A framework for mutational signature analysis based on DNA shape parameters

The mutation risk of a DNA locus depends on its oligonucleotide context. In turn, mutability of oligonucleotides varies across individuals, due to exposure to mutagenic agents or due to variable efficiency and/or accuracy of DNA repair. Such variability is captured by mutational signatures, a mathematical construct obtained by a deconvolution of mutation frequency spectra across individuals. There is a need to enhance methods for inferring mutational signatures to make better use of sparse mutation data (e.g., resulting from exome sequencing of cancers), to facilitate insight into underlying biological mechanisms, and to provide more accurate mutation rate baselines for inferring positive and negative selection. We propose a conceptualization of mutational signatures that represents oligonucleotides via descriptors of DNA conformation: base pair, base pair step, and minor groove width parameters. We demonstrate how such DNA structural parameters can accurately predict mutation occurrence due to DNA repair failures or due to exposure to diverse mutagens such as radiation, chemical exposure, and the APOBEC cytosine deaminase enzymes. Furthermore, the mutation frequency of DNA oligomers classed by structural features can accurately capture systematic variability in mutagenesis of >1,000 tumors originating from diverse human tissues. A nonnegative matrix factorization was applied to mutation spectra stratified by DNA structural features, thereby extracting novel mutational signatures. Moreover, many of the known trinucleotide signatures were associated with an additional spectrum in the DNA structural descriptor space, which may aid interpretation and provide mechanistic insight. Overall, we suggest that the power of DNA sequence motif-based mutational signature analysis can be enhanced by drawing on DNA shape features.

Frequency and Spectrum of Mutations Induced by Gamma Rays Revealed by Phenotype Screening and Whole-Genome Re-Sequencing in Arabidopsis thaliana

Genetic variations are an important source of germplasm diversity, as it provides an allele resource that contributes to the development of new traits for plant breeding. Gamma rays have been widely used as a physical agent for mutation creation in plants, and their mutagenic effect has attracted extensive attention. However, few studies are available on the comprehensive mutation profile at both the large-scale phenotype mutation screening and whole-genome mutation scanning. In this study, biological effects on M1 generation, large-scale phenotype screening in M2 generation, as well as whole-genome re-sequencing of seven M3 phenotype-visible lines were carried out to comprehensively evaluate the mutagenic effects of gamma rays on Arabidopsis thaliana. A total of 417 plants with visible mutated phenotypes were isolated from 20,502 M2 plants, and the phenotypic mutation frequency of gamma rays was 2.03% in Arabidopsis thaliana. On average, there were 21.57 single-base substitutions (SBSs) and 11.57 small insertions and deletions (InDels) in each line. Single-base InDels accounts for 66.7% of the small InDels. The genomic mutation frequency was 2.78 × 10−10/bp/Gy. The ratio of transition/transversion was 1.60, and 64.28% of the C > T events exhibited the pyrimidine dinucleotide sequence; 69.14% of the small InDels were located in the sequence with 1 to 4 bp terminal microhomology that was used for DNA end rejoining, while SBSs were less dependent on terminal microhomology. Nine genes, on average, were predicted to suffer from functional alteration in each re-sequenced line. This indicated that a suitable mutation gene density was an advantage of gamma rays when trying to improve elite materials for one certain or a few traits. These results will aid the full understanding of the mutagenic effects and mechanisms of gamma rays and provide a basis for suitable mutagen selection and parameter design, which can further facilitate the development of more controlled mutagenesis methods for plant mutation breeding.

Increased mutation efficiency of CRISPR/Cas9 genome editing in banana by optimized construct

The CRISPR/Cas9-mediated genome editing system has been used extensively to engineer targeted mutations in a wide variety of species. Its application in banana, however, has been hindered because of the species’ triploid nature and low genome editing efficiency. This has delayed the development of a DNA-free genome editing approach. In this study, we reported that the endogenous U6 promoter and banana codon-optimized Cas9 apparently increased mutation frequency in banana, and we generated a method to validate the mutation efficiency of the CRISPR/Cas9-mediated genome editing system based on transient expression in protoplasts. The activity of the MaU6c promoter was approximately four times higher than that of the OsU6a promoter in banana protoplasts. The application of this promoter and banana codon-optimized Cas9 in CRISPR/Cas9 cassette resulted in a fourfold increase in mutation efficiency compared with the previous CRISPR/Cas9 cassette for banana. Our results indicated that the optimized CRISPR/Cas9 system was effective for mutating targeted genes in banana and thus will improve the applications for basic functional genomics. These findings are relevant to future germplasm improvement and provide a foundation for developing DNA-free genome editing technology in banana.

Evaluating Methotrexate Toxicity and Its Association with ABCB1 Genetic Polymorphism in Children with Acute Lymphoblastic Leukemia

Background: Acute lymphoblastic leukemia (ALL) is among the most prevalent type of hematologic malignancy in children. The Children’s Oncology Group protocol recognizes methotrexate (MTX) as a therapy for this problem in children, despite its several complications. The relationship between MTX toxicity and ATP-binding cassette subfamily B member 1 (ABCB1) SNPs in ALL children patients has been investigated in many studies. Objectives: Regarding the controversial findings reported by these studies, the present work aims to evaluate Methotrexate toxicity and its association with ABCB1 Genetic Polymorphism in ALL pediatric patients. Methods: Blood samples were collected from pediatric ALL patients. Next, DNA was extracted and polymerase chain reaction (PCR) was conducted using 300 μMol/μL of direct primers in 50 µL as the ultimate volume. ABCB1 gene was amplified using the PCR technique, and 0.5% agarose gel electrophoresis was used to identify reaction products. Afterward, the PCR fragments’ length was proved by observing through UV-transilluminator. Finally, liver and blood toxicity was studied in all cases under treatment with MTX. Results: In the present study, 81 children with ALL (36 females and 45 males) with a mean age of 6.32 ± 3.08 years old were examined. The ABCB1 1199 G->A gene mutation frequency and the ABCB1 3435 C->T gene mutation frequency was 4.9 and 70.4%, respectively. The results showed no statistically significant difference between leukopenia, gastrointestinal toxicity, renal toxicity, hepatotoxicity, anemia, thrombocytopenia, and neutropenia in cases having homozygous heterozygous ABCB1 3435 C->T and ABCB1 1199 G->A mutant polymorphisms than those having ordinary polymorphism. Conclusions: Overall, it seems that C3435 T, G1199A, and ABCB1 are not significant MTX toxicity markers in pediatric ALL cases.

Detection of Ramularia collo-cygni DMI- and SDHI-resistant field populations in Austria and the effect of fungicides on the population and genetic diversity

Winter barley (Hordeum vulgare L.) is the third most cultivated crop after corn and wheat in Austria but one of the most challenging for disease control. The foliar pathogen Ramularia collo-cygni B. Sutton and J.M. Waller, causing Ramularia leaf spots (RLS), is one of the most important diseases in barley. In the recent years, control has only been achieved using fungicide mixtures including the multi-site inhibitor chlorothalonil, however this compound is totally banned in the EU. The objective of this study was to assess fungicide dose-rates and spray mixtures for RLS control. Furthermore, a field monitoring within the main barley growing areas of Austria was carried out, to analyse the current resistance situation to DMI and SDHI fungicides, which are still the backbone in RLS control. The results indicate that only the mixture with chlorothalonil achieved a good RLS control. Prothioconazole or benzovindiflupyr (alone or additively) decrease the severity of RLS but increase the local frequency of Cyp51 and sdhC mutations, especially the high dose rates. Based on a low Cyp51 mutation frequency of 16% in untreated control this frequency increased over 3.8 times following an application with 300 g ha−1 prothioconazole. The cumulative-sdhC mutations were even more increased after an application with benzovindiflupyr. This study showed that Ramularia collo-cygni is present in 91% of barley fields presented in this field survey. Widespread use of chlorothalonil fungicide maintained a low to moderate mutation frequency (Cyp51-I325T, Cyp51-I328L, sdhC-H146R and sdhC-H153R) in Austrian barley regions with no increase between 2017 and 2019.

Characterization of HIV-1 molecular epidemiology and Transmitted drug-resistance in newly diagnosed HIV-infected patients in Sichuan, China

Background Sichuan Province is one of the highest AIDS epidemic provinces in China, with a large number of floating population. The annual number of cases of HIV/AIDS reported in Sichuan has been the highest province in China for several successive years. There is a lack of widespread and representative data on the distribution of HIV subtypes in Sichuan. We aim to investigate the characteristics of HIV-1 molecular epidemiology and transmitted drug-resistance in newly diagnosed HIV-infected patients in Sichuan, China. Method Archived plasma samples (n = 1524) from HIV-1 newly-diagnosed individuals in April 2019 were selected by cross-sectional investigation from all 21 cities in Sichuan Province. Phylogenetic relationship, transmission cluster, and genotypic drug resistance analyses were performed using HIV-1 polymerase (pol) gene sequences. We also analysed the association of demographic and virological factors with transmitted drug-resistance (TDR). Results Partial pol gene sequences were obtained from 1297 cases. HIV-1 epidemic strains in Sichuan province: the majority of subtypes were circulating recombinant form (CRF) 07_BC (675, 52.04%), CRF01_AE (343, 26.45%), CRF08_BC (115, 8.87%), CRF85_BC (67, 5.17%), subtype B (33, 2.54%), the other subtypes only accounted for 4.93%, and circulating recombinant forms (URFs) (23, 1.77%) were observed in the study, and the difference of age, ethnicity, education, occupation, region and transmission pathway of different subtypes were statistically significant, CRF08_BC was significantly drug-resistant. A total of 205 (43.78%) pol sequences were involved in the genetic transmission network, with 76 clusters ranging in size from 2 to 24 pol sequences. In addition, the level of TDR has reached a medium level, with 72 of 1297 (5.55%) cases carrying drug-resistance mutation sites, TDR mutation frequency to nonnucleoside reverse transcriptase inhibitors (NNRTIs, 3.85%) was much higher than nucleoside reverse transcriptase inhibitors (NRTIs, 0.31%) and protease inhibitors (PIs, 1.70%). The most common HIV-1 mutation pattern for NNRTI was V106 (1.31%, 17/1297) and E138 (1.16%, 15/1297), and for PI was M46 (0.69%, 9/1297). Conclusion The distribution of HIV-1 genotypes in Sichuan is more diverse and complex, and the Men who have sex with men (MSM) is underrated, arguing for behavior scaling up intervention in this specific population besides the elderly people with heterosexual transmission risk groups. The risk of TDR mutation frequency increased in newly diagnosed patients highlights the significance of genotypic drug resistance monitoring and molecular surveillance of pretreatment HIV-1 drug resistance. The regimen composed of TDF, 3TC and EFV was still currently the preferred solution used free first-line therapy.

Genome Instability-Related miRNAs Predict Survival, Immune Landscape, and Immunotherapy Responses in Gastric Cancer

Background. Increasing evidence suggests that microRNAs (miRNAs) are involved in genome instability (GI) and drive the occurrence of tumors. However, the role of GI-related miRNAs in gastric cancer (GC) remains largely unknown. Herein, we developed a novel GI-related miRNA signature (GIMiSig) and further investigated its role in prognosis, the immune landscape, and immunotherapy responses in GC patients. Methods. An analysis of somatic mutation data on 434 gastric cancer cases from The Cancer Genome Atlas (TCGA) database was performed, thereby generating genome stability (GS) and GI groups. By detecting differentially expressed miRNAs between the GS and GI groups that were associated with overall survival, 8 miRNAs were identified and used to construct the GIMiSig. Results. The GIMiSig showed high accuracy in detecting GC patients. Using GIMiSig to stratify the patients into the high- and low-risk subgroups to predict survival outperformed the use of regular clinical features such as age, gender, or disease stage. Patients with low risk had a more favorable survival time than those with high risk. More importantly, the high-risk patients were associated with decreased UBQLN4 expression, higher accumulation of immune cells, lower Titin (TTN) mutation frequency, worse immunotherapy efficacy, and cancer-associated pathways. Conversely, the low-risk patients were characterized by UBQLN4 overexpression, lower fraction of immune cells, higher TTN mutation frequency, better response to immunotherapy, and GI-related pathways. Conclusion. In summary, we constructed a novel GIMiSig that could stratify GC patients into distinct risk groups that have different survival outcomes and immunotherapy efficacy. The results may provide new clues for improving GC outcomes.

Transcriptional Response of Mycobacterium tuberculosis to Cigarette Smoke Condensate

Smoking is known to be an added risk factor for tuberculosis (TB), with nearly a quarter of the TB cases attributed to cigarette smokers in the 22 countries with the highest TB burden. Many studies have indicated a link between risk of active TB and cigarette smoke. Smoking is also known to significantly decrease TB cure and treatment completion rate and increase mortality rates. Cigarette smoke contains thousands of volatile compounds including carcinogens, toxins, reactive solids, and oxidants in both particulate and gaseous phase. Yet, to date, limited studies have analyzed the impact of cigarette smoke components on Mycobacterium tuberculosis (Mtb), the causative agent of TB. Here we report the impact of cigarette smoke condensate (CSC) on survival, mutation frequency, and gene expression of Mtb in vitro. We show that exposure of virulent Mtb to cigarette smoke increases the mutation frequency of the pathogen and strongly induces the expression of the regulon controlled by SigH—a global transcriptional regulator of oxidative stress. SigH has previously been shown to be required for Mtb to respond to oxidative stress, survival, and granuloma formation in vivo. A high-SigH expression phenotype is known to be associated with greater virulence of Mtb. In patients with pulmonary TB who smoke, these changes may therefore play an important, yet unexplored, role in the treatment efficacy by potentially enhancing the virulence of tubercle bacilli.