Base Pair Substitutions Research Articles

A splice-site variant (c.3289-1G>T) in OTOF underlies profound hearing loss in a Pakistani kindred

BackgroundHearing loss/deafness is a common otological disorder found in the Pakistani population due to the high prevalence of consanguineous unions, but the full range of genetic causes is still unknown.MethodsA large consanguineous Pakistani kindred with hearing loss was studied. Whole-exome sequencing and Sanger sequencing were performed to search for the candidate gene underlying the disease phenotype. A minigene assay and reverse transcription polymerase chain reaction was used to assess the effect of splicing variants.ResultsThe splicing variants of OTOF (NM_194248, c.3289-1G>T) cosegregated with the disease phenotype in this Pakistani family. The substitution of a single base pair causes the deletion of 10 bp (splicing variant 1) or 13 bp (splicing variant 2) from exon 27, which results in truncated proteins of 1141 and 1140 amino acids, respectively.ConclusionOur findings reveal an OTOF splice-site variant as pathogenic for profound hearing loss in this family.

Label-Free Oligonucleotide-Based SPR Biosensor for the Detection of the Gene Mutation Causing Prothrombin-Related Thrombophilia.

Prothrombin-related thrombophilia is a genetic disorder produced by a substitution of a single DNA base pair, replacing guanine with adenine, and is detected mainly by polymerase chain reaction (PCR). A suitable alternative that could detect the single point mutation without requiring sample amplification is the surface plasmon resonance (SPR) technique. SPR biosensors are of great interest: they offer a platform to monitor biomolecular interactions, are highly selective, and enable rapid analysis in real time. Oligonucleotide-based SPR biosensors can be used to differentiate complementary sequences from partially complementary or noncomplementary strands. In this work, a glass chip covered with an ultrathin (50 nm) gold film was modified with oligonucleotide strands complementary to the mutated or normal (nonmutated) DNA responsible for prothrombin-related thrombophilia, forming two detection platforms called mutated thrombophilia (MT) biosensor and normal thrombophilia (NT) biosensor. The results show that the hybridization response is obtained in 30 min, label free and with high reproducibility. The sensitivity obtained in both systems was approximately 4 ΔμRIU/nM. The dissociation constant and limits of detection calculated were 12.2 nM and 20 pM (3 fmol), respectively, for the MT biosensor, and 8.5 nM and 30 pM (4.5 fmol) for the NT biosensor. The two biosensors selectively recognize their complementary strand (mutated or normal) in buffer solution. In addition, each platform can be reused up to 24 times when the surface is regenerated with HCl. This work contributes to the design of the first SPR biosensor for the detection of prothrombin-related thrombophilia based on oligonucleotides with single point mutations, label-free and without the need to apply an amplification method.

Profile of genetic variations in severely calcified carotid plaques by whole-exome sequencing.

Background: The precise mechanisms of carotid calcification and its clinical significance have not been established.Methods: We classified ten plaques from carotid endarterectomy patients into high- and low-calcified plaques based on the Agatston calcium scores. We performed whole-exome sequencing for genetic profiles with single nucleotide variations (SNVs), insertions, and deletions. Bioinformatic data mining was then conducted to disclose specific gene variations to either high- or low-calcified carotid plaques.Results: In the carotid plaques, G:C>A:T/C:G>T:A transitions as SNVs, insT after C/insC after A as insertions, and delA after G/delT after C as deletions were most frequently observed, but no significant difference was observed between the high- and low-calcified plaque groups in their proportion of base-pair substitution types. In the bioinformatic analysis, SNVs of ATP binding cassette subfamily C member 6 (ADCC6) were more commonly found in high-calcified plaques and SNVs of KLKB1 were more commonly found in low-calcified plaques compared to the other group. No new genetic variants related to calcification or atherosclerosis among those not registered in dbSNP was detected.Conclusion: Our findings clarified the features of base-pair substitutions in carotid plaques, showing no relation to calcification. However, genetic variants in ADCC6 relating to vascular calcification for high-calcified plaques, and in KLKB1 encoding kallikrein associated with vascular regulation of atherosclerosis for low-calcified plaques were more specifically extracted. These results contribute to a better understanding of the genetic basis of molecular activity and calcium formation in carotid plaques.

Role of epigenetic regulators in single-base substitution patterns in AML.

e19500 Background: Incidence of Acute Myeloid Leukemia (AML) increases with age, as does Clonal Hematopoiesis of indeterminate Potential (CHiP), wherein specific genes are mutated in healthy individuals. Single Base pair Substitutions (SBS) are associated with aging and AML, most commonly C>T. G>A transitions are found in a similar prevalence in all age groups, although the number of mutations are higher in older age groups and associated with therapy. Determining a pattern of SBS in leukemia that is distinct from aging has been a challenge. Methods: We performed mutational signature analysis on 10,152 mutations in 3,575 unique suspected AML patient bone marrow, peripheral blood, or FFPE tissue samples. Samples were evaluated using an all exon amplicon based 27 gene next generation sequencing panel. The cohort was divided into 5 groups based on age (0-14, 15-39, 40-54, 55-65, and >65 years), as well as into three gene mutation groups 1) TET2 WT (n=7,479 mutations), 2) TET2/DNMT3A WT (n=6,701) and 3) TET2/DNMT3A WT with chromatin regulators WT (ASXL1, ATRX, BCOR, BCORL1, EZH2, PHF6, RAD21, SMC1A, SMC3, STAG2; n=3627 mutations). Results: Predominant SBS were C>T (42.7%) and G>A (59.0%) transitions across all age groups. The percentage of C>T SBS increased with age: 0-14 years cohort (18.0% of cases; 1.5% of mutations) to >65 years cohort (58.8% of cases; 39.6% of mutations). There was no significant change in the prevalence of G>A mutations with age, however, the total number of mutations did increase with age (0-14 0.2% vs >65 39.6%). In each of the gene mutation groups, the number of C>T and G>A total mutations decreases as TET2 MT (42.7% and 59.0%), TET2 WT (27.0% and 45.1%), TET2/DNMT3A WT (20.8% and 34.3%), and TET2/DNMT3A WT with WT chromatin regulators (10.1% and 25.5%). Conclusions: In this study, both the prevalence and number of mutations in AML patients increases with age for C>T transitions, but for G>A transitions only the number of mutations increases with age, consistent with previously reported studies. In this data set, by removing mutations in TET2, TET2 and DNMT3A, TET2 and DNMT3A with genes that regulate chromatin accessibility, the number of C>T and G>A transitions decreases in order of effect, with the chromatin modifiers having the largest effect. The effect of removing these mutations suggests a mechanism driving leukemogenesis whereby DNMT3A methylates cytosine, TET2 deaminates this methylated cytosine, and finally chromatin modifiers regulate accessibility to these methylation sites, thereby modulating downstream oncologic pathways.

SUN-008 Loss of Antimullerian Hormone Immunoreactivity Due to a Homozygous AMH Gene Mutation Rs10417628 in a Woman with Classical Polycystic Ovary Syndrome

Anti-Müllerian hormone (AMH), an inhibitor of primordial/small antral follicle development and Leydig cell androgen synthesis in mice, could exaggerate the polycystic ovary syndrome (PCOS) phenotype, given reports of PCOS-specific AMH loss-of-function mutations (1–3). This report describes a normal-weight PCOS woman with severely reduced AMH levels (index PCOS woman). It examines the molecular basis for her reduced serum AMH levels and also compares her endocrine characteristics to similar-weight PCOS women with detectable AMH. Twenty normo-androgenic ovulatory (control) and 13 age- and body mass index-matched PCOS women (19–35 years; 19–25 kg/m2) underwent transvaginal sonography and serum hormone measures. Wilcoxon rank-sum test compared clinical features of control and PCOS women with detectable AMH, which were then individually ranked by magnitude in all PCOS women. DNA analysis was performed by PCR amplification with direct gene sequencing. The identified mutation was introduced in hAMH-expression plasmids for functional analysis of AMH processing in HEK293 cells by Western blot and ELISA (pico-AMH assay, Ansh Labs, Webster, TX), and for bioactivity in KK-1/AMHR2 cells using a luciferase reporter. Unpaired t-test compared AMH-induced luciferase activity between wild type and mutant AMH. A homozygous AMH gene mutation rs10417628 involving a single base pair substitution in exon 5 (NG_012190.1:g.7705C>T, p.(Ala515Val)) was identified in the index PCOS woman. PCOS women with detectable AMH had higher serum AMH (10.82 [6.74–13.40] ng/mL, Median [IQR]), total/free testosterone (T) (total T: 55.5 [49.5–62.5] ng/dL; fT: 5.65 [4.75–6.6] pg/mL) levels and greater total antral follicle numbers (AFNs) (46 [39–59] follicles) than controls (AMH: 4.03 [2.47–6.11] ng/ml; total T: 30 [24.5–34.5] ng/dL; fT: 2.2 [1.8–2.45] pg/mL; AFNs 16 [14.5–21.5] follicles, P<0.05, all values), along with a trend toward LH hypersecretion (P=0.06). The index PCOS woman with the lowest AMH levels (0.1 ng/ml) did not have the highest serum total T/fT (total T: 89 ng/dL; fT: 7 pg/mL,) or LH levels nor the greatest AFN (43 follicles). In vitro analysis of cells expressing hAMH-515Val or hAMH-515Ala showed that hAMH515-Val, in contrast to hAMH515-Ala, was undetectable and severely reduced in the pico-AMH assay in cell lysates and supernatants, respectively. AMH protein processing and AMH-induced luciferase activity, however, did not differ between hAMH515Val and hAMH515Ala. Thus, homozygous AMH mutation rs10417628 in a PCOS woman can impair serum AMH immunoreactivity without affecting AMH bioactivity, perhaps because of conformational changes from the mutation that only interfere with its immunodetection but not its function.

New complexities of SOS-induced “untargeted” mutagenesis in Escherichia coli as revealed by mutation accumulation and whole-genome sequencing

When its DNA is damaged, Escherichia coli induces the SOS response, which consists of about 40 genes that encode activities to repair or tolerate the damage. Certain alleles of the major SOS-control genes, recA and lexA, cause constitutive expression of the response, resulting in an increase in spontaneous mutations. These mutations, historically called “untargeted”, have been the subject of many previous studies. Here we re-examine SOS-induced mutagenesis using mutation accumulation followed by whole-genome sequencing (MA/WGS), which allows a detailed picture of the types of mutations induced as well as their sequence-specificity. Our results confirm previous findings that SOS expression specifically induces transversion base-pair substitutions, with rates averaging about 60-fold above wild-type levels. Surprisingly, the rates of G:C to C:G transversions, normally an extremely rare mutation, were induced an average of 160-fold above wild-type levels. The SOS-induced transversion showed strong sequence specificity, the most extreme of which was the G:C to C:G transversions, 60% of which occurred at the middle base of 5′GGC3′+5′GCC3′ sites, although these sites represent only 8% of the G:C base pairs in the genome. SOS-induced transversions were also DNA strand-biased, occurring, on average, 2- to 4- times more often when the purine was on the leading-strand template and the pyrimidine on the lagging-strand template than in the opposite orientation. However, the strand bias was also sequence specific, and even of reverse orientation at some sites. By eliminating constraints on the mutations that can be recovered, the MA/WGS protocol revealed new complexities of SOS “untargeted” mutations.

Mitochondrial genetic variation is enriched in G-quadruplex regions that stall DNA synthesis in vitro.

As the powerhouses of the eukaryotic cell, mitochondria must maintain their genomes which encode proteins essential for energy production. Mitochondria are characterized by guanine-rich DNA sequences that spontaneously form unusual three-dimensional structures known as G-quadruplexes (G4). G4 structures can be problematic for the essential processes of DNA replication and transcription because they deter normal progression of the enzymatic-driven processes. In this study, we addressed the hypothesis that mitochondrial G4 is a source of mutagenesis leading to base-pair substitutions. Our computational analysis of 2757 individual genomes from two Italian population cohorts (SardiNIA and InCHIANTI) revealed a statistically significant enrichment of mitochondrial mutations within sequences corresponding to stable G4 DNA structures. Guided by the computational analysis results, we designed biochemical reconstitution experiments and demonstrated that DNA synthesis by two known mitochondrial DNA polymerases (Pol γ, PrimPol) in vitro was strongly blocked by representative stable G4 mitochondrial DNA structures, which could be overcome in a specific manner by the ATP-dependent G4-resolving helicase Pif1. However, error-prone DNA synthesis by PrimPol using the G4 template sequence persisted even in the presence of Pif1. Altogether, our results suggest that genetic variation is enriched in G-quadruplex regions that impede mitochondrial DNA replication.

Specificity and mutagenesis bias of the mycobacterial alternative mismatch repair analyzed by mutation accumulation studies.

The postreplicative mismatch repair (MMR) is an almost ubiquitous DNA repair essential for maintaining genome stability. It has been suggested that Mycobacteria have an alternative MMR in which NucS, an endonuclease with no structural homology to the canonical MMR proteins (MutS/MutL), is the key factor. Here, we analyze the spontaneous mutations accumulated in a neutral manner over thousands of generations by Mycobacterium smegmatis and its MMR-deficient derivative (ΔnucS). The base pair substitution rates per genome per generation are 0.004 and 0.165 for wild type and ΔnucS, respectively. By comparing the activity of different bacterial MMR pathways, we demonstrate that both MutS/L- and NucS-based systems display similar specificity and mutagenesis bias, revealing a functional evolutionary convergence. However, NucS is not able to repair indels in vivo. Our results provide an unparalleled view of how this mycobacterial system works in vivo to maintain genome stability and how it may affect Mycobacterium evolution.

Generation of mouse model of TGFBI-R124C corneal dystrophy using CRISPR/Cas9-mediated homology-directed repair

Mutations in transforming growth factor-beta-induced (TGFBI) gene cause clinically distinct types of corneal dystrophies. To delineate the mechanisms driving these dystrophies, we focused on the R124C mutation in TGFBI that causes lattice corneal dystrophy type1 (LCD1) and generated novel transgenic mice harbouring a single amino acid substitution of arginine 124 with cysteine in TGFBI via ssODN-mediated base-pair substitution using CRISPR/Cas9 technology. Eighty percent of homozygous and 9.1% of heterozygous TGFBI-R124C mice developed a corneal opacity at 40 weeks of age. Hematoxylin and eosin and Masson trichrome staining showed eosinophilic deposits in subepithelial corneal stroma that stained negative for Congo-red. Although amyloid deposition was not observed in TGFBI-R124C mice, irregular amorphous deposits were clearly observed via transmission electron microscopy near the basement membrane. Interestingly, we found that the corneal deposition of TGFBI protein (TGFBIp) was significantly increased in homozygous TGFBI-R124C mice, suggesting a pathogenic role for the mutant protein accumulation. Furthermore, as observed in the LCD1 patients, corneal epithelial wound healing was significantly delayed in TGFBI-R124C mice. In conclusion, our novel mouse model of TGFBI-R124C corneal dystrophy reproduces features of the human disease. This mouse model will help delineate the pathogenic mechanisms of human corneal dystrophy.

High Fidelity of Mouse Models Mimicking Human Genetic Skeletal Disorders.

The 2019 International Skeletal Dysplasia Society nosology update lists 441 genes for which mutations result in rare human skeletal disorders. These genes code for enzymes (33%), scaffolding proteins (18%), signal transduction proteins (16%), transcription factors (14%), cilia proteins (8%), extracellular matrix proteins (5%), and membrane transporters (4%). Skeletal disorders include aggrecanopathies, channelopathies, ciliopathies, cohesinopathies, laminopathies, linkeropathies, lysosomal storage diseases, protein-folding and RNA splicing defects, and ribosomopathies. With the goal of evaluating the ability of mouse models to mimic these human genetic skeletal disorders, a PubMed literature search identified 260 genes for which mutant mice were examined for skeletal phenotypes. These mouse models included spontaneous and ENU-induced mutants, global and conditional gene knockouts, and transgenic mice with gene over-expression or specific base-pair substitutions. The human X-linked gene ARSE and small nuclear RNA U4ATAC, a component of the minor spliceosome, do not have mouse homologs. Mouse skeletal phenotypes mimicking human skeletal disorders were observed in 249 of the 260 genes (96%) for which comparisons are possible. A supplemental table in spreadsheet format provides PubMed weblinks to representative publications of mutant mouse skeletal phenotypes. Mutations in 11 mouse genes (Ccn6, Cyp2r1, Flna, Galns, Gna13, Lemd3, Manba, Mnx1, Nsd1, Plod1, Smarcal1) do not result in similar skeletal phenotypes observed with mutations of the homologous human genes. These discrepancies can result from failure of mouse models to mimic the exact human gene mutations. There are no obvious commonalities among these 11 genes. Body BMD and/or radiologic dysmorphology phenotypes were successfully identified for 28 genes by the International Mouse Phenotyping Consortium (IMPC). Forward genetics using ENU mouse mutagenesis successfully identified 37 nosology gene phenotypes. Since many human genetic disorders involve hypomorphic, gain-of-function, dominant-negative and intronic mutations, future studies will undoubtedly utilize CRISPR/Cas9 technology to examine transgenic mice having genes modified to exactly mimic variant human sequences. Mutant mice will increasingly be employed for drug development studies designed to treat human genetic skeletal disorders.SignificanceGreat progress is being made identifying mutant genes responsible for human rare genetic skeletal disorders and mouse models for genes affecting bone mass, architecture, mineralization and strength. This review organizes data for 441 human genetic bone disorders with regard to heredity, gene function, molecular pathways, and fidelity of relevant mouse models to mimic the human skeletal disorders. PubMed weblinks to citations of 249 successful mouse models are provided.

ACB-PCR Quantification of Low-Frequency Hotspot Cancer-Driver Mutations.

Allele-specific competitive blocker PCR (ACB-PCR) is a sensitive and quantitative approach for the selective amplification of a specific base substitution. Using the ACB-PCR technique, hotspot cancer-driver mutations (tumor-relevant mutations in oncogenes and tumor suppressor genes, which confer a selective growth advantage) are being developed as quantitative biomarkers of cancer risk. ACB-PCR employs a mutant-specific primer (with a 3'-penultimate mismatch relative to the mutant DNA sequence, but a double 3'-terminal mismatch relative to the wild-type DNA sequence) to selectively amplify rare mutant DNA molecules. A blocker primer having a non-extendable 3'-end and a 3'-penultimate mismatch relative to the wild-type DNA sequence, but a double 3'-terminal mismatch relative to the mutant DNA sequence is included in ACB-PCR to selectively repress amplification from abundant wild-type molecules. Consequently, ACB-PCR can quantify the level of a single base pair substitution mutation in a DNA population when present at a mutant:wild-type ratio of 1×10-5 or greater. Quantification of rare mutant alleles is achieved by parallel analysis of unknown samples and mutant fraction (MF) standards (defined mixtures of mutant and wild-type DNA sequences). The ability to quantify specific mutations with known association to cancer has several important applications in evaluating the carcinogenic potential of chemical exposures in rodent models. Further, the measurement of cancer-driver mutant subpopulations is important for precision cancer treatment (selecting the most appropriate targeted therapy and predicting the development of therapeutic resistance). This chapter provides a step-by-step description of the ACB-PCR methodology as it has been used to measure human PIK3CA codon 1047, CAT→CGT (H1047R) mutation.

Nonclinical evaluation of IQG-607, an anti-tuberculosis candidate with potential use in combination drug therapy

New effective compounds to treat tuberculosis are urgently needed. IQG-607 is an orally active anti-tuberculosis drug candidate, with promising preliminary safety profile and anti-mycobacterial activity in both in vitro and in vivo models of tuberculosis infection. Here, we evaluated the mutagenic and genotoxic effects of IQG-607, and its interactions with CYP450 isoforms. Moreover, we describe for the first time a combination study of IQG-607 in Mycobacterium tuberculosis-infected mice. Importantly, IQG-607 had additive effects when combined with the first-line anti-tuberculosis drugs rifampin and pyrazinamide in mice. IQG-607 presented weak to moderate inhibitory potential against CYP450 isoforms 3A4, 1A2, 2C9, 2C19, 2D6, and 2E1. The Salmonella mutagenicity test revealed that IQG-607 induced base pair substitution mutations in the strains TA100 and TA1535. However, in the presence of human metabolic S9 fraction, no mutagenic effect was detected in any strain. Additionally, IQG-607 did not increase micronucleus frequencies in mice, at any dose tested, 25, 100, or 250 mg/kg. The favorable activity in combination with first-line drugs and mild to moderate toxic events described in this study suggest that IQG-607 represents a candidate for clinical development.

Induction of Micronuclei, Base-pair Substitution Mutation and Excision-repair Deficient by Polluted Water from Asa River in Nigeria

Abstract Asa river is a major river designated to supply millions of people of Ilorin, Kwara State, Nigeria potable water for drinking but its managements is of grave concern due to anthropogenic activities. Thus, evaluation of genotoxicity of this river was carried out by subjecting the water samples and fish therein to three bioassays (Micronucleus (MN) assay, Ames test and SOS-chromo test). Physicochemical parameters and heavy metals were analysed at three different stations (Aliara (SI), Unity (SII) and Tuyil (SIII)) of the river. In SII, most of the heavy metals analysed were above the acceptable limits compare to SI and SIII. The peripheral erythrocyte of the fishes (Oreochromis niloticus, Synodontis batensoda, Synodontis eupterus, Clarias gariepinus and Clarias angullaris) at SI and SII stations showed a significant (p&lt;0.05) induction of MN and different nuclear abnormalities (NA). Water samples from the three stations subjected to Ames test (Salmonella typhimurium TA100) and SOS chromotests (Escherichia coli PQ37) at 25%, 50% and 100% concentrations showed statistically significant (p&lt;0.05) induction of DNA damage at all concentrations in the two tester strains, thus indicating base-pair substitution mutation and excision-repairdeficient, respectively, by the water samples. Therefore, drinking of this water and/or consumption of fish from this river should be taken with caution to avoid a carcinogenic risk.

STUDY OF MUTAGENIC ACTIVITY NANO- AND MICROPARTICLES IN THE AMES TEST (SALMONELLA / MICROSOME)

Introduction. One of the important steps in assessing the nanoparticles (NP) safety is the analysis of mutagenic activity, including the evaluation of gene, chromosomal, and genomic mutations. Material and methods. The purpose of this investigation is to study the ability of different NP aqueus suspensions and the same compounds in microforms to unduce gene mutations in Salmonella/microsome test (Ames test). Anatase titanium dioxide NP coated with simethicone (33.16 ± 16.7 nm, 5-50000 μg/ml), magnetite NP coated with silicate (10 nm, 0.92-575 μg/ml), silver NP coated with аrabian gum (14 ± 0.2 nm, 5-50000 μg/ml), aluminum hydroxide nanofibres (50-70 nm, 24-3000 μg/ml) and multi-walled carbon nanotubes (Taunit MWСNTs, outer diameter 15-40 nm, inner diameter 3-8 nm, length 2 and more microns, 5-50000 μg/ml). In parallel, the mutagenic activity of equivalent microparticles was evaluated in experiments. Ames test (Salmonella/microsomes) registers gene mutations induced by a different mechanism of action, in the variant with preincubation. A set of Salmonella typhimurium indicator strains: TA 100 (base pair substitution mutations), TA 98 and TA 97 (mutations of the frameshift type of the genetic code) were used. Using addition the S9 microsomal activating mixture during the experiment makes it is possible to determine the effect not only of the substances themselves, but also of their metabolites. Conclusion. The investigated nanomaterials as well as their micro analogs in the studied dose range did not induce gene mutations in the Ames test both in presence and absence microsomal activating mixture.

Pod indehiscence is a domestication and aridity resilience trait in common bean.

Plant domestication has stronglymodifiedcrop morphology and development. Nevertheless, many crops continueto display atavistic characteristics that were advantageous to their wild ancestors but are deleterious under cultivation, such as pod dehiscence (PD). Here, we provide the first comprehensive assessment of the inheritance of PD in the common bean (Phaseolus vulgaris), a major domesticated grain legume. Using three methods to evaluate the PD phenotype, we identifiedmultiple, unlinked genetic regions controlling PD in a biparental population and two diversity panels. Subsequently, we assessed patterns of orthology among these loci and those controlling the trait in other species. Our results show that different genes were selected in each domestication and ecogeographic race. A chromosome Pv03 dirigent-like gene, involved in lignin biosynthesis, showeda base-pair substitution that is associated with decreased PD. This haplotype may underlie the expansion of Mesoamerican domesticates into northern Mexico, where arid conditions promote PD. The rise in frequency of the decreased-PD haplotype may be a consequence of the markedly different fitness landscape imposed by domestication. Environmental dependencyandgenetic redundancy can explain the maintenance of atavistic traits under domestication.

Real-time PCR detection of a 16S rRNA single mutation of Helicobacter pylori isolates associated with reduced susceptibility and resistance to tetracycline in the gastroesophageal mucosa of individual hosts.

The molecular mechanism of Helicobacter pylori resistance to tetracycline involves mutations in the primary binding site of the ribosome. A resistance or reduced susceptibility to tetracycline could be the result of single, double or triple mutations in the 16S rRNA gene of H. pylori. We investigated if the genotype was correlated to tetracycline resistance as determined phenotypically in vitro for 96 H. pylori isolates in the gastroesophageal mucosa of Venezuelan individual hosts. E-test for antimicrobial susceptibility test and real-time PCR for the detection of 16S rRNA gene mutations were performed in 96 H. pylori isolates (48 obtained from antrum, and 48 from oesophagus) from eight dyspeptic patients. In the gastric mucosa, 38 isolates were identified sensitive and 10 resistant to tetracycline by E-test, whereas 44 sensitive and 4 resistant isolates were found in the oesophagus. Real-time PCR detection of the 16S rRNA gene exhibited mutants with a single base-pair substitution (AGA926GGA) in six antrum isolates and seven oesophagus isolates, whereas only three harboured a low level of tetracycline resistance in vitro. Our results indicate that real-time PCR detection of 16S rRNA is a reliable method to classify among tetracycline-resistant genotypes and useful in patients who have experienced a first-line treatment failure with triple therapy.

The Symmetrical Wave Pattern of Base-Pair Substitution Rates across the Escherichia coli Chromosome Has Multiple Causes.

Mutation accumulation experiments followed by whole-genome sequencing have revealed that, for several bacterial species, the rate of base-pair substitutions (BPSs) is not constant across the chromosome but varies in a wave-like pattern that is symmetrical about the origin of replication. The experiments reported here demonstrated that, in Escherichia coli, several interacting factors determine the wave. The origin is a major driver of BPS rates. When it is relocated, the BPS rates in a 1,000-kb region surrounding the new origin reproduce the pattern that surrounds the normal origin. However, the pattern across distant regions of the chromosome is unaltered and thus must be determined by other factors. Increasing the deoxynucleoside triphosphate (dNTP) concentration shifts the wave pattern away from the origin, supporting the hypothesis that fluctuations in dNTP pools coincident with replication firing contribute to the variations in the mutation rate. The nucleoid binding proteins (HU and Fis) and the terminus organizing protein (MatP) are also major factors. These proteins alter the three-dimensional structure of the DNA, and results suggest that mutation rates increase when highly structured DNA is replicated. Biases in error correction by proofreading and mismatch repair, both of which may be responsive to dNTP concentrations and DNA structure, also are major determinants of the wave pattern. These factors should apply to most bacterial and, possibly, eukaryotic genomes and suggest that different areas of the genome evolve at different rates.IMPORTANCE It has been found in several species of bacteria that the rate at which single base pairs are mutated is not constant across the genome but varies in a wave-like pattern that is symmetrical about the origin of replication. Using Escherichia coli as our model system, we show that this pattern is the result of several interconnected factors. First, the timing and progression of replication are important in determining the wave pattern. Second, the three-dimensional structure of the DNA is also a factor, and the results suggest that mutation rates increase when highly structured DNA is replicated. Finally, biases in error correction, which may be responsive both to the progression of DNA synthesis and to DNA structure, are major determinants of the wave pattern. These factors should apply to most bacterial and, possibly, eukaryotic genomes and suggest that different areas of the genome evolve at different rates.

Abstract 4888: Nuclear localization of antizyme inhibitor may be a marker for aggressiveness of prostate cancer

Abstract Antizyme suppresses cell cycle by inhibiting the polyamine synthesis through binding to the rate limiting enzyme ornithine decarboxylases (ODC). High levels of ODC have been reported in several forms of cancer, among them prostate cancer. Antizyme Inhibitor (AZIN) binds to antizyme and thereby blocks its inhibitory effect on ODC. Here, we have measured the expression and localization of AZIN in 202 prostate cancer specimens, along with 26 adjacent benign samples and found that nuclear localization of AZIN is associated with significantly lower survival. Upregulation and nuclear localization of AZIN have been observed in several cancers, as has editing of the AZIN1 mRNA. Others have hypothesized that the RNA- edited AZIN (edAZIN) may have an increased affinity to antizyme and that could explain the association of edAZIN to the various cancers. We have studied the mechanism behind the nuclear localization of AZIN and found the single base pair substitution caused by RNA editing is sufficient to result in nuclear localization of the protein in all cell types tested. To determine if the nuclear localization might result from increased antizyme-edAZIN affinity, we developed fluorescent protein FRET sensor for protein-protein interaction using Clover-AZIN and antizyme-mRuby2 fusion proteins. Unexpectedly, we found that the editing event modestly decreases edAZIN affinity for antizyme, notwithstanding increased interaction in vivo. Thus, the data indicate that the change in protein localization to the nucleus may be more important to oncogenic function than the actual degree of binding to antizyme. Other functional differences between edAZIN and AZIN might be explained by altered kinetics of binding, by the contribution of an additional adapter protein which modulates the intracellular antizyme:AZIN complex, or by competition for AZIN binding by other partners whose interaction is affected by the editing event. Finally, we identified AZIN and edAZIN interacting proteins by using tandem affinity purification and LC-MS-MS analysis. Among interacting proteins, we identified a complex containing two isoforms of nuclear actins (ACTG1 and ACTA2) and Myosin-9 that may be the driving force behind the nuclear shuttling of edAZIN. Citation Format: Aram Ghalali, James M. Rice, Liangzhe Wang, Chin Lee Wu, Michael S. Rogers, Bruce Zetter. Nuclear localization of antizyme inhibitor may be a marker for aggressiveness of prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4888.

Influence of chicken growth hormone (cGH) SNP genotypes on morphometric and growth traits of three chicken breeds in Nigeria.

This study was conducted to identify the diversity of chicken growth hormone (cGH) single nucleotide polymorphs’ (SNP) and their association on morphometric and growth traits of three Nigerian chicken breeds namely Funaab Alpha (FA), Shikabrown (SB) and Nigerian local chicken (NLC). Morphometric traits measured include shank length (cm), breast girth (cm) and breast width (cm) while growth traits measured were final body weight, av. weight gain, feed intake and feed conversion ratio all at laying stages respectively. Morphometric and growth traits were significantly influenced (P less than 0.05) between the different breeds. The SNP panel showed two base pair substitution mutation (GT and CA) on locus 5 chromosome for the FA and locus 6 chromosome for the SB and NLC respectively. The G – T alignment produced genotypes GG and GT, with TT not observed. Also C – A alignment produced genotypes CC and CA, with AA not observed. The NLC breed had the highest number of haplotypes of 6 while FA and SB had 4 haplotypes each implying that the local breed had more allelic variation than the other two breeds. Also FA had heterozygosity of 0.48 while SB and NLC had a value of 0.44, indicating higher genetic variability of FA breed than the rest with negative theta value (-0.0843) of F-statistics indicating high level of outbreeding. The conducted association of SNP genotypes showed no significant association effects (P&gt;0.05) on the morphometric traits, except for SB breed where CA genotypes was significantly associated with CC for bodyweight, and in NLC breed where CC genotype was also significantly associated with CA genotypes for shank length.

Oxidative stress, mutagenic effects, and cell death induced by retene

Retene (RET) is the most abundant polycyclic aromatic hydrocarbon (PAH) released upon burning of cellulose, although it is not considered as one of the priority PAHs and is not included for risk assessments by the US Environmental Protection Agency (US-EPA). There are only a few studies concerning the toxic effects of RET. To the best of our knowledge, this study is the first one to examine whether RET, in an environmental concentration, plays a crucial role in the induction of oxidative stress in A549 lung cell line, and its consequence as such as mutagenicity and cell death. Our results revealed that RET was able to significantly decrease cell viability only at 72 h of exposure, increase oxidative stress, mitochondrial membrane potential and mitochondrial contents, leading an increased reactive oxygen species (ROS) production. Mutagenic activity was not detected in Salmonella strains, suggesting that RET does not induce base-pair substitution (TA100), frameshift (TA98 and TA97a) and transition/transversion (TA102) mutations. However, exposure to RET led to a significant increase in micronuclei (MN), nucleoplasmic bridges (NPBs), and nuclear buds (NBUDs) frequency, as well as cell death, mainly due to necrosis. Taken together, the results of our study provide new evidence suggesting that RET promotes oxidative stress, contributes to the processes of genomic instability, and favors necrosis. Thus, we highlight the importance of including RET in routine environmental analyses in the future as a potential risk factor involved in complex diseases and carcinogenesis.