Disease-specific variant pathogenicity prediction significantly improves variant interpretation in inherited cardiac conditions.

PURPOSE: Accurate discrimination of benign and pathogenic rare variation remains a priority for clinical genome interpretation. State-of-the-art machine learning variant prioritization tools are imprecise and ignore important parameters defining gene-disease relationships, e.g., distinct consequences of gain-of-function versus loss-of-function variants. We hypothesized that incorporating disease-specific information would improve tool performance. METHODS: We developed a disease-specific variant classifier, CardioBoost, that estimates the probability of pathogenicity for rare missense variants in inherited cardiomyopathies and arrhythmias. We assessed CardioBoost's ability to discriminate known pathogenic from benign variants, prioritize disease-associated variants, and stratify patient outcomes. RESULTS: CardioBoost has high global discrimination accuracy (precision recall area under the curve [AUC] 0.91 for cardiomyopathies; 0.96 for arrhythmias), outperforming existing tools (4-24% improvement). CardioBoost obtains excellent accuracy (cardiomyopathies 90.2%; arrhythmias 91.9%) for variants classified with >90% confidence, and increases the proportion of variants classified with high confidence more than twofold compared with existing tools. Variants classified as disease-causing are associated with both disease status and clinical severity, including a 21% increased risk (95% confidence interval [CI] 11-29%) of severe adverse outcomes by age 60 in patients with hypertrophic cardiomyopathy. CONCLUSIONS: A disease-specific variant classifier outperforms state-of-the-art genome-wide tools for rare missense variants in inherited cardiac conditions ( https://www.cardiodb.org/cardioboost/ ), highlighting broad opportunities for improved pathogenicity prediction through disease specificity.

Nationwide study on hypertrophic cardiomyopathy in Iceland: evidence of a MYBPC3 founder mutation.

BACKGROUND: The geographic isolation and homogeneous population of Iceland are ideally suited to ascertain clinical and genetic characteristics of hypertrophic cardiomyopathy (HCM) at the population level. METHODS AND RESULTS: Medical records and cardiac imaging studies obtained between 1997 and 2010 were reviewed to identify Icelandic patients with HCM. Surviving patients were recruited for clinical and genetic studies. A previously identified Icelandic mutation, MYBPC3 c.927-2A>G, was genotyped, and mutation-negative samples were sequenced for HCM genes and other hypertrophic genes. Record review identified 180 patients with HCM. Genetic analyses of 151 patients defined pathogenic mutations in 101 (67%), including MYBPC3 c.927-2A>G (88 patients, 58%), 4 other MYBPC3 or MYH7 mutations (5 patients, 3.3%), and 2 GLA mutations (8 patients, 5.3%). Haplotype and genetic genealogical data defined MYBPC3 c.927-2A>G as a founder mutation, introduced into the Icelandic population in the 15th century, with a current population prevalence of 0.36%. MYBPC3 c.927-2A>G mutation carriers exhibited phenotypic diversity but were younger at diagnosis (42 versus 49 years; P=0.001) and sustained more adverse events (15% versus 2%; P=0.02) than mutation-negative patients. All-cause mortality for patients with HCM was similar to that of an age-matched Icelandic population (hazard ratio, 0.98; P=0.9). HCM-related mortality (0.78%/y) occurred at a mean age of 68 compared with 81 years for non-HCM-related mortality (P=0.02). CONCLUSIONS: A founder MYBPC3 mutation that arose >550 years ago is the predominant cause of HCM in Iceland. The MYBPC3 c.927-2A>G mutation is associated with low adverse event rates but earlier cardiovascular mortality, illustrating the impact of genotype on outcomes in HCM.

Designing small, nonsugar activators of antithrombin using hydropathic interaction analyses.

Conformational activation of antithrombin is a critical mechanism for the inhibition of factor Xa, a proteinase of the blood coagulation cascade, and is typically achieved with heparin, a polyanionic polysaccharide clinically used for anticoagulation. Although numerous efforts have been directed toward the design of better activators, a fundamental tenet of these studies has been the assumed requirement of an oligo- or a polysaccharide backbone. We demonstrate here a concept that small nonsaccharidic nonpolymeric molecules may be rationally designed to interact with and activate antithrombin for enhanced inhibition of factor Xa. The rational design strategy is based on a study of complexes of natural and mutant antithrombins with heparin-based oligosaccharides using hydropathic interaction (HINT) technique, a quantitative computerized tool for analysis of molecular interactions. A linear correlation was observed between the free energy of binding for antithrombinminus signoligosaccharide complexes and the HINT score over a wide range of approximately 13 kcal/mol, indicating strong predictive capability of the HINT technique. Using this approach, a small, nonsugar, aromatic molecule, (minus sign)-epicatechin sulfate (ECS), was designed to mimic the nonreducing end trisaccharide unit DEF of the sequence specific heparin pentasaccharide DEFGH. HINT suggested a comparable antithrombin-binding geometry and interaction profile for ECS and trisaccharide DEF. Biochemical studies indicated that ECS binds antithrombin with equilibrium dissociation constants of 10.5 and 66 microM at pH 6.0, I 0.025, and pH 7.4, I 0.035, respectively, that compare favorably with 2 and 80 microM observed for the natural activator DEF. ECS accelerates the antithrombin inhibition of factor Xa nearly 8-fold demonstrating for the first time that conformational activation of antithrombin is feasible with appropriately designed small nonsugar organic molecules. The results present unique opportunities for de novo activator design based on this first-generation lead.

Interaction of designed sulfated flavanoids with antithrombin: lessons on the design of organic activators.

Recently, we designed (-)-epicatechin sulfate (ECS), the first small nonsaccharide molecule, as an activator of antithrombin for the accelerated inhibition of factor Xa, a key proteinase of the coagulation cascade (Gunnarsson, G. T.; Desai, U. R. J. Med. Chem. 2002, 45, 1233-1243). Although sulfated flavanoid ECS was found to bind antithrombin with an affinity ( approximately 10.7 microM) comparable to the reference trisaccharide DEF ( approximately 4.5 microM), it accelerated the inhibition of factor Xa only 10-fold as compared to the approximately 300-fold observed with DEF. To determine whether this conformational activation of the inhibitor is dependent on the structure of the organic activator and to probe the basis for the deficiency in activation, we studied the interaction of similar sulfated flavanoids with antithrombin. (+)-Catechin sulfate (CS), a chiral stereoisomer of ECS, bound plasma antithrombin with a 3-fold higher affinity (K(D) = 3.5 microM) and a 2-fold higher second-order rate constant for factor Xa inhibition (k(ACT) = 6750 M(-1) s(-1)). On the contrary, the K(D) and k(ACT) were found to be lower approximately 7.4- and approximately 2.4-fold, respectively, for its racemic counterpart, (+/-)-catechin sulfate. Dependence of the equilibrium dissociation constant on the ionic strength of the medium at pH 6.0 and 7.4 suggests that nonionic interactions contribute a major proportion ( approximately 55-73%) of the total binding energy, and only 1-2 ion pairs, in comparison to the expected approximately 4 ion pairs for the reference trisaccharide, are formed in the interaction. Competitive binding experiments indicate that activator CS does not compete with a saccharide ligand that binds antithrombin in the pentasaccharide binding site, while it competes with full-length low-affinity heparin. A molecular docking study suggests plausible binding of CS in the extended heparin binding site, which is adjacent to the binding domain for the reference trisaccharide DEF. In combination, the results demonstrate that although conformational activation of antithrombin with small sulfated flavanoids is dependent on the structure of the activator, the designed activators do not bind in the pentasaccharide binding site in antithrombin resulting in weak activation. The mechanistic investigation highlights plausible directions to take in the rational design of specific high-affinity organic antithrombin activators.

On designing non-saccharide, allosteric activators of antithrombin.

Antithrombin, a plasma glycoprotein serpin, requires conformational activation by heparin to induce an anticoagulant effect, which is mediated through accelerated factor Xa inhibition. Heparin, a highly charged polymer and an allosteric activator of the serpin, is associated with major adverse effects. To design better, but radically different activators of antithrombin from heparin, we utilized a pharmacophore-based approach. A tetrahydroisoquinoline-based scaffold was designed to mimic four critical anionic groups of the key trisaccharide DEF constituting the sequence-specific pentasaccharide DEFGH in heparin. Activator IAS(5) containing 5,6-disulfated tetrahydroisoquinoline and 3,4,5-trisulfated phenyl rings was found to bind antithrombin at pH 7.4 with an affinity comparable to the reference trisaccharide DEF. IAS(5) activated the inhibitor nearly 30-fold, nearly 2- to 3-fold higher than our first generation flavanoid-based designs. This work advances the concept of antithrombin activation through non-saccharide, organic molecules and pinpoints a direction for the design of more potent molecules.

Synthesis of per-sulfated flavonoids using 2,2,2-trichloro ethyl protecting group and their factor Xa inhibition potential.

The synthesis of per-sulfated flavonoids, organic compounds with multiple sulfate groups, is challenging. We present here a two-step synthesis of fully sulfated flavonoids in high overall yields using the 2,2,2-trichloroethyl moiety as a protecting group. The two-step synthesis results in exclusive formation of the per-sulfated product in contrast to common sulfating agents that yield differentially sulfated mixture of compounds. Most per-sulfated flavonoids studied are activators of antithrombin for accelerated inhibition of factor Xa, a key enzyme of the blood coagulation cascade. As a group the per-sulfated flavonoids possess a range of factor Xa inhibition potential.

Capillary electrophoresis of highly sulfated flavanoids and flavonoids.

Flavanoids and flavonoids are natural products present in our diet and known to possess multiple biological activities. Sulfated species of these natural products represent highly charged water-soluble organic molecules that possess unique biochemical properties. We describe here the first studies on capillary electrophoresis of these highly charged molecules. Fully sulfated flavanoids and flavonoids can be electrophoresed and resolved under reverse polarity at pH 3.5 using 5-10 kV in less than 20 min. In contrast, at high pH under normal polarity these species can be electrophoresed only if a pressurized capillary is employed. (+/-)-Catechin sulfate, a racemic sulfated flavanoid, was resolved into its enantiomers using 15% beta-cyclodextrin, a chiral selector, but not with alpha- or gamma-cyclodextrins. Yet, the high charge density of these molecules challenges the resolving capability of capillary electrophoresis as diastereomers (-)-epicatechin sulfate and (+)-catechin sulfate do not resolve, even in the presence of cyclodextrins or chiral positively charged amino acids. Overall, capillary electrophoresis of highly sulfated flavanoids and flavonoids is expected to be useful in rapid structure analysis of sulfated flavonoids, either synthetic or natural.

Structural characterization of a serendipitously discovered bioactive macromolecule, lignin sulfate.

The herpes simplex virus-1 (HSV-1) utilizes cell-surface glycosaminoglycan, heparan sulfate, to gain entry into cells and cause infection. In a search for synthetic mimics of heparan sulfate to prevent HSV infection, we discovered potent inhibitory activity arising from sulfation of a monomeric flavonoid. Yet, detailed screening indicated that the sulfated flavonoid was completely inactive and the potent inhibitory activity arose from a macromolecular substance present in the parent flavonoid. The active principle was identified through a battery of biophysical and chemical analyses as a sulfated form of lignin, a three-dimensional network polymer composed of substituted phenylpropanoid monomers. Mass spectral analysis of the parent lignin and its sulfated derivative indicates the presence of p-coumaryl monomers interconnected through uncondensed beta-O-4-linkages. Elemental analysis of lignin sulfate correlates primarily with a polymer of p-coumaryl alcohol containing one sulfate group. High-performance size exclusion chromatography shows a wide molecular weight distribution from 1.5 to 40 kDa suggesting significant polydispersity. Polyacrylamide gel electrophoresis (PAGE) analysis indicates a highly networked polymer that differs significantly from linear charged polymers with respect to its electrophoretic mobility. Overall, macromolecular lignin sulfate presents a multitude of substructures that can interact with biomolecules, including viral glycoproteins, using hydrophobic, hydrogen-bonding, and ionic forces. Thus, lignin sulfate represents a large number of interesting structures with potential medicinal benefits.

Exploring new non-sugar sulfated molecules as activators of antithrombin.

New non-sugar, small, sulfated molecules, based on our de novo rationally designed activator (-)-epicatechin sulfate (ECS), were investigated to bind and activate antithrombin, an inhibitor of plasma coagulation enzyme factor Xa. For the activators studied, the equilibrium dissociation constant (K(D)) of the interaction with plasma antithrombin varies nearly 53-fold, with the highest affinity of 1.8 microM observed for morin sulfate, while the acceleration in factor Xa inhibition varies 2.6-fold. The results demonstrate that antithrombin binding and activation is a common property of these small sulfated molecules and suggests plausible directions for designing more potent activators.

Hydropathic interaction analyses of small organic activators binding to antithrombin.

Recently we designed the first small organic ligands, sulfated flavanoids and flavonoids, that act as activators of antithrombin for accelerated inhibition of factor Xa, a key proteinase of the coagulation cascade [Gunnarsson and Desai, Bioorg. Med. Chem. Lett. (2003) 13:579]. To better understand the binding properties of these activators at a molecular level, we have utilized computerized hydropathic interaction (HINT) analyses of the sulfated molecules interacting in two plausible electropositive regions, the pentasaccharide- and extended heparin-binding sites, of antithrombin in its native and activated forms. HINT analyses indicate favorable multi-point interactions of the activators in both binding sites of the two forms of antithrombin. Yet, HINT predicts better interaction of most activators, except for (-)-catechin sulfate, with the activated form of antithrombin than with the native form supporting the observation in solution that these molecules function as activators of the inhibitor. Further, whereas (+)-catechin sulfate recognized the activated form of antithrombin better in both the pentasaccharide- and extended heparin- binding sites, the native form was better recognized by (-)-catechin sulfate, thus explaining its weaker binding and activation potential in solution. A reasonable linear correlation between the overall HINT score and the solution free energy of binding of the sulfated activators was evident. This investigation indicates that HINT is a useful tool in understanding interactions of antithrombin with small sulfated organic ligands at a molecular level, has some good predictive properties, and is likely to be useful for rational design purposes.