoded sorcin have been associated with HCM, although more studies are required to validate initial findings. While a relatively uncommon cause of HCM, mutations in genes that encode calcium-handling proteins represent an emerging genetic subset of HCM. Furthermore, these naturally occurring disease-associated mutations have provided useful molecular tools for uncovering novel mechanisms of disease pathogenesis, increasing our understanding of basic cardiac physiology, and dissecting important structure-function relationships within these proteins.     

Disruption of Sarcoendoplasmic Reticulum Calcium ATPase Function in Drosophila Leads to Cardiac Dysfunction

Abnormal sarcoendoplasmic reticulum Calcium ATPase (SERCA) function has been associated with poor cardiac function in humans. While modifiers of SERCA function have been identified and studied using animal models, further investigation has been limited by the absence of a model system that is amenable to large-scale genetic screens. Drosophila melanogaster is an ideal model system for the investigation of SERCA function due to the significant homology to human SERCA and the availability of versatile genetic screening tools. To further the use of Drosophila as a model for examining the role of SERCA in cardiac function, we examined cardiac function in adult flies. Using optical coherence tomography (OCT) imaging in awake, adult Drosophila, we have been able to characterize cardiac chamber dimensions in flies with disrupted in Drosophila SERCA (CaP60A). We found that the best studied CaP60A mutant, the conditional paralytic mutant CaP60A^kum170, develops marked bradycardia and chamber enlargement that is closely linked to the onset of paralysis and dependent on extra cardiac CaP60A. In contrast to prior work, we show that disruption of CaP60A in a cardiac specific manner results in cardiac dilation and dysfunction rather than alteration in heart rate. In addition, the co-expression of a calcium release channel mutation with CaP60A ^kum170 is sufficient to rescue the cardiac phenotype but not paralysis. Finally, we show that CaP60A overexpression is able to rescue cardiac function in a model of Drosophila cardiac dysfunction similar to what is observed in mammals. Thus, we present a cardiac phenotype associated with Drosophila SERCA dysfunction that would serve as additional phenotyping for further large-scale genetic screens for novel modifiers of SERCA function.