Single-step Mutations Research Articles

Mutational analysis of 23 autosomal short tandem repeats based on trio paternity testing in the Korean population

This study aimed to estimate A-STR mutation rates in 2,317 Korean parent–child trios by examining 20 Combined DNA Index System (CODIS) core loci (D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, CSF1PO, FGA, TH01, TPOX, vWA, D1S1656, D2S441, D2S1338, D10S1248, D12S391, D19S433, and D22S1045) and three non-CODIS loci (Penta E, Penta D, and SE33). Locus-specific mutation rate estimates varied from 0.00 to 8.63 × 10-3 per generation, with an average mutation rate of 1.62 × 10-3 (95 % CI, 1.39–1.88 × 10-3). We also combined data from previous studies to obtain comprehensive genetic values for the Korean population, and the average mutation rate was 1.59 × 10-3 (95 % CI, 1.38–1.82 × 10-3). Single-step mutations (95.69 %) and double-step mutations (3.35 %) were observed in the mutation pattern analysis, and cases expected to have multi-step mutations (0.96 %) were also observed. Large-sized alleles exhibited more loss mutations than gain mutations, and paternal mutations (62.68 %) were more frequently observed than maternal mutations (19.62 %). The calculated values and features of the 23 A-STRs explored in this study are expected to play a crucial role in establishing criteria for forensic genetic interpretation.

A posteriori parameters from paternity tests of a Mexican laboratory with the powerplex fusion system

Population studies regarding Human identification (HID) systems report a priori forensic parameters, but rarely they describe a posteriori parameters from concluded paternity tests. We analyzed data from the PowerPlex® Fusion System in 1503 paternity tests from a Mexican laboratory for five years (2016–2020). The motherless duo paternity tests (89.8%) were more frequent than the standard trio tests (10.2%). A notable increase in motherless tests was noted regarding our previous report (89.8% vs 77.3%), probably explained by the COVID-19 pandemic. The estimated exclusion frequency in Mexico ranged from 30.1 (trio) to 32.1% (duo). For paternity exclusions, we report the number of mismatches and the frequency at which each STR was involved. The PowerPlex® Fusion system showed more than five mismatches in 100% of the standard trio tests excluding paternity, and the majority of motherless-duo tests (98.1%). In positive paternity tests, PowerPlex® Fusion offered a higher combined paternity index (PI) (average 1.18 E + 10) regarding HID systems with 15 and 20 STRs, even without the inclusion of the Y-linked locus DYS391 to the kinship interpretation. Individual and global STR mutation rates were estimated from 17 paternal mutations (μ = 0.0017), the majority involving a single-step mutation (94.11%). Five independent null alleles were detected, most of them involving the Penta E locus (80%), which suggests caution to the users working with DNA databases or kinship analysis, to avoid false exclusions with Penta E. In brief, our results provide a better overview of a posteriori informativeness offered by the PowerPlex® Fusion system for paternity testing in Mexico.

A Case of Single-step Mutations at Two Short Tandem Repeat loci (D13S317 and DXS10148) among Three Generations of a Korean Family

A Case of Single-step Mutations at Two Short Tandem Repeat loci (D13S317 and DXS10148) among Three Generations of a Korean Family

Relative frequency of cytomegalovirus UL56 gene mutations detected in genotypic letermovir resistance testing

Genotypic testing for letermovir (LMV) resistance was performed by Sanger sequencing of cytomegalovirus terminase gene UL56 (codons 202–412) in 1165 diagnostic specimens, disclosing 36 sequence variants among 173 (14.8%) of the specimens, including one or more LMV resistance mutations in 134 specimens. Codon 325 mutations (C325Y/F/W/R) were the most common (108 specimens), followed by those at codon 369 (R369 S/G/T/K, 13 specimens) and V236M (11 specimens). Mutations V231L, N232Y, Q234R, L257F and V363I were detected in 1–3 specimens each. Combinations of codon 325 mutation and those at codons 236 or 369 were found in 6 specimens. Eleven novel sequence variants were phenotyped, validating Q234R, V363I and R369K as conferring 2- to 5-fold increased LMV 50% inhibitory concentrations (EC50). These findings indicate that UL56 codon 325 mutations conferring >3000-fold LMV EC50 are detected much more frequently in clinical practice than those conferring lower grade resistance, and suggest that a single step mutation to absolute LMV resistance is an ongoing concern in its therapeutic use.

Mutational robustness changes during long-term adaptation in laboratory budding yeast populations.

As an adapting population traverses the fitness landscape, its local neighborhood (i.e., the collection of fitness effects of single-step mutations) can change shape because of interactions with mutations acquired during evolution. These changes to the distribution of fitness effects can affect both the rate of adaptation and the accumulation of deleterious mutations. However, while numerous models of fitness landscapes have been proposed in the literature, empirical data on how this distribution changes during evolution remains limited. In this study, we directly measure how the fitness landscape neighborhood changes during laboratory adaptation. Using a barcode-based mutagenesis system, we measure the fitness effects of 91 specific gene disruption mutations in genetic backgrounds spanning 8000-10,000 generations of evolution in two constant environments. We find that the mean of the distribution of fitness effects decreases in one environment, indicating a reduction in mutational robustness, but does not change in the other. We show that these distribution-level patterns result from differences in the relative frequency of certain patterns of epistasis at the level of individual mutations, including fitness-correlated and idiosyncratic epistasis.

Mutation rate evaluation at 21 autosomal STR loci: Paternity testing experience

The short tandem repeats (STRs) or microsatellites are used for paternity testing and these sequences mutate more rapidlythanbulkDNAsequences. A total of 746 paternity cases were analysed to understand the mutation rate of 21 autosomal STR loci. We identified 41 mutations in 11 STR Loci with a maximum at SE33. No mutations occurred in the remaining 10 STR loci. The overall average mutation rate was estimated as 0.004523 and the estimated locus-specific mutation rate varied between 0.001214 and 0.016990. Among these 90.24% was accounted for single-step mutation, 2.44% for two steps, and 7.32 % for three or muti steps. The obtained data is crucial and could be helpful for ensuring the accuracy of DNA testing and interpretation.

Historical Development of the Puccinia triticina Population in South Africa.

In contrast to many other countries, the virulence and genetic diversity of the South African Puccinia triticina population before 1980 is unknown, because of the absence of regular and systematic race analysis data and viable rust cultures. Herbarium specimens housed at the National Collection of Fungi, Biosystematics Unit, Plant Health and Protection, Agricultural Research Council, Pretoria, South Africa (SA), provided the opportunity to investigate the genetic development of the population using isolates collected between 1906 and 2010. Five subpopulations that survived between 21 and 82 years in the field were found. While three of these could represent the original races that entered SA during European settlement, two appear to be recent exotic introductions into SA, most probably from other African countries. The demise of the three oldest subpopulations might be from the release of resistant wheat cultivars. The population is clonal, where new virulence develops through single step mutations and selection for virulence. Although a possible case of somatic hybridization was found, sexual reproduction appears to be absent in SA. This study confirmed the importance of annual surveys in SA and its neighboring countries for the timely detection of new virulent races that could threaten wheat production in SA.

Races of Puccinia graminis on barley, oat, and wheat in Canada from 2015 to 2019

Stem rust is a major disease on barley, oat, and wheat crops worldwide. The primary means of control is by host resistance genes that are incorporated into commercial varieties and have been effective against most races of Puccinia graminis Pers., the causal pathogen. Since this pathogen continually mutates, analyzing the population virulence structure is crucial to detect new races with increased virulence. Stem rust samples were collected from 2015 to 2019 from the Canadian provinces of Manitoba, Ontario, Quebec, and Saskatchewan to find the prevalence of stem rust in barley (Hordeum vulgare L.), oat (Avena sativa L.), and wheat (Triticum aestivum L.) fields and the virulence dynamics in the two pathogen populations. Stem rust was absent in cultivated wheat and at trace (<1%) levels of incidence in barley and oat fields in all years. While the dominant races of P. graminis f. sp. tritici were MCCDC (54.5%) and TPMKC (27.3%) in 2015, and TMRTF (29.2%) in 2016, race QFCSC was dominant in 2017 (52.0%), 2018 (88.4%), and 2019 (81.3%). The dominant race of P. graminis f. sp. avenae was TJS from 2015 to 2018. Race TJS declined in frequency across the years, from 89.5% (2015) to 73.7% (2016), 42.6% (2017), 49.1% (2018), and only 14.3% in 2019. The dominant races in 2019 were SGB (51.7%) and TGN (19.6%). One new race (TJQ) of P. graminis f. sp. avenae was detected in 2016 at one location near Emerson, Manitoba, which may have arisen from a single-step mutation to avirulence to Pg15 from race TJS.

Underestimation and misclassification of mutations at X chromosome STRs depend on population’s allelic profile

Short tandem repeats are extremely abundant in the human genome and widely used in population and forensic genetics. The conventional approach to estimate mutation rates for STRs involves the detection of Mendelian incompatibilities and their frequency quantification, in family duos or trios. In this work, we aim to measure the impact this estimation methodology implies in the underestimation of mutation rates.We computed two independent experiences simulating one single-step mutation and one double step mutation in each pedigree. The pedigree with the highest number of compatibilities observed (hidden mutations) was mother-daughter duo, followed by the father-daughter duo, mother-son duo and, finally, mother-father-child trio, for the one and two-steps simulations. The estimation of X-STRs mutation rates from simple counting proved to be biased through systematic underestimation in an extent which depends on allele frequencies distribution of the marker, type of pedigree and number of repeat units involved.

Isoallelic frequency estimation for short tandem repeat markers from massive parallel sequencing data

Massive parallel sequencing (MPS) continues to receive increasing interest in the forensic community, among other things because it allows for more markers than other technologies and an increased resolution of the STR (short tandem repeat) regions. For evidential weight calculations, estimates of each compositional variant of an allele (isoalleles) needs to be estimated. Some isoalleles may not be present in the used reference database, but they may still exist in the population. We present a statistical model for estimating isoallele frequencies based on MPS data. We demonstrate our approach on the D12S391 marker included in the Illumina ForenSeq multiplex. Based on a single step mutation model, we link the isoalleles across the varying allele lengths to estimate underlying parameters used to predict the isoallelic frequencies, both those observed in the reference database and unobserved isoalleles.

A general framework for moment-based analysis of genetic data.

In population genetics, the Dirichlet (also called the Balding-Nichols) model has for 20years been considered the key model to approximate the distribution of allele fractions within populations in a multi-allelic setting. It has often been noted that the Dirichlet assumption is approximate because positive correlations among alleles cannot be accommodated under the Dirichlet model. However, the validity of the Dirichlet distribution has never been systematically investigated in a general framework. This paper attempts to address this problem by providing a general overview of how allele fraction data under the most common multi-allelic mutational structures should be modeled. The Dirichlet and alternative models are investigated by simulating allele fractions from a diffusion approximation of the multi-allelic Wright-Fisher process with mutation, and applying a moment-based analysis method. The study shows that the optimal modeling strategy for the distribution of allele fractions depends on the specific mutation process. The Dirichlet model is only an exceptionally good approximation for the pure drift, Jukes-Cantor and parent-independent mutation processes with small mutation rates. Alternative models are required and proposed for the other mutation processes, such as a Beta-Dirichlet model for the infinite alleles mutation process, and a Hierarchical Beta model for the Kimura, Hasegawa-Kishino-Yano and Tamura-Nei processes. Finally, a novel Hierarchical Beta approximation is developed, a Pyramidal Hierarchical Beta model, for the generalized time-reversible and single-step mutation processes.

Forensic parameters and mutation analysis of 23 short tandem repeat (PowerPlex® Fusion System) loci in Fujian Han Chinese population.

Kinship testing based on genetic markers has valuable practical applications. Short tandem repeat polymorphisms (STRPs) can have large number of alleles, and become the dominant marker for kinship identification. However, the high mutation rates affect the identification accuracy. Thus, accurate investigation of the mutation rate of STR loci in different populations is crucial for the reliability of phylogenetic relationships. In present study, forensic parameters and mutation rates (include 95% CI) of 23 short tandem repeats (STR) loci (D3S1358, D1S1656, D2S441, D10S1248, D13S317, D16S539, D18S51, D2S1338, CSF1PO, TH01, vWA, D21S11, D7S820, D5S818, TPOX, D8S1179, D12S391, D19S433, FGA, D22S1045, PentaE, PentaD and DYS391) were investigated through PowerPlex® Fusion System in Fujian Han population. The high level of CDP (0.999999999999999999999999992) and CPE (0.999999993) indicated the panel was high efficiency in forensic DNA identification and paternity testing. In mutation analysis, 43 mutation cases were found through 54,124 parent-child meiotic transfers. The observed mutation rates ranged from 0 (D3S1358, D1S1656, D13S317, TH01, D19S433 and D22S1045) to 0.0025 (PentaE and FGA). The overall mutation rate across all loci was 0.0008 and the average mutation rate for the 23 loci was estimated to be 0.00078 per meiosis. The vast majority of mutations were single-step (88.4%) mutation and also include double-step (9.3%) and triple-step (2.3%) mutations. Paternal mutation rate was more common than maternal mutation rate with a ratio of 7.2:1. In addition, mutation rates indicated positive correlation (r = 0.633, p = 0.009) with the expected heterozygosity (He).

Mutation Analysis of Autosomal STR Loci Commonly Used in Paternity Testing in Bosnia and Herzegovina

Mutation analysis in forensic genetics and occurrence of mutations at short-tandem repeat (STR) loci, are very important in paternity testing and precise elucidation of obtained genetic profiles. To determine these locus-specific mutations in Bosnian-Herzegovinian population and their rate, 15 or 22 autosomal loci were typed using PowerPlex 16 and PowerPlex Fusion systems. In total, 1253 individuals within 583 parenthood testing cases were profiled at the Institute for Genetic Engineering and Biotechnology, University of Sarajevo during the period from 2009-2018. Out of total cases, in 13 cases 14 mutations were discovered at 11 loci. Among all tested DNA profiles two mutations occurred at D8S1179, D18S51 and FGA loci each, and one mutation at PENTA D, D3S1358, CSF1P0, D21S11, D5S818, vWA, D16S539, PENTA E. Mutation rates were calculated for 11 loci and were in consistency with mutation rates reported for correspondent locus. In our study, one mutation at locus PENTA D derives from maternal source. Also in one trio paternity case two single-step mutations at loci D16S539 and D18S51 were observed. Our results confirmed mutation analysis is important in paternity testing and therefore much attention should be directed at their analysis.

A Single Step Mutation at D3S1358 Locus in a DNA Paternity Testing with 2 Alleged Fathers.

Genetic information is used very frequently in human identification in civil or judicial cases. Establishing the kinship relationship between a child and his biological father involves many ethical facts. We describe a DNA paternity case with two alleged fathers and an inconsistency between alleged father-2 and the child at D3S1358 locus. As biological samples we used saliva collected from inside the cheek of each person using buccal swabs (Copan, Italy). We collected the biological samples from each of person after each person gave the consent. In order to find the concentration of salivary DNA, the DNA samples were quantified by 7500 ABI Real-time PCR using the Quantifiler Human DNA kit (Applied Biosystems, USA). The next step was the amplification of the Salivary DNA samples by polymerase chain reaction (PCR). It was performed on a ProFlex PCR System (Applied Biosystems, USA) using the multiplex STR markers from the AmpFlSTR® Identifiler Plus Amplification Kit (Applied Biosystems, USA). After amplification, the PCR products were run on capillary electrophoresis on an ABI 3500 Genetic Analyzer (Applied Biosystems, USA). AF-1 was excluded as biological father. The DNA profiles of AF-2 and the child had one mismatch at D3S1358 locus. Further, we amplified the Y-STR markers to confirm the mutation, obtaining a perfect match between the 2 persons. In paternity testing, where one or two inconsistencies are present between the child and the alleged father on autosomal STR markers, the use of haploid markers X-STR or Y-STRs is needed for the confirmation or exclusion of paternity.

Meropenem-Vaborbactam Resistance Selection, Resistance Prevention, and Molecular Mechanisms in Mutants of KPC-Producing Klebsiella pneumoniae.

ABSTRACTVaborbactam (formerly RPX7009) is a new β-lactamase inhibitor based on a cyclic boronic acid pharmacophore with potent inhibitory activity against Klebsiella pneumoniae carbapenemases (KPC). It has been developed in combination with meropenem. The objective of these studies was to identify the concentrations of both agents associated with the selection or prevention of single-step mutations leading to reduced sensitivity to the combination and to characterize the selected mutations. Eighteen strains of KPC-producing Klebsiella pneumoniae with various degrees of sensitivity to meropenem (MICs, 8 to 512 μg/ml) and meropenem-vaborbactam (MICs, ≤0.06 to 32 μg/ml) and preexisting resistance mechanisms were selected from a worldwide collection of isolates recovered from surveillance studies, emphasizing strains for which MICs were in the upper range of the meropenem-vaborbactam MIC distribution. Meropenem and vaborbactam at 8 μg/ml each suppressed the drug resistance mutation frequency to <1 × 10−8 in 77.8% (14/18) of strains, and all strains were inhibited when the meropenem concentration was increased to 16 μg/ml. Mutants selected at lower drug concentrations showed phenotypes associated with previously described carbapenem resistance mechanisms, including ompK36 inactivation in mutants selected from OmpK36-proficient strains and an increased blaKPC gene copy number in strains with partially functional ompK36. No mutations in the coding region of blaKPC were identified. These data indicate that the selection of mutants with reduced sensitivity to meropenem-vaborbactam from KPC-producing Klebsiella pneumoniae strains is associated with previously described mechanisms involving porin mutations and the increase in the blaKPC gene copy number and not changes in the KPC enzyme and can be prevented by the drug concentrations achieved with optimal dosing of the combination.

Multistep microsatellite mutation at D18S51 locus in a parentage testing case

Mutations of short tandem repeats (STRs) loci might cause allelic mismatchs between the child and the parents, which entangled the forensic inference in paternity testing. Most of the reported microsatellite mutations are confined to single-step mutations, and multistep mutations were rarely reported and only account for a very limited number of STR mutation events in previously studied cases. Here we reported a paternity case with a mismatch in locus D18S51. The genotypes of the alleged father, the mother and the child in D18S51 locus were 14/23, 15/16, and 15/20, respectively. Examination of 38 autosomal STR loci revealed no mismatches, and the paternity index is above 1.58E+15. These results suggested that the alleged father is the biological father of the child that a rare three-steps mutation had occurred in the paternal allele of D18S51.

Population data and mutation rates of 20 autosomal STR loci in a Chinese Han population from Yunnan Province, Southwest China.

The genetic polymorphisms of 20 autosomal short tandem repeat (STR) loci included in the PowerPlex® 21 kit were evaluated from 2068 unrelated, healthy individuals from the Chinese Han population of Yunnan Province in southwest China. All of the loci reached Hardy-Weinberg equilibrium. These loci were examined to determine allele frequencies and forensic statistical parameters. The genetic relationships among the Yunnan Han and other Chinese populations were also estimated. The combined discrimination power and probability of excluding paternity of the 20 STR loci were 0.99999999999999999999999126 and 0.999999975, respectively. In addition, mutation rates from 4363 parentage cases (2215 trios and 2148 duos) were investigated in this study. A total of 164 mutations were observed in 6578 meioses from the 20 loci. The highest mutation rate was observed in D12S391 (0.30%), and the lowest mutation rates were observed in D13S317 (0.03%) and TPOX (0.03%). The average mutation rate for the 20 loci was estimated to be 1.246×10-3 per meiosis. The mutations were primarily single-step and paternal mutations.

Microsatellite Mutation Rate in Atlantic Sturgeon (Acipenser oxyrinchus).

Understanding mutation rates can greatly extend the utility of population and conservation genetic analyses. Herein, we present an estimate of genome-wide microsatellite mutation rate in Atlantic sturgeon (Acipenser oxyrinchus) based on parent-offspring transmission patterns. We screened 307 individuals for parentage and mutation-rate analysis applying 43 variable markers. Out of 13228 allele transfers, 11 mutations were detected, producing a mutation rate of 8.3 × 10-4 per locus per generation (95% confidence interval: 1.48 × 10-3, 4.15 × 10-4). Single-step mutations predominated and there were trends toward mutations in loci with greater polymorphism and allele length. Two of the detected mutations were most probably cluster mutations, being identified in 12 and 28 sibs, respectively. Finally, we observed evidences of polyploidy based on the sporadic presence of 3 or 4 alleles per locus in the genotyped individuals, supporting previous reports of incomplete diploidization in Atlantic sturgeon.

Molecular Characterization of Australian Isolates of Puccinia graminis f. sp. tritici Supports Long-Term Clonality but also Reveals Cryptic Genetic Variation.

Long-term surveys of pathogenicity in Puccinia graminis f. sp. tritici in Australia have implicated mutation as a major source of virulence, at times leading to the demise of stem-rust-resistant wheat cultivars and substantial yield losses. Since 1925, these surveys have identified at least four occasions on which exotic isolates of P. graminis f. sp. tritici appeared in Australia, with each acting as a founding isolate that gave rise sequentially to derivative pathotypes via presumed single-step mutation. The current study examined the relationship between virulence and molecular patterns using simple-sequence repeat (SSR) markers on selected isolates of P. graminis f. sp. tritici collected in Australia during a 52-year period in order to propose an evolutionary pathway involving these isolates. Studies of SSR variability among this collection of isolates within a putative clonal lineage based on pathotype 21-0, first detected in 1954 (the "21/34 lineage"), provided compelling evidence of clonality over the 52-year period, coupled with single-step acquisition of virulence for resistance genes. It also supported the postulation that two triticale-attacking pathotypes (34-2,12 and 34-2,12,13) detected in the early 1980s were derived from pathotype 21-0 via stepwise sequential acquisition of virulence for Sr5, Sr11, Sr27, and then SrSatu. Some of the isolates examined that were regarded as members of the race 21/34 lineage based on pathogenicity differed significantly in their SSR genotypes, indicating that they may have originated from processes more complex than simple mutation. This included two isolates of pathotype 21-0, which were collected in 1994 and 2006. Given that sexual recombination in P. graminis is rare or absent in Australia, the cryptic complexity observed could indicate that one or more of these isolates arose as a consequence of asexual recombination.

The phenotypic and molecular resistance induced by a single-exposure to sub-mutant prevention concentration of marbofloxacin in Salmonella Typhimurium isolates from swine

In the present study, the molecular mechanisms of antibiotic resistance in Salmonella Typhimurium clinical isolates from pigs were investigated using a single-step mutation model of exposure to sub-mutant prevention concentrations (MPCs) of marbofloxacin. The minimum inhibitory concentrations (MICs) of seven antibacterial drugs were evaluated against 30 S. Typhimurium clinical isolates from different pigs. MPCs of marbofloxacin were also determined. The mechanism of marbofloxacin-resistance was investigated by sequencing analysis of target gene mutations and quantifying the overexpression of efflux pumps and their regulators by quantitative RT-PCR. Marbofloxacin showed the highest potency against all isolates (23.3%), including multi-drug resistant isolates. The MPC50 (0.5μg/mL) and MPC90 (2μg/mL) of marbofloxacin were determined, as were MPC/MIC ratios of 2.5 to 8. A gyrA mutation (Ser83Phe or Asp87His) was detected in isolates with an MIC>0.06μg/mL and all single-step mutants. Moreover, expression of acrAB-tolC and marA/soxS/ramA increased following a single-step mutation, but only ramA expression showed a positive correlation with the resistance phenotype of clinical isolates and single-step mutants (p<0.05). Furthermore, the acrR mutation was detected in two clinical isolates and 50% of single-step mutants, regardless of whether the gyrA mutation was present. This is the first report of acrR mutations in S. Typhimurium isolates from pigs in Korea. Our findings suggest that a single-exposure to sub-MPCs of marbofloxacin was sufficient to reduce the susceptibility of Salmonella isolates. Therefore, optimized dosing based on application with the MPC concept is required to reduce the chances of marbofloxacin resistance.