DNA Base Changes Research Articles

Foes to friends: do pathogens hold the key to gene therapy?

Imagine starting life with a disease where the chances of reaching a fifth birthday are unlikely, let alone making it to adulthood, and the only treatments available are palliative at best. This is the reality facing millions of individuals born with a rare genetic disease. Sometimes, due to a single DNA base change – out of a possible 3 billion – occurring in the wrong place. A simple spelling mistake! However, recent years have seen the first effective gene therapies being developed, based on supplementing patient cells with functional copies of the faulty genes, or using antisense oligonucleotides or small molecules to alter pre-mRNA processing. The challenges were how to deliver genes or alter gene expression in the diseased cells and how to do it safely, without negatively affecting other endogenous genes. The answers came in the form of viruses, small non-coding RNAs and bacterial proteins, which is somewhat ironic, because we are using infectious agents or mimetics of their products to help cure diseases. With old enemies turned allies, tool sets are now being assembled to tackle the most challenging of genetic diseases. This article explores some of those tool chests in further detail, using ataxia telangiectasia, spinal muscular atrophy and several other rare diseases to highlight progress and challenges.

Functional and biological heterogeneity of KRASQ61 mutations.

Missense mutations at the three hotspots in the guanosine triphosphatase (GTPase) RAS-Gly12, Gly13, and Gln61 (commonly known as G12, G13, and Q61, respectively)-occur differentially among the three RAS isoforms. Q61 mutations in KRAS are infrequent and differ markedly in occurrence. Q61H is the predominant mutant (at 57%), followed by Q61R/L/K (collectively 40%), and Q61P and Q61E are the rarest (2 and 1%, respectively). Probability analysis suggested that mutational susceptibility to different DNA base changes cannot account for this distribution. Therefore, we investigated whether these frequencies might be explained by differences in the biochemical, structural, and biological properties of KRASQ61 mutants. Expression of KRASQ61 mutants in NIH 3T3 fibroblasts and RIE-1 epithelial cells caused various alterations in morphology, growth transformation, effector signaling, and metabolism. The relatively rare KRASQ61E mutant stimulated actin stress fiber formation, a phenotype distinct from that of KRASQ61H/R/L/P, which disrupted actin cytoskeletal organization. The crystal structure of KRASQ61E was unexpectedly similar to that of wild-type KRAS, a potential basis for its weak oncogenicity. KRASQ61H/L/R-mutant pancreatic ductal adenocarcinoma (PDAC) cell lines exhibited KRAS-dependent growth and, as observed with KRASG12-mutant PDAC, were susceptible to concurrent inhibition of ERK-MAPK signaling and of autophagy. Our results uncover phenotypic heterogeneity among KRASQ61 mutants and support the potential utility of therapeutic strategies that target KRASQ61 mutant-specific signaling and cellular output.

T Cell Recognition of Tumor Neoantigens and Insights Into T Cell Immunotherapy.

In cancer, non-synonymous DNA base changes alter protein sequence and produce neoantigens that are detected by the immune system. For immune detection, neoantigens must first be presented on class I or II human leukocyte antigens (HLA) followed by recognition by peptide-specific receptors, exemplified by the T-cell receptor (TCR). Detection of neoantigens represents a unique challenge to the immune system due to their high similarity with endogenous ‘self’ proteins. Here, we review insights into how TCRs detect neoantigens from structural studies and delineate two broad mechanistic categories: 1) recognition of mutated ‘self’ peptides and 2) recognition of novel ‘non-self’ peptides generated through anchor residue modifications. While mutated ‘self’ peptides differ only by a single amino acid from an existing ‘self’ epitope, mutations that form anchor residues generate an entirely new epitope, hitherto unknown to the immune system. We review recent structural studies that highlight these structurally distinct mechanisms and discuss how they may lead to differential anti-tumor immune responses. We discuss how T cells specific for neoantigens derived from anchor mutations can be of high affinity and provide insights to their use in adoptive T cell transfer-based immunotherapy.

The Point of Base Editors: Correcting Point Mutations

Genetic Engineering & Biotechnology NewsVol. 41, No. S4 CRISPR & Genome EditingThe Point of Base Editors: Correcting Point MutationsSpecific and precise base changes in genomic DNA and in RNA offer hope to the millions of people suffering genetic diseases associated with point mutationsMaryAnn LabantMaryAnn LabantSearch for more papers by this authorPublished Online:12 Sep 2021https://doi.org/10.1089/gen.41.S4.06AboutSectionsView articleView Full TextPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail View articleFiguresReferencesRelatedDetailsCited byOne Health: Animal Models of Heritable Human Bleeding Diseases26 December 2022 | Animals, Vol. 13, No. 1 Volume 41Issue S4Sep 2021 Information© 2021 by GEN PublishingTo cite this article:MaryAnn Labant.The Point of Base Editors: Correcting Point Mutations.Genetic Engineering & Biotechnology News.Sep 2021.S17-S19.http://doi.org/10.1089/gen.41.S4.06Published in Volume: 41 Issue S4: September 12, 2021PDF download

Programmable base editing in zebrafish using a modified CRISPR-Cas9 system.

The use of CRISPR/Cas9 to knockout genes in zebrafish has been well established. However, to better model many human diseases that are caused by point mutations, a robust methodology for generating desirable DNA base changes is still needed. Recently, Cas9-linked cytidine deaminases (base editors) evolved as a strategy to introduce single base mutations in model organisms. They have been used to convert cytidine to thymine at specific genomic loci. Here we describe a protocol for using the base editing system in zebrafish and its application to reproduce a single base mutation observed in human Ablepharon-Macrostomia Syndrome.

Beam launches for base editing

It’s shaping up to be another year full of cash flowing for CRISPR gene editing. In just the past two months, four companies have launched based on newly discovered or designed versions of CRISPR. Of all those variants, the base editors developed by David R. Liu’s lab at the Broad Institute of MIT & Harvard might be causing the most excitement. They’ve certainly brought in the most money. Just two years ago, Liu debuted his lab’s first base editors, which can make single nucleotide base changes in DNA. Since then, those base editors, along with advanced iterations, have been shared with research labs worldwide more than 3,000 times. They’ve been used to edit DNA of bacteria, fungi, plants, insects, fish, mice, and even human embryos. Amid that whirlwind of development, Liu has been quietly building a company that will try to transform the base editors into treatments for genetic diseases.

Oxidation Products of 5-Methylcytosine are Decreased in Senescent Cells and Tissues of Progeroid Mice.

5-Hydroxymethylcytosine and 5-formylcytosine are stable DNA base modifications generated from 5-methylcytosine by the ten-eleven translocation protein family that function as epigenetic markers. 5-Hydroxymethyluracil may also be generated from thymine by ten-eleven translocation enzymes. Here, we asked if these epigenetic changes accumulate in senescent cells, since they are thought to be inversely correlated with proliferation. Testing this in ERCC1-XPF-deficient cells and mice also enabled discovery if these DNA base changes are repaired by nucleotide excision repair. Epigenetic marks were measured in proliferating, quiescent and senescent wild-type (WT) and Ercc1-/- primary mouse embryonic fibroblasts. The pattern of epigenetic marks depended more on the proliferation status of the cells than their DNA repair capacity. The cytosine modifications were all decreased in senescent cells compared to quiescent or proliferating cells, whereas 5-(hydroxymethyl)-2'-deoxyuridine was increased. In vivo, both 5-(hydroxymethyl)-2'-deoxyuridine and 5-(hydroxymethyl)-2'-deoxycytidine were significantly increased in liver tissues of aged WT mice compared to young adult WT mice. Livers of Ercc1-deficient mice with premature senescence and aging had reduced level of 5-(hydroxymethyl)-2'-deoxycytidine and 5-formyl-2'-deoxycytidine compared to aged-matched WT controls. Taken together, we demonstrate for the first time, that 5-(hydroxymethyl)-2'-deoxycytidine is significantly reduced in senescent cells and tissue, potentially yielding a novel marker of senescence.

TALEN-Mediated Homologous Recombination Produces Site-Directed DNA Base Change and Herbicide-Resistant Rice

Over the last decades, much endeavor has been made to advance genome editing technology due to its promising role in both basic and synthetic biology. The breakthrough has been made in recent years with the advent of sequence-specific endonucleases, especially zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPRs) guided nucleases (e.g., Cas9). In higher eukaryotic organisms, site-directed mutagenesis usually can be achieved through non-homologous end-joining (NHEJ) repair to the DNA double-strand breaks (DSBs) caused by the exogenously applied nucleases. However, site-specific gene replacement or genuine genome editing through homologous recombination (HR) repair to DSBs remains a challenge. As a proof of concept gene replacement through TALEN-based HR in rice (Oryza sativa), we successfully produced double point mutations in rice acetolactate synthase gene (OsALS) and generated herbicide resistant rice lines by using TALENs and donor DNA carrying the desired mutations. After ballistic delivery into rice calli of TALEN construct and donor DNA, nine HR events with different genotypes of OsALS were obtained in T0 generation at the efficiency of 1.4%–6.3% from three experiments. The HR-mediated gene edits were heritable to the progeny of T1 generation. The edited T1 plants were as morphologically normal as the control plants while displayed strong herbicide resistance. The results demonstrate the feasibility of TALEN-mediated genome editing in rice and provide useful information for further genome editing by other nuclease-based genome editing platforms.

Methanol-based fixation is superior to buffered formalin for next-generation sequencing of DNA from clinical cancer samples

Next-generation sequencing (NGS) of tumour samples is a critical component of personalised cancer treatment, but it requires high-quality DNA samples. Routine neutral-buffered formalin (NBF) fixation has detrimental effects on nucleic acids, causing low yields, as well as fragmentation and DNA base changes, leading to significant artefacts. We have carried out a detailed comparison of DNA quality from matched samples isolated from high-grade serous ovarian cancers from 16 patients fixed in methanol and NBF. These experiments use tumour fragments and mock biopsies to simulate routine practice, ensuring that results are applicable to standard clinical biopsies. Using matched snap-frozen tissue as gold standard comparator, we show that methanol-based fixation has significant benefits over NBF, with greater DNA yield, longer fragment size and more accurate copy-number calling using shallow whole-genome sequencing (WGS). These data also provide a new approach to understand and quantify artefactual effects of fixation using non-negative matrix factorisation to analyse mutational spectra from targeted and WGS data. We strongly recommend the adoption of methanol fixation for sample collection strategies in new clinical trials. This approach is immediately available, is logistically simple and can offer cheaper and more reliable mutation calling than traditional NBF fixation.

Biochemical and Biological Studies of Mouse APOBEC3

Many murine leukemia viruses (MLVs) are partially resistant to restriction by mouse APOBEC3 (mA3) and essentially fully resistant to induction of G-to-A mutations by mA3. In contrast, Vif-deficient HIV-1 (ΔVif HIV-1) is profoundly restricted by mA3, and the restriction includes high levels of G-to-A mutation. Human APOBEC3G (hA3G), unlike mA3, is fully active against MLVs. We produced a glutathione S-transferase-mA3 fusion protein in insect cells and demonstrated that it possesses cytidine deaminase activity, as expected. This activity is localized within the N-terminal domain of this 2-domain protein; the C-terminal domain is enzymatically inactive but required for mA3 encapsidation into retrovirus particles. We found that a specific arginine residue and several aromatic residues, as well as the zinc-coordinating cysteines in the C-terminal domain, are necessary for mA3 packaging; a structural model of this domain suggests that these residues line a potential nucleic acid-binding interface. Mutation of a few potential phosphorylation sites in mA3 drastically reduces its antiviral activity by impairing either deaminase activity or its encapsidation. mA3 deaminates short single-stranded DNA oligonucleotides preferentially toward their 3' ends, whereas hA3G exhibits the opposite polarity. However, when packaged into infectious ΔVif HIV-1 virions, both mA3 and hA3G preferentially induce deaminations toward the 5' end of minus-strand viral DNA, presumably because of the sequence of events during reverse transcription in vivo. Despite the fact that mA3 in MLV particles does not induce detectable deaminations upon infection, its deaminase activity is easily detected in virus lysates. We still do not understand how MLV resists mA3-induced G-to-A mutation. One way that mammalian cells defend themselves against infection by retroviruses is with APOBEC3 proteins. These proteins convert cytidine bases to uridine bases in retroviral DNA. However, mouse APOBEC3 protein blocks infection by murine leukemia viruses without catalyzing this base change, and the mechanism of inhibition is not understood in this case. We have produced recombinant mouse APOBEC3 protein for the first time and characterized it here in a number of ways. Our mutational studies shed light on the mechanism by which mouse APOBEC3 protein is incorporated into retrovirus particles. While mouse APOBEC3 does not catalyze base changes in murine leukemia virus DNA, it can be recovered from these virus particles in enzymatically active form; it is still not clear why it fails to induce base changes when these viruses infect new cells.

Conditionally fluorescent molecular probes for detecting single base changes in double-stranded DNA

Small variations in nucleic acid sequences can have far-reaching phenotypic consequences. Reliably distinguishing closely related sequences is therefore important for research and clinical applications. Here, we demonstrate that conditionally fluorescent DNA probes are capable of distinguishing variations of a single base in a stretch of target DNA. These probes use a novel programmable mechanism in which each single nucleotide polymorphism generates two thermodynamically destabilizing mismatch bubbles rather than the single mismatch formed during typical hybridization-based assays. Up to a 12,000-fold excess of a target that contains a single nucleotide polymorphism is required to generate the same fluorescence as one equivalent of the intended target, and detection works reliably over a wide range of conditions. Using these probes we detected point mutations in a 198 base-pair subsequence of the Escherichia coli rpoB gene. That our probes are constructed from multiple oligonucleotides circumvents synthesis limitations and enables long continuous DNA sequences to be probed.

Local Delivery of Gene-Modifying Triplex-Forming Molecules to the Epidermis

Epidermal keratinocytes are particularly suitable candidates for in situ gene correction. Intraperitoneal administration of a triplex-forming oligonucleotide (TFO) was shown previously to introduce DNA base changes in a reporter gene in skin, without identifying which cells had been targeted. We extend those previous experiments using two triplex-forming molecules (TFMs), a peptide nucleic acid (PNA-Antp) and a TFO (AG30), and two lines of transgenic mice that have the chromosomally integrated λsupFG1 shuttle-reporter transgene. Successful in vivo genomic modification occurs in epidermis and dermis in CD1 transgenic mice following either intraperitoneal or intradermal delivery of the PNA-Antennapedia conjugate. FITC-PNA-Antp accumulates in nuclei of keratinocytes and, after intradermal delivery of the PNA-Antp, chromosomally modified, keratin 5 positive basal keratinocytes persist for at least 10 days. In hairless (SKH1) mice with the λsupFG1 transgene, intradermal delivery of the TFO, AG30, introduces gene modifications in both tail and back skin and those chromosomal modifications persist in basal keratinocytes for 10 days. Hairless mice should facilitate comparison of various targeting agents and methods of delivery. Gene targeting by repeated local administration of oligonucleotides may prove clinically useful for judiciously selected disease-causing genes in the epidermis.

μ-eLCR: a microfabricated device for electrochemical detection of DNA base changes in breast cancer cell lines

Microfabricated devices for the electrochemical detection of single DNA base changes in cancer cell lines are highly desirable due to the inherent advantages such as portability, simplicity, and the rapid and inexpensive nature of electrochemical readout methods. Moreover, molecular sensors that use microscale-footprint working electrodes have shown high signal-to-noise ratio. Herein we report a microdevice-based electrochemical assay (μ-eLCR) measuring ligase chain reaction (LCR)-amplified long and short "knife" motifs which reflect the presence or absence of a DNA base change of interest in a target sequence. This μ-eLCR approach has higher sensitivity (4.4 to 10 fold improvement over macrodisk electrodes) and good reproducibility (%RSD 6.5%, n = 12) for the detection of LCR-amplified DNA bases. The devices also exhibited excellent sensitivity for the detection of DNA methylation (C to T base change in a locus associated with cancer metastasis) in two cell lines and serum derived DNA samples. We believe that the μ-eLCR device may be a useful diagnostic tool for inexpensive and rapid detection of single DNA base change applications such as DNA methylation and single nucleotide polymorphism (SNP) detection.

ELCR: electrochemical detection of single DNA base changes via Ligase Chain Reaction

Simple, inexpensive and wide-scaled analysis of single DNA base changes ('point mutations' which include SNP's, somatic mutations and epigenetic changes) is one of the holy grail's of point-of-care diagnostics. Herein, we present eLCR: a simple methodology that fuses Ligase Chain Reaction (LCR) with electrochemical detection based on DNA-mediated charge transport. LCR generates long and short "knife" motifs representing the presence or absence of single DNA base changes, which are then detected electrochemically by either methylene blue intercalation or horseradish peroxidase labelling.

Reversible Isolated Hypogonadotropic Hypogonadism due to Mutations in the Neurokinin B Regulation of Gonadotropin-Releasing Hormone Release

In this commentary, congenital isolated hypogonadotropic hypogonadism (IHH) is defined as absent, incomplete, or arrested isosexual development when both chronological and bone ages are at least 18 yr, together with low concentrations of gonadotropins and sex hormones in the absence of systemic disease, syndromic malformations, nutritional deprivation, and other functional or anatomic pituitary abnormalities. To date, the identified forms of IHH are primarily due to inactivating mutations in the genes whose products control: 1) the differentiation and development (NROB1 or DAX1, CHD7, FGFR1, FGF8); 2) the subsequent migration of neurons that synthesize and secrete GnRH from their embryological site of origin in the nasal (olfactory) placode to their network in the hypothalamic arcuate nucleus (infundibulum); 3) the synthesis, release, and action of GnRH; and 4) the synthesis and secretion of the gonadotropins (LH, FSH) (Table 1) (1, 2). The migration of GnRH-synthesizing nerve cells requires the coordinated activity of the products encoded by KAL1, FGFR1, FGF8, NELF, PROK2, PROKR2, and likely many other factors yet to be identified. Inasmuch as loss-of-function mutations in these genes are often associated with abnormalities of olfaction (anosmia, hyposmia), they constitute the defined forms of Kallmann syndrome. Disrupted expression of factors (LEP, LEPR, KISS1R) that regulate the synthesis of GnRH (GNRH1), its storage in the median eminence, its release into the hypophyseal-portal vasculature, its action upon the pituitary gonadotrope (GHAHR), or the expression and synthesis of the gonadotropins (LH , FSH , PCSK1) themselves also leads to IHH (3). To the list of modulators of GnRH synthesis and secretion have recently been added the products of TAC3 (tachykinin 3 or neurokinin B) and TACR3 (the G proteincoupled receptor for tachykinin 3) (4, 5). Gianetti et al. (6) have now genotyped TAC3 and TACR3 in 345 patients (247 males) with IHH and have identified both nonsense and missense nonsynonymous and synonymous mutations in TACR3 in 19 patients (four females) (5.5%) as well as a frameshift mutation in TAC3 in one woman (0.3%). In nine subjects, the TACR3 mutation was biallelic and in eight of nine homozygotic, whereas in 10 patients the mutations in TACR3 were monoallelic. Both recessive and dominant modes of IHH transmission were present in patients with mutations in TACR3. Patients with heterozygous mutations in TACR3 were clinically similar to the subjects with homozygous mutations. In none of the patients with heterozygous mutations in TACR3 were mutations in other examined IHH genes identified, suggesting that digenic transmission of IHH was less likely in these subjects (7). Functional characterization of several of the missense mutations in TACR3 revealed that the DNA base changes inactivated the product except for two monoallelic mutations (Gly18Asp and Ile249Val) in which in vitro bioactivity was normal, leaving unexplained the mechanism by which these mutations resulted in IHH in their carriers. Clinically, 14 of 15 males with IHH due to mutations in TACR3 had phallic lengths that were less than 10.5 cm and were classified as micropenis. As anticipated, at diagnosis of IHH, most affected males had very small testes, but in some patients testicular volumes were a bit larger than prepubertal, a size increase attributable to endogenous mechanisms. After discontinuation of sex hormone replacement therapy, testicular volume and/or gonadotropin and/or testosterone secretion increasedspontaneously infivemales, twoofwhomreported

Hydrogen peroxide induces a specific DNA base change profile in the presence of the iron chelator 2,2’ dipyridyl in Escherichia coli

Hydrogen peroxide induces a specific DNA base change profile in the presence of the iron chelator 2,2’ dipyridyl in Escherichia coli

Hydrogen peroxide induces a specific DNA base change profile in the presence of the iron chelator 2,2’ dipyridyl in Escherichia coli

Pretreatment of Escherichia coli cultures with the iron chelator 2,2'-dipyridyl (1 mM) protects against the lethal effects of low concentrations of hydrogen peroxide (<15 mM). However, at H(2)O(2) concentrations equal to or greater than 15 mM, dipyridyl pretreatment increases lethality and mutagenesis, which is attributed to the formation of different types of DNA lesions. We show here that pretreatment with dipyridyl (1 mM) prior to challenge with high H(2)O(2) concentrations (>or=15 mM) induced mainly G:C-->A:T transitions (more than 100X with 15 mM and more than 250X with 20 mM over the spontaneous mutagenesis rate) in E. coli. In contrast, high H(2)O(2) concentrations in the absence of dipyridyl preferentially induced A:T-->T:A transversions (more than 1800X and more than 300X over spontaneous mutagenesis for 15 and 20 mM, respectively). We also show that in the fpg nth double mutant, the rpoB gene mutation (RifS-RifR) induced by 20 mM H(2)O(2) alone (20X higher) was increased in 20 mM H(2)O(2) and dipyridyl-treated cultures (110X higher), suggesting additional and/or different lesions in cells treated with H(2)O(2) under iron deprivation. It is suggested that, upon iron deprivation, cytosine may be the main damaged base and the origin of the pre-mutagenic lesions induced by H(2)O(2).

Identification of six new HLA‐G alleles with non‐coding DNA base changes

We identified six novel human leukocyte antigen-G alleles with synonymous mutations in Caucasian and/or African populations.

Different effects of novel mtDNA G3242A and G3244A base changes adjacent to a common A3243G mutation in patients with mitochondrial disorders

Two novel mitochondrial DNA base changes were identified at both sides of the 3243A > G mutation, the most common mutation associated with mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS). One was a 3244G > A transition in a girl with MELAS. The other was a 3242G > A transition in a girl with a mitochondrial disorder without a MELAS phenotype. Although the two base changes were adjacent to the 3243A > G mutation, they had different effects on the clinical phenotype, muscle pathology, and respiratory chain enzyme activity. Investigations of the different effects of the 3244G > A and 3242G > A base changes may provide a better understanding of tRNA dysfunction in mitochondrial disorders.

Epigenetic and Pharmacoepigenomic Studies of Major Psychoses and Potentials for Therapeutics

Individuals with neuropsychiatric diseases have epigenetic programming disturbances, both in the brain, which is the primary affected organ, and in secondary tissues. Epigenetic modulations are molecular modifications made to DNA, RNA and proteins that fine-tune genotype into phenotype and do not include DNA base changes. For instance, gene-expression modulation is linked to epigenetic codes in chromatin that consist of post-replication DNA methylation and histone protein modifications (e.g., methylation, acetylation and so on), particularly in gene-promoter regions. Epigenetic coding is modulated globally, and in a gene-specific manner by environmental exposures that include nutrition, toxins, drugs and so on. Analysis of epigenetic aberrations in diseases helps to identify dysfunctional genes and pathways, establish more robust cause-effect relationships than genetic studies alone, and identify new pharmaceutical targets and drugs, including nucleic acid reagents such as inhibitory RNAs. The emerging science of pharmacoepigenomics can impact the treatment of psychiatric and other complex diseases. In fact, some therapeutics now in use target epigenetic programming. In the near future, epigenetic interventions should help stabilize affected individuals and lead to prevention strategies.