Correction: DNA breakpoint assay reveals a majority of gross duplications occur in tandem reducing VUS classifications in breast cancer predisposition genes.

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DNA breakpoint assay reveals a majority of gross duplications occur in tandem reducing VUS classifications in breast cancer predisposition genes.

PURPOSE: Gross duplications are ambiguous in terms of clinical interpretation due to the limitations of the detection methods that cannot infer their context, namely, whether they occur in tandem or are duplicated and inserted elsewhere in the genome. We investigated the proportion of gross duplications occurring in tandem in breast cancer predisposition genes with the intent of informing their classifications. METHODS: The DNA breakpoint assay (DBA) is a custom, paired-end, next-generation sequencing (NGS) method designed to capture and detect deep-intronic DNA breakpoints in gross duplications in BRCA1, BRCA2, ATM, CDH1, PALB2, and CHEK2. RESULTS: DBA allowed us to ascertain breakpoints for 44 unique gross duplications from 147 probands. We determined that the duplications occurred in tandem in 114 (78%) carriers from this cohort, while the remainder have unknown tandem status. Among the tandem gross duplications that were eligible for reclassification, 95% of them were upgraded to pathogenic. CONCLUSION: DBA is a novel, high-throughput, NGS-based method that informs the tandem status, and thereby the classification of, gross duplications. This method revealed that most gross duplications in the investigated genes occurred in tandem and resulted in a pathogenic classification, which helps to secure the necessary treatment options for their carriers.

Postmortem Diagnostic Exome Sequencing Identifies a De Novo TUBB3 Alteration in a Newborn With Prenatally Diagnosed Hydrocephalus and Suspected Walker-Warburg Syndrome.

Objective Herein, we report a case of a deceased newborn with prenatally detected hydrocephalus. Postnatal findings included abnormal brain imaging and electroencephalogram, optic nerve abnormalities, and elevated creatine kinase (CK). No underlying genetic etiology had been previously identified for the proband, despite testing with a congenital muscular dystrophy gene panel. Methods Diagnostic exome sequencing (DES) was performed on the proband-parents trio, and candidate alterations were confirmed using automated fluorescence dideoxy sequencing. Results Exome sequencing of the proband, mother and father identified a previously unreported apparently de novo heterozygous tubulin, beta-3 ( TUBB3) c.523G>C (p.V175L) alteration in the proband. Conclusion Overall, DES established a likely molecular genetic diagnosis for a postmortem case after traditional testing methods were uninformative. The DES results allowed for reproductive options, such as preimplantation genetic diagnosis and/or prenatal diagnosis, to be available to the parents in future pregnancies.

A recurrent mutation in KCNA2 as a novel cause of hereditary spastic paraplegia and ataxia.

The hereditary spastic paraplegias (HSPs) are heterogeneous neurodegenerative disorders with over 50 known causative genes. We identified a recurrent mutation in KCNA2 (c.881G>A, p.R294H), encoding the voltage-gated K(+) -channel, KV 1.2, in two unrelated families with HSP, intellectual disability (ID), and ataxia. Follow-up analysis of > 2,000 patients with various neurological phenotypes identified a de novo p.R294H mutation in a proband with ataxia and ID. Two-electrode voltage-clamp recordings of Xenopus laevis oocytes expressing mutant KV 1.2 channels showed loss of function with a dominant-negative effect. Our findings highlight the phenotypic spectrum of a recurrent KCNA2 mutation, implicating ion channel dysfunction as a novel HSP disease mechanism. Ann Neurol 2016.

Current state of theoretical and experimental studies of the voltage-dependent anion channel (VDAC).

Voltage-dependent anion channel (VDAC), the major channel of the mitochondrial outer membrane provides a controlled pathway for respiratory metabolites in and out of the mitochondria. In spite of the wealth of experimental data from structural, biochemical, and biophysical investigations, the exact mechanisms governing selective ion and metabolite transport, especially the role of titratable charged residues and interactions with soluble cytosolic proteins, remain hotly debated in the field. The computational advances hold a promise to provide a much sought-after solution to many of the scientific disputes around solute and ion transport through VDAC and hence, across the mitochondrial outer membrane. In this review, we examine how Molecular Dynamics, Free Energy, and Brownian Dynamics simulations of the large ß-barrel channel, VDAC, advanced our understanding. We will provide a short overview of non-conventional techniques and also discuss examples of how the modeling excursions into VDAC biophysics prospectively aid experimental efforts. This article is part of a Special Issue entitled: Membrane Proteins edited by J.C. Gumbart and Sergei Noskov.

Two-Dimensional Potentials of Mean Force of Nile Red in Intact and Damaged Model Bilayers. Application to Calculations of Fluorescence Spectra.

Fluorescent dyes revolutionized and expanded our understanding of biological membranes. The interpretation of experimental fluorescence data in terms of membrane structure, however, requires detailed information about the molecular environment of the dyes. Nile red is a fluorescent molecule whose excitation and emission maxima depend on the polarity of the solvent. It is mainly used as a probe to study lipid microenvironments, for example in imaging the progression of damage to the myelin sheath in multiple sclerosis. In this study, we determine the position and orientation of Nile red in lipid bilayers by calculating two-dimensional Potential of Mean Force (2D-PMF) profiles in a defect-free 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayer and in damaged bilayers containing two mixtures of the oxidized lipid 1-palmitoyl-2-(9'-oxo-nonanoyl)-sn-glycero-3-phosphocholine and POPC. From 2D-PMF simulations we obtain positions and orientations of Nile Red corresponding to the minimum on the binding free energy surface in three different membrane environments with increasing amounts of water, mimicking damage in biological tissue. Using representative snapshots from the simulations, we use combined quantum mechanical/molecular mechanical (QM/MM) models to calculate the emission spectrum of Nile red as a function of its local solvation environment. The results of QM and QM/MM computations are in qualitative agreement with the experimentally observed shift in fluorescence for the dye moving from aqueous solution to the more hydrophobic environment of the lipid interiors. The range of the conformation dependent values of the computed absorption-emission spectra and the lack of solvent relaxation effects in the QM/MM calculations made it challenging to delineate specific differences between the intact and damaged bilayers.

Molecular strategies to achieve selective conductance in NaK channel variants.

A recent crystallization of several ion channels has provided strong impetus for efforts aimed at understanding the different strategies employed by nature for selective ion transport. In this work, we used two variants of the selectivity filter of NaK channel to explore molecular mechanisms that give rise to K(+)-selectivity. We computed one-dimensional (1D) and two-dimensional (2D) potentials of mean force (PMFs) for ion permeation across the channel. The results indicate that the energies for Na(+) and K(+) permeation across the selectivity filter display significant differences in positions of the binding sites and barriers. One characteristic signature of a K(+)-selective channel is the apparent preservation of the site analogous to that of S2 in KcsA. The S2-bound ion can be almost ideally dehydrated and coordinated by 6 to 8 carbonyls. In a striking contrast, the PMFs controlling transport of ions in a nonselective variant show almost identical profiles for either K(+) or Na(+) and significant involvement of water molecules in ion coordination across the entire selectivity filter. An analysis of differences in 1D PMFs for Na(+) and K(+) suggests that coordination number alone is an insufficient predictor of site selectivity, while chemical composition (ratio of carbonyls and water molecules) correlates well with preference for K(+). Multi-ion effects such as dependence of the barriers and wells for permeant ion on the type of copermeant ion were found to play a significant role in the selectivity signature of the channel as well.

Iron porphyrin dications with neutral axial ligands: DFT calculations delineate similarities with heme protein compound II intermediates.

OLYP/TZP calculations on two symmetrized model complexes [Fe(TPP)(py)(2)](2+) and [Fe(TPP)(PhNC)(2)](2+) (TPP = meso-tetraphenylporphyrin, py = pyridine, PhNC = phenylisocyanide) reveal dense manifolds of low-energy electronic states. For the latter complex, broken-symmetry calculations successfully reproduce the unique S = 0 ground state that is expected on the basis of experimental measurements on a closely related complex; the S = 0 state arises from antiferromagnetic coupling between a low-spin d(xy)(1)(d(xz),d(yz))(4) Fe(III) center and a porphyrin "a(2u)" radical. Furthermore, the calculations indicate low-energy Fe(IV) states for both complexes. Overall, the results contribute to our deepening understanding of the factors contributing to the stability of iron(IV) centers. Thus, a dianionic π-donor oxo ligand is no longer deemed a requirement for the stability of heme-based Fe(IV) centers; iron(IV) intermediates of heme proteins such as chloroperoxidase, catalase, and MauG, having only monanionic ligands such as hydroxide, thiolate, and phenolate and/or (in the case of MauG) a neutral histidine as axial ligands, are now firmly established.

Consistent van der Waals radii for the whole main group.

Atomic radii are not precisely defined but are nevertheless widely used parameters in modeling and understanding molecular structure and interactions. The van der Waals radii determined by Bondi from molecular crystals and data for gases are the most widely used values, but Bondi recommended radius values for only 28 of the 44 main-group elements in the periodic table. In the present Article, we present atomic radii for the other 16; these new radii were determined in a way designed to be compatible with Bondi's scale. The method chosen is a set of two-parameter correlations of Bondi's radii with repulsive-wall distances calculated by relativistic coupled-cluster electronic structure calculations. The newly determined radii (in A) are Be, 1.53; B, 1.92; Al, 1.84; Ca, 2.31; Ge, 2.11; Rb, 3.03; Sr, 2.49; Sb, 2.06; Cs, 3.43; Ba, 2.68; Bi, 2.07; Po, 1.97; At, 2.02; Rn, 2.20; Fr, 3.48; and Ra, 2.83.

Performance of SM8 on a test to predict small-molecule solvation free energies.

The SM8 quantum mechanical aqueous continuum solvation model is applied to a 17-molecule test set proposed by Nicholls et al. (J. Med. Chem. 2008, 51, 769) to predict free energies of solvation. With the M06-2X density functional, the 6-31G(d) basis set, and CM4M charge model, the root-mean-square error (RMSE) of SM8 is 1.08 kcal mol(-1) for aqueous geometries and 1.14 kcal mol(-1) for gas-phase geometries. These errors compare favorably with optimal explicit and continuum models reported by Nicholls et al., having RMSEs of 1.33 and 1.87 kcal mol(-1), respectively. Other models examined by these workers had RMSEs of 1.5-2.6 kcal mol(-1). We also explore the use of other density functionals and charge models with SM8 and the RMSE increases to 1.21 kcal mol(-1) for mPW1/CM4 with gas-phase geometries, to 1.50 kcal mol(-1) for M06-2X/CM4 with gas-phase geometries, and to 1.27-1.64 kcal mol(-1) with three different models at B3LYP gas-phase geometries.

Extension of a temperature-dependent aqueous solvation model to compounds containing nitrogen, fluorine, chlorine, bromine, and sulfur.

Most methods for predicting free energies of solvation have been developed or validated exclusively for room temperature. Recently, we developed a model called SM6T for predicting aqueous solvation free energies as a function of temperature for solutes composed of C, H, or O, and here we present solvation model 8 with temperature dependence (SM8T) for predicting the temperature dependence of aqueous free energies of solvation for compounds containing H, C, N, O, F, S, Cl, and Br in the range 273-373 K. We also describe the database of experimental aqueous free energies of solvation used to parametrize the model. SM8T partitions the temperature dependence of the free energy of solvation into two components: the temperature dependence of the bulk electrostatic contribution to the free energy of solvation, which is computed using the generalized Born equation, and the temperature dependence of first-solvation-shell effects, which is modeled by terms proportional to the solvent-exposed surface areas of atoms in functional groups determined entirely by geometry. SM8T predicts the temperature dependence of aqueous free energies of solvation with a mean unsigned error of 0.08 kcal/mol over a database of 4403 measurements on 348 compounds at various temperatures. We also discuss the accuracy of SM8T for predicting the temperature dependence of aqueous free energies of solvation for ions and present free energies of solvation as a function of temperature for two sample ions.

Modeling free energies of solvation in olive oil.

Olive oil partition coefficients are useful for modeling the bioavailability of drug-like compounds. We have recently developed an accurate solvation model called SM8 for aqueous and organic solvents (Marenich, A. V.; Olson, R. M.; Kelly, C. P.; Cramer, C. J.; Truhlar, D. G. J. Chem. Theory Comput. 2007, 3, 2011) and a temperature-dependent solvation model called SM8T for aqueous solution (Chamberlin, A. C.; Cramer, C. J.; Truhlar, D. G. J. Phys. Chem. B 2008, 112, 3024). Here we describe an extension of SM8T to predict air-olive oil and water-olive oil partitioning for drug-like solutes as functions of temperature. We also describe the database of experimental partition coefficients used to parametrize the model; this database includes 371 entries for 304 compounds spanning the 291-310 K temperature range.

Polarization Effects in Aqueous and Nonaqueous Solutions.

Polarization effects in aqueous and nonaqueous solutions were analyzed for nine neutral and three charged organic solutes by the SM8 universal implicit solvation model and class IV partial atomic charges based on Charge Model 4M (CM4M) with the M06-2X density functional. The CM4M partial atomic charges in neutral and ionic solutes and in the corresponding clustered solutes (supersolutes), which included one solute molecule and one or two solvent molecules, were modeled in three solvents (benzene, methylene chloride, and water) and compared to those in the gas phase. The use of the supersolute approach (microsolvation) allows one to account for charge transfer from the solute to the solvent, and we find charge transfers as large as 0.06 atomic units for neutral solutes (pyridine in water) and 0.32 atomic units for ions (methoxide anion in water). Relaxation of the electronic structure of the solute in the presence of solvent increases the polarization free energy of the neutral solutes studied here, on average, by 16% in benzene, 30% in methylene chloride, and 43% in water. The increase for the ions in water averaged 43%.

Predicting aqueous free energies of solvation as functions of temperature.

This work introduces a model, solvation model 6 with temperature dependence (SM6T), to predict the temperature dependence of aqueous free energies of solvation for compounds containing H, C, and O in the range 273-373 K. In particular, we extend solvation model 6 (SM6), which was previously developed (Kelly, C. P.; Cramer, C. J.; Truhlar, D. G. J. Chem. Theory Comput. 2005, 1, 1133) for predicting aqueous free energies of solvation at 298 K, to predict the variation of the free energy of solvation relative to 298 K. Also, we describe the database of experimental aqueous free energies of solvation for compounds containing H, C, and O that was used to parametrize and test the new model. SM6T partitions the temperature dependence of the free energy of solvation into two components: the temperature dependence of the bulk electrostatic contribution to the free energy of solvation, which is computed using the generalized Born equation, and the temperature dependence of first-solvation-shell effects which is modeled using a parametrized solvent-exposed surface-area-dependent term. We found that SM6T predicts the temperature dependence of aqueous free energies of solvation with a mean unsigned error of 0.08 kcal/mol over our entire database, whereas using the experimental value at 298 K produces a mean unsigned error of 0.53 kcal/mol.

Deprotonation of lithiated benzenes.

The deprotonation energies of all possible lithiobenzenes (C(6)Li(n)H(6-n), n = 0-5) were computed at B3LYP/6-311+G(d,p). Based on natural population analysis, the conjugate bases can be thought of as salts between a polyanionic phenyl core and associated lithium cations. The most stable structures maximize the electrostatic attraction between these two species, typically by positioning the lithium cations to bridge in the ring plane across two adjacent carbanion centers. Favorable deprotonation occurs when the formal carbanion centers are adjacent to each other and then the proton is removed from an adjacent carbon. The deprotonation free energies range from 365.0 to 397.2 kcal mol(-1), with most of them less than the deprotonation free energy of benzene (391.8 kcal mol(-1)).

Theoretical study of nucleophilic substitution at the disulfide bridge of cyclo-l-cystine.

The reaction of cyclo-l-cystine with thiolate is examined at the B3LYP/6-31+G level. The two isomers of cyclo-l-cystine differ in their dihedral angle about the disulfide bond; the M isomer (with dihedral angle of -90.1 degrees) is found to be slightly lower in energy. The nucleophilic substitution reaction at sulfur follows the addition-elimination mechanism, exemplified by the hypercoordinate sulfur intermediate on the reaction surface. The reaction is exergonic (DeltaG = -6.16 kcal mol(-1)), and both the entrance and exit transition state lie below the reactant energies.