Mutant Frequency Values Research Articles

SARS-CoV-2 Mutant Spectra at Different Depth Levels Reveal an Overwhelming Abundance of Low Frequency Mutations.

Populations of RNA viruses are composed of complex and dynamic mixtures of variant genomes that are termed mutant spectra or mutant clouds. This applies also to SARS-CoV-2, and mutations that are detected at low frequency in an infected individual can be dominant (represented in the consensus sequence) in subsequent variants of interest or variants of concern. Here we briefly review the main conclusions of our work on mutant spectrum characterization of hepatitis C virus (HCV) and SARS-CoV-2 at the nucleotide and amino acid levels and address the following two new questions derived from previous results: (i) how is the SARS-CoV-2 mutant and deletion spectrum composition in diagnostic samples, when examined at progressively lower cut-off mutant frequency values in ultra-deep sequencing; (ii) how the frequency distribution of minority amino acid substitutions in SARS-CoV-2 compares with that of HCV sampled also from infected patients. The main conclusions are the following: (i) the number of different mutations found at low frequency in SARS-CoV-2 mutant spectra increases dramatically (50- to 100-fold) as the cut-off frequency for mutation detection is lowered from 0.5% to 0.1%, and (ii) that, contrary to HCV, SARS-CoV-2 mutant spectra exhibit a deficit of intermediate frequency amino acid substitutions. The possible origin and implications of mutant spectrum differences among RNA viruses are discussed.

Longitudinal study of t-cell somatic mutations conferring glycosylphosphatidylinositol-anchor deficiency in gulf war I veterans exposed to depleted uranium.

Fifty Veterans of the first Gulf War in 1991 exposed to depleted uranium (DU) were studied for glycosylphosphatidylinositol-anchor (GPIa) deficient T-cell mutants on three occasions during the years 2009, 2011, and 2013. GPIa deficiency was determined in two ways: cloning assays employing aerolysin selection and cytometry using the FLAER reagent for positive staining of GPIa cell surface proteins. Subsequent molecular analyses of deficient isolates recovered from cloning assays (Nicklas JA et al. [2019]: Environ Mol Mutagen) revealed apparent incomplete selection in some cloning assays, necessitating correction of original data to afford a more realistic estimate of GPIa deficient mutant frequency (MF) values. GPIa deficient variant frequencies (VFs) determined by cytometry were determined in the years 2011 and 2013. A positive but nonsignificant association was observed between MF and VF values determined on the same blood samples during 2013. Exposure to DU had no effect on either GPIa deficient MF or VFs. Environ. Mol. Mutagen. 60:494-504, 2019. © 2019 Wiley Periodicals, Inc.

A High Frequency of Blasts with the PNH Phenotype in Patients with ALL.

Abstract 1575Poster Board I-601It has been proposed that if more than 2 mutations are required for leukemia to arise, then an elevation in the mutation rate would be necessary for these mutations to coincide in the same cell, based on the rarity of spontaneous mutations (Loeb 1991). Hypermutability could occur transiently, for example, due to mutagens, or permanently, due to inactivation of repair pathways. Genome-wide analysis has indicated that in acute lymphoblastic leukemia (ALL), more than 2 somatic mutations are often found (Mullighan et al 2007), suggesting that hypermutability is likely to occur at least in some cases. To investigate this hypothesis, we have used our method for the detection of rare cells that have acquired a spontaneous inactivating mutation in the PIG-A gene. Expanded populations of stem cells with PIG-A mutations in the setting of bone marrow failure are the hallmark of paroxysmal nocturnal hemoglobinuria (PNH), but under most other circumstances, PIG-A mutations are thought to be growth neutral in vitro and in vivo. PIG-A is on the X-chromosome, and therefore, a single inactivating mutation is sufficient to produce the PNH phenotype, characterized by a lack of glycosylphosphatidylinositol (GPI)-linked proteins and an inability to bind to the FLAER reagent. Furthermore, it is known from patients with PNH and from cell lines that a very broad spectrum of mutations can inactivate PIG-A. This combination of features makes PIG-A uniquely suited as a sentinel gene for spontaneous mutagenesis in humans, which is otherwise very difficult to quantitate. Here we have analyzed a panel of blast cells derived from ficolled marrow samples from patients with B-lineage ALL, obtained from the Children's Oncology Group ALL Cell Bank. To determine the frequency of spontaneous PIG-A mutations, samples were stained sequentially with an Alexa-488 conjugate of the FLAER reagent, a mixture of murine anti-CD55 and anti-CD59 antibodies, FITC-conjugated rabbit anti-mouse immunoglobulin secondary antibody, and PE-conjugated anti-CD45 antibody. The blast population was identified based on forward and side scatter, dim expression of CD45, and exclusion of propidium iodide. Control GPI (+) and GPI (-) cells from a patient with PNH served as a control. Among 19 cases of B lineage ALL studied, the frequency of blast cells with the PNH phenotype varied over a very wide range, and the distribution of mutant frequency values was seen to be bimodal. One group, representing about 40% of the cases of ALL, had a median mutant frequency of 6.8 × 10-6, which is very similar to the proportion of granulocytes that acquire the PNH phenotype due to spontaneous PIG-A mutations in normal individuals (Araten et al, 1999). These values are also close to the frequency of mutants reported for the HPRT, GPA, and XK genes in normal individuals. The other 60% of the samples had a median mutant frequency of 465 × 10-6, which is far above the range of this parameter in normal individuals. Among 6 established cell lines derived from T-ALL, the mutant frequency ranged from 4.1 ×10-6 to 265 × 10-6. Here as well, the distribution suggested that in this subtype of ALL, there may be two distinct phenotypes with respect to this parameter. Based on this data, we believe that an increase in inactivating mutations is not essential for the development of ALL, but that it is a common feature of this condition. We cannot rule out the possibility that samples with a low mutant frequency would demonstrate some form of genomic instability that would not be detected by our assay, such as an increase in gene duplications or translocations. Further investigations are needed to determine whether hypermutability as detected by this assay is associated with distinct cytogenetic abnormalities, clinical features at presentation, and outcome. DisclosuresNo relevant conflicts of interest to declare.

Tissue‐specific metabolic activation and mutagenicity of 3‐nitrobenzanthrone in Muta™Mouse

3-Nitrobenzanthrone (3-NBA) is a mutagen and suspected human carcinogen detected in diesel exhaust, airborne particulate matter, and urban soil. We investigated the tissue specific mutagenicity of 3-NBA at the lacZ locus of transgenic MutaMouse following acute single dose or 28-day repeated-dose oral administration. In the acute high dose (50 mg/kg) exposure, increased lacZ mutant frequency was observed in bone marrow and colonic epithelium, but not in liver and bladder. In the repeated-dose study, a dose-dependent increase in lacZ mutant frequency was observed in bone marrow and liver (2- and 4-fold increase above control), but not in lung or intestinal epithelium. In addition, a concentration-dependent increase in mutant frequency (8.5-fold above control) was observed for MutaMouse FE1 lung epithelial cells exposed in vitro. 1-Nitropyrene reductase, 3-NBA reductase, and acetyltransferase activities were measured in a variety of MutaMouse specimens in an effort to link metabolic activation and mutagenicity. High 3-NBA nitroreductase activities were observed in lung, liver, colon and bladder, and detectable N-acetyltransferase activities were found in all tissues except bone marrow. The relatively high 3-NBA nitroreductase activity in MutaMouse tissues, as compared with those in Salmonella TA98 and TA100, suggests that 3-NBA is readily reduced and activated in vivo. High 3-NBA nitroreductase levels in liver and colon are consistent with the elevated lacZ mutant frequency values, and previously noted inductions of hepatic DNA adducts. Despite an absence of induced lacZ mutations, the highest 3-NBA reductase activity was detected in lung. Further studies are warranted, especially following inhalation or intratracheal exposures.

Phenotypic mixing and hiding may contribute to memory in viral quasispecies

BackgroundIn a number of recent experiments with food-and-mouth disease virus, a deleterious mutant, RED, was found to avoid extinction and remain in the population for long periods of time. Since RED characterizes the past evolutionary history of the population, this observation was called quasispecies memory. While the quasispecies theory predicts the existence of these memory genomes, there is a disagreement between the expected and observed mutant frequency values. Therefore, the origin of quasispecies memory is not fully understood.ResultsWe propose and analyze a simple model of complementation between the wild type virus and a mutant that has an impaired ability of cell entry, the likely cause of fitness differences between wild type and RED mutants. The mutant will go extinct unless it is recreated from the wild type through mutations. However, under phenotypic mixing-and-hiding as a mechanism of complementation, the time to extinction in the absence of mutations increases with increasing multiplicity of infection (m.o.i.). If the RED mutant is constantly recreated by mutations, then its frequency at equilibrium under selection-mutation balance also increases with increasing m.o.i. At high m.o.i., a large fraction of mutant genomes are encapsidated with wild-type protein, which enables them to infect cells as efficiently as the wild type virions, and thus increases their fitness to the wild-type level. Moreover, even at low m.o.i. the equilibrium frequency of the mutant is higher than predicted by the standard quasispecies model, because a fraction of mutant virions generated from wild-type parents will also be encapsidated by wild-type protein.ConclusionsOur model predicts that phenotypic hiding will strongly influence the population dynamics of viruses, particularly at high m.o.i., and will also have important effects on the mutation-selection balance at low m.o.i. The delay in mutant extinction and increase in mutant frequencies at equilibrium may, at least in part, explain memory in quasispecies populations.

Comparison of the mutant frequencies and mutation spectra of three non-genotoxic carcinogens, oxazepam, phenobarbital, and Wyeth 14,643, at the λcII locus in Big Blue® transgenic mice

Oxazepam (OX), a widely used benzodiazepine anxiolytic, phenobarbital (PHE), a drug used for convulsive disorders, and Wyeth 14,643 (WY; [4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio]acetic acid), a hypolipidemic agent, are all hepatocarcinogenic in B6C3F1 mice. They have been classified as “non-genotoxic” carcinogens since they are non-DNA reactive in in vitro assays and are either negative or weakly positive in Salmonella typhimurium (Ames assay). Male B6C3F1 Big Blue® transgenic mice were fed 2500 ppm of OX or PHE or 500 ppm of WY in their diet, while a control group of mice received diet alone for 180 days. The mutant frequency (MF) of cII in the control mice, after correction for clonality, was 6.2 ± 2.8 × 10−5. The MF values for mice fed OX, PHE, and WY were 10.0 ± 3.6 × 10−5 (P < 0.05), 7.9 ± 1.3 × 10−5 (P = 0.1) and 17.4 ± 4.2 × 10−5 (P < 0.01), respectively. The mutation spectrum (MS) at cII from the PHE-fed mice was significantly different (P < 0.05) from that of the control mice even though the MF was not, whereas the MS spectra of mice fed OX (P = 0.4) and WY (P = 0.7) were not significantly different. The PHE-derived spectrum differed from the spontaneous spectrum in the lower occurrence of G:C>C:G transversions (17 vs 1.6%) and the higher incidence of A:T>T:A transversions (3.4 vs 9.5%). Prior to correction for clonal expansion, each treated group exhibited a high incidence of frameshift mutations at the homopolymeric run of guanines at bp 179–184 (OX 21%, PHE 21%, WY 16% of the total mutations); this was not the case with the control group (6%). Even after clonal correction, more than 10% of the mutations were frameshifts in the treated mice, while 5% were frameshifts in the control mice. Despite this hypersensitive region of the gene, our findings suggest that the cII locus is less sensitive than the lacI locus to mutation induction by non-DNA reactive carcinogens.

Genetic instability in human T-lymphocytes

Mutations arising in vivo in the hypoxanthine-guanine phosphoribosyltransferase ( HPRT) gene of T-lymphocytes provide a measure of mutation induction in human somatic cells. Studies of measured background HPRT mutant frequency (MF) values show wide inter-individual variation. At the extremes are individual with ‘outlier’ MF values, i.e., non-exposed individuals with MF > 100 × 10 −6 [Robinson et al., Mutation Res. 313 (1994) 227–247.]. The elevated HPRT MF in one well-studied outlier is due to the in vivo expansion of mutant cells possessing an identical T-cell receptor (TCR) gene rearrangement pattern. We report here that this in vivo expanding TCR clone shows multiple different HPRT mutations and thus possesses a mutator phenotype. Other individuals with T-cell mutator phenotypes have been found, suggesting that this phenomenon may contribute to the extremes of variation in HPRT MFs in the human population.

Genotoxicity of agaritine in the lacI transgenic mouse mutation assay: Evaluation of the health risk of mushroom consumption

The mutagenic potency of the common mushroom Agaricus bisporus and crude agaritine extracted from mushrooms was determined in vivo using a new mutagenesis assay with lacI transgenic mice (Big Blue mice). Pairs of female lacl mice were fed one of three diets for 15 wk: (1) fresh mushrooms 3 days/wk followed by normal lab chow for 4 days/wk; (2) freeze-dried mushrooms mixed at 25% (w/w) into powdered chow; or (3) a mushroom extract containing 30% agaritine (w/w) mixed into powdered chow. The corresponding daily doses of agaritine were 30 (averaged over the whole week), 80 and 120 mg/kg body weight, respectively. Positive control animals received N-nitrosodimethylamine, N-nitrosomethylurea or urethane, mixed into powdered chow at concentrations corresponding to daily doses of 0.3, 3 and 130 mg/kg body weight, respectively. DNA of the forestomach, kidney, liver, lung and glandular stomach of the lacl mice was examined for increases in mutant frequency (MF). Control MFs ranged from 5 × 10−5 to 10 × 10−5. Positive control substances induced a two- to seven-fold increase in MF in their respective target organs. Of the mushroom diets, significant effects were seen only with the crude agaritine extract: it induced an increase in MF of 100% in the kidney and 50% in the forestomach. The other two A. bisporus diets, with lower agaritine doses, showed slightly but not significantly, raised MF values in the kidney alone. Thus, agaritine was weakly genotoxic in vivo; no genotoxic activity other than that attributable to agaritine was detected in A. bisporus. Substances or processes that might influence carcinogenicity by means of non-genotoxic mechanisms (e.g. increase in fibre, or decrease in calorie intake) are not detected in the lacI assay. Using a previously derived quantitative correlation between mutagenicity in the lacl test and carcinogenic potency, the carcinogenicity of agaritine in mushrooms was estimated: the average Swiss mushroom consumption of 4 g/day would be expected to contribute a lifetime cumulative cancer risk of about two cases per 100,000 lives.

Studies on the activities of benzo[ &lt;italic&gt;a&lt;/italic&gt; ]pyrene, benzidine and ethyl methanesulphonate in the L5178Y TK &lt;sup&gt;+/-&lt;/sup&gt; mouse lymphoma mutagenicity assay using standardized and non-standardized protocols

As part of the third UKEMS collaborative trial, ethylmethane sulphonate (EMS), benzo[a]pyrene (B[a]P) and benzidine (BZD) were assayed for mutagenicity using the L5178Y mouse lymphoma TK +/- forward mutation system. Exogenous metabolic activation was achieved with two different sources of rat liver S9 which were used under optimized conditions for B[a]P and BZD, the latter being tested also without S9. EMS was assayed in the absence of S9 only. Mutants were selected for trifluorothymidine (TFT) resistance after 48 and 72 h expression time. Mutant frequency (MF) data were subjected to ANOVA analysis and t-tests for differences between replicate cultures, linear trend and differences from solvent controls. Large TFT resistant colonies are believed to be due to induction of small amounts of genetic damage (point mutations, small deletions) at the tk locus, whilst small colonies are thought to be a result of larger effects. The relative proportions of the two colony types were determined in some experiments. All three compounds induced dose-related increases in MF under all conditions. B[a]P induced equal proportions of large and small colonies at all doses. EMS induced predominantly large colonies at all doses. The effect of BZD on colony size was variable. The source of S9 did not exert any consistent effect on toxicity, mutagenicity or mutant colony size. Maximum MF values occurred at 72 h for B[a]P and BZD but the results with EMS were variable. No consistent differences were apparent between estimates of toxicities made either by cloning immediately after treatment (day 0 cloning) or by estimating the relative total growth (RTG) at expression time.

T Cells Responsive to Myelin Basic Protein in Patients with Multiple Sclerosis

Gene mutation in vivo in human T lymphocytes appears to occur preferentially in dividing cells. Individuals with multiple sclerosis (MS) are assumed to have one or more populations of diving T cells that are being stimulated by autoantigens. Mutant T cell clones from MS patients were isolated and tested for reactivity to myelin basic protein, an antigen that is thought to participate in the induction of the disease. The hypoxanthine guanine phosphoribosyltransferase (hprt) clonal assay was used to determine mutant frequency values in MS patients with chronic progressive disease. Eleven of 258 thioguanine-resistant (hprt-) T cell clones from five of the six MS patients who were tested proliferated in response to human myelin basic protein without prior in vitro exposure to this antigen. No wild-type clones from these patients, nor any hprt- or wild-type clones from three healthy individuals responded to myelin basic protein. Thus, T cell clones that react with myelin basic protein can be isolated from the peripheral blood of MS patients.

In vivo hprt mutant frequencies in T-cells of normal human newborns

Mutation at the hypoxanthine-guanine phosphoribosyl transferase locus ( hprt; HPRT enzyme) in the human fetus was studied by clonal assay of placental cord blood samples from full-term newborns. Conditions for determining hprt mutant frequencies, as defined for adults, were also optimal for studies in newborns. The mean mutant frequency for 45 normal human newborns (37 male, 8 female) was 0.64 × 10 −6 (SD = 0.41 × 10 −6; median value = 0.58 × 10 −6). These values are approx. 10-fold lower than corresponding adult hprt mutant frequency values. Factors such as limiting-dilution cloning efficiencies, delay prior to study of sample, sex, cryopreservation or technician performing the assay did not significantly affect assay results. Maternal smoking did not result in elevated mutant frequency values. Most wild-type and mutant clones studied were CD4 surface antigen positive (helper/inducer). All hprt mutants analyzed lacked HPRT activity.

Longitudinal study of the in vivo hprt mutant frequency in human T-lymphocytes as determined by a cell cloning assay.

The in vivo frequency of mutants resulting from mutation at the hprt locus in human T-lymphocytes can be determined by a cloning assay. This assay quantifies the frequency of 6-thioguanine-resistant (TGr) T-cells through growth of colonies in 96-well microtiter dishes. The reproducibility of the TGr mutant frequency values has now been assessed in a longitudinal study of six individuals (three male, three female, aged 22-33 years) employing 4-5 blood samples over a 26-37 week time period. Cloning assays were performed with both fresh and cryopreserved cell samples. No significant differences were found among the mutant frequency values for multiple samples from each individual with both fresh and cryopreserved cell samples. These results demonstrate the reproducibility of this cloning assay for in vivo mutant frequency determinations in human T-lymphocytes.

Molecular analyses of in vivo hprt mutations in human T-lymphocytes. I. Studies of low frequency 'spontaneous' mutants by Southern blots.

Fifty wild-type and 164 in vivo-derived hprt mutant T-cell clones obtained from eight non-mutagen-exposed adult males with mutant frequency values in the normal range (usually less than 10 X 10(-6) were studied by Southern blot analyses to determine frequency and extent of gross structural alterations in the hprt gene. Sixteen (9.8%) of the mutant clones showed hprt changes. No site or type of lesion predominated. Relative frequencies of gross structural alterations in the recovered hprt mutants did not differ among the eight individuals, within limits detectable by the study. DNA from 201 of these 214 clones was also studied with a T-cell receptor (TCR) beta gene probe as a marker for independence of in vivo-derived clones. Some clones were also studied with a TCR gamma gene probe. Ninety-four percent of wild-type and 89% of the hprt mutants were found to originate from independent in vivo precursors. Therefore, most of the recovered hprt mutants in the study were presumably derived from separate in vivo mutations. For non-mutagenized adults with normal mutant frequencies, in vivo mutant frequencies are thus reasonable approximations of in vivo mutation frequencies, although elsewhere we show that this is not necessarily true for individuals with grossly elevated mutant frequencies.