Mutational Mechanisms Research Articles

Properties and Mechanisms of Deletions, Insertions, and Substitutions in the Evolutionary History of SARS-CoV-2.

SARS-CoV-2 has accumulated many mutations since its emergence in late 2019. Nucleotide substitutions leading to amino acid replacements constitute the primary material for natural selection. Insertions, deletions, and substitutions appear to be critical for coronavirus's macro- and microevolution. Understanding the molecular mechanisms of mutations in the mutational hotspots (positions, loci with recurrent mutations, and nucleotide context) is important for disentangling roles of mutagenesis and selection. In the SARS-CoV-2 genome, deletions and insertions are frequently associated with repetitive sequences, whereas C>U substitutions are often surrounded by nucleotides resembling the APOBEC mutable motifs. We describe various approaches to mutation spectra analyses, including the context features of RNAs that are likely to be involved in the generation of recurrent mutations. We also discuss the interplay between mutations and natural selection as a complex evolutionary trend. The substantial variability and complexity of pipelines for the reconstruction of mutations and the huge number of genomic sequences are major problems for the analyses of mutations in the SARS-CoV-2 genome. As a solution, we advocate for the development of a centralized database of predicted mutations, which needs to be updated on a regular basis.

TP63 truncating mutation causes increased cell apoptosis and premature ovarian insufficiency by enhanced transcriptional activation of CLCA2

BackgroundPremature ovarian insufficiency (POI) is a severe disorder leading to female infertility. Genetic mutations are important factors causing POI. TP63-truncating mutation has been reported to cause POI by increasing germ cell apoptosis, however what factors mediate this apoptosis remains unclear.MethodsNinety-three patients with POI were recruited from Beijing Obstetrics and Gynecology Hospital, Capital Medical University. Whole-exome sequencing (WES) was performed for each patient. Sanger sequencing was used to confirm potential causative genetic variants. A minigene assay was performed to determine splicing effects of TP63 variants. A TP63-truncating plasmid was constructed. Real-time quantitative PCR, western blot analyses, dual luciferase reporter assays, immunofluorescence staining, and cell apoptosis assays were used to study the underlying mechanism of a TP63-truncating mutation causing POI.ResultsBy WES of 93 sporadic patients with POI, we found a 14-bp deletion covering the splice site in the TP63 gene. A minigene assay demonstrated that the 14-bp deletion variant led to exon 13 skipping during TP63 mRNA splicing, resulting in the generation of a truncated TP63 protein (TP63-mut). Overexpression of TP63-mut accelerated cell apoptosis. Mechanistically, the TP63-mut protein could bind to the promoter region of CLCA2 and activate the transcription of CLCA2 several times compared to that of the TP63 wild-type protein. Silencing CLCA2 using a specific small interfering RNA (siRNA) or inhibiting the Ataxia Telangiectasia Mutated (ATM) pathway using the KU55933 inhibitor attenuated cell apoptosis caused by TP63-mut protein expression.ConclusionOur findings revealed a crucial role for CLCA2 in mediating apoptosis in POI pathogenesis, and suggested that CLCA2 is a potential therapeutic target for POI.

Abstract 5444: EGFR missense mutation(s) induce OLIG2 expression to regulate glioma stem cells maintenance and therapeutic resistance

Abstract Glioblastoma (GBM) tumors contain a subpopulation of glioma stem cells (GSC) which can escape therapeutic effects through self-renewal and invasion. Epidermal growth factor receptor (EGFR) mutations are the most common alteration in GBM (50%). One example includes extracellular domain missense mutations like G598V, A289V, and R108K (10-15%). These extracellular domain missense mutations increase ligand binding affinity, inducing a highly active receptor. Earlier reports have shown these EGFR missense mutations exhibit therapeutic resistance; however, the molecular mechanism of these missense mutations in GBM remains unknown. In this study, we show high oligodendrocyte transcription factor 2 (OLIG2) expression in GSCs with EGFR-G598V and EGFR-A289V missense mutations. OLIG2 is universally expressed in glioma and essential for reprogramming GBM cells into stem-like tumor cells. Here, we report GSCs with these point mutations display increased self-renewal, growth, invasion, and therapeutic resistance. In addition, EGFR-G598V and EGFR-A289V trigger distinct downstream signaling pathways in GSCs to regulate OLIG2 function. These missense mutation(s) also represent different pathway-based GBM subclassifications, where A289V induces a Neuronal subtype and G598V induces a Glycolytic/Plurimetabolic subtype. We hypothesize that GBM tumors harboring EGFR missense mutations (G598V, A289V) influence stem cell properties via multiple pathways to evade therapeutic effects and inhibiting these different signaling pathways will decrease therapeutic resistance in GBM. Understanding the mechanism of these EGFR mutations may lead to improved therapeutic strategies for GBM patients. Citation Format: Sidney Weaver, Aunay Miller, Mylan Blomquist, James McNamara, Keely Orndorff, Chris Sereduk, Jean Kloss, Joseph Loftus, Shwetal Mehta, Nhan L. Tran. EGFR missense mutation(s) induce OLIG2 expression to regulate glioma stem cells maintenance and therapeutic resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5444.

Abstract 4337: Illuminating the complex rearrangement landscape of sarcoma

Abstract Structural rearrangements and in particular, gene fusions, are key mutational processes in bone and soft tissue tumors, used for both disease classification and as biomarkers. However, the mutational processes and rearrangement processes underlying many of these events remain poorly characterized. Recent data indicate that sarcomas show particularly high frequencies of complex rearrangement events, including patterns which do not fit those of known mutational mechanisms. As the largest whole genome sequencing (WGS) cohort of sarcomas to date, the Genomics England (GE) 100,000 Genomes project represents a unique dataset in which to profile these events. Structural variants (SVs) were identified in whole genome sequencing data for 978 samples from the Genomics England 100,000 Genomes Project using an optimized approach based on 5 specialized SV callers. SVs identified by at least 2 callers were filtered to retain only those which passed the quality filter, and to remove variants present in either matched germline samples or >1% of a panel of normal variants. Chromothriptic events, extrachromosomal DNA (ecDNA) and genes enriched for SVs were identified using established algorithms. The prevalence of SVs varies across sarcoma, in keeping with previous reports, with particularly high rates observed in myxofibrosarcoma and liposarcoma. As described previously, liposarcomas showed the highest rates of chromothripsis. We have examined some tumor types at higher granularity than previously available, demonstrating directly that the rates of complex rearrangement events vary across subtypes. For example, chromothriptic events are identified in 100% of well-differentiated liposarcomas, but less than 10% of myxoid liposarcomas. Similarly, the presence of ecDNA varies by tumor type, with the novel observation of particularly high rates in angiosarcoma. Using these data, we have catalogued the rearrangement patterns generating canonical gene fusions in fusion-driven soft tissue tumor types. Despite more recent advances in histological classification, survival for patients with sarcoma has remained largely unchanged for 40 years. Characterization of the mutational processes underlying these rearrangements will shed light on the pathogenesis of these tumors. Citation Format: Sara Waise, Tom Lesluyes, Jonas Demeulemeester, Maxime Tarabichi, Adrienne Flanagan, Nischalan Pillay, Peter Van Loo. Illuminating the complex rearrangement landscape of sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4337.

Abstract 4887: Unveiling the functional impact of glioma non-coding germline variants using large language models

Abstract Efforts to identify the risk for developing gliomas through Genome-Wide Association Studies (GWAS) face relevant challenges, such as accurately pinpointing the genomic positions of germline variants, primarily found in non-coding regions, with unclear functional implications in glioma development. In this context, the GET method, a large language model designed to unveil regulatory grammars across 213 human cell types, emerges as a valuable tool for elucidating the mechanisms of non-coding mutations. Our aims include: I) Validate the efficacy of the GET method using a well-documented case and prioritize relevant variants for analysis; II) Refine the genomic positions of these variants and exploring their linkage disequilibrium (LD); III) Identify potential motifs and genes that might be influenced by these high-impact variants. After examining published GWAS, 39 non-coding SNPs in gliomas were found. Adding 316 variants in high LD (R2 > 0.8) expanded the dataset to 355 variants for analysis. Using the GET method, we calculated impact scores (ISs) for each variant, considering changes in motif binding score (ΔBM) and motif gradient (GM) across human astrocytes. We focused on rare variants (risk allele frequency <0.25) with the higher odds ratios (OR). The table presents GET results for a well-known case-control, showing its role in increasing glioma risk through OCT4 motif disruption and enhanced MYC expression, as previously was confirmed experimentally. Moreover, it details results concerning the 5 rare germline variants exhibiting the highest ISs, including precise genomic positions and the most affected motifs and genes that could potentially be impacted by each variant. Using a large language model, this study overcomes the challenge of mere variant identification, aiming to establish a framework for uncovering the underlying mechanism of risk variants. While further validation is essential, this is a promising approach to explore the impact of germline mutations. Variant ID Genomic Position Altered Motif Affected Gene Ref allele Alt allele Case-control rs55705857 Chr8:129633445 OCT4+SOX2:POU MYC A G H-I variant #1 rs78378222 Chr17:7668433 HD/18 PER1 T G H-I variant #2 rs518394 Chr9:22019673 KLF/SP/2 CDKN2A G C H-I variant #3 rs79495512 Chr15:76199995 KLF/SP/2 LINGO1 T C H-I variant #4 rs78185702 Chr15:76317893 SMAD LINGO1 C T H-I variant #5 rs2538067 Chr7:54907724 CTCF EGFR G A Citation Format: Maria del Mar Alvarez-Torres, Xi Fu, Romella K Sagatelian, Alejandro L Buendia, Raul Rabadan. Unveiling the functional impact of glioma non-coding germline variants using large language models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4887.

Abstract 1270: p53-R172H mutation confers gain-of-function properties and promotes metastasis in squamous cell carcinoma

Abstract INTRODUCTION: Metastasis is associated with more than 90% of cancer-related mortality, and thus, there is a compelling need for innovative therapeutic breakthroughs. TP53 mutations are present in 60-70% of human cancers, especially in squamous cell cancers. For example, TP53 mutations are detected in up to 80% of esophageal squamous cell carcinomas (ESCCs). Accumulating evidence suggests that certain missense mutant forms of p53 such as R175 (R172 in mouse), R273 and R282 can acquire neomorphic pro-oncogenic activities that are referred to as gain-of-function (GOF). To elucidate novel mutant p53-dependent mechanisms in promoting metastasis, we conducted RNA-Seq, p53 ChIP-Seq and H3K27ac CUT&RUN-Seq on primary and metastatic tumor cells harvested from our mouse model of ESCC harboring Trp53R172H/−, Trp53−/− and Trp53+/+. Herein, we have identified p53-R172H gene targets in metastatic ESCC. One of the dominant effectors of p53-R172H-mediated metastasis was Colony stimulating factor-1 (Csf-1) signaling through its cognate receptor (Csf-1r), which we recently published in Cancer Discovery (PMID: 37676642). Overall, this study aims to investigate the GOF properties and mediators of mutant p53 in promoting ESCC metastasis. RESULTS/DISCUSSION: Using ex vivo cultures and in vivo tail-vein injections, we demonstrated increased colonization and metastatic capabilities of ESCC cells harboring p53-R172H in comparison to the tumor cells with null and wild-type p53. p53-R172H occupies more genes in the metastatic ESCCs than in the primary tumors that is independent of p53 expression levels. We identified 72 unique targets of p53-R172H with altered gene occupancy and expression that occurs during the transition from primary tumor to metastasis. Such targets in metastatic ESCC are exemplified by Csf-1 and Birc5 (encoding anti-apoptosis protein Survivin) that are also enriched with H3K27ac in their promoters. They are upregulated during metastasis compared to the primary tumors dependent upon p53 mutation status, which is reinforced by TCGA data and ESCC patient-derived tissue microarrays (TMAs). Interestingly, other frequently detected “hotspot” p53 mutations including R282W and R273C, which we introduced into the ESCC cells using base editing promote differential pro-tumorigenic activities. Indeed, the analysis of SCC datasets in TCGA reveals that specific p53 mutations are associated with altered overall survival, as well as differences in the enriched pathways. CONCLUSION: We have demonstrated GOF properties and mediators of p53-R172H in promoting ESCC metastasis that may be applicable to other SCCs. We are further expanding our analyses to additional “hotspot” p53 mutations to dissect the mechanisms of distinct p53 mutations in fostering ESCC metastasis, which can open up new avenues for therapeutic applications. Citation Format: Gizem Efe, Katherine Cunningham, Raul Navaridas Fernandez de Bobadilla, Karen J. Dunbar, Kensuke Sugiura, Noriyuki Nishiwaki, Saul Carcamo, Lois Resnick-Silverman, Dan Hasson, Andres J. Klein-Szanto, Alison M. Taylor, James J. Manfredi, Carol Prives, Anil K. Rustgi. p53-R172H mutation confers gain-of-function properties and promotes metastasis in squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1270.

Exploring the potential of bacterial consortium for the treatment of paper mill effluent treatment through various treatment strategies and evaluation of their toxicity

Paper mill effluent containing kraft lignin (KL) and other toxic compounds poses serious environmental health concerns due to its potential for bioaccumulation. A prospective bacterial consortium is still needed for the remediation of paper mill effluent. Therefore, the current work aims to explore the potential of consortium, i.e. Bacillus cereus, B. paramycoides, and B. aryabhattai via different treatment strategies: batch, continuous, multipulse, and dissolved oxygen (DO) stat fed-batch for maximum detoxification of effluent under optimised conditions. A minimal salt medium containing 1.0 % glucose, 0.5 % ammonium nitrate, and pH 8.0 was the optimum condition for the consortium to degrade 1000 mg/L of kraft lignin in the effluent. Laboratory adaptation evolution (LAE) studies showed that the maximum number of bacterial generations was observed within 178 h at 5th cycle for 3000 mg/L kraft lignin. The maximum removal of KL, color, chemical oxygen demand (COD), biological oxygen demand (BOD), and total phenol by the dissolved oxygen stat fed-batch treatment strategy was 94 %, 86 %, 91 %, 90 %, and 97 %, respectively. Phytotoxicity testing of the consortium-treated effluent on Vigna radiata L., and Trigonella foenum-graecum L. revealed a reduction in phytotoxicity by ∼66 % and ∼68 %, respectively. The genotoxicity study on Allium cepa L. showed that the percent of aberrant cells with different chromosomal aberrations was decreased in consortium-treated effluent as compared to untreated effluent. This work provides a new insight and strategy for the paper mill effluent treatment at an industrial scale. Statement of environmental implicationThe paper mill effluent contains various hazardous compounds; chlorinated phenols, dioxins, kraft lignin, and endocrine disrupting compounds, when discharge from industries without proper treatment. This effluent causes mutagenic, clastogenic, carcinogenic, and endocrine-disrupting mechanisms in flora and fauna. This study provides a comparative performance evaluation of different treatment strategies: batch, continuous fed-batch, multipulse fed-batch, and dissolved oxygen stat fed-batch, for paper mill effluent treatment using a bacterial consortium for maximum removal of kraft lignin, color, chemical oxygen demand, total phenol concentration, and quantified ligninolytic enzyme production. Treated effluent showed a lesser phytotoxic and genotoxic effect. Therefore, the results of this study are suitable for environmental restoration. Significance of studyContrastingly, throughout this study, found that bacterial consortium have superior detoxifying qualities of paper mill effluent compared to pure cultures, via different treatment strategies: batch, continuous, multipulse, and dissolved oxygen stat fed-batch strategies under optimised conditions at lab scale. According to these results, such dissolved oxygen stat fed-batch strategy mediated treated effluent that promotes seed germination and showed a higher mitotic index of root cells of the plant in contrast to untreated effluent. Therefore, these treatment strategies are adequate, effective, and sustainable to reduce the significant pollution load in effluent.

Reservoir Porosity Prediction Based on BiLSTM-AM Optimized by Improved Pelican Optimization Algorithm

To accurately predict reservoir porosity, a method based on bi-directional long short-term memory with attention mechanism (BiLSTM-AM) optimized by the improved pelican optimization algorithm (IPOA) is proposed. Firstly, the nonlinear inertia weight factor, Cauchy mutation, and sparrow warning mechanism are introduced to improve the pelican optimization algorithm (POA). Secondly, the superiority of IPOA is verified by using the CEC–2022 benchmark test functions. In addition, the Wilcoxon test is applied to evaluate the experimental results, which proves the superiority of IPOA against other popular algorithms. Finally, BiLSTM-AM is optimized by IPOA, and IPOA-BiLSTM-AM is used for porosity prediction in the Midlands basin. The results show that IPOA-BiLSTM-AM has the smallest prediction error for the verification set samples (RMSE and MAE were 0.5736 and 0.4313, respectively), which verifies its excellent performance.

A Novel Artificial Electric Field Algorithm for Solving Global Optimization and Real-World Engineering Problems.

The Artificial Electric Field Algorithm (AEFA) stands out as a physics-inspired metaheuristic, drawing inspiration from Coulomb's law and electrostatic force; however, while AEFA has demonstrated efficacy, it can face challenges such as convergence issues and suboptimal solutions, especially in high-dimensional problems. To overcome these challenges, this paper introduces a modified version of AEFA, named mAEFA, which leverages the capabilities of Lévy flights, simulated annealing, and the Adaptive s-best Mutation and Natural Survivor Method (NSM) mechanisms. While Lévy flights enhance exploration potential and simulated annealing improves search exploitation, the Adaptive s-best Mutation and Natural Survivor Method (NSM) mechanisms are employed to add more diversity. The integration of these mechanisms in AEFA aims to expand its search space, enhance exploration potential, avoid local optima, and achieve improved performance, robustness, and a more equitable equilibrium between local intensification and global diversification. In this study, a comprehensive assessment of mAEFA is carried out, employing a combination of quantitative and qualitative measures, on a diverse range of 29 intricate CEC'17 constraint benchmarks that exhibit different characteristics. The practical compatibility of the proposed mAEFA is evaluated on five engineering benchmark problems derived from the civil, mechanical, and industrial engineering domains. Results from the mAEFA algorithm are compared with those from seven recently introduced metaheuristic algorithms using widely adopted statistical metrics. The mAEFA algorithm outperforms the LCA algorithm in all 29 CEC'17 test functions with 100% superiority and shows better results than SAO, GOA, CHIO, PSO, GSA, and AEFA in 96.6%, 96.6%, 93.1%, 86.2%, 82.8%, and 58.6% of test cases, respectively. In three out of five engineering design problems, mAEFA outperforms all the compared algorithms, securing second place in the remaining two problems. Results across all optimization problems highlight the effectiveness and robustness of mAEFA compared to baseline metaheuristics. The suggested enhancements in AEFA have proven effective, establishing competitiveness in diverse optimization problems.

An ensemble method with a hybrid of genetic algorithm and K-prototypes algorithm for mixed data classification

Due to challenges posed by mixed data clustering, this study aims to introduce an innovative clustering-based classification algorithm that possesses the advantages of both classification and clustering techniques for mixed data analysis. The proposed algorithm employs the K-prototypes algorithm with a genetic algorithm to optimize weights and centroids and utilizes the bagging method to build multiple classifiers, thereby enhancing classification performance. Furthermore, it incorporates four mutation mechanisms, including Gaussian, Cauchy, Levy, and single-point mutations, to explore optimal solutions. This study suggests using a 20% sampling ratio for the bootstrap sampling in the proposed algorithm. This ratio has been proven to be sufficient for achieving good classification performance while reducing computational time. Experimental results indicate that the proposed algorithm outperforms benchmark classifiers, demonstrating superior classification performance across five performance indicators. In addition, the loan eligibility case study offers valuable insights into applying the proposed algorithm in real-world scenarios, demonstrating that the proposed algorithm can achieve superior classification performance compared to other algorithms. It also offers managerial implications to help different industries and fields understand the appropriate timing and scenarios for implementing the algorithm.

Assessing the mechanism of fast-cycling cancer-associated mutations of Rac1 small Rho GTPase.

Rho-GTPases proteins function as molecular switches alternating from an active to an inactive state upon Guanosine triphosphate (GTP) binding and hydrolysis to Guanosine diphosphate (GDP). Among them, Rac subfamily regulates cell dynamics, being overexpressed in distinct cancer types. Notably, these proteins are object of frequent cancer-associated mutations at Pro29 (P29S, P29L, and P29Q). To assess the impact of these mutations on Rac1 structure and function, we performed extensive all-atom molecular dynamics simulations on wild-type (wt) and oncogenic isoforms of this protein in GDP- and GTP-bound states. Our results unprecedentedly elucidate that P29Q/S-induced structural and dynamical perturbations of Rac1 core domain weaken the binding of the catalytic site Mg2+ ion, and reduce the GDP residence time within protein, enhancing the GDP/GTP exchange rate and Rac1 activity. This broadens our knowledge of the role of cancer-associated mutations on small GTPases mechanism supplying valuable information for future drug discovery efforts targeting specific Rac1 isoforms.

No safe haven: Loss of avirulence in the plant pathogen Leptosphaeria maculans by DNA duplication and repeat‐induced point mutation

AbstractMicrobes can overcome the ability of plant resistance genes to confer protection against disease by mutating their corresponding avirulence genes. The fungus Leptosphaeria maculans causes blackleg disease on canola crops and subverts Brassica napus resistance genes through several DNA mutation mechanisms. One of these is repeat‐induced point (RIP) mutation, which can ‘leak’ into the avirulence genes from the adjacent repetitive sequences that the mutation process is targeting. Here, we identified populations of L. maculans in Australia that have extensive RIP mutations in the avirulence gene AvrLm2 and show that this has been triggered by a duplication of the gene and surrounding DNA that includes the distant (>55 kb in total) AvrLm6 gene. This finding provides another mechanism of mutation by which pathogens can overcome host resistance, and more broadly contributes to understanding the complex balance between gene duplication and genome defence.

Enhancing colistin efficacy against Salmonella infections with a quinazoline-based dual therapeutic strategy

Colistin remains one of the last-resort therapies for combating infections caused by multidrug-resistant (MDR) Enterobacterales, despite its adverse nephro- and neuro-toxic effects. This study elucidates the mechanism of action of a non-antibiotic 4-anilinoquinazoline-based compound that synergistically enhances the effectiveness of colistin against Salmonella enterica. The quinazoline sensitizes Salmonella by deactivating intrinsic, mutational, and transferable resistance mechanisms that enable Salmonella to counteract the antibiotic impact colistin, together with an induced disruption to the electrochemical balance of the bacterial membrane. The attenuation of colistin resistance via the combined treatment approach also proves efficacious against E. coli, Klebsiella, and Acinetobacter strains. The dual therapy reduces the mortality of Galleria mellonella larvae undergoing a systemic Salmonella infection when compared to individual drug treatments. Overall, our findings unveil the potential of the quinazoline-colistin combined therapy as an innovative strategy against MDR bacteria.

Identification of mutations in the CYP19 gene in patients with polycystic ovary syndrome in Thi-Qar city

This study was conducted to diagnose the possible relationship between genetic variations in the CYP19 gene with the progression of polycystic ovary disease in Thi- Qar city. This study included ten samples, and a genetic fragment with a length of 202 base pairs was amplified in the 3′ untranslatable region (3′UTR) of the CYP19 gene. These samples were subjected to experiments to determine the sequence from both directions of the amplified fragment. Sequence identification experiments found that there was one previously undiagnosed point mutation, g.27933770C>G, while these experiments found that there were two previously known point mutations, that is, previously discovered in the same amplified fragment, namely rs1201251894 and rs1163221780. Of the total of three-point mutations discovered, the current results indicate the presence of two of those point mutations observed in all the study samples, namely the new g.27933770C>G and the previously known rs1201251894, while the mutation rs1163221780 was observed in only three samples, which are samples No. 3 and 5 and 10. As a result, this observation may contribute to revealing a specific intervention of these discovered mutations, or one of them, in the development of polycystic ovary disease in Thi -Qar. This research is the first to describe the presence of an unprecedented point mutation distributed in all samples subject to sequencing experiments for the CYP19 genetic segment in all patients with polycystic ovary syndrome. This study recommends investigating the pattern and mechanisms of this unprecedented mutation in its possible relationship with polycystic ovary syndrome within this geographical region.

A 76-base pair duplication within the enhancer region of the HMX1 gene causes sheep microtia

Sheep congenital microtia is characterized by underdeveloped ears and provides an ideal basis for studying human microtia. This study identified the causal mutation and regulatory mechanisms underlying this disorder. Whole-genome association analysis was conducted using 23 ear tissue samples from sheep with microtia and 28 samples from normal-eared sheep. A significant correlation was found between microtia and a 76-base pair duplication in the enhancer region of the HMX1 gene. Further analysis of offspring phenotypes confirmed an autosomal dominant inheritance pattern. Genotypic analysis showed that individuals that are homozygous for this duplication were earless, heterozygous individuals exhibited shortened ears, and wild-type individuals had normal ears. Moreover, luciferase assays confirmed that this duplication increased HMX1 gene expression, and duplication knock-in mice also exhibited shorter and narrower external ears compared to wild-type mice. Transcriptomic analysis further demonstrated that this duplication enhanced HMX1 gene expression in animal models. This study characterized the causal regulatory mutation underlying sheep microtia.

Mechanisms of Nitrosamine Mutagenicity and Their Relationship to Rodent Carcinogenic Potency.

A thorough literature review was undertaken to understand how the pathways of N-nitrosamine transformation relate to mutagenic potential and carcinogenic potency in rodents. Empirical and computational evidence indicates that a common radical intermediate is created by CYP-mediated hydrogen abstraction at the α-carbon; it is responsible for both activation, leading to the formation of DNA-reactive diazonium species, and deactivation by denitrosation. There are competing sites of CYP metabolism (e.g., β-carbon), and other reactive species can form following initial bioactivation, although these alternative pathways tend to decrease rather than enhance carcinogenic potency. The activation pathway, oxidative dealkylation, is a common reaction in drug metabolism and evidence indicates that the carbonyl byproduct, e.g., formaldehyde, does not contribute to the toxic properties of N-nitrosamines. Nitric oxide (NO), a side product of denitrosation, can similarly be discounted as an enhancer of N-nitrosamine toxicity based on carcinogenicity data for substances that act as NO-donors. However, not all N-nitrosamines are potent rodent carcinogens. In a significant number of cases, there is a potency overlap with non-N-nitrosamine carcinogens that are not in the Cohort of Concern (CoC; high-potency rodent carcinogens comprising aflatoxin-like-, N-nitroso-, and alkyl-azoxy compounds), while other N-nitrosamines are devoid of carcinogenic potential. In this context, mutagenicity is a useful surrogate for carcinogenicity, as proposed in the ICH M7 (R2) (2023) guidance. Thus, in the safety assessment and control of N-nitrosamines in medicines, it is important to understand those complementary attributes of mechanisms of mutagenicity and structure-activity relationships that translate to elevated potency versus those which are associated with a reduction in, or absence of, carcinogenic potency.

Mutation-driven resistance development in wastewater E. coli upon low-level cephalosporins: Pharmacophore contribution and novel mechanism

Cephalosporins have been widely applied in clinical and veterinary settings and detected at increasing concentrations in water environments. They potentially induce high-level antibiotic resistance at environmental concentrations. This study characterized how typical wastewater bacteria developed heritable antibiotic resistance under exposure to different cephalosporins, including pharmacophore-resistance correlation, resistance mechanism, and occurrence of resistance-relevant mutations in different water environments. Wastewater-isolated E. coli JX1 was exposed to eight cephalosporins individually at 25 µg/L for 60 days. Multidrug resistance developed and diverse mutations arose in selected mutants, where a single mutation in ATP phosphoribosyltransferase encoding gene (hisG) resulted in up to 128-fold increase in resistance to meropenem. Molprint2D pharma RQSAR analysis revealed that hydrogen-bond acceptors and hydrophobic groups in the R1 and R2 substituents of cephalosporins contributed positively to antibiotic resistance. Some of these pharmacophores may persist during bio- or photo-degradation in the environment. hisG mutation confers a novel resistance mechanism by inhibiting fatty acid degradation, and its variants were more abundant in water-related E. coli (especially in the effluent of wastewater treatment plants) compared with those in non-water environments. These results suggest that specific degradation of particular pharmacophores in cephalosporins could be useful for controlling resistance development, and mutations in previously unreported resistance genes (e.g., hisG) can lead to overlooked antibiotic resistance risks in water environments.

Abstract A013: Cell-extrinsic mechanisms of accelerated therapy resistance mediated by altered pharmacokinetics

Abstract Background: Targeted therapies directed against oncogenic signaling addictions, including one resulting from EML4-ALK fusions, can induce strong and durable clinical responses. However, some tumors do not respond (intrinsic resistance), while the initially responsive advanced cancers eventually relapse (acquired resistance). Our earlier studies with experimental models of ALK+ lung cancer revealed that acquired resistance to ALK inhibitors represents a gradual, multifactorial adaptation that progresses through multiple intermediate stages. These evolutionary intermediates can display unique collateral sensitivities to pharmacological inhibitors, including high sensitivity to a KDM histone demethylase inhibitor JIB-04. Results: To explore the utility of using JIB04 to suppress the evolution of resistance in vivo, we examined its ability to enhance responses to clinical ALKi alectinib and lorlatinib in mouse xenograft models. Surprisingly, JIB04 blunted the in vivo tumor responses and accelerated their relapse. The discrepancy between the in vitro and in vivo effects of JIB04 implied environmental mediation. Therefore, we examined the potential involvement of several mechanisms of environment-mediated resistance. We found that rather than directly decreasing cell-intrinsic therapy sensitivity, JIB04 altered ALKi pharmacokinetics. Using mass spectrometry measurements, we found a strong reduction in systemic and intratumor levels of the ALKi. This reduction is attributable to an accelerated metabolic conversion and excretion of metabolites. Our ongoing and planned efforts focus on the elucidation of mechanistic details and the exploration of clinical correlation. Significance: Both intrinsic and acquired resistance are assumed to reflect reduced sensitivities of tumor cells due to mutational, plasticity-related, and microenvironmental mechanisms. Pharmacokinetics studies are the essential component of drug development and clinical trial pipelines. However, after clinical approval, drug concentrations are not routinely examined in clinics. Whereas JIB04 is not used in clinics, its effects are mediated by mechanisms that might be triggered by more relevant dietary or pharmacological factors. Our results highlight the potential danger of blind spots in consideration of causes of therapy resistance and warrant the inclusion of pharmacokinetics analyses in diagnosing/treating therapy resistance. Citation Format: Pragya Kumar, Robert Vander Velde, Virginia Turati, Andriy Marusyk. Cell-extrinsic mechanisms of accelerated therapy resistance mediated by altered pharmacokinetics [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Cancer Evolution and Data Science: The Next Frontier; 2023 Dec 3-6; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_2):Abstract nr A013.

Mutational mechanisms related to sleep disturbance in patients with SYNGAP1–associated syndrome

Mutational mechanisms related to sleep disturbance in patients with SYNGAP1–associated syndrome

Effects of parental age and polymer composition on short tandem repeat de novo mutation rates.

Short tandem repeats (STRs) are hotspots of genomic variability in the human germline because of their high mutation rates, which have long been attributed largely to polymerase slippage during DNA replication. This model suggests that STR mutation rates should scale linearly with a father's age, as progenitor cells continually divide after puberty. In contrast, it suggests that STR mutation rates should not scale with a mother's age at her child's conception, since oocytes spend a mother's reproductive years arrested in meiosis II and undergo a fixed number of cell divisions that are independent of the age at ovulation. Yet, mirroring recent findings, we find that STR mutation rates covary with paternal and maternal age, implying that some STR mutations are caused by DNA damage in quiescent cells rather than polymerase slippage in replicating progenitor cells. These results echo the recent finding that DNA damage in oocytes is a significant source of de novo single nucleotide variants and corroborate evidence of STR expansion in postmitotic cells. However, we find that the maternal age effect is not confined to known hotspots of oocyte mutagenesis, nor are postzygotic mutations likely to contribute significantly. STR nucleotide composition demonstrates divergent effects on de novo mutation (DNM) rates between sexes. Unlike the paternal lineage, maternally derived DNMs at A/T STRs display a significantly greater association with maternal age than DNMs at G/C-containing STRs. These observations may suggest the mechanism and developmental timing of certain STR mutations and contradict prior attribution of replication slippage as the primary mechanism of STR mutagenesis.