Myocardial Alternative RNA Splicing and Gene Expression Profiling in Early Stage Hypoplastic Left Heart Syndrome

Hypoplastic Left Heart Syndrome (HLHS) is a congenital defect characterized by underdevelopment of the left ventricle and pathological compensation of the right ventricle. If untreated, HLHS is invariably lethal due to the extensive increase in right ventricular workload and eventual failure. Despite the clinical significance, little is known about the molecular pathobiological state of HLHS. Splicing of mRNA transcripts is an important regulatory mechanism of gene expression. Tissue specific alterations of this process have been associated with several cardiac diseases, however, transcriptional signature profiles related to HLHS are unknown. In this study, we performed genome-wide exon array analysis to determine differentially expressed genes and alternatively spliced transcripts in the right ventricle (RV) of six neonates with HLHS, compared to the RV and left ventricle (LV) from non-diseased control subjects. In HLHS, over 180 genes were differentially expressed and 1800 were differentially spliced, leading to changes in a variety of biological processes involving cell metabolism, cytoskeleton, and cell adherence. Additional hierarchical clustering analysis revealed that differential gene expression and mRNA splicing patterns identified in HLHS are unique compared to non-diseased tissue. Our findings suggest that gene expression and mRNA splicing are broadly dysregulated in the RV myocardium of HLHS neonates. In addition, our analysis identified transcriptome profiles representative of molecular biomarkers of HLHS that could be used in the future for diagnostic and prognostic stratification to improve patient outcome

Arrhythmogenic Calmodulin Mutations Impede Activation of Small-conductance Calcium-Activated Potassium Current

Background Apamin sensitive small-conductance Ca2+-activated K+ (SK) channels are gated by intracellular Ca2+ through a constitutive interaction with calmodulin. Objective We hypothesize that arrhythmogenic human calmodulin mutations impede activation of SK channels. Methods We studied 5 previously published calmodulin mutations (N54I, N98S, D96V, D130G and F90L). Plasmids encoding either wild type (WT) or mutant calmodulin were transiently transfected into human embryonic kidney (HEK) 293 cells that stably express SK2 channels (SK2 Cells). Whole-cell voltage-clamp recording was used to determine apamin-sensitive current (IKAS) densities. We also performed optical mapping studies in normal murine hearts to determine the effects of apamin in hearts with (N=7) or without (N=3) pretreatment with sea anemone toxin (ATX II). Results SK2 cells transfected with WT calmodulin exhibited IKAS density (in pA/pF) of 33.6 [31.4;36.5] (median and confidence interval 25%-75%), significantly higher than that observed for cells transfected with N54I (17.0 [14.0;27.7], p=0.016), F90L (22.6 [20.3;24.3], p=0.011), D96V (13.0 [10.9;15.8], p=0.003), N98S (13.7 [8.8;20.4], p=0.005) and D130G (17.6 [13.8;24.6], p=0.003). The reduction of SK2 current was not associated with a decrease in membrane protein expression or intracellular distribution of the channel protein. Apamin increased the ventricular APD80 (from 79.6 ms [63.4-93.3] to 121.8 ms [97.9-127.2], p=0.010) in hearts pre-treated with ATX-II but not in control hearts. Conclusion Human arrhythmogenic calmodulin mutations impede the activation of SK2 channels in HEK 293 cells

Genetic Evaluation of Cardiomyopathy - a Heart Failure Society of America Practice Guideline

This guideline describes the approach and expertise needed for the genetic evaluation of cardiomyopathy. First published in 2009 by the Heart Failure Society of America (HFSA), this guidance has now been updated in collaboration with the American College of Medical Genetics and Genomics (ACMG). The writing group, composed of cardiologists and genetics professionals with expertise in adult and pediatric cardiomyopathy, reflects the emergence and increased clinical activity devoted to cardiovascular genetic medicine. The genetic evaluation of cardiomyopathy is a rapidly emerging key clinical priority, as high throughput sequencing is now feasible for clinical testing, and conventional interventions can improve survival, reduce morbidity, and enhance quality of life. Moreover, specific interventions may be guided by genetic analysis. A systematic approach is recommended: always a comprehensive family history; an expert phenotypic evaluation of the proband and at-risk family members to confirm a diagnosis and guide genetic test selection and interpretation; referral to expert centers as needed; genetic testing, with pre- and post-test genetic counseling; and specific guidance as indicated for drug and device therapies. The evaluation of infants and children demands special expertise. The approach to manage secondary and incidental sequence findings as recommended by the ACMG is provided

Distribution of killer cell immunoglobulin-like receptors genes in the Italian Caucasian population

BACKGROUND: Killer cell immunoglobulin-like receptors (KIRs) are a family of inhibitory and activatory receptors that are expressed by most natural killer (NK) cells. The KIR gene family is polymorphic: genomic diversity is achieved through differences in gene content and allelic polymorphism. The number of KIR loci has been reported to vary among individuals, resulting in different KIR haplotypes. In this study we report the genotypic structure of KIRs in 217 unrelated healthy Italian individuals from 22 immunogenetics laboratories, located in the northern, central and southern regions of Italy. METHODS: Two hundred and seventeen DNA samples were studied by a low resolution PCR-SSP kit designed to identify all KIR genes. RESULTS: All 17 KIR genes were observed in the population with different frequencies than other Caucasian and non-Caucasian populations; framework genes KIR3DL3, KIR3DP1, KIR2DL4 and KIR3DL2 were present in all individuals. Sixty-five different profiles were found in this Italian population study. Haplotype A remains the most prevalent and genotype 1, with a frequency of 28.5%, is the most commonly observed in the Italian population. CONCLUSION: The Italian Caucasian population shows polymorphism of the KIR gene family like other Caucasian and non-Caucasian populations. Although 64 genotypes have been observed, genotype 1 remains the most frequent as already observed in other populations. Such knowledge of the KIR gene distribution in populations is very useful in the study of associations with diseases and in selection of donors for haploidentical bone marrow transplantation

Medium-Chain Polyprenols Influence Chloroplast Membrane Dynamics In Solanum Lycopersicum

The widespread occurrence of polyprenols throughout the plant kingdom is well documented, yet their functional role is poorly understood. These lipophilic compounds are known to be assembled from isoprenoid precursors by a class of enzymes designated as cisprenyltransferases (CPTs), which are encoded by small CPT gene families in plants. In this study, we report that RNAi-mediated knockdown of one member of the tomato CPT family (SlCPT5) reduced polyprenols in leaves by ~70%. Assays with recombinant SlCPT5 produced in E. coli determined that the enzyme synthesizes polyprenols of approximately 50-55 carbons (Pren-10, Pren-11) in length and accommodates a variety of trans-prenyldiphosphate precursors as substrates. Introduction of SlCPT5 into the polyprenol-deficient yeast Δrer2 mutant resulted in the accumulation of Pren-11 in yeast cells, restored proper protein Nglycosylation, and rescued the temperature sensitive growth phenotype that is associated with its polyprenol deficiency. Subcellular fractionation studies together with in vivo localization of SlCPT5 fluorescent protein fusions demonstrated that SlCPT5 resides in the chloroplast stroma and that its enzymatic products accumulate into both thylakoid and envelope membranes. Transmission electron microscopy images of polyprenol-deficient leaves revealed alterations in chloroplast ultrastructure and anisotropy measurements revealed a more disordered state of their envelope membranes. In polyprenol-deficient leaves, CO2 assimilation was hindered and their thylakoid membranes exhibited lower phase transition temperatures and calorimetric enthalpies, which coincided with a decreased photosynthetic electron transport rate. Taken together, these results uncover a role for polyprenols in governing chloroplast membrane dynamics

Clusters of Sudden Unexplained Death Associated with the Mushroom, Trogia venenata, in Rural Yunnan Province, China

INTRODUCTION: Since the late 1970's, time-space clusters of sudden unexplained death (SUD) in northwest Yunnan, China have alarmed the public and health authorities. From 2006-2009, we initiated enhanced surveillance for SUD to identify a cause, and we warned villagers to avoid eating unfamiliar mushrooms. METHODS: We established surveillance for SUD, defined as follows: sudden onset of serious, unexplained physical impairment followed by death in <24 hours. A mild case was onset of any illness in a member of the family or close socially related group of a SUD victim within 1 week of a SUD. We interviewed witnesses of SUD and mild case-persons to identify exposures to potentially toxic substances. We tested blood from mild cases, villagers, and for standard biochemical, enzyme, and electrolyte markers of disease. RESULTS: We identified 33 SUD, a 73% decline from 2002-2005, distributed among 21 villages of 11 counties. We found a previously undescribed mushroom, Trogia venenata, was eaten by 5 of 7 families with SUD clusters compared to 0 of 31 other control-families from the same villages. In T. venenata-exposed persons SUD was characterized by sudden loss of consciousness during normal activities. This mushroom grew nearby 75% of 61 villages that had time-space SUD clusters from 1975 to 2009 compared to 17% of 18 villages with only single SUD (p<0.001, Fisher's exact test). DISCUSSION: Epidemiologic data has implicated T. venenata as a probable cause of clusters of SUD in northwestern Yunnan Province. Warnings to villagers about eating this mushroom should continue

Standardisation of magnetic nanoparticles in liquid suspension

Suspensions of magnetic nanoparticles offer diverse opportunities for technology innovation, spanning a large number of industry sectors from imaging and actuation based applications in biomedicine and biotechnology, through large-scale environmental remediation uses such as water purification, to engineering-based applications such as position-controlled lubricants and soaps. Continuous advances in their manufacture have produced an ever-growing range of products, each with their own unique properties. At the same time, the characterisation of magnetic nanoparticles is often complex, and expert knowledge is needed to correctly interpret the measurement data. In many cases, the stringent requirements of the end-user technologies dictate that magnetic nanoparticle products should be clearly defined, well characterised, consistent and safe; or to put it another way—standardised. The aims of this document are to outline the concepts and terminology necessary for discussion of magnetic nanoparticles, to examine the current state-of-the-art in characterisation methods necessary for the most prominent applications of magnetic nanoparticle suspensions, to suggest a possible structure for the future development of standardisation within the field, and to identify areas and topics which deserve to be the focus of future work items. We discuss potential roadmaps for the future standardisation of this developing industry, and the likely challenges to be encountered along the way