Mutational Analysis Research Articles

Genetic etiological spectrum of sperm morphological abnormalities.

Male infertility manifests in the form of a reduction in sperm count, sperm motility, or the loss of fertilizing ability. While the loss of sperm production can have mixed reasons, sperm structural defects, cumulatively known as teratozoospermia, have predominantly genetic bases. The aim of the present review is to undertake a comprehensive analysis of the genetic mutations leading to sperm morphological deformities/teratozoospermia. We undertook literature review for genes involved in sperm morphological abnormalities. The genes were classified according to the type of sperm defects they cause and on the basis of the level of evidence determined by the number of human studies and the availability of a mouse knockout. Mutations in the SUN5, CEP112, BRDT, DNAH6, PMFBP1, TSGA10, and SPATA20 genes result in acephalic sperm; mutations in the DPY19L2, SPATA16, PICK1, CCNB3, CHPT1, PIWIL4, and TDRD9 genes cause globozoospermia; mutations in the AURKC gene cause macrozoospermia; mutations in the WDR12 gene cause tapered sperm head; mutations in the RNF220 and ADCY10 genes result in small sperm head; mutations in the AMZ2 gene lead to vacuolated head formation; mutations in the CC2D1B and KIAA1210 genes lead to pyriform head formation; mutations in the SEPT14, ZPBP1, FBXO43, ZCWPW1, KATNAL2, PNLDC1, and CCIN genes cause amorphous head; mutations in the SEPT12, RBMX, and ACTL7A genescause deformed acrosome formation; mutations in the DNAH1, DNAH2, DNAH6, DNAH17, FSIP2, CFAP43, AK7, CHAP251, CFAP65, ARMC2 and several other genes result in multiple morphological abnormalities of sperm flagella (MMAF). Altogether, mutations in 31 genes have been reported to cause head defects and mutations in 62 genes are known to cause sperm tail defects.

Spatial Dynamics of T and B Cell Response Predicts Clinical Outcome of Resectable and Unresectable Hepatocellular Carcinoma.

Immunotherapies have led to a paradigm shift in the treatment of hepatocellular carcinoma (HCC). Studies have revealed the single-cell catalogues of tumor-infiltrating immune cells and the trajectories of their differentiation. Nevertheless, the spatial distribution of these immune cells with distinct phenotypes in tumor microenvironment and their clinicopathological significance in resectable and unresectable HCCs are still largely unclear. We analyzed the spatial dynamics of intratumoral CD4 and CD8 T cells and their association with B and plasma cells using 283 surgically resected HCCs, 58 unresectable HCC samples before combined immunotherapy (atezolizumab plus bevacizumab [Atezo+Bev]), and autopsy specimens from 50 cases of advanced-stage HCCs through multiplex immunohistochemistry combined with transcriptomic and driver gene mutation analysis. Classification based on the spatial dynamics of T and B cell responses (refined immunosubtype) was developed and its clinicopathological significance was analyzed. We found that stem-like CD4 and CD8 T cells were mainly observed in T cell aggregates. Differentiation of T follicular helper cells were associated with the development of tertiary lymphoid structures, whereas differentiation of CD4 TCXCL13 cells with phenotype resembling T peripheral helper cells were associated with the development of lymphoplasmacytic microenvironment. The refined immunosubtype could predict clinical outcomes of resectable HCC after surgery and unresectable HCC after Atezo+Bev therapy. The immune microenvironment of metastatic lesions tended to reflect those of primary lesions. We revealed the spatial dynamics of T and B cell response in HCC, which is closely associated with the clinical outcome after surgical resection or Atezo+Bev therapy.

Phosphorylation regulates the chromatin remodeler SMARCAD1 in nucleosome binding, ATP hydrolysis, and histone exchange

Maintaining the dynamic structure of chromatin is critical for regulating the cellular processes that require access to the DNA template, such as DNA damage repair, transcription, and replication. Histone chaperones and ATP-dependent chromatin remodeling factors facilitate transitions in chromatin structure by assembling and positioning nucleosomes through a variety of enzymatic activities. SMARCAD1 is a unique chromatin remodeler that combines the ATP-dependent ability to exchange histones, with the chaperone-like activity of nucleosome deposition. We have shown previously that phosphorylated SMARCAD1 exhibits reduced binding to nucleosomes. However, it is unknown how phosphorylation affects SMARCAD1's ability to perform its various enzymatic activities. Here we use mutational analysis, activity assays, and mass spectrometry, to probe SMARCAD1 regulation and to investigate the role of its flexible N-terminal region. We show that phosphorylation affects SMARCAD1 binding to nucleosomes, DNA, and histones H2A-H2B as well as ATP hydrolysis and histone exchange. Conversely, we report only a marginal effect of phosphorylation for histone H3-H4 binding and nucleosome assembly. In addition, the SMARCAD1 N-terminal region is revealed to be critical for nucleosome assembly and histone exchange. Together, this work examines the intricacies of how phosphorylation governs SMARCAD1 activity and provides insight into its complex regulation and diverse activities.

TP53 mutations and survival in ovarian carcinoma patients receiving first-line chemotherapy plus bevacizumab: Results of the MITO16A/MaNGO OV-2 study.

To date, there are no biomarkers that define a patient subpopulation responsive to bevacizumab (BEV), an effective treatment option for advanced ovarian carcinoma (OC). In the context of the MITO16A/MaNGO OV-2 trial, a Phase IV study of chemotherapy combined with BEV in first-line treatment of advanced OC, we evaluated TP53 mutations by next-generation sequencing and p53 expression by immunohistochemistry (IHC) on 202 and 311 cases, respectively. We further correlated TP53 mutations in terms of type, function, and site, and IHC data with patients' clinicopathological characteristics and survival. TP53 missense mutations of unknown function (named unclassified) represented the majority of variants in our population (44.4%) and were associated with a significantly improved overall survival (OS) both in univariable (hazard ratio [HR] = 0.43, 95% confidence interval [CI] = 0.20-0.92, p = .03) and multivariable analysis (HR = 0.39, 95% CI = 0.18-0.86, p = .02). Concordance between TP53 mutational analysis and IHC was 91%. We observed an HR of 0.70 for OS in patients with p53 IHC overexpression compared to p53 wild-type, which however did not reach statistical significance (p = .31, 95% CI = 0.36-1.38). Our results indicate that the presence of unclassified TP53 mutations has favorable prognostic significance in patients with OC receiving upfront BEV plus chemotherapy. In particular, unclassified missense TP53 mutations characterize a subpopulation of patients with a significant survival advantage, independently of clinicopathological characteristics. Our findings warrant future investigations to confirm the prognostic impact of TP53 mutations in BEV-treated OC patients and deserve to be assessed for their potential predictive role in future randomized clinical studies.

Familial adenomatous polyposis gene MUTYH mutations are identified in anaplastic thyroid carcinoma

Abstract Introduction/Objective Anaplastic thyroid carcinoma (ATC) is a rare aggressive disease unresponsive to treatment. Next-Generation Sequencing (NGS) allows identification of targets for precision therapies, such as BRAF, with implications in treating ATC. Our prior work identified ATC clusters with characteristic gene mutations and multiple previously unreported mutations. One of these is MUTYH, which is associated with colon (CRC), ovarian, breast, bladder, and duodenal carcinomas in MUTYH-associated polyposis (MAP). Its association with ATC is not defined. Methods/Case Report Exploratory analysis of pathogenic mutations in 727 ATC cases using a 324-gene panel (FoundationOneCDx) and an interactive visualization of association rules was conducted for informative maximal frequent patterns among mutated gene combinations to identify gene clusters including genes not commonly associated with ATC. TCGA database was reviewed for frequency of MUTYH mutations. My Cancer Genome and the Drug-Gene Interaction Database were used to identify potential targeted drugs for MUTYH gene-mutated cancers. Results (if a Case Study enter NA) NGS showed MUTYH short-variant mutations in 17 cases (2.34%). Germline MUTYH variants incidence is 1.43% in normal samples. 5 cases harbored only Y165C and 7 had only G382D, with 1 case having both Y165C and G382D. The other 4 cases contained sporadic truncating events in MUTYH. All instances of Y165C and G382D were predicted to be germline. 6 patients (35%) had BRAF co-mutations, correlating with expected frequency of ATC thought to transform from papillary carcinoma (PC). One case had only MUTYH mutation while a second had MUTYH with TERT and TP53. TCGA database contained 6(0.2%) cases of PC with MUTYH mutations (total 2871 MUTYH-mutated carcinomas), but no cases of ATC or other thyroid tumors were described. Conclusion MUTYH is a DNA repair enzyme linked to CRC and other cancers. MUTYH mutations occur in 6.7% of MAP patients and 1–2.2% of sporadic CRC cases that are mutually exclusive of BRAF mutations. While MAP increases the lifetime risk of CRC by over 60%, the link of MUTYH mutations to PC has only been observed in MAP and not been described outside of it. To our knowledge this is the first description of MUTYH mutations in ATC, especially without concurrent BRAF mutation (11 cases), suggesting MUTYH may independently contribute to pathogenesis of ATC. PARP inhibitors, PD-1/PD-L1 inhibitors, and ATR inhibitors demonstrate promising results in clinical trials of MUTYH-mutated cancers, warranting further research.

NANOPORE-BASED INTRA-OPERATIVE CLASSIfiCATION OF CENTRAL NERVOUS SYSTEM TUMOURS IN THE NHS

Abstract AIMS The 2021 WHO classification of CNS tumours necessitates the use of molecular testing in order to reach a confident diagnosis of many tumour types. At present, this is achieved using array-based technology delivered by a centralised model. This approach has inherent delays that mean it is often several weeks before the treating clinical team is informed of diagnosis, delaying the start of adjuvant therapy and causing significant distress for patients. As such, there is an urgent need to rethink this pathway and improve time to diagnosis. Nanopore longread sequencing technology has the potential to enable a paradigm shift in diagnostic and therapeutic pathways, offering rapid, comprehensive molecular diagnosis that can be performed in the intra-operative setting. We aimed to test the hypothesis that intra-operative methylation-based classification can be performed using nanopore sequencing and thereby expediently inform surgical and oncological decision-making. METHOD Oxford Nanopore Technology (ONT) is a native-strand, long-read sequencing platform that can perform copy- number profiling along with parallel single-gene methylation, structural variant, mutational and methylation analyses. Capital and consumable costs are relatively low, and testing can be performed in the local setting on single samples. We have optimised DNA extraction and library preparation for rapid tissue processing. We have developed a robust bioinformatic pipeline which is able to produce intraoperative classification within minutes of sequencing and full molecular profiling after 24 hours. RESULTS We demonstrate the feasibility of using ONT to deliver intra-operative methylation-based classification of CNS tumours in ‘realtime’ within an NHS hospital setting and will discuss the challenges of implementing this testing and the opportunities to inform neuro-oncological practice. CONCLUSION By giving a summary of our novel protocol of rapid DNA extraction, library preparation and bioinformatic analysis, we will show that this approach can revolutionise the diagnosis of brain tumours and has the potential for rapid adoption by non-expert laboratories.

Effect of C-to-T transition at CpG sites on tumor suppressor genes in tumor development in cattle evaluated by somatic mutation analysis in enzootic bovine leukosis.

Oncogenic transformation of normal cells is caused by mutations and chromosomal abnormalities in cancer-related genes. Enzootic bovine leukosis (EBL) is a malignant B-cell lymphoma caused by bovine leukemia virus (BLV) infection in cattle. Although a small fraction of BLV-infected cattle develops EBL after a long latent period, the mechanisms for oncogenesis in EBL cattle remain largely unknown. In this study, we analyzed the types and patterns of somatic mutations in cancer cells from 36 EBL cases, targeting 21 cancer-related genes. Various somatic mutations were identified in eight genes, TP53, KMT2D, CREBBP, KRAS, PTEN, NOTCH1, MYD88, and CARD11. In addition, TP53 gene was found to be mutated in 69.4% of EBL cases, with most being biallelic mutations. In some cases, associations were observed between the ages at which cattle had developed EBL and somatic mutation patterns; young onset of EBL possibly occurs due to high impact mutations affecting protein translation and biallelic mutations. Furthermore, nucleotide substitution patterns indicated that cytosine at CpG sites tended to be converted to thymine in many EBL cases, which was considered to be the result of spontaneous deamination of 5-methylcytosine. These results demonstrate how somatic mutations have occurred in cancer cells leading to EBL development, thereby explaining its pathogenic mechanism. These findings will contribute to a better understanding and future elucidation of disease progression in BLV infection.IMPORTANCEEnzootic bovine leukosis (EBL) is a malignant and lethal disease in cattle. Currently, there are no effective vaccines or therapeutic methods against bovine leukemia virus (BLV) infection, resulting in severe economic losses in livestock industry. This study provides a renewed hypothesis to explain the general mechanisms of EBL onset by combining the previous finding that several integration sites of BLV provirus can affect the increase in survival and proliferation of infected cells. We demonstrate that two additional random events are necessary for oncogenic transformation in infected cell clones, elucidating the reason why only few infected cattle develop EBL. Further exploration of somatic mutation and BLV integration sites could support this hypothesis more firmly, potentially contributing to the development of novel control methods for EBL onset.

Report of 3 Cases Diagnosed with Basal Cell Carcinoma and Systemic Mastocytosis with Favorable Prognosis

Abstract Introduction/Objective The co-existence of basal cell carcinoma (BCC) and systemic mastocytosis is an exceptionally rare clinical occurrence, and the prognosis is not well-documented. Understanding the clinical and histopathological features of this uncommon association is relevant for accurate diagnosis, management, and prognostication. Methods/Case Report We conducted a retrospective analysis of three cases of BCC with concurrent systemic mastocytosis that were identified in the pathology database of the University of Pennsylvania from 2010 to 2023. Patient demographics, clinical and histopathologic characteristics, and treatment outcomes were reviewed. Histopathologic assessment, immunohistochemistry, and C-KIT D816V mutation analysis (with an allele-specific PCR assay) were performed on biopsy material. Next-Generation Sequencing (NGS) was used to identify other hematologic neoplasm-associated mutations. Results (if a Case Study enter NA) All three cases were female with a median age of 55.3 years (50, 69, 47). In all three cases, atypical CD117+ tryptase+ CD25+ mast cells with mild to moderate pleomorphism were found in the dermis adjacent to the BCC cells. All three cases had bone marrow involvement. Serum tryptase levels were mildly elevated in all 3 patients (maximum 65.5 μg/L). All three cases harbor a C-KIT D816V mutation and a history of systemic glucocorticosteroids before the diagnosis of BCC. One of the cases was treated with phototherapy. NGS detected the following somatic mutations: Case #2 DNMT3A p.R882H c.2645G>A; Case #3 JAK2 V617F. Case #1 had multiple relapses of BCC, but a subsequent marrow biopsy did not identify any involvement by mastocytosis. Case #2 had mast cell leukemia, which typically has a poor prognosis; however, this patient has survived for several years. Case #3 had essential thrombocythemia and systemic mastocytosis with an associated hematological neoplasm, and her symptoms were stable. Treatment modalities for the three patients ranged from surgical excision to anti-mastocytosis therapy, with favorable outcomes (Table 1). Conclusion The coexistence of BCC with mastocytosis poses diagnostic challenges due to its infrequency and varied clinical presentations. Previous literature reports on treatments of mastocytosis such as phototherapy and steroids, which have been identified as risk factors for the development of basal cell carcinoma. However, recognizing this unique combination in clinical practice may aid in providing appropriate treatment strategies, ultimately leading to favorable patient outcomes.

Recent Advances in the Molecular Biology of Chronic Lymphocytic Leukemia: How to Define Prognosis and Guide Treatment

Chronic Lymphocytic Leukemia (CLL) is the most frequent type of leukemia in Western countries. In recent years, there have been important advances in the knowledge of molecular alterations that underlie the disease’s pathogenesis. Very heterogeneous prognostic subgroups have been identified by the mutational status of immunoglobulin heavy variable genes (IGVH), FISH analysis and molecular evaluation of TP53 mutations. Next-generation sequencing (NGS) technologies have provided a deeper characterization of the genomic and epigenomic landscape of CLL. New therapeutic targets have led to a progressive reduction of traditional chemoimmunotherapy in favor of specific biological agents. Furthermore, in the latest clinical trials, the minimal residual disease (MRD) has emerged as a potent marker of outcome and a guide to treatment duration. This review focuses on recent insights into the understanding of CLL biology. We also consider the translation of these findings into the development of risk-adapted and targeted therapeutic approaches.

Genome sequencing analysis of the pncA, rpsA and panD genes responsible for pyrazinamide resistance of Mycobacterium tuberculosis from Indonesian isolates.

Developing the most suitable treatment against tuberculosis based on resistance profiles is imperative to effectively cure tuberculosis patients. Whole-genome sequencing is a molecular method that allows for the rapid and cost-effective detection of mutations in multiple genes associated with anti-tuberculosis drug resistance. This sequencing approach addresses the limitations of culture-based methods, which may not apply to certain anti-TB drugs, such as pyrazinamide, because of their specific culture medium requirements, potentially leading to biased resistance culture results. Thirty-four M. tuberculosis isolates were subcultured on a Lowenstein-Jensen medium. The genome of these bacteria was subsequently isolated using cetyltrimethylammonium bromide. Genome sequencing was performed with Novaseq Illumina 6000 (Illumina), and the data were analysed using the GenTB and Mykrobe applications. We also conducted a de novo analysis to compare the two methods and performed mutation analysis of other genes encoding pyrazinamide resistance, namely rpsA and panD. The results revealed mutations in the pncA gene, which were identified based on the databases accessed through GenTB and Mykrobe. Two discrepancies between the drug susceptibility testing and sequencing results may suggest potential instability in the drug susceptibility testing culture, specifically concerning PZA. Meanwhile, the results of the de novo analysis showed the same result of pncA mutation to the GenTB or Mykrobe; meanwhile, there were silent mutations in rpsA in several isolates and a point mutation; no mutations were found in the panD gene. However, the mutations in the genes encoding pyrazinamide require further and in-depth study to understand their relationship to the phenotypic profile. Compared to the conventional culture method, the whole-genome sequencing method has advantages in determining anti-tuberculosis resistance profiles, especially in reduced time and bias.

Analysis of low-density lipoprotein receptor gene mutations in a family with familial hypercholesterolemia.

Familial hypercholesterolemia (FH) is a common monogenic autosomal dominant disorder, primarily mainly caused by pathogenic mutations in the low-density lipoprotein receptor (LDLR) gene. Through phenotypic-genetic linkage analysis, two LDLR pathogenic mutations were identified in FH families: c.G1027A (p.Gly343Ser) and c.G1879A (p.Ala627Thr). Whole exome sequencing was conducted on the proband with familial hypercholesterolemia to identify the target gene and screen for potential pathogenic mutations. The suspicious responsible mutation sites in 14 family members were analyzed using Sanger sequencing to assess genotype-phenotype correlations. Mutant and wild type plasmids were constructed and transfected into HEK293T cells to evaluate LDLR mRNA and protein expression. In parallel, bioinformatics tools were employed to predict structural and functional changes in the mutant LDLR. Immunofluorescence analysis revealed no significant difference in the intracellular localization of the p.Gly343Ser mutation, whereas protein expression of the p.Ala627Thr mutation was decreased and predominantly localized in the cytoplasm. Western blotting has showed that protein expression levels of the mutant variants were markedly declined in both cell lysates and supernatants. Enzyme linked immunosorbent assay has demonstrated that LDLR protein levels in the supernatant of cell culture medium was not significant different from those of the wild-type group. However, LDLR protein levels in the cell lysate of both the Gly343Ser and Ala627Thr variants groups were significantly lower than those in the wild-type group. Bioinformatic predictions further suggested that these mutations may affect post-translational modifications of the protein, providing additional insight into the mechanisms underlying the observed reduction in protein expression. In this study, we identified two heterozygous pathogenic variants in the LDLR gene, c.G1027A (p.Gly343Ser) and c.G1879A (p.Ala627Thr), in a family with familial hypercholesterolemia. We also conducted preliminary investigations into the mechanisms by which these mutations contribute to disease pathology.

Are Ameloblastic Fibroma-related Lesions True Tumors?: Evidence Through CNA and BRAF Mutation Analysis.

Ameloblastic fibroma (AF) and related lesions, namely ameloblastic fibrodentinoma (AFD) and ameloblastic fibro-odontoma (AFO), span a spectrum from true neoplasms to hamartomas. The 2017 World Health Organization classification proposes that AFD and AFO are precursors to odontomas, yet their precise nature remains uncertain. This study examined 19 AF cases, 4 AFD, 15 AFO, 19 odontomas (OD, 14 complex, 5 compound), and 2 ameloblastic fibrosarcomas (AFS), focusing on clinical characteristics, recurrence, and molecular profiles. AF primarily affected individuals under 20 years (60.0% of cases), mainly in the mandible (68.4%), with a recurrence rate of 21.1% in the followed cases. AFD and AFO appeared in younger patients (average age 15.7y) without any recurrence observed. Notable differences in site and size distribution were observed between AF, its related lesions, and odontomas. Copy number alterations (CNAs) were detected in the mesenchymal component in 9 of 19 AF (47.4%), 2 of 4 AFD (50.0%), 6 of 14 AFO (42.9%), and 2 of 2 AFS (100%). In contrast, all odontomas exhibited normal CNAs, highlighting the specificity of CNAs in mesenchymal elements of AF and related lesions. BRAF p.V600E mutation was identified in the mesenchymal component in 13 of 19 AF (68.4%), 2 of 4 AFD (50.0%), 8 of 15 AFO (53.3%), and 2 of 2 AFS (100%), whereas all 19 odontomas were BRAF wild type. No mutations were found in the epithelial component. Our analysis reveals that AF and its related lesions present a spectrum of biological behaviors, from true neoplasms to hamartomas. The presence of BRAF p.V600E mutations and CNAs in their mesenchymal components, as opposed to odontomas, indicates potential neoplastic characteristics. Profiling copy number alterations in AF and related lesions emerge as a valuable tool for enhancing their differential diagnosis and facilitating the anticipation of disease progression. Our findings underscore the efficacy of copy number alteration analysis in determining the nature of lesions within AF and related lesions.

Simultaneous co-circulation of two genotypes of dengue virus serotype 3 causing a large outbreak in Sri Lanka in year 2023.

We observed a discrepancy between dengue NS1 antigen test and molecular diagnostics, with the emergence of (DENV) serotype 3 in Sri Lanka and sought to understand the cause for the rise in cases and high failure rates of molecular diagnostics. Whole genomic sequencing was carried out in 22 DENV-3 samples. Phylogenetic and molecular clock analysis were done for genotype assignment and to understand the rate of evolution. Mutation analysis was done to understand the reasons for PCR non-detection. We identified two DENV-3 genotypes (I and III) co-circulating. DENV-3 genotype III strains shared a common ancestor with a sequence from India collected in 2022, while DENV-3 genotype I, was found to share a common ancestor with DENV-3 sequences from China. DENV-3 genotype III was detected by the modified CDC DENV-3 primers whereas, genotype I evaded detection due to key mutations at forward and reverse primer binding sites. We identified point mutations, C744T and A756G of the forward primer binding sites and in position G795A of the reverse primer binding sites which were not identified in DENV-3 genotype III. Furthermore, our Sri Lankan DENV-3 strains demonstrated a high root to tip ratio, compared to the previous DENV-3 sequences, indicating a high mutation rate during the points of sampling (year 2017 to 2023). The co-circulation of multiple genotypes associated with an increase in cases highlights the importance of continuous surveillance of DENVs to identify mutations resulting in non-detection by diagnostics and differences in virulence.

The distinct roles of NEIL1 and XPA in limiting aflatoxin B₁-induced mutagenesis in mice.

Dietary exposure to aflatoxin B₁ (AFB₁) is a risk factor for the development of hepatocellular carcinomas (HCCs). Following metabolic activation, AFB₁ reacts with guanines to form covalent DNA adducts, which induce high-frequency G > T transversions. The molecular signature associated with these mutational events aligns with the single base substitution signature 24 (SBS24) in the Catalog of Somatic Mutations in Cancer (COSMIC) database. Deficiencies in either base excision repair (BER) due to the absence of Nei-like DNA glycosylase 1 (NEIL1) or nucleotide excision repair (NER) due to the absence of xeroderma complementation group A protein (XPA) contribute to HCCs in murine models. In the current study, ultra-low error duplex sequencing was used to characterize mutational profiles in liver DNAs of NEIL1-deficient, XPA-deficient, and DNA repair-proficient mice following neonatal injection of 1 mg/kg AFB₁. Analyses of AFB₁-induced mutations showed high cosine similarity to SBS24, regardless of repair proficiency status. The absence of NEIL1 resulted in an approximately 30% increase in the frequency of mutations, with distribution suggesting preferential NEIL1-dependent repair of AFB₁ lesions in open chromatin regions. A trend of increased mutagenesis was also observed in the absence of XPA. Consistent with the role of XPA in transcription-coupled repair, mutational profiles in XPA-deficient mice showed disruption of the transcriptional bias in mutations associated with SBS24. Implications: Our findings define the roles of DNA repair pathways in AFB₁-induced mutagenesis and carcinogenesis in murine models, with these findings having implications in human health for those with BER and NER deficiencies.

KLF14 directly downregulates the expression of GPX4 to exert antitumor effects by promoting ferroptosis in cervical cancer

BackgroundCervical cancer is the fourth leading cause of cancer-related death among women worldwide, and effective therapeutic strategies for its treatment are limited. Recent studies have indicated that ferroptosis, a form of regulated cell death, is a promising therapeutic strategy. KLF14 has been shown to regulate both cell proliferation and apoptosis in cervical cancer. However, its role in modulating lipid peroxidation and ferroptosis remains largely unexplored and enigmatic.MethodsSiHa and HeLa cells were transduced with lentiviral vectors to overexpress KLF14. Protein levels were analyzed via western blotting and immunohistochemistry (IHC). LDH assays, calcein-AM/propidium iodide (PI) staining, and generation of cell growth curves using a real-time cell analysis (RTCA) system were used to detect cell damage and proliferation. Cellular ROS, lipid ROS, transmission electron microscopy (TEM), and Fe2+ assays and a xenograft mouse model were used to measure the level of ferroptosis. Proteomics combined with bioinformatics methods was used to screen target genes regulated by KLF14, and CUT&Tag and dual-luciferase assays confirmed the repression of GPX4 by KLF14 via direct binding to its promoter.ResultsKLF14 is abnormally expressed in various tumors and downregulated in cervical cancer. Overexpression of KLF14 induced ferroptosis and inhibited cell proliferation in vitro as well as xenograft tumorigenicity in vivo. Mechanistic studies revealed that KLF14 binds to the promoter of GPX4, suppressing its transcriptional activity and thereby decreasing its expression, which contributes to the induction of ferroptosis. Truncation and point mutation analyses of the GPX4 promoter revealed multiple binding sites for KLF14 within the − 1000 bp to + 35 bp region, which are responsible for its inhibitory effect on GPX4 transcription. Additionally, deletion of the zinc finger motif in KLF14 abolished its inhibitory effect on GPX4 promoter activity and cell proliferation.ConclusionOur data revealed a previously unidentified function of KLF14 in promoting ferroptosis, which results in the suppression of cell proliferation. Mechanistically, we revealed a novel regulatory mechanism by which KLF14 targets GPX4. These findings suggest a novel strategy to induce ferroptosis through the targeting of KLF14 in human cervical cancer cells.

Mutational analysis differentiating sporadic carcinomas from colitis-associated colorectal carcinomas

BackgroundUlcerative colitis (UC) is a chronic inflammatory bowel disease (IBD) that is associated with increased risk of developing colitis-associated carcinoma (CAC). The genetic profile of CACs is fairly similar to the sporadic colorectal carcinomas (sCRCs), although showing certain differences in the timing and sequence of alterations that contribute to carcinogenesis. Also, both cancer types typically show a strong histological resemblance, which complicates the pathologists’ diagnosis. Due to the different clinical consequences, it is of utmost importance to categorize the corresponding cancer type correctly.MethodsIn this study, we determined the mutation profiles of 64 CACs and sCRCs in the hotspot regions of 50 cancer-associated genes and compared them to 29 controls to identify genetic gene variants that can facilitate the pathologists’ diagnosis. Pearson Chi-Square or Fisher’s exact tests were used for statistical analyses.ResultsWe found that sCRCs tend to mutate more frequently in APC and PIK3CA genes than CACs and that mainly males were affected. Our CAC cohort identified the KRAS G12D mutation as group-specific variant that was not detected in the sCRCs. When separating conventional from non-conventional CACs, it was discovered that the conventional type shows significantly more mutations for ATM.ConclusionsTaken together, our data highlights genetic differences between sCRC and CAC and enables the possibility to utilize specific gene alterations to support the pathologist’s diagnosis.

Mutation Analysis for Hemoglobinopathies in Eastern Jharkhand

Mutation Analysis for Hemoglobinopathies in Eastern Jharkhand

Prognostic modeling of hepatocellular carcinoma based on T-cell proliferation regulators: a bioinformatics approach.

The prognostic value and immune significance of T-cell proliferation regulators (TCRs) in hepatocellular carcinoma (HCC) have not been previously reported. This study aimed to develop a new prognostic model based on TCRs in patients with HCC. This study used The Cancer Genome Atlas-Liver Hepatocellular Carcinoma (TCGA-LIHC) and International Cancer Genome Consortium-Liver Cancer-Riken, Japan (ICGC-LIRI-JP) datasets along with TCRs. Differentially expressed TCRs (DE-TCRs) were identified by intersecting TCRs and differentially expressed genes between HCC and non-cancerous samples. Prognostic genes were determined using Cox regression analysis and were used to construct a risk model for HCC. Kaplan-Meier survival analysis was performed to assess the difference in survival between high-risk and low-risk groups. Receiver operating characteristic curve was used to assess the validity of risk model, as well as for testing in the ICGC-LIRI-JP dataset. Additionally, independent prognostic factors were identified using multivariate Cox regression analysis and proportional hazards assumption, and they were used to construct a nomogram model. TCGA-LIHC dataset was subjected to tumor microenvironment analysis, drug sensitivity analysis, gene set variation analysis, and immune correlation analysis. The prognostic genes were analyzed using consensus clustering analysis, mutation analysis, copy number variation analysis, gene set enrichment analysis, and molecular prediction analysis. Among the 18 DE-TCRs, six genes (DCLRE1B, RAN, HOMER1, ADA, CDK1, and IL1RN) could predict the prognosis of HCC. A risk model that can accurately predict HCC prognosis was established based on these genes. An efficient nomogram model was also developed using clinical traits and risk scores. Immune-related analyses revealed that 39 immune checkpoints exhibited differential expression between the high-risk and low-risk groups. The rate of immunotherapy response was low in patients belonging to the high-risk group. Patients with HCC were further divided into cluster 1 and cluster 2 based on prognostic genes. Mutation analysis revealed that HOMER1 and CDK1 harbored missense mutations. DCLRE1B exhibited an increased copy number, whereas RAN exhibited a decreased copy number. The prognostic genes were significantly enriched in tryptophan metabolism pathways. This bioinformatics analysis identified six TCR genes associated with HCC prognosis that can serve as diagnostic markers and therapeutic targets for HCC.

Pan-cancer analysis of TP53 mutations and their association with liver cancer and aristolochic acids

Liver cancer is a major global health concern, as its incidence continues to rise in numerous countries, making it a leading cause of cancer-related deaths. The TP53 gene, which encodes the p53 tumor suppressor protein, plays a crucial role in regulating cell growth and division. Mutations in TP53 have been implicated in increased risk for liver cancer development. In this study, we aim to conduct a pan-cancer analysis to further investigate the potential role of TP53 mutations in various cancer types and their relationship with liver cancer. Additionally, we will explore the interplay between TP53 gene mutations, liver cancer, and exposure to Aristolochic acids (AAs). To achieve these goals, we will utilize data from The Cancer Genome Atlas (TCGA) project and the Gene Expression Omnibus (GEO) databases, enabling a comprehensive analysis of TP53 in the context of multiple cancer types.

Insecticide binding mode analysis and biological effects of acetylcholinesterase target-site resistance mutations in Spodoptera frugiperda

It is urgent to solve insecticide resistance issues for fall armyworm (FAW), Spodoptera frugiperda. Some acetylcholinesterase-1 (Ace-1) mutations (A201S, G227A and F290V) have been identified as a cause of FAW resistance to organophosphates (OPs) and carbamates insecticides (CXs). However, the structural biological mechanisms on the relationship between the Ace-1 mutations and resistance to OPs and CXs still remain elusive. In this study, the A201S and F290V mutaions were found in eight fields populations of FAW except the G227A. Molecular docking revealed that the four Ace-1 proteins (Ace1-WT, Ace1-A201S, Ace1-G227A and Ace1-F290V) had the same binding modes and the same binding energies with acetylcholine (Ach), trichlorfon, chlorpyrifos, methomyl, carbaryl and chlorpyrifos oxide. The structural biological analysis revealed that the A201S mutations can enhance enzyme catalytic efficiency by introducing the hydroxyl group (-OH) from serine which performed the same function as the main-chain -NH and enhanced the interaction with the carboxy oxygen of acetylcholine (Ach), and the F290V mutation can effectively improve FAW resistance to insecticides by increasing the likelihood of Ach to enter the enzyme's active center for phenylalanine replaced by smaller valine under insecticide inhibition conditions. The bioassays and age-stage-specific life table analysis of FAW-SS and FAW-F290V populations revealed that F290V mutation effectively contributed to FAW resistance with a low fitness cost. This study provides a theoretical basis for future pest resistance management.