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Nucleic Acids Research Jul 2023Understanding the effects of mutations on protein stability is crucial for variant interpretation and prioritisation, protein engineering, and biotechnology. Despite...
Understanding the effects of mutations on protein stability is crucial for variant interpretation and prioritisation, protein engineering, and biotechnology. Despite significant efforts, community assessments of predictive tools have highlighted ongoing limitations, including computational time, low predictive power, and biased predictions towards destabilising mutations. To fill this gap, we developed DDMut, a fast and accurate siamese network to predict changes in Gibbs Free Energy upon single and multiple point mutations, leveraging both forward and hypothetical reverse mutations to account for model anti-symmetry. Deep learning models were built by integrating graph-based representations of the localised 3D environment, with convolutional layers and transformer encoders. This combination better captured the distance patterns between atoms by extracting both short-range and long-range interactions. DDMut achieved Pearson's correlations of up to 0.70 (RMSE: 1.37 kcal/mol) on single point mutations, and 0.70 (RMSE: 1.84 kcal/mol) on double/triple mutants, outperforming most available methods across non-redundant blind test sets. Importantly, DDMut was highly scalable and demonstrated anti-symmetric performance on both destabilising and stabilising mutations. We believe DDMut will be a useful platform to better understand the functional consequences of mutations, and guide rational protein engineering. DDMut is freely available as a web server and API at https://biosig.lab.uq.edu.au/ddmut.
Topics: Deep Learning; Mutation; Point Mutation; Protein Stability; Software; Proteins
PubMed: 37283042
DOI: 10.1093/nar/gkad472 -
Homology directed correction, a new pathway model for point mutation repair catalyzed by CRISPR-Cas.Scientific Reports May 2022Gene correction is often referred to as the gold standard for precise gene editing and while CRISPR-Cas systems continue to expand the toolbox for clinically relevant...
Gene correction is often referred to as the gold standard for precise gene editing and while CRISPR-Cas systems continue to expand the toolbox for clinically relevant genetic repair, mechanistic hurdles still hinder widespread implementation. One of the most prominent challenges to precise CRISPR-directed point mutation repair centers on the prevalence of on-site mutagenesis, wherein insertions and deletions appear at the targeted site following correction. Here, we introduce a pathway model for Homology Directed Correction, specifically point mutation repair, which enables a foundational analysis of genetic tools and factors influencing precise gene editing. To do this, we modified an in vitro gene editing system which utilizes a cell-free extract, CRISPR-Cas RNP and donor DNA template to catalyze point mutation repair. We successfully direct correction of four unique point mutations which include two unique nucleotide mutations at two separate targeted sites and visualize the repair profiles resulting from these reactions. This extension of the cell-free gene editing system to model point mutation repair may provide insight for understanding the factors influencing precise point mutation correction.
Topics: CRISPR-Cas Systems; Catalysis; Gene Editing; Mutagenesis; Mutation; Point Mutation
PubMed: 35581233
DOI: 10.1038/s41598-022-11808-2 -
Genes Oct 2021The OTUD6B and ZMIZ1 genes were recently identified as causes of syndromic intellectual disability (ID) with shared phenotypes of facial dysmorphism, distal limb...
The OTUD6B and ZMIZ1 genes were recently identified as causes of syndromic intellectual disability (ID) with shared phenotypes of facial dysmorphism, distal limb anomalies, and seizure disorders. OTUD6B- and ZMIZ1-related ID are inherited in autosomal recessive and autosomal dominant patterns, respectively. We report a 5-year-old girl with developmental delay, facial phenotypes resembling Williams syndrome, and cardiac defects. The patient also had terminal broadening of the fingers and polydactyly. Cytogenomic microarray (CMA), whole exome sequencing (WES), and mRNA analysis were performed. The CMA showed a paternally inherited 0.118 Mb deletion of 8q21.3, chr8:92084087-92202189, with OTUD6B involved. The WES identified a hemizygous OTUD6B variant, c.873delA (p.Lys291AsnfsTer3). The mother was heterozygous for this allele. The WES also demonstrated a heterozygous ZMIZ1 variant, c.1491 + 2T > C, in the patient and her father. This ZMIZ1 variant yielded exon 14 skipping, as evidenced by mRNA study. We suggest that Williams syndrome-like phenotypes, namely, periorbital edema, hanging cheek, and long and smooth philtrum represent expanded phenotypes of OTUD6B-related ID. Our data expand the genotypic spectrum of OTUD6B- and ZMIZ1-related disorders. This is the first reported case of a compound heterozygote featuring point mutation, chromosomal microdeletion of OTUD6B, and the unique event of OTUD6B, coupled with ZMIZ1 variants.
Topics: Alleles; Child, Preschool; Chromosome Deletion; Endopeptidases; Exome; Female; Genetic Predisposition to Disease; Heterozygote; Humans; Infant; Intellectual Disability; Male; Pedigree; Phenotype; Point Mutation; Transcription Factors; Exome Sequencing
PubMed: 34680978
DOI: 10.3390/genes12101583 -
Scientific Reports Jun 2023Protein-protein interactions (PPIs) play a critical role in all biological processes. Menin is tumor suppressor protein, mutated in multiple endocrine neoplasia type 1...
Protein-protein interactions (PPIs) play a critical role in all biological processes. Menin is tumor suppressor protein, mutated in multiple endocrine neoplasia type 1 syndrome and has been shown to interact with multiple transcription factors including (RPA2) subunit of replication protein A (RPA). RPA2, heterotrimeric protein required for DNA repair, recombination and replication. However, it's still remains unclear the specific amino acid residues that have been involved in Menin-RPA2 interaction. Thus, accurately predicting the specific amino acid involved in interaction and effects of MEN1 mutations on biological systems is of great interests. The experimental approaches for identifying amino acids in menin-RPA2 interactions are expensive, time-consuming, and challenging. This study leverages computational tools, free energy decomposition and configurational entropy scheme to annotate the menin-RPA2 interaction and effect on menin point mutation, thereby proposing a viable model of menin-RPA2 interaction. The menin-RPA2 interaction pattern was calculated on the basis of different 3D structures of menin and RPA2 complexes, constructed using homology modeling and docking strategy, generating three best-fit models: Model 8 (- 74.89 kJ/mol), Model 28 (- 92.04 kJ/mol) and Model 9 (- 100.4 kJ/mol). The molecular dynamic (MD) was performed for 200 ns and binding free energies and energy decomposition analysis were calculated using Molecular Mechanics Poisson-Boltzmann Surface Area (MM/PBSA) in GROMACS. From binding free energy change, model 8 of Menin-RPA2 exhibited most negative binding energy of - 205.624 kJ/mol, followed by model 28 of Menin-RPA2 with - 177.382 kJ/mol. After S606F point mutation in Menin, increase of BFE (ΔG) by - 34.09 kJ/mol in Model 8 of mutant Menin-RPA2 occurs. Interestingly, we found a significant reduction of BFE (ΔG) and configurational entropy by - 97.54 kJ/mol and - 2618 kJ/mol in mutant model 28 as compared the o wild type. Collectively, this is the first study to highlight the configurational entropy of protein-protein interactions thereby strengthening the prediction of two significant important interaction sites in menin for the binding of RPA2. These predicted sites could be vulnerable for structural alternation in terms of binding free energy and configurational entropy after missense mutation in menin.
Topics: Humans; Point Mutation; Mutation; Transcription Factors; Binding Sites; Multiple Endocrine Neoplasia Type 1; Amino Acids; Replication Protein A
PubMed: 37291166
DOI: 10.1038/s41598-023-35599-2 -
Nature Communications Mar 2022Retroviruses utilize the viral integrase (IN) protein to integrate a DNA copy of their genome into host chromosomal DNA. HIV-1 integration sites are highly biased...
Retroviruses utilize the viral integrase (IN) protein to integrate a DNA copy of their genome into host chromosomal DNA. HIV-1 integration sites are highly biased towards actively transcribed genes, likely mediated by binding of the IN protein to specific host factors, particularly LEDGF, located at these gene regions. We here report a substantial redirection of integration site distribution induced by a single point mutation in HIV-1 IN. Viruses carrying the K258R IN mutation exhibit a high frequency of integrations into centromeric alpha satellite repeat sequences, as assessed by deep sequencing, a more than 10-fold increase over wild-type. Quantitative PCR and in situ immunofluorescence assays confirm this bias of the K258R mutant virus for integration into centromeric DNA. Immunoprecipitation studies identify host factors binding to IN that may account for the observed bias for integration into centromeres. Centromeric integration events are known to be enriched in the latent reservoir of infected memory T cells, as well as in elite controllers who limit viral replication without intervention. The K258R point mutation in HIV-1 IN is also present in databases of latent proviruses found in patients, and may reflect an unappreciated aspect of the establishment of viral latency.
Topics: Centromere; HIV Integrase; Humans; Point Mutation; Proviruses; Virus Integration; Virus Replication
PubMed: 35304442
DOI: 10.1038/s41467-022-29097-8 -
International Journal of Molecular... Oct 2020Among the natural bases, guanine is the most oxidizable base. The damage caused by oxidation of guanine, commonly referred to as oxidative guanine damage, results in the... (Review)
Review
Among the natural bases, guanine is the most oxidizable base. The damage caused by oxidation of guanine, commonly referred to as oxidative guanine damage, results in the formation of several products, including 2,5-diamino-4-imidazol-4-one (Iz), 2,2,4-triamino-5(2)-oxazolone (Oz), guanidinoformimine (Gf), guanidinohydantoin/iminoallantoin (Gh/Ia), spiroiminodihydantoin (Sp), 5-carboxamido-5-formamido-2-iminohydantoin (2Ih), urea (Ua), 5-guanidino-4-nitroimidazole (NI), spirodi(iminohydantoin) (5-Si and 8-Si), triazine, the M+7 product, other products by peroxynitrite, alkylated guanines, and 8,5'-cyclo-2'-deoxyguanosine (cG). Herein, we summarize the present knowledge about base pairs containing the products of oxidative guanine damage and guanine. Of these products, Iz is involved in G-C transversions. Oz, Gh/Ia, and Sp form preferably Oz:G, Gh/Ia:G, and Sp:G base pairs in some cases. An involvement of Gf, 2Ih, Ua, 5-Si, 8-Si, triazine, the M+7 product, and 4-hydroxy-2,5-dioxo-imidazolidine-4-carboxylic acid (HICA) in G-C transversions requires further experiments. In addition, we describe base pairs that target the RNA-dependent RNA polymerase (RdRp) of RNA viruses and describe implications for the 2019 novel coronavirus (SARS-CoV-2): When products of oxidative guanine damage are adapted for the ribonucleoside analogs, mimics of oxidative guanine damages, which can form base pairs, may become antiviral agents for SARS-CoV-2.
Topics: Animals; Base Pairing; Betacoronavirus; DNA Damage; Guanine; Humans; Oxidation-Reduction; Point Mutation; SARS-CoV-2
PubMed: 33076559
DOI: 10.3390/ijms21207645 -
Nature Chemical Biology Jan 2022Glutathione peroxidase 4 (GPX4), as the only enzyme in mammals capable of reducing esterified phospholipid hydroperoxides within a cellular context, protects cells from...
Glutathione peroxidase 4 (GPX4), as the only enzyme in mammals capable of reducing esterified phospholipid hydroperoxides within a cellular context, protects cells from ferroptosis. We identified a homozygous point mutation in the GPX4 gene, resulting in an R152H coding mutation, in three patients with Sedaghatian-type spondylometaphyseal dysplasia. Using structure-based analyses and cell models, including patient fibroblasts, of this variant, we found that the missense variant destabilized a critical loop, which disrupted the active site and caused a substantial loss of enzymatic function. We also found that the R152H variant of GPX4 is less susceptible to degradation, revealing the degradation mechanism of the GPX4 protein. Proof-of-concept therapeutic treatments, which overcome the impaired R152H GPX4 activity, including selenium supplementation, selective antioxidants and a deuterated polyunsaturated fatty acid were identified. In addition to revealing a general approach to investigating rare genetic diseases, we demonstrate the biochemical foundations of therapeutic strategies targeting GPX4.
Topics: Humans; Phospholipid Hydroperoxide Glutathione Peroxidase; Point Mutation; Precision Medicine; Proof of Concept Study
PubMed: 34931062
DOI: 10.1038/s41589-021-00915-2 -
Bioinformatics (Oxford, England) Jun 2020Next-generation sequencing technologies have accelerated the discovery of single nucleotide variants in the human genome, stimulating the development of predictors for...
MOTIVATION
Next-generation sequencing technologies have accelerated the discovery of single nucleotide variants in the human genome, stimulating the development of predictors for classifying which of these variants are likely functional in disease, and which neutral. Recently, we proposed CScape, a method for discriminating between cancer driver mutations and presumed benign variants. For the neutral class, this method relied on benign germline variants found in the 1000 Genomes Project database. Discrimination could, therefore, be influenced by the distinction of germline versus somatic, rather than neutral versus disease driver. This motivates this article in which we consider predictive discrimination between recurrent and rare somatic single point mutations based solely on using cancer data, and the distinction between these two somatic classes and germline single point mutations.
RESULTS
For somatic point mutations in coding and non-coding regions of the genome, we propose CScape-somatic, an integrative classifier for predictively discriminating between recurrent and rare variants in the human cancer genome. In this study, we use purely cancer genome data and investigate the distinction between minimal occurrence and significantly recurrent somatic single point mutations in the human cancer genome. We show that this type of predictive distinction can give novel insight, and may deliver more meaningful prediction in both coding and non-coding regions of the cancer genome. Tested on somatic mutations, CScape-somatic outperforms alternative methods, reaching 74% balanced accuracy in coding regions and 69% in non-coding regions, whereas even higher accuracy may be achieved using thresholds to isolate high-confidence predictions.
AVAILABILITY AND IMPLEMENTATION
Predictions and software are available at http://CScape-somatic.biocompute.org.uk/.
CONTACT
[email protected] or [email protected].
SUPPLEMENTARY INFORMATION
Supplementary data are available at Bioinformatics online.
Topics: Genome, Human; Genomics; Humans; Mutation; Neoplasms; Point Mutation; Software
PubMed: 32282885
DOI: 10.1093/bioinformatics/btaa242 -
Aging Jun 2021
Topics: Extracellular Vesicles; Gastrointestinal Microbiome; Gene Editing; Humans; Molecular Targeted Therapy; Point Mutation; Progeria
PubMed: 34176790
DOI: 10.18632/aging.203254 -
PloS One 2021Macrolides are generally considered to be the drugs of choice for treatment of patients with Mycoplasma pneumoniae infection. However, macrolide-resistant M. pneumoniae...
OBJECTIVES
Macrolides are generally considered to be the drugs of choice for treatment of patients with Mycoplasma pneumoniae infection. However, macrolide-resistant M. pneumoniae has been emerging since about 2000. The Smart Gene® system (MIZUHO MEDY Co., Ltd., Tosu, Japan) is a novel fully automated system for detection of pathogens using the method of quantitative polymerase chain reaction (qPCR) with QProbe (QProbe PCR). The entire procedure is completed within 50 min and the size of the instrument is small (15 x 34 x 30 cm). The purpose of this study was to evaluate the usefulness of the Smart Gene® system for detection of M. pneumoniae and detection of a point mutation at domain V of the 23S rRNA gene of M. pneumoniae.
MATERIALS
Pharyngeal swab samples were collected from 154 patients who were suspected of having respiratory tract infections associated with M. pneumoniae.
RESULTS
Compared with the results of qPCR, the sensitivity and specificity of the Smart Gene® system were 98.7% (78/79) and 100.0% (75/75), respectively. A point mutation at domain V of the 23S rRNA gene was detected from 7 (9.0%) of 78 M. pneumoniae-positive samples by the Smart Gene® system and these results were confirmed by direct sequencing. The minimum inhibitory concentrations of clarithromycin among the 5 isolates of M. pneumoniae with a point mutation at domain V of the 23S rRNA gene were >64 μg/ml and those among the 33 isolates without a mutation in the 23S rRNA gene were <0.0625 μg/ml.
CONCLUSION
The Smart Gene® system is a rapid and accurate assay for detection of the existence of M. pneumoniae and a point mutation at domain V of the 23S rRNA gene of M. pneumoniae at the same time. The Smart Gene® system is suitable for point-of-care testing in both hospital and outpatient settings.
Topics: Adolescent; Adult; Aged; Child; Child, Preschool; Clarithromycin; DNA, Bacterial; DNA, Ribosomal; Female; Humans; Infant; Male; Microbial Sensitivity Tests; Microbial Viability; Middle Aged; Mycoplasma pneumoniae; Pharynx; Pneumonia, Mycoplasma; Point Mutation; Point-of-Care Testing; RNA, Ribosomal, 23S; Reagent Kits, Diagnostic; Real-Time Polymerase Chain Reaction; Sensitivity and Specificity; Young Adult
PubMed: 34648603
DOI: 10.1371/journal.pone.0258694