Saturation-scale functional evidence supports clinical variant interpretation in Lynch syndrome.

BACKGROUND: Lynch syndrome (LS) is a cancer predisposition syndrome affecting more than 1 in every 300 individuals worldwide. Clinical genetic testing for LS can be life-saving but is complicated by the heavy burden of variants of uncertain significance (VUS), especially missense changes. RESULT: To address this challenge, we leverage a multiplexed analysis of variant effect (MAVE) map covering >94% of the 17,746 possible missense variants in the key LS gene MSH2. To establish this map's utility in large-scale variant reclassification, we overlay it on clinical databases of >15,000 individuals with LS gene variants uncovered during clinical genetic testing. We validate these functional measurements in a cohort of individuals with paired tumor-normal test results and find that MAVE-based function scores agree with the clinical interpretation for every one of the MSH2 missense variants with an available classification. We use these scores to attempt reclassification for 682 unique missense VUS, among which 34 scored as deleterious by our function map, in line with previously published rates for other cancer predisposition genes. Combining functional data and other evidence, ten missense VUS are reclassified as pathogenic/likely pathogenic, and another 497 could be moved to benign/likely benign. Finally, we apply these functional scores to paired tumor-normal genetic tests and identify a subset of patients with biallelic somatic loss of function, reflecting a sporadic Lynch-like Syndrome with distinct implications for treatment and relatives' risk. CONCLUSION: This study demonstrates how high-throughput functional assays can empower scalable VUS resolution and prospectively generate strong evidence for variant classification.

Three Newly Recognized Likely Pathogenic Gene Variants Associated with Hereditary Transthyretin Amyloidosis.

INTRODUCTION: Hereditary transthyretin amyloidosis (ATTRv [variant]) is a clinically heterogeneous, progressively debilitating, fatal disease resulting from the deposition of insoluble amyloid fibrils in various organs and tissues. Early diagnosis of ATTRv can be facilitated with genetic testing; however, such testing of the TTR gene identifies variants of uncertain significance (VUS) in a minority of cases, a small percentage of which have the potential to be pathogenic. The Akcea/Ambry VUS Initiative is dedicated to gathering molecular, clinical, and inheritance data for each TTR VUS identified by genetic testing programs to reclassify TTR variants to a clinically actionable status (e.g., variant likely pathogenic [VLP]) where appropriate. METHODS: Classification criteria used here, based on recommendations from the American College of Medical Genetics and Genomics, are stringent and comprehensive, requiring distinct lines of evidence supporting pathogenesis. RESULTS: Three TTR variants have been reclassified from VUS to VLP, including c.194C>T (p.A65V), c.172G>C (p.D58H), and c.239C>T (p.T80I). In each case, the totality of genetic, structural, and clinical evidence provided strong support for pathogenicity. CONCLUSIONS: Based on several lines of evidence, three TTR VUS were reclassified as VLP, resulting in a high likelihood of disease diagnosis for those and subsequent patients as well as at-risk family members.

REVEL and BayesDel outperform other in silico meta-predictors for clinical variant classification.

Many in silico predictors of genetic variant pathogenicity have been previously developed, but there is currently no standard application of these algorithms for variant assessment. Using 4,094 ClinVar-curated missense variants in clinically actionable genes, we evaluated the accuracy and yield of benign and deleterious evidence in 5 in silico meta-predictors, as well as agreement of SIFT and PolyPhen2, and report the derived thresholds for the best performing predictor(s). REVEL and BayesDel outperformed all other meta-predictors (CADD, MetaSVM, Eigen), with higher positive predictive value, comparable negative predictive value, higher yield, and greater overall prediction performance. Agreement of SIFT and PolyPhen2 resulted in slightly higher yield but lower overall prediction performance than REVEL or BayesDel. Our results support the use of gene-level rather than generalized thresholds, when gene-level thresholds can be estimated. Our results also support the use of 2-sided thresholds, which allow for uncertainty, rather than a single, binary cut-point for assigning benign and deleterious evidence. The gene-level 2-sided thresholds we derived for REVEL or BayesDel can be used to assess in silico evidence for missense variants in accordance with current classification guidelines.

Mutations in MAP3K1 that cause 46,XY disorders of sex development disrupt distinct structural domains in the protein.

Missense mutations in the gene, MAP3K1, are a common cause of 46,XY gonadal dysgenesis, accounting for 15-20% of cases [Ostrer, 2014, Disorders of sex development (DSDs): an update. J. Clin. Endocrinol. Metab., 99, 1503-1509]. Functional studies demonstrated that all of these mutations cause a protein gain-of-function that alters co-factor binding and increases phosphorylation of the downstream MAP kinase pathway targets, MAPK11, MAP3K and MAPK1. This dysregulation of the MAP kinase pathway results in increased CTNNB1, increased expression of WNT4 and FOXL2 and decreased expression of SRY and SOX9. Unique and recurrent pathogenic mutations cluster in three semi-contiguous domains outside the kinase region of the protein, a newly identified N-terminal domain that shares homology with the Guanine Exchange Factor (residues Met164 to Glu231), a Plant HomeoDomain (residues Met442 to Trp495) and an ARMadillo repeat domain (residues Met566 to Glu862). Despite the presence of the mutation clusters and clinical data, there exists a dearth of mechanistic insights behind the development imbalance. In this paper, we use structural modeling and functional data of these mutations to understand alterations of the MAP3K1 protein and the effects on protein folding, binding and downstream target phosphorylation. We show that these mutations have differential effects on protein binding depending on the domains in which they occur. These mutations increase the binding of the RHOA, MAP3K4 and FRAT1 proteins and generally decrease the binding of RAC1. Thus, pathologies in MAP3K1 disrupt the balance between the pro-kinase activities of the RHOA and MAP3K4 binding partners and the inhibitory activity of RAC1.

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.

Biallelic mutations in FDXR cause neurodegeneration associated with inflammation.

Mitochondrial dysfunction lies behind many neurodegenerative disorders, owing largely to the intense energy requirements of most neurons. Such mitochondrial dysfunction may work through a variety of mechanisms, from direct disruption of the electron transport chain to abnormal mitochondrial biogenesis. Recently, we have identified biallelic mutations in the mitochondrial flavoprotein "ferredoxin reductase" (FDXR) gene as a novel cause of mitochondriopathy, peripheral neuropathy, and optic atrophy. In this report, we expand upon those results by describing two new cases of disease-causing FDXR variants in patients with variable severity of phenotypes, including evidence of an inflammatory response in brain autopsy. To investigate the underlying pathogenesis, we examined neurodegeneration in a mouse model. We found that Fdxr mutant mouse brain tissues share pathological changes similar to those seen in patient autopsy material, including increased astrocytes. Furthermore, we show that these abnormalities are associated with increased levels of markers for both neurodegeneration and gliosis, with the latter implying inflammation as a major factor in the pathology of Fdxr mutations. These data provide further insight into the pathogenic mechanism of FDXR-mediated central neuropathy, and suggest an avenue for mechanistic studies that will ultimately inform treatment.

Whole-exome sequencing uncovers oxidoreductases DHTKD1 and OGDHL as linkers between mitochondrial dysfunction and eosinophilic esophagitis.

Eosinophilic esophagitis (EoE) is an allergic inflammatory esophageal disorder with a complex underlying genetic etiology often associated with other comorbidities. Using whole-exome sequencing (WES) of 63 patients with EoE and 60 unaffected family members and family-based trio analysis, we sought to uncover rare coding variants. WES analysis identified 5 rare, damaging variants in dehydrogenase E1 and transketolase domain-containing 1 (DHTKD1). Rare variant burden analysis revealed an overabundance of putative, potentially damaging DHTKD1 mutations in EoE (P = 0.01). Interestingly, we also identified 7 variants in the DHTKD1 homolog oxoglutarate dehydrogenase-like (OGDHL). Using shRNA-transduced esophageal epithelial cells and/or patient fibroblasts, we further showed that disruption of normal DHTKD1 or OGDHL expression blunts mitochondrial function. Finally, we demonstrated that the loss of DHTKD1 expression increased ROS production and induced the expression of viperin, a gene previously shown to be involved in production of Th2 cytokines in T cells. Viperin had increased expression in esophageal biopsies of EoE patients compared with control individuals and was upregulated by IL-13 in esophageal epithelial cells. These data identify a series of rare genetic variants implicating DHTKD1 and OGDHL in the genetic etiology of EoE and underscore a potential pathogenic role for mitochondrial dysfunction in EoE.

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.

De Novo Variants in GRIA4 Lead to Intellectual Disability with or without Seizures and Gait Abnormalities.

Using trio whole-exome sequencing, we have identified de novo heterozygous pathogenic variants in GRIA4 in five unrelated individuals with intellectual disability and other symptoms. GRIA4 encodes an AMPA receptor subunit known as GluR4, which is found on excitatory glutamatergic synapses and is important for learning and memory. Four of the variants are located in the highly conserved SYTANLAAF motif in the transmembrane protein M3, and the fifth is in an extra-cellular domain. Molecular modeling of the altered protein showed that three of the variants in the SYTANLAAF motif orient toward the center of the pore region and most likely lead to disturbance of the gating mechanism. The fourth variant in the SYTANLAAF motif most likely results in reduced permeability. The variant in the extracellular domain potentially interferes with the binding between the monomers. On the basis of clinical information and genetic results, and the fact that other subunits of the AMPA receptor have already been associated with neurodevelopmental disorders, we suggest that pathogenic de novo variants in GRIA4 lead to intellectual disability with or without seizures, gait abnormalities, problems of social behavior, and other variable features.

Biallelic mutations in the ferredoxin reductase gene cause novel mitochondriopathy with optic atrophy.

Iron-sulfur (Fe-S) clusters are ubiquitous cofactors essential to various cellular processes, including mitochondrial respiration, DNA repair, and iron homeostasis. A steadily increasing number of disorders are being associated with disrupted biogenesis of Fe-S clusters. Here, we conducted whole-exome sequencing of patients with optic atrophy and other neurological signs of mitochondriopathy and identified 17 individuals from 13 unrelated families with recessive mutations in FDXR, encoding the mitochondrial membrane-associated flavoprotein ferrodoxin reductase required for electron transport from NADPH to cytochrome P450. In vitro enzymatic assays in patient fibroblast cells showed deficient ferredoxin NADP reductase activity and mitochondrial dysfunction evidenced by low oxygen consumption rates (OCRs), complex activities, ATP production and increased reactive oxygen species (ROS). Such defects were rescued by overexpression of wild-type FDXR. Moreover, we found that mice carrying a spontaneous mutation allelic to the most common mutation found in patients displayed progressive gait abnormalities and vision loss, in addition to biochemical defects consistent with the major clinical features of the disease. Taken together, these data provide the first demonstration that germline, hypomorphic mutations in FDXR cause a novel mitochondriopathy and optic atrophy in humans.

Molecular mechanism of Zn2+ inhibition of a voltage-gated proton channel.

Voltage-gated proton (Hv1) channels are involved in many physiological processes, such as pH homeostasis and the innate immune response. Zn2+ is an important physiological inhibitor of Hv1. Sperm cells are quiescent in the male reproductive system due to Zn2+ inhibition of Hv1 channels, but become active once introduced into the low-Zn2+-concentration environment of the female reproductive tract. How Zn2+ inhibits Hv1 is not completely understood. In this study, we use the voltage clamp fluorometry technique to identify the molecular mechanism of Zn2+ inhibition of Hv1. We find that Zn2+ binds to both the activated closed and resting closed states of the Hv1 channel, thereby inhibiting both voltage sensor motion and gate opening. Mutations of some Hv1 residues affect only Zn2+ inhibition of the voltage sensor motion, whereas mutations of other residues also affect Zn2+ inhibition of gate opening. These effects are similar in monomeric and dimeric Hv1 channels, suggesting that the Zn2+-binding sites are localized within each subunit of the dimeric Hv1. We propose that Zn2+ binding has two major effects on Hv1: (i) at low concentrations, Zn2+ binds to one site and prevents the opening conformational change of the pore of Hv1, thereby inhibiting proton conduction; and (ii) at high concentrations, Zn2+, in addition, binds to a second site and inhibits the outward movement of the voltage sensor of Hv1. Elucidating the molecular mechanism of how Zn2+ inhibits Hv1 will further our understanding of Hv1 function and might provide valuable information for future drug development for Hv1 channels.

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.

Mapping the gating and permeation pathways in the voltage-gated proton channel Hv1.

Voltage-gated proton channels (Hv1) are ubiquitous throughout nature and are implicated in numerous physiological processes. The gene encoding for Hv1, however, was only identified in 2006. The lack of sufficient structural information of this channel has hampered the understanding of the molecular mechanism of channel activation and proton permeation. This study uses both simulation and experimental approaches to further develop existing models of the Hv1 channel. Our study provides insights into features of channel gating and proton permeation pathway. We compare open- and closed-state structures developed previously with a recent crystal structure that traps the channel in a presumably closed state. Insights into gating pathways were provided using a combination of all-atom molecular dynamics simulations with a swarm of trajectories with the string method for extensive transition path sampling and evolution. A detailed residue-residue interaction profile and a hydration profile were studied to map the gating pathway in this channel. In particular, it allows us to identify potential intermediate states and compare them to the experimentally observed crystal structure of Takeshita et al. (Takeshita K, Sakata S, Yamashita E, Fujiwara Y, Kawanabe A, Kurokawa T, et al. X-ray crystal structure of voltage-gated proton channel. Nature 2014). The mechanisms governing ion transport in the wild-type and mutant Hv1 channels were studied by a combination of electrophysiological recordings and free energy simulations. With these results, we were able to further refine ideas about the location and function of the selectivity filter. The refined structural models will be essential for future investigations of this channel and the development of new drugs targeting cellular proton transport.

Acidification asymmetrically affects voltage-dependent anion channel implicating the involvement of salt bridges.

The voltage-dependent anion channel (VDAC) is the major pathway for ATP, ADP, and other respiratory substrates through the mitochondrial outer membrane, constituting a crucial point of mitochondrial metabolism regulation. VDAC is characterized by its ability to "gate" between an open and several "closed" states under applied voltage. In the early stages of tumorigenesis or during ischemia, partial or total absence of oxygen supply to cells results in cytosolic acidification. Motivated by these facts, we investigated the effects of pH variations on VDAC gating properties. We reconstituted VDAC into planar lipid membranes and found that acidification reversibly increases its voltage-dependent gating. Furthermore, both VDAC anion selectivity and single channel conductance increased with acidification, in agreement with the titration of the negatively charged VDAC residues at low pH values. Analysis of the pH dependences of the gating and open channel parameters yielded similar pKa values close to 4.0. We also found that the response of VDAC gating to acidification was highly asymmetric. The presumably cytosolic (cis) side of the channel was the most sensitive to acidification, whereas the mitochondrial intermembrane space (trans) side barely responded to pH changes. Molecular dynamic simulations suggested that stable salt bridges at the cis side, which are susceptible to disruption upon acidification, contribute to this asymmetry. The pronounced sensitivity of the cis side to pH variations found here in vitro might provide helpful insights into the regulatory role of VDAC in the protective effect of cytosolic acidification during ischemia in vivo.

Rehabilitating drug-induced long-QT promoters: in-silico design of hERG-neutral cisapride analogues with retained pharmacological activity.

BACKGROUND: The human ether-a-go-go related gene 1 (hERG1), which codes for a potassium ion channel, is a key element in the cardiac delayed rectified potassium current, IKr, and plays an important role in the normal repolarization of the heart's action potential. Many approved drugs have been withdrawn from the market due to their prolongation of the QT interval. Most of these drugs have high potencies for their principal targets and are often irreplaceable, thus "rehabilitation" studies for decreasing their high hERG1 blocking affinities, while keeping them active at the binding sites of their targets, have been proposed to enable these drugs to re-enter the market. METHODS: In this proof-of-principle study, we focus on cisapride, a gastroprokinetic agent withdrawn from the market due to its high hERG1 blocking affinity. Here we tested an a priori strategy to predict a compound's cardiotoxicity using de novo drug design with molecular docking and Molecular Dynamics (MD) simulations to generate a strategy for the rehabilitation of cisapride. RESULTS: We focused on two key receptors, a target interaction with the (adenosine) receptor and an off-target interaction with hERG1 channels. An analysis of the fragment interactions of cisapride at human A2A adenosine receptors and hERG1 central cavities helped us to identify the key chemical groups responsible for the drug activity and hERG1 blockade. A set of cisapride derivatives with reduced cardiotoxicity was then proposed using an in-silico two-tier approach. This set was compared against a large dataset of commercially available cisapride analogs and derivatives. CONCLUSIONS: An interaction decomposition of cisapride and cisapride derivatives allowed for the identification of key active scaffolds and functional groups that may be responsible for the unwanted blockade of hERG1.

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.