Publications by authors named "Shozeb Haider"

112 Publications

Structural basis of the effect of activating mutations on the EGF receptor.

Elife 2021 Jul 28;10. Epub 2021 Jul 28.

Department of Chemistry, University College London, London, United Kingdom.

Mutations within the kinase domain of the epidermal growth factor receptor (EGFR) are common oncogenic driver events in non-small cell lung cancer. Although the activation of EGFR in normal cells is primarily driven by growth-factor-binding-induced dimerization, mutations on different exons of the kinase domain of the receptor have been found to affect the equilibrium between its active and inactive conformations giving rise to growth-factor-independent kinase activation. Using molecular dynamics simulations combined with enhanced sampling techniques, we compare here the conformational landscape of the monomers and homodimers of the wild-type and mutated forms of EGFR ΔELREA and L858R, as well as of two exon 20 insertions, D770-N771insNPG, and A763-Y764insFQEA. The differences in the conformational energy landscapes are consistent with multiple mechanisms of action including the regulation of the hinge motion, the stabilization of the dimeric interface, and local unfolding transitions. Overall, a combination of different effects is caused by the mutations and leads to the observed aberrant signaling.
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http://dx.doi.org/10.7554/eLife.65824DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8318590PMC
July 2021

DIAPH1 Variants in Non-East Asian Patients With Sporadic Moyamoya Disease.

JAMA Neurol 2021 Jun 14. Epub 2021 Jun 14.

Yale Center for Genome Analysis, West Haven, Connecticut.

Importance: Moyamoya disease (MMD), a progressive vasculopathy leading to narrowing and ultimate occlusion of the intracranial internal carotid arteries, is a cause of childhood stroke. The cause of MMD is poorly understood, but genetic factors play a role. Several familial forms of MMD have been identified, but the cause of most cases remains elusive, especially among non-East Asian individuals.

Objective: To assess whether ultrarare de novo and rare, damaging transmitted variants with large effect sizes are associated with MMD risk.

Design, Setting, And Participants: A genetic association study was conducted using whole-exome sequencing case-parent MMD trios in a small discovery cohort collected over 3.5 years (2016-2019); data were analyzed in 2020. Medical records from US hospitals spanning a range of 1 month to 1.5 years were reviewed for phenotyping. Exomes from a larger validation cohort were analyzed to identify additional rare, large-effect variants in the top candidate gene. Participants included patients with MMD and, when available, their parents. All participants who met criteria and were presented with the option to join the study agreed to do so; none were excluded. Twenty-four probands (22 trios and 2 singletons) composed the discovery cohort, and 84 probands (29 trios and 55 singletons) composed the validation cohort.

Main Outcomes And Measures: Gene variants were identified and filtered using stringent criteria. Enrichment and case-control tests assessed gene-level variant burden. In silico modeling estimated the probability of variant association with protein structure. Integrative genomics assessed expression patterns of MMD risk genes derived from single-cell RNA sequencing data of human and mouse brain tissue.

Results: Of the 24 patients in the discovery cohort, 14 (58.3%) were men and 18 (75.0%) were of European ancestry. Three of 24 discovery cohort probands contained 2 do novo (1-tailed Poisson P = 1.1 × 10-6) and 1 rare, transmitted damaging variant (12.5% of cases) in DIAPH1 (mammalian diaphanous-1), a key regulator of actin remodeling in vascular cells and platelets. Four additional ultrarare damaging heterozygous DIAPH1 variants (3 unphased) were identified in 3 other patients in an 84-proband validation cohort (73.8% female, 77.4% European). All 6 patients were non-East Asian. Compound heterozygous variants were identified in ena/vasodilator-stimulated phosphoproteinlike protein EVL, a mammalian diaphanous-1 interactor that regulates actin polymerization. DIAPH1 and EVL mutant probands had severe, bilateral MMD associated with transfusion-dependent thrombocytopenia. DIAPH1 and other MMD risk genes are enriched in mural cells of midgestational human brain. The DIAPH1 coexpression network converges in vascular cell actin cytoskeleton regulatory pathways.

Conclusions And Relevance: These findings provide the largest collection to date of non-East Asian individuals with sporadic MMD harboring pathogenic variants in the same gene. The results suggest that DIAPH1 is a novel MMD risk gene and impaired vascular cell actin remodeling in MMD pathogenesis, with diagnostic and therapeutic ramifications.
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http://dx.doi.org/10.1001/jamaneurol.2021.1681DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8204259PMC
June 2021

Dynamic Profiling of β-Coronavirus 3CL M Protease Ligand-Binding Sites.

J Chem Inf Model 2021 06 14;61(6):3058-3073. Epub 2021 Jun 14.

UCL School of Pharmacy, London WC1N 1AX, U.K.

β-coronavirus (CoVs) alone has been responsible for three major global outbreaks in the 21st century. The current crisis has led to an urgent requirement to develop therapeutics. Even though a number of vaccines are available, alternative strategies targeting essential viral components are required as a backup against the emergence of lethal viral variants. One such target is the main protease (M) that plays an indispensable role in viral replication. The availability of over 270 M X-ray structures in complex with inhibitors provides unique insights into ligand-protein interactions. Herein, we provide a comprehensive comparison of all nonredundant ligand-binding sites available for SARS-CoV2, SARS-CoV, and MERS-CoV M. Extensive adaptive sampling has been used to investigate structural conservation of ligand-binding sites using Markov state models (MSMs) and compare conformational dynamics employing convolutional variational auto-encoder-based deep learning. Our results indicate that not all ligand-binding sites are dynamically conserved despite high sequence and structural conservation across β-CoV homologs. This highlights the complexity in targeting all three M enzymes with a single pan inhibitor.
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http://dx.doi.org/10.1021/acs.jcim.1c00449DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8230960PMC
June 2021

An Integrated Computational and Experimental Approach to Identifying Inhibitors for SARS-CoV-2 3CL Protease.

Front Mol Biosci 2021 17;8:661424. Epub 2021 May 17.

Department of Chemical and Biological Engineering, Villanova University, Villanova, PA, United States.

The newly evolved SARS-CoV-2 has caused the COVID-19 pandemic, and the SARS-CoV-2 main protease 3CLpro is essential for the rapid replication of the virus. Inhibiting this protease may open an alternative avenue toward therapeutic intervention. In this work, a computational docking approach was developed to identify potential small-molecule inhibitors for SARS-CoV-2 3CLpro. Totally 288 potential hits were identified from a half-million bioactive chemicals via a protein-ligand docking protocol. To further evaluate the docking results, a quantitative structure activity relationship (QSAR) model of 3CLpro inhibitors was developed based on existing small molecule inhibitors of the 3CLpro and their corresponding IC data. The QSAR model assesses the physicochemical properties of identified compounds and estimates their inhibitory effects on 3CLpro. Seventy-one potential inhibitors of 3CLpro were selected through these computational approaches and further evaluated via an enzyme activity assay. The results show that two chemicals, i.e., 5-((1-([1,1'-biphenyl]-4-yl)-2,5-dimethyl-1H-pyrrol-3-yl)methylene)pyrimidine-2,4,6(1H,3H,5H)-trione and N-(4-((3-(4-chlorophenylsulfonamido)quinoxalin-2-yl)amino)phenyl)acetamide, effectively inhibited 3CLpro SARS-CoV-2 with IC's of 19 ± 3 μM and 38 ± 3 μM, respectively. The compounds contain two basic structures, pyrimidinetrione and quinoxaline, which were newly found in 3CLpro inhibitor structures and are of high interest for lead optimization. The findings from this work, such as 3CLpro inhibitor candidates and the QSAR model, will be helpful to accelerate the discovery of inhibitors for related coronaviruses that may carry proteases with similar structures to SARS-CoV-2 3CLpro.
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http://dx.doi.org/10.3389/fmolb.2021.661424DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8166273PMC
May 2021

Conformational Flexibility of A Highly Conserved Helix Controls Cryptic Pocket Formation in FtsZ.

J Mol Biol 2021 Jul 21;433(15):167061. Epub 2021 May 21.

Department of Pharmaceutical and Biological Chemistry, School of Pharmacy, University College London, 29-39 Brunswick Square, London WC1N 1AX, UK. Electronic address:

Mycobacterium tuberculosis is responsible for more than 1.6 million deaths each year. One potential antibacterial target in M. tuberculosis is filamentous temperature sensitive protein Z (FtsZ), which is the bacterial homologue of mammalian tubulin, a validated cancer target. M. tuberculosis FtsZ function is essential, with its inhibition leading to arrest of cell division, elongation of the bacterial cell and eventual cell death. However, the development of potent inhibitors against FtsZ has been a challenge owing to the lack of structural information. Here we report multiple crystal structures of M. tuberculosis FtsZ in complex with a coumarin analogue. The 4-hydroxycoumarin binds exclusively to two novel cryptic pockets in nucleotide-free FtsZ, but not to the binary FtsZ-GTP or GDP complexes. Our findings provide a detailed understanding of the molecular basis for cryptic pocket formation, controlled by the conformational flexibility of the H7 helix, and thus reveal an important structural and mechanistic rationale for coumarin antibacterial activity.
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http://dx.doi.org/10.1016/j.jmb.2021.167061DOI Listing
July 2021

Allosteric communication in class A β-lactamases occurs via cooperative coupling of loop dynamics.

Elife 2021 Mar 23;10. Epub 2021 Mar 23.

University College London School of Pharmacy, Pharmaceutical and Biological Chemistry, London, United Kingdom.

Understanding allostery in enzymes and tools to identify it offer promising alternative strategies to inhibitor development. Through a combination of equilibrium and nonequilibrium molecular dynamics simulations, we identify allosteric effects and communication pathways in two prototypical class A β-lactamases, TEM-1 and KPC-2, which are important determinants of antibiotic resistance. The nonequilibrium simulations reveal pathways of communication operating over distances of 30 Å or more. Propagation of the signal occurs through cooperative coupling of loop dynamics. Notably, 50% or more of clinically relevant amino acid substitutions map onto the identified signal transduction pathways. This suggests that clinically important variation may affect, or be driven by, differences in allosteric behavior, providing a mechanism by which amino acid substitutions may affect the relationship between spectrum of activity, catalytic turnover, and potential allosteric behavior in this clinically important enzyme family. Simulations of the type presented here will help in identifying and analyzing such differences.
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http://dx.doi.org/10.7554/eLife.66567DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8060031PMC
March 2021

Selective Modulation of α5 GABA Receptors Exacerbates Aberrant Inhibition at Key Hippocampal Neuronal Circuits in Mouse Model of Alzheimer's Disease.

Front Cell Neurosci 2020 11;14:568194. Epub 2020 Nov 11.

UCL School of Pharmacy, London, United Kingdom.

Selective negative allosteric modulators (NAMs), targeting α5 subunit-containing GABA receptors (GABARs) as potential therapeutic targets for disorders associated with cognitive deficits, including Alzheimer's disease (AD), continually fail clinical trials. We investigated whether this was due to the change in the expression of α5 GABARs, consequently altering synaptic function during AD pathogenesis. Using medicinal chemistry and computational modeling, we developed aqueous soluble hybrids of 6,6-dimethyl-3-(2-hydroxyethyl) thio-1-(thiazol-2-yl)-6,7-dihydro-2-benzothiophene-4(5H)-one, that demonstrated selective binding and high negative allosteric modulation, specifically for the α5 GABAR subtypes in constructed HEK293 stable cell-lines. Using a knock-in mouse model of AD ( ), which expresses a mutant form of human amyloid-β (Aβ), we performed immunofluorescence studies combined with electrophysiological whole-cell recordings to investigate the effects of our key molecule, α5-SOP002 in the hippocampal CA1 region. In aged mice, selective preservation of α5 GABARs was observed in, calretinin- (CR), cholecystokinin- (CCK), somatostatin- (SST) expressing interneurons, and pyramidal cells. Previously, we reported that CR dis-inhibitory interneurons, specialized in regulating other interneurons displayed abnormally high levels of synaptic inhibition in the mouse model, here we show that this excessive inhibition was "normalized" to control values with bath-applied α5-SOP002 (1 μM). However, α5-SOP002, further inhibition onto CCK and pyramidal cells that were already largely compromised by exhibiting a of inhibition in the AD model. In summary, using a multi-disciplinary approach, we show that exposure to α5 GABAR NAMs may further compromise aberrant synapses in AD. We, therefore, suggest that the α5 GABAR is not a suitable therapeutic target for the treatment of AD or other cognitive deficits due to the widespread neuronal-networks that use α5 GABARs.
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http://dx.doi.org/10.3389/fncel.2020.568194DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7686552PMC
November 2020

First-in-class humanized FSH blocking antibody targets bone and fat.

Proc Natl Acad Sci U S A 2020 11 30;117(46):28971-28979. Epub 2020 Oct 30.

The Mount Sinai Bone Program, Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, NY 10029.

Blocking the action of FSH genetically or pharmacologically in mice reduces body fat, lowers serum cholesterol, and increases bone mass, making an anti-FSH agent a potential therapeutic for three global epidemics: obesity, osteoporosis, and hypercholesterolemia. Here, we report the generation, structure, and function of a first-in-class, fully humanized, epitope-specific FSH blocking antibody with a of 7 nM. Protein thermal shift, molecular dynamics, and fine mapping of the FSH-FSH receptor interface confirm stable binding of the Fab domain to two of five receptor-interacting residues of the FSHβ subunit, which is sufficient to block its interaction with the FSH receptor. In doing so, the humanized antibody profoundly inhibited FSH action in cell-based assays, a prelude to further preclinical and clinical testing.
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http://dx.doi.org/10.1073/pnas.2014588117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7682550PMC
November 2020

Exome Sequencing Implicates Impaired GABA Signaling and Neuronal Ion Transport in Trigeminal Neuralgia.

iScience 2020 Oct 11;23(10):101552. Epub 2020 Sep 11.

Yale Center for Genome Analysis, West Haven, CT, USA.

Trigeminal neuralgia (TN) is a common, debilitating neuropathic face pain syndrome often resistant to therapy. The familial clustering of TN cases suggests that genetic factors play a role in disease pathogenesis. However, no unbiased, large-scale genomic study of TN has been performed to date. Analysis of 290 whole exome-sequenced TN probands, including 20 multiplex kindreds and 70 parent-offspring trios, revealed enrichment of rare, damaging variants in GABA receptor-binding genes in cases. Mice engineered with a TN-associated mutation (p.Cys188Trp) in the GABA receptor Cl channel γ-1 subunit () exhibited trigeminal mechanical allodynia and face pain behavior. Other TN probands harbored rare damaging variants in Na and Ca channels, including a significant variant burden in the α-1H subunit of the voltage-gated Ca channel Ca3.2 (). These results provide exome-level insight into TN and implicate genetically encoded impairment of GABA signaling and neuronal ion transport in TN pathogenesis.
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http://dx.doi.org/10.1016/j.isci.2020.101552DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7554653PMC
October 2020

BMPQ-1 binds selectively to (3+1) hybrid topologies in human telomeric G-quadruplex multimers.

Nucleic Acids Res 2020 11;48(20):11259-11269

State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China.

A single G-quadruplex forming sequence from the human telomere can adopt six distinct topologies that are inter-convertible under physiological conditions. This presents challenges to design ligands that show selectivity and specificity towards a particular conformation. Additional complexity is introduced in differentiating multimeric G-quadruplexes over monomeric species, which would be able to form in the single-stranded 3' ends of telomeres. A few ligands have been reported that bind to dimeric quadruplexes, but their preclinical pharmacological evaluation is limited. Using multidisciplinary approaches, we identified a novel quinoline core ligand, BMPQ-1, which bound to human telomeric G-quadruplex multimers over monomeric G-quadruplexes with high selectivity, and induced the formation of G-quadruplex DNA along with the related DNA damage response at the telomere. BMPQ-1 reduced tumor cell proliferation with an IC50 of ∼1.0 μM and decreased tumor growth rate in mouse by half. Biophysical analysis using smFRET identified a mixture of multiple conformations coexisting for dimeric G-quadruplexes in solution. Here, we showed that the titration of BMPQ-1 shifted the conformational ensemble of multimeric G-quadruplexes towards (3+1) hybrid-2 topology, which became more pronounced as further G-quadruplex units are added.
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http://dx.doi.org/10.1093/nar/gkaa870DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7672424PMC
November 2020

Exome sequencing implicates genetic disruption of prenatal neuro-gliogenesis in sporadic congenital hydrocephalus.

Nat Med 2020 11 19;26(11):1754-1765. Epub 2020 Oct 19.

Departments of Neurosurgery, Engineering Science & Mechanics, and Physics; Center for Neural Engineering and Infectious Disease Dynamics, The Pennsylvania State University, University Park, PA, USA.

Congenital hydrocephalus (CH), characterized by enlarged brain ventricles, is considered a disease of excessive cerebrospinal fluid (CSF) accumulation and thereby treated with neurosurgical CSF diversion with high morbidity and failure rates. The poor neurodevelopmental outcomes and persistence of ventriculomegaly in some post-surgical patients highlight our limited knowledge of disease mechanisms. Through whole-exome sequencing of 381 patients (232 trios) with sporadic, neurosurgically treated CH, we found that damaging de novo mutations account for >17% of cases, with five different genes exhibiting a significant de novo mutation burden. In all, rare, damaging mutations with large effect contributed to ~22% of sporadic CH cases. Multiple CH genes are key regulators of neural stem cell biology and converge in human transcriptional networks and cell types pertinent for fetal neuro-gliogenesis. These data implicate genetic disruption of early brain development, not impaired CSF dynamics, as the primary pathomechanism of a significant number of patients with sporadic CH.
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http://dx.doi.org/10.1038/s41591-020-1090-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7871900PMC
November 2020

Mutations disrupting neuritogenesis genes confer risk for cerebral palsy.

Nat Genet 2020 10 28;52(10):1046-1056. Epub 2020 Sep 28.

Henan Key Laboratory of Child Brain Injury, Third Affiliated Hospital of Zhengzhou University, Zhengzhou, China.

In addition to commonly associated environmental factors, genomic factors may cause cerebral palsy. We performed whole-exome sequencing of 250 parent-offspring trios, and observed enrichment of damaging de novo mutations in cerebral palsy cases. Eight genes had multiple damaging de novo mutations; of these, two (TUBA1A and CTNNB1) met genome-wide significance. We identified two novel monogenic etiologies, FBXO31 and RHOB, and showed that the RHOB mutation enhances active-state Rho effector binding while the FBXO31 mutation diminishes cyclin D levels. Candidate cerebral palsy risk genes overlapped with neurodevelopmental disorder genes. Network analyses identified enrichment of Rho GTPase, extracellular matrix, focal adhesion and cytoskeleton pathways. Cerebral palsy risk genes in enriched pathways were shown to regulate neuromotor function in a Drosophila reverse genetics screen. We estimate that 14% of cases could be attributed to an excess of damaging de novo or recessive variants. These findings provide evidence for genetically mediated dysregulation of early neuronal connectivity in cerebral palsy.
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http://dx.doi.org/10.1038/s41588-020-0695-1DOI Listing
October 2020

Role of in silico structural modeling in predicting immunogenic neoepitopes for cancer vaccine development.

JCI Insight 2020 09 3;5(17). Epub 2020 Sep 3.

The Sidney Kimmel Comprehensive Cancer Center, The Skip Viragh Center for Pancreatic Cancer, The Bloomberg-Kimmel Institute for Cancer Immunotherapy, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

In prior studies, we delineated the landscape of neoantigens arising from nonsynonymous point mutations in a murine pancreatic cancer model, Panc02. We developed a peptide vaccine by targeting neoantigens predicted using a pipeline that incorporates the MHC binding algorithm NetMHC. The vaccine, when combined with immune checkpoint modulators, elicited a robust neoepitope-specific antitumor immune response and led to tumor clearance. However, only a small fraction of the predicted neoepitopes induced T cell immunity, similarly to that reported for neoantigen vaccines tested in clinical studies. While these studies have used binding affinities to MHC I as surrogates for T cell immunity, this approach does not include spatial information on the mutated residue that is crucial for TCR activation. Here, we investigate conformational alterations in and around the MHC binding groove induced by selected minimal neoepitopes, and we examine the influence of a given mutated residue as a function of its spatial position. We found that structural parameters, including the solvent-accessible surface area (SASA) of the neoepitope and the position and spatial configuration of the mutated residue within the sequence, can be used to improve the prediction of immunogenic neoepitopes for inclusion in cancer vaccines.
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http://dx.doi.org/10.1172/jci.insight.136991DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7526456PMC
September 2020

Structure-based enzyme engineering improves donor-substrate recognition of Arabidopsis thaliana glycosyltransferases.

Biochem J 2020 08;477(15):2791-2805

Pharmaceutical and Biological Chemistry, UCL School of Pharmacy, London WC1N 1AX, U.K.

Glycosylation of secondary metabolites involves plant UDP-dependent glycosyltransferases (UGTs). UGTs have shown promise as catalysts in the synthesis of glycosides for medical treatment. However, limited understanding at the molecular level due to insufficient biochemical and structural information has hindered potential applications of most of these UGTs. In the absence of experimental crystal structures, we employed advanced molecular modeling and simulations in conjunction with biochemical characterization to design a workflow to study five Group H Arabidopsis thaliana (76E1, 76E2, 76E4, 76E5, 76D1) UGTs. Based on our rational structural manipulation and analysis, we identified key amino acids (P129 in 76D1; D374 in 76E2; K275 in 76E4), which when mutated improved donor substrate recognition than wildtype UGTs. Molecular dynamics simulations and deep learning analysis identified structural differences, which drive substrate preferences. The design of these UGTs with broader substrate specificity may play important role in biotechnological and industrial applications. These findings can also serve as basis to study other plant UGTs and thereby advancing UGT enzyme engineering.
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http://dx.doi.org/10.1042/BCJ20200477DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7419078PMC
August 2020

In silico drug repositioning of FDA-approved drugs to predict new inhibitors for alpha-synuclein aggregation.

Comput Biol Chem 2020 Oct 24;88:107308. Epub 2020 Jun 24.

Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran. Electronic address:

One of the hallmarks of Parkinson's disease (PD), a long-term neurodegenerative syndrome, is the accumulation of alpha-synuclein (α-syn) fibrils. Despite numerous studies and efforts, inhibition of α-syn protein aggregation is still a challenge. To overcome this issue, we propose an in silico pharmacophore-based repositioning strategy, to find a pharmaceutical drug that, in addition to their defined role, can be used to prevent aggregation of the α-syn protein. Ligand-based pharmacophore modeling was developed and the best model was selected with validation parameters including 72 % sensitivity, 98 % specificity and goodness score about 0.7. The optimal model has three groups of hydrogen bond donor (HBD), three groups of hydrogen bond acceptor (HBA), and two aromatic rings (AR). The FDA-Approved reports in the ZINC15 database were screened with the pharmacophore model taken from inhibitor compounds. The model identified 22 hits, as promising candidate drugs for Parkinson's therapy. It is noteworthy that among these, 10 drugs have been reported to inhibition of α-syn aggregation or treat/reduce Parkinson's pathogenesis. This model was used to virtual screen ZINC, NCI databases, and natural products from the pomegranate. The results of this screen were filtered for their inability to cross the blood-brain barrier, poor oral bioavailability, etc. Finally, the selected compounds of two ZINC and NCI databases were combined and structurally clustered. Remained compounds were clustered in 28 different clusters, and the 17 compounds were introduced as final candidates.
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http://dx.doi.org/10.1016/j.compbiolchem.2020.107308DOI Listing
October 2020

Beyond bone biology: Lessons from team science.

J Orthop Res 2020 11 22;38(11):2331-2338. Epub 2020 Jul 22.

The Mount Sinai Bone Program, Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, New York.

Today, research in biomedicine often requires the knowledge and technologies in diverse fields. Therefore, there is an increasing need for collaborative team science that crosses traditional disciplines. Here, we discuss our own lessons from both interdisciplinary and transdisciplinary teams, which ultimately ushered us to expand our research realm beyond bone biology.
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http://dx.doi.org/10.1002/jor.24771DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7722176PMC
November 2020

Repurposing erectile dysfunction drugs tadalafil and vardenafil to increase bone mass.

Proc Natl Acad Sci U S A 2020 06 8;117(25):14386-14394. Epub 2020 Jun 8.

The Mount Sinai Bone Program, Icahn School of Medicine at Mount Sinai, New York, NY 10029.

We report that two widely-used drugs for erectile dysfunction, tadalafil and vardenafil, trigger bone gain in mice through a combination of anabolic and antiresorptive actions on the skeleton. Both drugs were found to enhance osteoblastic bone formation in vivo using a unique gene footprint and to inhibit osteoclast formation. The target enzyme, phosphodiesterase 5A (PDE5A), was found to be expressed in mouse and human bone as well as in specific brain regions, namely the locus coeruleus, raphe pallidus, and paraventricular nucleus of the hypothalamus. Localization of PDE5A in sympathetic neurons was confirmed by coimmunolabeling with dopamine β-hydroxylase, as well as by retrograde bone-brain tracing using a sympathetic nerve-specific pseudorabies virus, PRV152. Both drugs elicited an antianabolic sympathetic imprint in osteoblasts, but with net bone gain. Unlike in humans, in whom vardenafil is more potent than tadalafil, the relative potencies were reversed with respect to their osteoprotective actions in mice. Structural modeling revealed a higher binding energy of tadalafil to mouse PDE5A compared with vardenafil, due to steric clashes of vardenafil with a single methionine residue at position 806 in mouse PDE5A. Collectively, our findings suggest that a balance between peripheral and central actions of PDE5A inhibitors on bone formation together with their antiresorptive actions specify the osteoprotective action of PDE5A blockade.
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http://dx.doi.org/10.1073/pnas.2000950117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7321982PMC
June 2020

Native de novo structural determinations of non-canonical nucleic acid motifs by X-ray crystallography at long wavelengths.

Nucleic Acids Res 2020 09;48(17):9886-9898

State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China.

Obtaining phase information remains a formidable challenge for nucleic acid structure determination. The introduction of an X-ray synchrotron beamline designed to be tunable to long wavelengths at Diamond Light Source has opened the possibility to native de novo structure determinations by the use of intrinsic scattering elements. This provides opportunities to overcome the limitations of introducing modifying nucleotides, often required to derive phasing information. In this paper, we build on established methods to generate new tools for nucleic acid structure determinations. We report on the use of (i) native intrinsic potassium single-wavelength anomalous dispersion methods (K-SAD), (ii) use of anomalous scattering elements integral to the crystallization buffer (extrinsic cobalt and intrinsic potassium ions), (iii) extrinsic bromine and intrinsic phosphorus SAD to solve complex nucleic acid structures. Using the reported methods we solved the structures of (i) Pseudorabies virus (PRV) RNA G-quadruplex and ligand complex, (ii) PRV DNA G-quadruplex, and (iii) an i-motif of human telomeric sequence. Our results highlight the utility of using intrinsic scattering as a pathway to solve and determine non-canonical nucleic acid motifs and reveal the variability of topology, influence of ligand binding, and glycosidic angle rearrangements seen between RNA and DNA G-quadruplexes of the same sequence.
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http://dx.doi.org/10.1093/nar/gkaa439DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7515729PMC
September 2020

A Self-Assembling Lipidic Peptide and Selective Partial V2 Receptor Agonist Inhibits Urine Production.

Sci Rep 2020 04 29;10(1):7269. Epub 2020 Apr 29.

UCL School of Pharmacy, 29-39 Brunswick Square, London, WC1N 1AX, UK.

Lipidised analgesic peptide prodrugs self-assemble into peptide nanofibers; with the nanofiber morphology protecting the peptide from plasma degradation and improving therapeutic efficacy. Extending this learning, we hypothesised that a self-assembling lipidized peptide arginine vasopressin (AVP) receptor agonist, that had not been designed as a prodrug, could prove pharmacologically active and control urine production. The only approved AVP receptor agonist, desmopressin is indicated for the treatment of central diabetes insipidus (DI), bedwetting, haemophilia A and von Willebrand disease. Desmopressin is well tolerated by most patients, however adverse effects, such as hyponatraemia and water intoxication necessitate a strict fluid intake, thus motivating the search for alternative DI treatments. Selective V2 receptor agonism is required for anti-DI activity and we hypothesised that our new lipidized peptide (METx) would lead to selective AVP receptor agonism. METx was synthesised and characterised and then tested for activity against the V2, V1a and OT uterine receptors and not tested against the V1b receptor as METx was not expected to cross the blood brain barrier. METx was also tested in vivo in a healthy rat model. METx forms nanofibers and is a partial V2 receptor agonist (determined by measuring MDCK cell line cAMP accumulation), producing 57% of AVP's maximal activity (EC50 = 2.7 nM) and is not a V1a agonist up to a concentration of 1 μM (determined by measuring A7r5 cell line D-myo-inositol-1-phosphate accumulation). METx is a weak OT receptor antagonist, reducing the frequency of OT induced contractions (EC50 = 350 nM) and increasing the OT EC50 from 0.081 nM to 21 nM at a concentration of 600 nM. METx (41 nM) had no effect on spontaneous uterine contractions and METx (100 nM) had no effect on OT induced uterine contractions. Simulated binding studies show that binding avidity to the receptors follows the trend: V2 > OT > V1a. On intravenous injection, a nanoparticle formulation of METx reduced urine production in a healthy rat model in a dose responsive manner, with 40 mg kg METx resulting in no urine production over 4 hours. The lipidized self-assembling peptide - METx - is a selective competitive V2 receptor agonist and an anti-diuretic.
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http://dx.doi.org/10.1038/s41598-020-64070-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7190706PMC
April 2020

Synergistic Effect of Chlorogenic Acid and Caffeic Acid with Fosfomycin on Growth Inhibition of a Resistant Strain.

ACS Omega 2020 Apr 23;5(13):7537-7544. Epub 2020 Mar 23.

Department of Chemical Engineering, Villanova University, 800 E Lancaster Avenue, Villanova, Pennsylvania 19085, United States.

, a human foodborne pathogen that causes listeriosis with high-rate mortality, has been reported to be resistant to commonly used antibiotics. New antibiotics or cocktails of existing antibiotics with synergistic compounds are in high demand for treating this multi-drug-resistant pathogen. Fosfomycin is one of the novel and promising therapeutic antibiotics for the treatment of listeriosis. However, some strains with the gene were recently reported to survive from the fosfomycin treatment. This work aims to identify FosX inhibitors that can revive fosfomycin in treating resistant . Since structures and activities of the FosX protein in have been well studied, we used an integrated computational and experimental approach to identify FosX inhibitors that show synergistic effect with fosfomycin in treating resistant . Specifically, automated ligand docking was implemented to perform virtual screening of the Indofine natural-product database and FDA-approved drugs to identify potential inhibitors. An in vitro bacterial growth inhibition test was then utilized to verify the effectiveness of identified compounds combined with fosfomycin in inhibiting the resistant strains. Two phenolic acids, i.e., caffeic acid and chlorogenic acid, were predicted as high-affinity FosX inhibitors from the ligand-docking platform. Experiments with these compounds indicated that the cocktail of either caffeic acid (1.5 mg/mL) or chlorogenic acid (3 mg/mL) with fosfomycin (50 mg/L) was able to significantly inhibit the growth of the pathogen. The finding of this work implies that the combination of fosfomycin with either caffeic acid or chlorogenic acid is of potential to be used in the clinical treatment of infections.
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http://dx.doi.org/10.1021/acsomega.0c00352DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7144146PMC
April 2020

Electrostatic Switching Controls Channel Dynamics of the Sensor Protein VirB10 in Type IV Secretion System.

ACS Omega 2020 Feb 4;5(7):3271-3281. Epub 2020 Feb 4.

Department of Pharmaceutical and Biological Chemistry, University College London School of Pharmacy, WC1N 1AX London, United Kingdom.

Type IV secretion systems are large nanomachines assembled across the bacterial cell envelope for effector translocation and conjugation. VirB10 traverses the inner and outer membranes, sensing cellular signals for coordinating the conformational switch for pilus biogenesis and/or secretion. Mutations uncoupling secretion from pilus biogenesis were identified in VirB10 including a gating defect mutation G272R that made VirB10 unresponsive to intracellular ATP, causing unregulated secretion of VirE2 in a contact-independent manner. Comparative long-timescale molecular dynamics of the wild type and G272R mutant of the VirB10 tetradecamer reveals how the G272R mutation locks the oligomer in a rigid conformation by swapping the ionic interactions between the loops from the β-barrel close to the inner leaflet of the outer membrane. This electrostatic switching changes the allosteric communication pathway from the extracellular loop to the base of the barrel, suggesting that the local conformational dynamics in the loops can gate information across VirB10.
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http://dx.doi.org/10.1021/acsomega.9b03313DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7045316PMC
February 2020

A Standard Numbering Scheme for Class C β-Lactamases.

Antimicrob Agents Chemother 2020 02 21;64(3). Epub 2020 Feb 21.

Department of Molecular Biology and Microbiology, Case Western Reserve University, Cleveland, Ohio, USA

Unlike for classes A and B, a standardized amino acid numbering scheme has not been proposed for the class C (AmpC) β-lactamases, which complicates communication in the field. Here, we propose a scheme developed through a collaborative approach that considers both sequence and structure, preserves traditional numbering of catalytically important residues (Ser, Lys, Tyr, and Lys), is adaptable to new variants or enzymes yet to be discovered and includes a variation for genetic and epidemiological applications.
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http://dx.doi.org/10.1128/AAC.01841-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7038296PMC
February 2020

Phosphorylation of Histone Deacetylase 8: Structural and Mechanistic Analysis of the Phosphomimetic S39E Mutant.

Biochemistry 2019 11 4;58(45):4480-4493. Epub 2019 Nov 4.

Department of Chemistry , University of Michigan , 930 North University Avenue , Ann Arbor , Michigan 48109 , United States.

Histone deacetylase (HDAC) enzymes that catalyze removal of acetyl-lysine post-translational modifications are frequently post-translationally modified. HDAC8 is phosphorylated within the deacetylase domain at conserved residue serine 39, which leads to decreased catalytic activity. HDAC8 phosphorylation at S39 is unique in its location and function and may represent a novel mode of deacetylation regulation. To better understand the impact of phosphorylation of HDAC8 on enzyme structure and function, we performed crystallographic, kinetic, and molecular dynamics studies of the S39E HDAC8 phosphomimetic mutant. This mutation decreases the level of deacetylation of peptides derived from acetylated nuclear and cytoplasmic proteins. However, the magnitude of the effect depends on the peptide sequence and the identity of the active site metal ion [Zn(II) vs Fe(II)], with the value of / for the mutant decreasing 9- to >200-fold compared to that of wild-type HDAC8. Furthermore, the dissociation rate constant of the active site metal ion increases by ∼10-fold. S39E HDAC8 was crystallized in complex with the inhibitor Droxinostat, revealing that phosphorylation of S39, as mimicked by the glutamate side chain, perturbs local structure through distortion of the L1 loop. Molecular dynamics simulations of both S39E and phosphorylated S39 HDAC8 demonstrate that the perturbation of the L1 loop likely occurs because of the lost hydrogen bond between D29 and S39. Furthermore, the S39 perturbation causes structural changes that propagate through the protein scaffolding to influence function in the active site. These data demonstrate that phosphorylation plays an important regulatory role for HDAC8 by affecting ligand binding, catalytic efficiency, and substrate selectivity.
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http://dx.doi.org/10.1021/acs.biochem.9b00653DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6903415PMC
November 2019

Discovery of a potent histone deacetylase (HDAC) 3/6 selective dual inhibitor.

Eur J Med Chem 2019 Dec 7;184:111755. Epub 2019 Oct 7.

Department of Pharmacy, National University of Singapore, 18 Science Drive 4, 117543, Singapore. Electronic address:

Herein, we report the discovery of a dual histone deacetylase inhibitor displaying a unique HDAC3/6 selectivity profile. An initial strategy to merge two epigenetic pharmacophores resulted in the discovery of potent HDAC6 inhibitors with selectivity over HDAC1. Screening in an HDAC panel revealed additional low nanomolar inhibition only against HDAC3. Low micromolar antiproliferative activities against two breast cancer and four hematological cancer cell lines was supported by pharmacodynamic studies on a preferred molecule, 24c, substantiating the HDAC inhibitory profile in cells. Apoptosis was identified as one of the main cell death pathways. Modelling studies of 24c against HDAC1,2,3 and 6 further provided insights on the orientation of specific residues relevant to compound potency, explaining the observed HDAC3/6 selectivity. A subset of the compounds also exhibited good antimalarial activities, particularly against the chloroquine-resistant strain K1 of P.falciparum. In vitro studies revealed a favourable DMPK profile warranting further investigation of the therapeutic potential of these compounds.
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http://dx.doi.org/10.1016/j.ejmech.2019.111755DOI Listing
December 2019

Recessive Inheritance of Congenital Hydrocephalus With Other Structural Brain Abnormalities Caused by Compound Heterozygous Mutations in .

Front Cell Neurosci 2019 26;13:425. Epub 2019 Sep 26.

Department of Neurosurgery, School of Medicine, Yale University, New Haven, CT, United States.

Background: encodes the α3 subunit of the Na/K ATPase, a fundamental ion-transporting enzyme. Primarily expressed in neurons, is mutated in several autosomal dominant neurological diseases. To our knowledge, damaging recessive genotypes in have never been associated with any human disease. deficiency in zebrafish results in hydrocephalus; however, no known association exists between and human congenital hydrocephalus (CH).

Methods: We utilized whole-exome sequencing (WES), bioinformatics, and computational modeling to identify and characterize novel mutations in a patient with CH. We performed immunohistochemical studies using mouse embryonic brain tissues to characterize expression during brain development.

Results: We identified two germline mutations in (p. Arg19Cys and p.Arg463Cys), each of which was inherited from one of the patient's unaffected parents, in a single patient with severe obstructive CH due to aqueductal stenosis, along with open schizencephaly, type 1 Chiari malformation, and dysgenesis of the corpus callosum. Both mutations are predicted to be highly deleterious and impair protein stability. Immunohistochemical studies demonstrate robust expression in neural stem cells (NSCs), differentiated neurons, and choroid plexus of the mouse embryonic brain.

Conclusion: These data provide the first evidence of a recessive human phenotype associated with mutations in , and implicate impaired Na/K ATPase function in the pathogenesis of CH.
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http://dx.doi.org/10.3389/fncel.2019.00425DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6775207PMC
September 2019

Inactivation of PP2A by a recurrent mutation drives resistance to MEK inhibitors.

Oncogene 2020 01 20;39(3):703-717. Epub 2019 Sep 20.

Department of Internal Medicine: Genetic Medicine, University of Michigan, Ann Arbor, MI, USA.

The serine/threonine Protein Phosphatase 2A (PP2A) functions as a tumor suppressor by negatively regulating multiple oncogenic signaling pathways. The canonical PP2A holoenzyme comprises a scaffolding subunit (PP2A Aα/β), which serves as the platform for binding of both the catalytic C subunit and one regulatory B subunit. Somatic heterozygous missense mutations in PPP2R1A, the gene encoding the PP2A Aα scaffolding subunit, have been identified across multiple cancer types, but the effects of the most commonly mutated residue, Arg-183, on PP2A function have yet to be fully elucidated. In this study, we used a series of cellular and in vivo models and discovered that the most frequent Aα R183W mutation formed alternative holoenzymes by binding of different PP2A regulatory subunits compared with wild-type Aα, suggesting a rededication of PP2A functions. Unlike wild-type Aα, which suppressed tumorigenesis, the R183W mutant failed to suppress tumor growth in vivo through activation of the MAPK pathway in RAS-mutant transformed cells. Furthermore, cells expressing R183W were less sensitive to MEK inhibitors. Taken together, our results demonstrate that the R183W mutation in PP2A Aα scaffold abrogates the tumor suppressive actions of PP2A, thereby potentiating oncogenic signaling and reducing drug sensitivity of RAS-mutant cells.
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http://dx.doi.org/10.1038/s41388-019-1012-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6980487PMC
January 2020

Is the Fate of Clinical Candidate Arry-520 Already Sealed? Predicting Resistance in Eg5-Inhibitor Complexes.

Mol Cancer Ther 2019 12 5;18(12):2394-2406. Epub 2019 Sep 5.

Université Grenoble Alpes, CNRS, CEA, Institut de Biologie Structurale (IBS), Grenoble, France.

Arry-520 is an advanced drug candidate from the Eg5 inhibitor class undergoing clinical evaluation in patients with relapsed or refractory multiple myeloma. Here, we show by structural analysis that Arry-520 binds stoichiometrically to the motor domain of Eg5 in the conventional allosteric loop L5 pocket in a complex that suggests the same structural mechanism as other Eg5 inhibitors. We have previously shown that acquired resistance through mutations in the allosteric-binding site located at loop L5 in the Eg5 structure appears to be independent of the inhibitors' scaffold, which suggests that Arry-520 will ultimately have the same fate. When Arry-520 was assessed in two cell lines selected for the expression of either Eg5(D130A) or Eg5(L214A) STLC-resistant alleles, mutations previously shown to convey resistance to this class of inhibitors, it was inactive in both. Surprisingly, when the cells were challenged with ispinesib, another Eg5 inhibitor, the Eg5(D130A) cells were resistant, but those expressing Eg5(L214A) were strikingly sensitive. Molecular dynamics simulations suggest that subtle differences in ligand binding and flexibility in both compound and protein may alter allosteric transmission from the loop L5 site that do not necessarily result in reduced inhibitory activity in mutated Eg5 structures. Although we predict that cells challenged with Arry-520 in the clinical setting are likely to acquire resistance through point mutations in the Eg5-binding site, the data for ispinesib suggest that this resistance mechanism is not scaffold independent as previously thought, and new inhibitors can be designed that retain inhibitory activity in these resistant cells.
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http://dx.doi.org/10.1158/1535-7163.MCT-19-0154DOI Listing
December 2019

Repurposing of Tranilast for Potential Neuropathic Pain Treatment by Inhibition of Sepiapterin Reductase in the BH Pathway.

ACS Omega 2019 Jul 10;4(7):11960-11972. Epub 2019 Jul 10.

Centre for Biomarker Research, School of Applied Sciences, Department of Pharmacy, School of Applied Sciences, Innovative Physical Organic Solutions (IPOS), Department of Chemical and Biological Sciences, and Department of Chemical Sciences, School of Applied Sciences, University of Huddersfield, Queensgate, Huddersfield HD1 3DH, U.K.

Tetrahydrobiopterin (BH) is a cofactor in the production of various signaling molecules including nitric oxide, dopamine, adrenaline, and noradrenaline. BH levels are critical for processes associated with cardiovascular function, inflammation, mood, pain, and neurotransmission. Increasing pieces of evidence suggest that BH is upregulated in chronic pain. Sepiapterin reductase (SPR) catalyzes both the reversible reduction of sepiapterin to dihydrobiopterin (BH) and 6-pyruvoyl-tetrahydrobiopterin to BH within the BH pathway. Therefore, inhibition of SPR by small molecules can be used to control BH production and ultimately alleviate chronic pain. Here, we have used various in silico and in vitro experiments to show that tranilast, licensed for use in bronchial asthma, can inhibit sepiapterin reduction by SPR. Docking and molecular dynamics simulations suggest that tranilast can bind to human SPR (hSPR) at the same site as sepiapterin including S157, one of the catalytic triad residues of hSPR. Colorimetric assays revealed that tranilast was nearly twice as potent as the known hSPR inhibitor, -acetyl serotonin. Tranilast was able to inhibit hSPR activity both intracellularly and extracellularly in live cells. Triple quad mass spectrophotometry of cell lysates showed a proportional decrease of BH in cells treated with tranilast. Our results suggest that tranilast can act as a potent hSPR inhibitor and therefore is a valid candidate for drug repurposing in the treatment of chronic pain.
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http://dx.doi.org/10.1021/acsomega.9b01228DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6682008PMC
July 2019

Mutations in and previously unimplicated genes of the BMP, Wnt, and Hedgehog pathways in syndromic craniosynostosis.

Proc Natl Acad Sci U S A 2019 07 10;116(30):15116-15121. Epub 2019 Jul 10.

Department of Genetics, Yale University School of Medicine, New Haven, CT 06510;

Craniosynostosis (CS) is a frequent congenital anomaly featuring the premature fusion of 1 or more sutures of the cranial vault. Syndromic cases, featuring additional congenital anomalies, make up 15% of CS. While many genes underlying syndromic CS have been identified, the cause of many syndromic cases remains unknown. We performed exome sequencing of 12 syndromic CS cases and their parents, in whom previous genetic evaluations were unrevealing. Damaging de novo or transmitted loss of function (LOF) mutations were found in 8 genes that are highly intolerant to LOF mutation ( = 4.0 × 10); additionally, a rare damaging mutation in , which has a lower level of intolerance, was identified. Four probands had rare damaging mutations (2 de novo) in , a transcription factor that orchestrates neural crest cell migration and differentiation; this mutation burden is highly significant ( = 8.2 × 10). Three probands had rare damaging mutations in , , or , which function in the Hedgehog, BMP, and Wnt signaling pathways; other genes in these pathways have previously been implicated in syndromic CS. Similarly, damaging de novo mutations were identified in genes encoding the chromatin modifier , and , encoding catenin α-1. These findings establish as a CS gene, have implications for assessing risk to subsequent children in these families, and provide evidence implicating other genes in syndromic CS. This high yield indicates the value of performing exome sequencing of syndromic CS patients when sequencing of known disease loci is unrevealing.
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http://dx.doi.org/10.1073/pnas.1902041116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6660739PMC
July 2019
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