Sudden unexplained death may be the first manifestation of an unknown inherited cardiac disease. Current genetic technologies may enable the unraveling of an etiology and the identification of ...relatives at risk. The aim of our study was to define the etiology of natural deaths, younger than 50 years of age, and to investigate whether genetic defects associated with cardiac diseases could provide a potential etiology for the unexplained cases.
Our cohort included a total of 789 consecutive cases (77.19% males) <50 years old (average 38.6±12.2 years old) who died suddenly from non-violent causes. A comprehensive autopsy was performed according to current forensic guidelines. During autopsy a cause of death was identified in most cases (81.1%), mainly due to cardiac alterations (56.87%). In unexplained cases, genetic analysis of the main genes associated with sudden cardiac death was performed using Next Generation Sequencing technology. Genetic analysis was performed in suspected inherited diseases (cardiomyopathy) and in unexplained death, with identification of potentially pathogenic variants in nearly 50% and 40% of samples, respectively.
Cardiac disease is the most important cause of sudden death, especially after the age of 40. Close to 10% of cases may remain unexplained after a complete autopsy investigation. Molecular autopsy may provide an explanation for a significant part of these unexplained cases. Identification of genetic variations enables genetic counseling and undertaking of preventive measures in relatives at risk.
All eukaryotic CLC Cl â channel subunits possess a long cytoplasmic carboxy-terminus that contains two so-called CBS (cystathionine β-synthase) domains.
These domains are found in various ...unrelated proteins from all phylae. The crystal structure of the CBS domains of inosine
monophosphate dehydrogenase (IMPDH) is known, but it is not known whether this structure is conserved in CLC channels. Working
primarily with ClC-1, we used deletion scanning mutagenesis, coimmunoprecipitation and electrophysiology to demonstrate that
its CBS domains interact. The replacement of CBS domains of ClC-1 with the corresponding CBS domains from other CLC channels
and even human IMPDH yielded functional channels, indicating a high degree of structural conservation. Based on a homology
model of the pair of CBS domains of CLC channels, we identified some residues that, when mutated, affected the common gate
which acts on both pores of the dimeric channel. Thus, we propose that the structure of CBS domains from CLC channels is highly
conserved and that they play a functional role in the common gate.
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.
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.
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.
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.
Highlights • Molecular autopsy should be implemented in forensic protocols. • Nearly 40% of sudden death young cases carry a cardiac potentially pathogenic variant. • It is crucial to undertake a ...careful genetic analysis in a clinical context. • Genetic analyses help to identify relatives at risk of sudden death.
Brugada syndrome (BrS) is a form of cardiac arrhythmia which may lead to sudden cardiac death. The recommended genetic testing (direct sequencing of SCN5A) uncovers disease-causing SNVs and/or indels ...in ~20% of cases. Limited information exists about the frequency of copy number variants (CNVs) in SCN5A in BrS patients, and the role of CNVs in BrS-minor genes is a completely unexplored field.
220 BrS patients with negative genetic results were studied to detect CNVs in SCN5A. 63 cases were also screened for CNVs in BrS-minor genes. Studies were performed by Multiplex ligation-dependent probe amplification or Next-Generation Sequencing (NGS).
The detection rate for CNVs in SCN5A was 0.45% (1/220). The detected imbalance consisted of a duplication from exon 15 to exon 28, and could potentially explain the BrS phenotype. No CNVs were found in BrS-minor genes.
CNVs in current BrS-related genes are uncommon among BrS patients. However, as these rearrangements may underlie a portion of cases and they undergo unnoticed by traditional sequencing, an appealing alternative to conventional studies in these patients could be targeted NGS, including in a single experiment the study of SNVs, indels and CNVs in all the known BrS-related genes.
Selection of amino acid substitutions associated with resistance to nucleos(t)ide-analog (NA) therapy in the hepatitis B virus (HBV) reverse transcriptase (RT) and their combination in a single viral ...genome complicates treatment of chronic HBV infection and may affect the overlapping surface coding region. In this study, the variability of an overlapping polymerase-surface region, critical for NA resistance, is investigated before treatment and under antiviral therapy, with assessment of NA-resistant amino acid changes simultaneously occurring in the same genome (linkage analysis) and their influence on the surface coding region.
Serum samples obtained from chronic HBV-infected patients at pre-treatment and during sequential NA treatment with lamivudine, adefovir, and entecavir were analyzed by ultra-deep pyrosequencing (UDPS) using the GS-FLX platform (454 Life Sciences-Roche). The pre-treatment HBV quasispecies was not enriched with NA-resistant substitutions. The frequencies of this type of substitutions at pre-treatment did not predict the frequencies observed during lamivudine treatment. On linkage analysis of the RT region studied, NA-resistant HBV variants (except for rtA181T) were present in combinations of amino acid substitutions that increased in complexity after viral breakthrough to entecavir, at which time the combined variant rtL180M-S202G-M204V-V207I predominated. In the overlapping surface region, NA-resistant substitutions caused selection of stop codons in a significant percentage of sequences both at pre-treatment and during sequential treatment; the rtA181T substitution, related to sW172stop, predominated during treatment with lamivudine and adefovir. A highly conserved RT residue (rtL155), even more conserved than the essential residues in the RT catalytic motif YMDD, was identified in all samples.
UDPS methodology enabled quantification of HBV quasispecies variants, even those harboring complex combinations of amino acid changes. The high percentage of potentially defective genomes, especially in the surface region, suggests effective trans-complementation of these variants.
Three asymptomatic members had inherited the 3 variants and had abnormal CMR: the mother's CMR (II.2) had areas of right ventricular aneurisms, dyskinesia, and patches of interstitial fibrosis; the ...youngest uncle's CMR (II.6) had biventricular dilation, with no fibrosis or fatty infiltration; and the grandmother's CMR (I.1) had areas of RV dyskinesia and aneurysms with patchy fibrosis. ...the only segregating pathogenic variant was DSP (p.E290K). Myocarditis is an inflammatory process, not suspected to be of familial origin. ...genetic analyses are not considered in myocarditis.
After decades of using urea as denaturant, the kinetic role of this molecule in the unfolding process is still undefined: does urea actively induce protein unfolding or passively stabilize the ...unfolded state? By analyzing a set of 30 proteins (representative of all native folds) through extensive molecular dynamics simulations in denaturant (using a range of force-fields), we derived robust rules for urea unfolding that are valid at the proteome level. Irrespective of the protein fold, presence or absence of disulphide bridges, and secondary structure composition, urea concentrates in the first solvation shell of quasi-native proteins, but with a density lower than that of the fully unfolded state. The presence of urea does not alter the spontaneous vibration pattern of proteins. In fact, it reduces the magnitude of such vibrations, leading to a counterintuitive slow down of the atomic-motions that opposes unfolding. Urea stickiness and slow diffusion is, however, crucial for unfolding. Long residence urea molecules placed around the hydrophobic core are crucial to stabilize partially open structures generated by thermal fluctuations. Our simulations indicate that although urea does not favor the formation of partially open microstates, it is not a mere spectator of unfolding that simply displaces to the right of the folded ←→ unfolded equilibrium. On the contrary, urea actively favors unfolding: it selects and stabilizes partially unfolded microstates, slowly driving the protein conformational ensemble far from the native one and also from the conformations sampled during thermal unfolding.
Sudden unexpected death in epilepsy is an unpredicted condition in patients with a diagnosis of epilepsy, and autopsy does not conclusively identify cause of death. Although the pathophysiological ...mechanisms that underlie this entity remain unknown, the fact that epilepsy can affect cardiac function is not surprising. The genetic factors involving ion channels co-expressed in the heart and brain and other candidate genes have been previously described. In the present study, 20 epilepsy patients with personal or family history of heart rhythm disturbance/cardiac arrhythmias/sudden death were sequenced using a custom re-sequencing panel. Twenty-six relatives were genetically analysed to ascertain the family segregation in ten individuals. Four subjects revealed variants with positive genotype-phenotype segregation: four missense variants in the CDKL5, CNTNAP2, GRIN2A and ADGRV1 genes and one copy number variant in KCNQ1. The potential pathogenic role of variants in new candidate genes will need further studies in larger cohorts, and the evaluation of the potential pathogenic role in the cardio-cerebral mechanisms requires in vivo/in vitro studies. In addition to family segregation, evaluation of the potential pathogenic roles of these variants in cardio-cerebral mechanisms by in vivo/in vitro studies should also be performed. The potential pathogenic role of variants in new candidate genes will need further studies in larger cohorts.
consensus view of protein dynamics Rueda, Manuel; Ferrer-Costa, Carles; Meyer, Tim ...
Proceedings of the National Academy of Sciences - PNAS,
01/2007, Letnik:
104, Številka:
3
Journal Article
Recenzirano
Odprti dostop
The dynamics of proteins in aqueous solution has been investigated through a massive approach based on "state of the art" molecular dynamics simulations performed for all protein metafolds using the ...four most popular force fields (OPLS, CHARMM, AMBER, and GROMOS). A detailed analysis of the massive database of trajectories (>1.5 terabytes of data obtained using almost equal to50 years of CPU) allowed us to obtain a robust-consensus picture of protein dynamics in aqueous solution.
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