Fast neutron mutagenesis in soybean enriches for small indels and creates frameshift mutations

The mutagenic effects of ionizing radiation have been used for decades to create novel variants in experimental populations. Fast neutron (FN) bombardment as a mutagen has been especially widespread in plants, with extensive reports describing the induction of large structural variants, i.e., deletions, insertions, inversions, and translocations. However, the full spectrum of FN-induced mutations is poorly understood. We contrast small insertions and deletions (indels) observed in 27 soybean lines subject to FN irradiation with the standing indels identified in 107 diverse soybean lines. We use the same populations to contrast the nature and context (bases flanking a nucleotide change) of single nucleotide variants. The accumulation of new single nucleotide changes in FN lines is marginally higher than expected based on spontaneous mutation. In FN treated lines and in standing variation, C→T transitions and the corresponding reverse complement G→A transitions are the most abundant and occur most frequently in a CpG local context. These data indicate that most SNPs identified in FN lines are likely derived from spontaneous de novo processes in generations following mutagenesis rather than from the FN irradiation mutagen. However, small indels in FN lines differ from standing variants. Short insertions, from 1–6 base pairs, are less abundant than in standing variation. Short deletions are more abundant and prone to induce frameshift mutations that should disrupt the structure and function of encoded proteins. These findings indicate that FN irradiation generates numerous small indels, increasing the abundance of loss of function mutations that impact single genes.

Loss of nr4a1 abrogates Fitness of asxl1-mutant Hematopoietic Clones

Clonal fitness of mutant hematopoietic stem and progenitor cells (HSPCs) underlies clonal hematopoiesis (CH), a state of clonal expansion associated with increased risk of blood malignancies and cardiovascular disease. Mechanisms by which acquired mutations lead to clonal fitness are not known. We used a zebrafish model to study the effect of acquired asxl1 mutations on HSPC clonality with TWISTR (Tissue editing With Inducible Stem cell Tagging via Recombination) that combined mosaic CRISPR-Cas9 mutagenesis with color labeling of HSPC clones. TWISTR asxl1 mutants showed clonal dominant states with the expansion of single-colored clones contributing to over 30% of myelopoiesis. These zebrafish had normal hematopoietic output and no major lineage skewing. Single-cell RNA sequencing analysis of TWISTR mutant marrow cells harboring asxl1 mutations showed >10-fold upregulation of inflammatory cytokines in mutant mature myeloid cells and >30-fold upregulation of anti-inflammatory modulators in immature progenitors. Increased inflammation has been widely documented in persons with CH with acquired mutations in DNMT3A, TET2, ASXL1 and other genes. Moreover, chronic inflammation due to infection was shown to promote relative clonal fitness in Dnmt3a mutant mice. Based on our results, we proposed the hypothesis that upregulation of the anti-inflammatory genes, including nr4a1, served as a mechanism of resistance to chronic inflammation created by the mutant HSPC progeny, resulting in a self-perpetuating cycle of clonal fitness in that environment. To test this, we used TWISTR to generate mosaic mutants of asxl1 and nr4a1 by injecting zebrafish with guide RNAs targeting exon 12 of asxl1 and exon 3 of nr4a1 together. Our model would predict that abrogation of nr4a1 expression in asxl1-mutant clones would weaken their fitness relative to clones that maintained nr4a1 expression. We sorted over 300 clones of various sizes from this cohort of zebrafish and sequenced the two targeted genes. We found that clones with frameshift mutations in asxl1 with either no nr4a1 mutations or heterozygous nr4a1 mutations had a clone size of 20%±14% or 19.7%±15% in myeloid cells, respectively. Asxl1-mutant clones with biallelic frameshift mutations in nr4a1 were significantly smaller with a clone size of 13.8±11.5% (p < 0.015). Clones with intact asxl1 did not differ in their clone size independent of nr4a1 genetic status (11.9% wildtype nr4a1 vs 11.5% homozygous nr4a1 mutant). Chemical inhibition of nr4a1 over 3 months resulted in reduced change of edited clones in asxl1-mutant zebrafish compared to vehicle-treated zebrafish, with median change in allelic fraction of 3%±4.8% vs 5.3%±7.5%, respectively. This suggested that upregulation of nr4a1 in asxl1-mutant clones maintains their fitness in inflammatory conditions, potentially by limiting HSPC exhaustion. We successfully used TWISTR to study asxl1 induced CH in zebrafish and identified nr4a1 upregulation as a critical pathway engaged for establishing clonal fitness. Disclosures Zon: Fate Therapeutics: Current equity holder in publicly-traded company, Other: Founder; CAMP4 Therapeutics: Current holder of individual stocks in a privately-held company, Other: Founder; Amagma Therapeutics: Current holder of individual stocks in a privately-held company, Other: Founder; Scholar Rock: Current equity holder in publicly-traded company, Other: Founder; Branch Biosciences: Current holder of individual stocks in a privately-held company, Other: Founder; Celularity: Consultancy; Cellarity: Consultancy.

Characterization of Tigecycline Resistance Among Tigecycline Non-susceptible Klebsiella pneumoniae Isolates From Humans, Food-Producing Animals, and in vitro Selection Assay

Emergence of extensively drug-resistant isolates of Klebsiella pneumoniae has prompted increased reliance on the last-resort antibiotics such as tigecycline (TGC) for treating infections caused by these pathogens. Consumption of human antibiotics in the food production industry has been found to contribute to the current antibiotic resistance crisis. In the current study, we aimed to investigate the mechanisms of TGC resistance among 18 TGC-non-susceptible (resistant or intermediate) K. pneumoniae (TGC-NSKP) isolates obtained from human (n = 5), food animals (n = 7), and in vitro selection experiment (n = 6). Isolates were genotyped by multilocus sequence typing (MLST). ramR, acrR, rpsJ, tetA, and mgrB (for colistin resistance) genes were sequenced. The presence of tetX, tetX1, and carbapenemase genes was examined by PCR. Susceptibility to different classes of antibiotics was evaluated by disc diffusion and broth macrodilution methods. The expression level of acrB was quantified by RT-qPCR assay. The 12 TGC-NSKP isolates [minimum inhibitory concentrations (MICs) = 4–32 mg/l] belonged to 10 distinct sequence types including ST37 (n = 2), ST11, ST15, ST45, ST1326 (animal isolates); ST147 (n = 2, human and animal isolates); and ST16, ST377, ST893, and ST2935 (human isolates). Co-resistance to TGC and colistin was identified among 57 and 40% of animal and human isolates, respectively. All human TGC-NSKP isolates carried carbapenemase genes (blaOXA–48, blaNDM–1, and blaNDM–5). tetX/X1 genes were not detected in any isolates. About 83% of TGC-NSKP isolates (n = 15) carried ramR and/or acrR alterations including missense/nonsense mutations (A19V, L44Q, I141T, G180D, A28T, R114L, T119S, Y59stop, and Q122stop), insertions (positions +205 and +343), or deletions (position +205) for ramR, and R90G substitution or frameshift mutations for acrR. In one isolate ramR amplicon was not detected using all primers used in this study. Among seven colistin-resistant isolates, five harbored inactivated/mutated MgrB due to premature termination by nonsense mutations, insertion of IS elements, and frameshift mutations. All isolates revealed wild-type RpsJ and TetA (if present). Increased expression of acrB gene was detected among all resistant isolates, with the in vitro selected mutants showing the highest values. A combination of RamR and AcrR alterations was involved in TGC non-susceptibility in the majority of studied isolates.

Species-wide phylogenomics of the Staphylococcus aureus agr operon reveals convergent evolution of frameshift mutations

Staphylococcus aureus is a prominent nosocomial pathogen that causes several life-threatening diseases such as pneumonia and bacteremia. S. aureus modulates expression of its arsenal of virulence factors through sensing and integrating responses to environmental signals. The agr (accessory gene regulator) quorum sensing (QS) system is a major regulator of virulence phenotypes in S. aureus. There are four agr specificity groups each with a different autoinducer peptide sequence (encoded by the agrD gene). Though agr is critical for expression of many toxins, paradoxically, S. aureus strains often have non-functional agr activity due to loss-of-function mutations in the four-gene agr operon. To understand patterns in agr variability across S. aureus, we undertook a species-wide genomic investigation. We developed a software tool (AgrVATE; https://github.com/VishnuRaghuram94/AgrVATE) for typing and detecting frameshift mutations in the agr operon. In an analysis of over 40,000 S. aureus genomes, we showed close association between agr type and S. aureus clonal complex. We also found strong linkage between agrBDC alleles (encoding the peptidase, the autoinducing peptide itself, and the peptide sensor respectively) but not agrA (encoding the -response regulator). More than five percent of genomes were found to have frameshift mutations in the agr operon. Though most mutations occur only once in the entire species, we observed a small number of recurring mutations evolving convergently across different clonal lineages. Phylogenetic patterns suggested that strains with agr frameshifts were evolutionary dead ends. Overall, genomic analysis of agr operon suggests evolution through multiple processes with functional consequences that are not fully understood.

Case Report: Two Novel Frameshift Mutations in SLC20A2 and One Novel Splice Donor Mutation in PDGFB Associated With Primary Familial Brain Calcification

Primary familial brain calcification (PFBC, OMIM#213600), also known as Fahr's disease, is characterized by bilateral and symmetric brain calcification in the basal ganglia (globus pallidus, caudate nucleus, and putamen), thalamus, subcortical white matter, and cerebellum. PFBC can be caused by loss-of-function mutations in any of the six known causative genes. The most common clinical manifestations include movement disorders, cognitive impairment, and neuropsychiatric signs that gradually emerge in middle-aged patients. To broaden the PFBC mutation spectrum, we examined nine members of a family with PFBC and two sporadic cases from clinical departments, and sequenced all PFBC-causative genes in the index case. Two novel frameshift mutations in SLC20A2 [NM_001257180.2; c.806delC, p.(Pro269Glnfs*49) and c.1154delG, p.(Ser385Ilefs*70)] and one novel splice donor site mutation (NM_002608.4, c.456+1G>C, r.436_456del) in PDGFB were identified in the patient cohort. c.806delC co-segregated with brain calcification and led to SLC20A2 haploinsufficiency among the affected family members. The c.456+1G>C mutation in PDGFB resulted in aberrant mRNA splicing, thereby forming mature transcripts containing an in-frame 21 base pair (bp) deletion, which might create a stably truncated protein [p.(Val146_Gln152del)] and exert a dominant negative effect on wild-type PDGFB. All three mutations were located in highly conserved regions among multiple species and predicted to be pathogenic, as evaluated by at least eight common genetic variation scoring systems. This study identified three novel mutations in SLC20A2 and PDGFB, which broadened and enriched the PFBC mutation spectrum.

Imbalance between Expression of FOXC2 and Its lncRNA in Lymphedema-Distichiasis Caused by Frameshift Mutations

Forkhead-box C2 (FOXC2) is a transcription factor involved in lymphatic system development. FOXC2 mutations cause Lymphedema-distichiasis syndrome (LD). Recently, a natural antisense was identified, called lncRNA FOXC2-AS1, which increases FOXC2 mRNA stability. No studies have evaluated FOXC2 and FOXC2-AS1 blood expression in LD and healthy subjects. Here, we show that FOXC2 and FOXC-AS1 expression levels were similar in both controls and patients, and a significantly higher amount of both RNAs was observed in females. A positive correlation between FOXC2 and FOXC2-AS1 expression was found in both controls and patients, excluding those with frameshift mutations. In these patients, the FOXC2-AS1/FOXC2 ratio was about 1:1, while it was higher in controls and patients carrying other types of mutations. The overexpression or silencing of FOXC2-AS1 determined a significant increase or reduction in FOXC2 wild-type and frameshift mutant proteins, respectively. Moreover, confocal and bioinformatic analysis revealed that these variations caused the formation of nuclear proteins aggregates also involving DNA. In conclusion, patients with frameshift mutations presented lower values of the FOXC2-AS1/FOXC2 ratio, due to a decrease in FOXC2-AS1 expression. The imbalance between FOXC2 mRNA and its lncRNA could represent a molecular mechanism to reduce the amount of FOXC2 misfolded proteins, protecting cells from damage.

Somatic Mutation of NLRP Genes in Gastric and Colonic Cancers

Nucleotide-binding and leucine-rich repeat protein (NLRP) genes are involved in inflammasome formation that plays a role in inflammation/host defense and cell death. Both cell death and inflammation are crucial for cancer development, but the roles of NLRPs in cancer are partially known. In this study, we analyzed mononucleotide repeats in coding sequences of NLRP1, NLRP2, NLRP4 and NLRP9, and found 1, 1, 1 and 8 frameshift mutation (s) in gastric (GC) and colonic cancers (CRC), respectively. Five of the 32 high microsatellite instability (MSI-H) GCs (15.5%) and 6 of 113 MSI-H CRCs (5.5%) exhibited the frameshift mutations. There was no NLRP frameshift mutations in microsatellite stable (MSS) GCs and CRCs. We also discovered that 2 of 16 CRCs (12.5%) harbored intratumoral heterogeneity (ITH) of the NLRP9 frameshift mutations in one or more areas. In both GC and CRC with MSI-H, NLRP9 expression in NLRP9-mutated cases was significantly lower than that in NLRP9-non-mutated cases. Our data indicate that NLRP9 is altered at multiple levels (frameshift mutation, mutational ITH and loss of expression), which together could contribute to pathogenesis of MSI-H GC and CRC.