Biophysical phenotypes of SCN5A mutations causing long QT and Brugada syndromes

Long QT and Brugada syndromes are two hereditary cardiac diseases. Brugada syndrome has so far been associated with only one gene, SCN5A, which encodes the cardiac sodium channel. However, in long QT syndrome (LQTS) at least six genes, including the SCN5A, are implicated. The substitution (D1790G) causes LQTS and the insertion (D1795) induces both LQTS and Brugada syndromes in carrier patients. hH1/insD1795 and hH1/D1790G mutant channels were expressed in the tsA201 human cell line and characterized using the patch clamp technique in whole-cell configuration. Our data revealed a persistent inward sodium current of about 6% at -30 mV for both D1790G and insD1795, and a reduction of 62% of channel expression for the insD1795. Moreover, a shift of steady-state inactivation curve in both mutants was also observed. Our findings uphold the idea that LQT3 is related to a persistent sodium current whereas reduction in the expression level of cardiac sodium channels is one of the biophysical characteristics of Brugada syndrome.     

A candidate gene for hemochromatosis: frequency of the C282Y and H63D mutations

The gene whose alteration causes hereditary hemochromatosis (HFE according to the international nomenclature) was, more than 20 years ago, shown to map to 6p21.3. It has since escaped all efforts to identify it by positional cloning strategies. Quite recently, a gene named HLA-H was reported as being responsible for the disease. Two missense mutations, Cys282Tyr (C282Y) and His63Asp (H63D), were observed, but no proof was produced that the gene described is the hemochromatosis gene. To validate this gene as the actual site of the alteration causing hemochromatosis, we decided to look for the two mutations in 132 unrelated patients from Brittany. Our results indicate that more than 92% of these patients are homozygous for the C282Y mutation, and that all 264 chromosomes but 5 carry either mutation. These findings confirm the direct implication of HLA-H in hemochromatosis. Received: 16 December 1996 / Accepted: 13 May 1997     

In silico Investigation of the Disease-Associated Retinoschisin C110Y and C219G Mutants

The juvenile X-linked retinoschisis (XLRS) is a retinal disease caused by mutations in the secretory protein, retinoschisin (RS1). Majority of the disease is resulted from single point mutations on the RS1 discoidin domain with cysteine mutations being related to some of the more severe cases of XLRS. Previous studies have indicated that two mutations (C110Y and C219G), which involve cysteines that form intramolecular disulfide bonds in the native discoidin domain, resulted in different oligomerization states of the proteins and did not correlate with the degree of protein stability as calculated by the change in folding free energy. Through homology modeling, bioinformatics predictions, molecular dynamics (MD) and docking simulations, we attempt to investigate the effects of these two mutations on the structure of the RS1 discoidin domain in relevance to the discrepancy found between structural stability and aggregation propensity. Based on our findings, this discrepancy can be explained by the ability of C110Y mutant to establish suitable modules for initiating amorphous aggregation and to expand the aggregating mass through predominantly hydrophobic interactions. The low capability of C219G mutant to oligomerize, on the other hand, may be due to its greater structural instability and lesser hydrophobic tendency, two properties that may be unsupportive of aggregation. The results, altogether, indicate that aggregation propensity in the RS1 C110Y mutant is dependent upon the formation of suitable aggregating substrates for propagation of aggregation and not directly related to or determined by overall structural instability. As for the wildtype protein, the binding specificity of the spikes for biological function and the formation of octameric structure are contributed by important loop interactions, as well as evolved structural and sequence-based properties that prevent aggregation.     

Geography of HFE C282Y and H63D mutations

Hereditary hemochromatosis (HH) is a common autosomal recessive disorder causing inappropriate dietary iron absorption that affects North Europeans. HH is associated with the C282Y mutation of the HFE gene, and the H63D mutation to a lesser degree. Both mutations are abundant in Europe, with H63D also appearing in North Africa, the Middle East, and Asia. Emigration from Europe over the past 500 years has introduced C282Y and H63D to America, Australia, New Zealand, and South Africa in an essentially predictable fashion. The distinctive characteristics of the population genetics of HH are the confined racial distribution and high frequency in North European peoples. C282Y frequencies in North Europeans are typically between 5% and 10%, with homozygotes accounting for between 1/100 and 1/400 of these populations. The scarcity of the C282Y mutation in other populations accounts for the lack of HH in non-Europeans.     

In silico risk assessment for drug-induction of cardiac arrhythmia

The main components of repolarization reserve for the ventricular action potential (AP) are the rapid (I_Kr) and slow (I_Ks) delayed outward K⁺ currents. While many drugs block I_Kr and cause life-threatening arrhythmias including torsades de pointes, the frequency of arrhythmias varies between different I_Kr-blockers. Different types of block of I_Kr cause distinct phenotypes of prolongation of action potential duration (APD), increase in transmural dispersion of repolarization (TDR) and, accordingly, occurrence of torsades de pointes. Therefore the assessment of a drug's proarrhythmic risk requires a method that provides quantitative and comprehensive comparison of the effects of different forms of I_Kr-blockade upon APDs and TDR. However, most currently available methods are not adapted to such an extensive comparison. Here, we introduce I_Kr–I_Ks two-dimensional maps of APD and TDR as a novel risk-assessment method. Taking the kinetics of I_Kr-blockade into account, APDs can be calculated upon a ventricular AP model which systematically alters the magnitudes of I_Kr and I_Ks. The calculated APDs are then plotted on a map where the x axis represents the conductance of I_Kr while the y axis represents that of I_Ks. TDR is simulated with models corresponding to APs in epicardial, midcardial and endocardial myocardium. These two-dimensional maps of APD and TDR successfully account for differences in the risk resulting from three distinct types of I_Kr-blockade which correspond to the effects of dofetilide, quinidine and vesnarinone. This method may be of use to assess the arrhythmogenic risk of various I_Kr-blockers.     

Frequency of hemochromatosis C282Y and H63D mutations in Sardinia

Hereditary hemochromatosis (HH) is one of the most common autosomal recessive disorders of iron metabolism among Caucasians, and it is associated with C282Y mutation of the HFE gene in populations of Celtic origins. A second mutation, H63D, shows a very high widespread frequency, although its role in iron metabolism is still inconclusive. There are no data on the frequencies of these two mutations in Sardinia, an island in the Mediterranean sea that has not been invaded by Celtic peoples. We examined 836 chromosomes from Sardinian subjects and tested for the mutation by restriction enzyme digestion of PCR products. Among the 836 analyzed chromosomes, we found a C282Y allele frequency of 0.0036 and an H63D allele frequency of 0.173. These data could explain the observed rarity of HH in Sardinia. The high allele frequency of H63D and the rarity of HH in Sardinia is suggestive that this mutation is not a major contributor to this disease.     

Frequency of HFE gene mutations C282Y and H63D in Bosnia and Herzegovina

Genetic epidemiology studies of hereditary hemochromatosis (HHC) have shown a high prevalence of the C282Y mutation in individuals of the North Western European origin, whereas lower prevalence of HFE gene mutations was detected in the populations from southern European countries. However, no HFE mutation prevalence data have been provided for the population of Bosnia-Herzegovina so far. Therefore, the aim of this study was to determine the frequency of the C282Y and H63D HFE gene mutations in the population of Bosnia-Herzegovina. Among 200 analysed subjects 8 (4%) were C282Y heterozygotes; no C282Y homozygotes were found. The frequency of the H63D allele was 11.5%. There were 33 (16.5%) heterozygotes and 6 (3%) homozygotes for the H63D mutation. One (0.5%) compound heterozygote C282Y/H63D was identified. The observed C282Y and H63D allele frequency was 2.25% (95% confidence interval: 1.2-4.2) and 11.5% (95% confidence interval: 8.7-14.9), respectively. The prevalence of the C282Y and H63D mutations was estimated in Bosnia-Herzegovina, which fit well in the European northwest-to-southeast gradient of the C282Y mutation distribution. In addition, these results have an important implication for clinical evaluation of HHC in Bosnia-Herzegovina.     

In silico analysis of SLC3A1 and SLC7A9 mutations in Iranian patients with Cystinuria

Molecular Biology Reports - Cystinuria is an autosomal recessive defect in reabsorptive transport of cystine and the dibasic amino acids ornithine, arginine, and lysine from renal tubule and small...     

Y1767C, a novel SCN5A mutation, induces a persistent Na+ current and potentiates ranolazine inhibition of Nav1.5 channels

Long QT syndrome type 3 (LQT3) has been traced to mutations of the cardiac Na(+) channel (Na(v)1.5) that produce persistent Na(+) currents leading to delayed ventricular repolarization and torsades de pointes. We performed mutational analyses of patients suffering from LQTS and characterized the biophysical properties of the mutations that we uncovered. One LQT3 patient carried a mutation in the SCN5A gene in which the cysteine was substituted for a highly conserved tyrosine (Y1767C) located near the cytoplasmic entrance of the Na(v)1.5 channel pore. The wild-type and mutant channels were transiently expressed in tsA201 cells, and Na(+) currents were recorded using the patch-clamp technique. The Y1767C channel produced a persistent Na(+) current, more rapid inactivation, faster recovery from inactivation, and an increased window current. The persistent Na(+) current of the Y1767C channel was blocked by ranolazine but not by many class I antiarrhythmic drugs. The incomplete inactivation, along with the persistent activation of Na(+) channels caused by an overlap of voltage-dependent activation and inactivation, known as window currents, appeared to contribute to the LQTS phenotype in this patient. The blocking effect of ranolazine on the persistent Na(+) current suggested that ranolazine may be an effective therapeutic treatment for patients with this mutation. Our data also revealed the unique role for the Y1767 residue in inactivating and forming the intracellular pore of the Na(v)1.5 channel.     

Characterization of the novel heterozygous SCN5A genetic variant Y739D associated with Brugada syndrome

Genetic variants in SCN5A gene were identified in patients with various arrhythmogenic conditions including Brugada syndrome. Despite significant progress of last decades in studying the molecular mechanism of arrhythmia-associated SCN5A mutations, the understanding of relationship between genetics, electrophysiological consequences and clinical phenotype is lacking. We have found a novel genetic variant Y739D in the SCN5A-encoded sodium channel Na_v1.5 of a male patient with Brugada syndrome (BrS). The objective of the study was to characterize the biophysical properties of Na_v1.5-Y739D and provide possible explanation of the phenotype observed in the patient. The WT and Y739D channels were heterologously expressed in the HEK-293T cells and the whole-cell sodium currents were recorded. Substitution Y739D reduced the sodium current density by 47 ± 2% at ?20 mV, positively shifted voltage-dependent activation, accelerated both fast and slow inactivation, and decelerated recovery from the slow inactivation. The Y739D loss-of-function phenotype likely causes the BrS manifestation. In the hNa_v1.5 homology models, which are based on the cryo-EM structure of rat Na_v1.5 channel, Y739 in the extracellular loop IIS1-S2 forms H-bonds with K1381 and E1435 and pi-cation contacts with K1397 (all in loop IIIS5-P1). In contrast, Y739D accepts H-bonds from K1397 and Y1434. Substantially different contacts of Y739 and Y739D with loop IIIS5-P1 would differently transmit allosteric signals from VSD-II to the fast-inactivation gate at the N-end of helix IIIS5 and slow-inactivation gate at the C-end of helix IIIP1. This may underlie the atomic mechanism of the Y739D channel dysfunction.     

Mutation analysis in congenital Long QT Syndrome--a case with missense mutations in KCNQ1 and SCN5A

Long QT Syndrome (LQTS) is a cardiac disease characterized by a prolonged QT interval on a surface electrocardiogram (ECG) and by clinical symptoms such as seizures, syncope, and cardiac sudden death. At present, causal mutations of LQTS have been identified in five cardiac ion-channel genes. Because a causal mutation is usually unique to a specific family and can be located in any region of any of these five genes, a mutation analysis effort may require screening of the complete coding regions of each of these genes. The causative nature of a detected mutation can then be determined either by family history or by functional studies, such as the electrophysiological signature of the mutation. Here we describe a mutation analysis of an LQTS patient who carries two heterozygous missense mutations in two different LQTS genes. The first mutation identified, A572D in SCN5A, was not linked with clinical LQTS features in the two other mutation carriers in the family; neither was it identified in 90 healthy controls. Therefore, this mutation most likely has either a mild effect on cardiac ion-channel function or represents a very rare polymorphism. The second mutation, V254M in KCNQ1, co-segregated with higher QT intervals and symptoms in other family members, and was previously reported in another LQTS family. Because the clinical LQTS symptoms are most pronounced in the proband, a combined effect of both mutations cannot be excluded, although no functional data are available to support such an hypothesis. We conclude that, for newly presented LQTS cases, a mutation analysis strategy should routinely screen the complete coding region of all LQTS genes, followed by an evaluation of the identified mutation(s) in conjunction with family or functional data.     

In silico assessment of S100A12 monomer and dimer structural dynamics: implications for the understanding of its metal-induced conformational changes

Changes in the concentration of different ions modulate several cellular processes, such as Ca²⁺ and Zn²⁺ in inflammation. Upon activation of immune system effector cells, the intracellular Ca²⁺ concentration rises propagating the activation signal, leading to degranulation and generation of reactive oxygen species, which increases the Zn²⁺ intracellular concentration as a consequence of the cellular antioxidant machinery. In this context, S100A12 is of special interest because it is a pro-inflammatory protein expressed in neutrophils whose structure and function are modulated by both Ca²⁺ and Zn²⁺. The current hypothesis about its mechanism of action was built based on biochemical and crystallographic data. However, there are missing connections between molecular structure and the way in which many events are concatenated at the triggering and along the inflammatory process. In this work we use molecular dynamics simulations to describe how variations in Zn²⁺ and Ca²⁺ concentrations modulate the structural dynamics of the calcium-free S100A12 dimer and monomer, which was not considered a part of the mechanism of action before. Our results suggest that (i) Zn²⁺ have a determinant role in the dimerization step, as well as in the unbinding of the Na⁺ complexed to the N-terminal EF-hand; (ii) the N-terminal EF-hand domain is the first to bind Ca²⁺, and not the C-terminal, as usually accepted; and that (iii) Ca²⁺ modulates the structural dynamics of H-III.     

In silico insights on tankyrase protein: A potential target for colorectal cancer

Abstract

The Wnt/β-catenin pathway plays an important regulatory role in cancer signaling and cell regenerative mechanisms. Its suppression has long been considered as an important challenge of anticancer treatment and management. The poly(ADP-ribose) polymerase (PARP) family represented as a new class of therapeutic targets with diverse potential disease indications. Tankyrase (TNKS) is considered to be a potential target for the intervention of various cancers. The main objective of the work is to explore the molecular and quantum mechanics of the drug-like compounds and to identify the potential inhibitors for TNKS protein using the structure and ligand-based virtual screening from several databases and to explore the binding pocket and interactions of active residues. The screened compounds were further filtered using binding-free energy calculation and molecular dynamics simulation studies. The results have provided a strong molecular knowledge of TNKS and offered top hit potent inhibitors. The identified lead compounds LC_40781, LC_40777, LC_39767, LC_8346, NCI_682438, and NCI_721141 were observed to have potent activity against TNKS protein. The hydrogen bonding of compounds with Asp1198, His1201, Tyr1203 in TNKS1 and Gly1032, Ser1068 in TNKS2 are the key interactions plays a major role in binding energy. Therefore, the outcome of the study would help for further validation and provides valuable information to guide the future TNKS-specific inhibitor designing.

Communicated by Ramaswamy H. Sarma     

Matrix protein 1: A comparative in silico study on different strains of influenza A H5N1 Virus

The importance of influenza viruses as worldwide infectious agents is well recognized. Specific mutations and evolution in influenza viruses is difficult to predict. We studied specific mutations in matrix protein 1 (M1) of H5N1 influenza A virus together with properties associated with it using prediction tools developed in Bioinformatics. Changes in hydrophobicity, polarity and secondary structure at the site of mutation were noticed and documented to gain insight towards its infection.     

In vitro and in silico analysis reveals an efficient algorithm to predict the splicing consequences of mutations at the 5' splice sites

We have found that two previously reported exonic mutations in the PINK1 and PARK7 genes affect pre-mRNA splicing. To develop an algorithm to predict underestimated splicing consequences of exonic mutations at the 5′ splice site, we constructed and analyzed 31 minigenes carrying exonic splicing mutations and their derivatives. We also examined 189 249 U2-dependent 5′ splice sites of the entire human genome and found that a new variable, the SD-Score, which represents a common logarithm of the frequency of a specific 5′ splice site, efficiently predicts the splicing consequences of these minigenes. We also employed the information contents (R_i) to improve the prediction accuracy. We validated our algorithm by analyzing 32 additional minigenes as well as 179 previously reported splicing mutations. The SD-Score algorithm predicted aberrant splicings in 198 of 204 sites (sensitivity = 97.1%) and normal splicings in 36 of 38 sites (specificity = 94.7%). Simulation of all possible exonic mutations at positions −3, −2 and −1 of the 189 249 sites predicts that 37.8, 88.8 and 96.8% of these mutations would affect pre-mRNA splicing, respectively. We propose that the SD-Score algorithm is a practical tool to predict splicing consequences of mutations affecting the 5′ splice site.     

Prevalence of C282Y and H63D mutations in the haemochromatosis (HFE) gene in Tunisian population

The studies of the HFE mutations: H63D and C282Y in North African populations have revealed the extreme rarity or even the absence of the C282Y mutation. We have examined 1140 chromosomes (570 Tunisian people) for the presence of the two HFE mutations by PCR-RFLP analysis. We have found that the allele frequencies are, respectively, 15.17% (+/-2.1%) for the H63D and 0.09% (+/-0.17%) for the C282Y. These results are consistent with the worldwide spread of the H63D mutation and the north European restriction of the C282Y. This study will be completed by determining whether homozygote trait for H63D and associated risk factors (beta thalassémia) can lead to iron overload in Tunisia.