Mutational Analysis Research Articles

Analyzing the role of TM4SF1 expression in pancreatic adenocarcinoma: understanding prognostic implications and therapeutic opportunities.

Pancreatic adenocarcinoma (PAAD) is a highly lethal malignancy characterized by aggressive growth and poor prognosis. Understanding the molecular mechanisms underlying PAAD is crucial for developing effective therapies. This study aimed to explore the role of TM4SF1 and other key genes in PAAD progression, their prognostic implications, and therapeutic opportunities. Differential gene expression analysis was performed using PAAD and normal tissue samples to identify upregulated genes, with TM4SF1 emerging as significantly elevated in PAAD. Functional enrichment analysis elucidated associated signaling pathways. A prognostic model comprising BPIFB4, PLEKHN1, CPTP, DVL1, and DDR1 was developed using least absolute shrinkage and selection operator (LASSO) regression and validated in an independent cohort. Genetic mutation analysis provided insights into the functional significance of identified genes. Pharmacogenomic analysis examined associations between gene expression and drug sensitivity. Experimental validation included quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blot analyses to confirm gene expression patterns and protein levels. Lower TM4SF1 expression correlated with enhanced anti-tumor immune activity in PAAD, suggesting a complex interplay between genetic expression and immune response. The prognostic model showed robust associations with patient survival outcomes, validated across diverse patient cohorts. Genetic mutation analysis highlighted potential therapeutic targets. Pharmacogenomic analysis revealed correlations between gene expression profiles and drug responsiveness, suggesting personalized treatment strategies. Experimental validation confirmed elevated TM4SF1 levels in tumor tissues and demonstrated its role in promoting cancer cell proliferation and colony formation. This study advances understanding of the molecular landscape of PAAD, emphasizing TM4SF1 as a key regulator and potential therapeutic target. The integration of genetic expression, immune response dynamics, and pharmacogenomics offers a multifaceted approach to personalized treatment strategies for PAAD, paving the way for improved patient outcomes and novel therapeutic interventions. Further research is warranted to elucidate the clinical utility of targeting TM4SF1 and other identified genes in PAAD management.

Cell-free analysis reveals the role of RG/RGG motifs in DDX3X phase separation and their potential link to cancer pathogenesis

The DEAD-box RNA helicase DDX3X is a multifunctional protein involved in RNA metabolism and stress responses. In this study, we investigated the role of RG/RGG motifs in the dynamic process of liquid-liquid phase separation (LLPS) of DDX3X using cell-free assays and explored their potential link to cancer development through bioinformatic analysis. Our results demonstrate that the number, location, and composition of RG/RGG motifs significantly influence the ability of DDX3X to undergo phase separation and form self-aggregates. Mutational analysis revealed that the spacing between RG/RGG motifs and the number of glycine residues within each motif are critical factors in determining the extent of phase separation. Furthermore, we found that DDX3X is co-expressed with the stress granule protein G3BP1 in several cancer types and can undergo co-phase separation with G3BP1 in a cell-free system, suggesting a potential functional interaction between these proteins in phase-separated structures. DDX3X and G3BP1 may interact through their RG/RGG domains and subsequently exert important cellular functions under stress situation. Collectively, our findings provide novel insights into the role of RG/RGG motifs in modulating DDX3X phase separation and their potential contribution to cancer pathogenesis.

Clinical features and genetic analysis of 5 cases of infantile-type glycogen storage disease type II: Case reports.

Clinical and genetic mutation analysis was performed on 5 infantile glycogen storage disease type II children in Chinese mainland. Clinical data of 5 children with infantile-type glycogen storage disease type II due to the acidic α-glucosidase (GAA) gene variants diagnosed and treated at Hebei Provincial Children's Hospital from January 2018 to April 2020 were retrospectively analyzed. Among the 5 cases, 1 was female and 4 were male, and the age at first diagnosis was from 2 months to 7 months. The first symptoms of all 5 cases showed progressive muscle weakness, hypotonia, and motor developmental backwardness, and all of them had abnormally elevated creatine kinase, and the echocardiograms suggested different degrees of myocardial hypertrophy, with ejection fractions ranging from 44% to 67%. Analysis of GAA gene variations: all 5 cases were compound heterozygous, and a total of 12 variant loci were detected, of which c.2024_2026delACA, c.2853G > A, c.1124G > T, c.574G > A, c.2509C > T, and c.2013G > A were new mutations that had not been reported. All 5 children died before 1 year of age, and the age of death ranged from 7 months to 11.5 months, with a mean survival time of 9.8 months. Peripheral blood GAA gene testing and alpha-glucosidase enzyme activity testing is an effective method for diagnosing this disease.

Next generation sequence-based targeted somatic mutation analysis in thyroid nodules with pathologically diagnosed as indeterminate cytology

PurposeThe management of indeterminate thyroid nodules remains a topic of ongoing debate, particularly regarding the differentiation of malignancy. Somatic mutation analysis offers crucial insights into tumor characteristics. This study aimed to assist the clinical management of indeterminate nodules with somatic mutation analysis. Methods: Aspiration samples from 20 indeterminate thyroid nodules were included in the study. A next-generation sequencing panel containing 67 genes was used for molecular profiling. The results were compared with pathology data from surgical material, which is considered the gold standard. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated. Results: Variants in six genes (NRAS, BRAF, TP53, TERT, PTEN, PIK3CA) were detected in 10 out of 20 samples. We identified nine Tier 1 or 2 variants in 10 (67 %) out of 15 malignant nodules (NRAS, BRAF, TP53, TERT, PTEN, PIK3CA) and one Tier 2 (PIK3CA) variant in one out of five benign nodules. The study demonstrated an NPV of 40 %, a PPV of 90 %, a specificity of 80 %, and a sensitivity of 60 %. Conclusion: Based on the detected molecular markers, at least nine patients (45 %) could be managed correctly without needing a repeat FNAB attempt. This study underscores the clinical practicality of molecular tests in managing nodules with indeterminate cytology. Additionally, this study emphasizes the importance of considering the patient's age when determining the DNA- or RNA-based genetic testing method. Finally, we discussed the significance of the somatic mutation profile and its impact on the current pathological classification.

Distinct clinical outcomes and biological features of specific KRAS mutants in human pancreatic cancer

KRAS mutations in pancreatic ductal adenocarcinoma (PDAC) are suggested to vary in oncogenicity but the implications for human patients have not been explored in depth. We examined 1,360 consecutive PDAC patients undergoing surgical resection and find that KRASG12R mutations are enriched in early-stage (stage I) disease, owing not to smaller tumor size but increased node-negativity. KRASG12R tumors are associated with decreased distant recurrence and improved survival as compared to KRASG12D. To understand the biological underpinnings, we performed spatial profiling of 20 patients and bulk RNA-sequencing of 100 tumors, finding enhanced oncogenic signaling and epithelial-mesenchymal transition (EMT) in KRASG12D and increased nuclear factor κB (NF-κB) signaling in KRASG12R tumors. Orthogonal studies of mouse KrasG12R PDAC organoids show decreased migration and improved survival in orthotopic models. KRAS alterations in PDAC are thus associated with distinct presentation, clinical outcomes, and biological behavior, highlighting the prognostic value of mutational analysis and the importance of articulating mutation-specific PDAC biology.

Mutational analysis reveals the importance of residues of the access tunnel inhibitor site to human P-glycoprotein (ABCB1)-mediated transport.

Human P-glycoprotein (P-gp) utilizes energy from ATP hydrolysis for the efflux of chemically dissimilar amphipathic small molecules and plays an important role in the development of resistance to chemotherapeutic agents in most cancers. Efforts to overcome drug resistance have focused on inhibiting P-gp-mediated drug efflux. Understanding the features distinguishing P-gp inhibitors from substrates is critical. Cryo-electron microscopy has revealed distinct binding patterns, emphasizing the role of the L-site or access tunnel in inhibition. We substituted 5-9 residues of the L-site with alanine to investigate whether the binding of a second inhibitor molecule to the L-site is required for inhibiting drug efflux. We reveal, for the first time, that mutations in the L-site affect the drug efflux activity of P-gp, despite their distance from the substrate-binding pocket (SBP). Surprisingly, after the mutations were introduced, inhibitors such as tariquidar and zosuquidar still inhibited drug efflux by mutant P-gps. Communication between the transmembrane helices (TMHs) and nucleotide-binding domains (NBDs) was evaluated using the ATPase assay, revealing distinct modulation patterns by inhibitors for the mutants, with zosuquidar exhibiting substrate-like stimulation of ATPase. Furthermore, L-site mutations abolished ATP-dependent thermal stabilization. In silico molecular docking studies corroborated the altered inhibitor binding due to mutations in the L-site residues, shedding light on their critical role in substrate transport and inhibitor interactions with P-gp. These findings suggest that inhibitors bind either to the SBP alone, and/or to alternate site(s) when the L-site is disabled by mutagenesis.

Real-world Comparison of P53 Immunohistochemistry and TP53 Mutation Analysis Using Next-generation Sequencing.

TP53 mutation in breast cancer (BC) is associated with chemoresistance, endocrine therapy resistance, and late recurrence, resulting in poor prognosis. Nuclear accumulation of p53 in immunohistochemistry (IHC) is a surrogate marker of TP53 mutation. This study analyzed the frequency, type, and distribution of TP53 mutations in BCs and assessed the efficacy of p53 IHC as a surrogate marker of TP53 mutation. We collected data from 112 BC cases, including the results of p53 IHC and next-generation sequencing (NGS). Over-expression of p53 IHC was observed in 36 patients (32.1%), complete absence in 19 patients (17.0%), aberrant cytoplasmic staining in 1 patient (0.9%), and wild-type in 56 (50.0%) patients. The concordance rate between TP53 mutation and p53 IHC was 88.4% in all BCs, 89.9% in luminal BCs, and 86.0% in triple-negative BCs (TNBC). TNBC, abnormal p53 IHC pattern, p53 IHC over-expression, neoadjuvant chemotherapy (NAC) history, TP53 mutation, and high pre-treatment ki-67 labeling index (≥50%) were significantly associated with worse distant metastasis-free survival (DMFS) and overall survival (OS) (p<0.05). Pre-NAC clinical stage III was associated with worse DMFS but not OS. Multivariate analysis showed that NAC history, TNBC, and p53 IHC over-expression were independent predictors of worse DMFS. An abnormal p53 IHC pattern and NAC history were independent predictors of worse OS. P53 IHC is a valid surrogate marker of TP53 mutation in BC. Accumulation of abnormal p53 alone, regardless of TP53 mutation, was associated with worse DMFS and can be used as an easily accessible biomarker to predict chemoresistance.

Analysis of gene mutation in a family with Muir-Torre syndrome accompanied with extraorbital cystic sebaceous carcinoma

This study reported a family of MLH1 mutation-induced Muir-Torre syndrome (MTS) and evaluated it's clinical and genetic characteristics. A 51 year-old patient with extraorbital cystic sebaceous and colon adenocarcinoma diagnosed in November 2021 in Zhongshan Hospital of Xiamen University was included. The clinical data of the family were collected and a pedigree chart was drawn, which was in line with the Chinese Lynch syndrome diagnostic criteria and was a typical MTS family. NM_000249.4:c.298C>T(p.R100*) of MLH1 gene in exon 3 was detected by whole exome sequencing and multiplex ligation dependent amplification, which is a pathogenic mutation. After the pathogenic mutation was identified, Sanger sequencing was performed on 4 direct members of the family for MLH1 gene, and 3 family members were found to have detected the mutation and included in MTS risk control. Until December 25 2023, follow-up showed the proband patients were not suffered from recurrence or new occurrence of skin or gastrointestinal tumors. The study reported a typical MTS family and found a possible pathogenic nonsense mutation in the MLH1 gene, which provides new evidence for the pathogenicity of this mutation.

Malassezia responds to environmental pH signals through the conserved Rim/Pal pathway.

During mammalian colonization and infection, microorganisms must be able to rapidly sense and adapt to changing environmental conditions including alterations in extracellular pH. The fungus-specific Rim/Pal signaling pathway is one process that supports microbial adaptation to alkaline pH. This cascading series of interacting proteins terminates in the proteolytic activation of the highly conserved Rim101/PacC protein, a transcription factor that mediates microbial responses that favor survival in neutral/alkaline pH growth conditions, including many mammalian tissues. We identified the putative Rim pathway proteins Rim101 and Rra1 in the human skin colonizing fungus Malassezia sympodialis. Gene deletion by transconjugation and homologous recombination revealed that Rim101 and Rra1 are required for M. sympodialis growth at higher pH. In addition, comparative transcriptional analysis of the mutant strains compared to wild-type suggested mechanisms for fungal adaptation to alkaline conditions. These pH-sensing signaling proteins are required for optimal growth in a murine model of atopic dermatitis, a pathological condition associated with increased skin pH. Together, these data elucidate both conserved and phylum-specific features of microbial adaptation to extracellular stresses.IMPORTANCEThe ability to adapt to host pH has been previously associated with microbial virulence in several pathogenic fungal species. Here we demonstrate that a fungal-specific alkaline response pathway is conserved in the human skin commensal fungus Malassezia sympodialis (Ms). This pathway is characterized by the pH-dependent activation of the Rim101/PacC transcription factor that controls cell surface adaptations to changing environmental conditions. By disrupting genes encoding two predicted components of this pathway, we demonstrated that the Rim/Pal pathway is conserved in this fungal species as a facilitator of alkaline pH growth. Moreover, targeted gene mutation and comparative transcriptional analysis support the role of the Ms Rra1 protein as a cell surface pH sensor conserved within the basidiomycete fungi, a group including plant and human pathogens. Using an animal model of atopic dermatitis, we demonstrate the importance of Ms Rim/Pal signaling in this common inflammatory condition characterized by increased skin pH.

The Salmonella Effector SspH2 Facilitates Spatially Selective Ubiquitination of NOD1 to Enhance Inflammatory Signaling.

As part of its pathogenesis, Salmonella enterica serovar Typhimurium delivers effector proteins into host cells. One effector is SspH2, a member of the so-called novel E3 ubiquitin ligase family, that interacts with and enhances, NOD1 pro-inflammatory signaling, though the underlying mechanisms are unclear. Here, we report that SspH2 interacts with multiple members of the NLRC family to enhance pro-inflammatory signaling by targeted ubiquitination. We show that SspH2 modulates host innate immunity by interacting with both NOD1 and NOD2 in mammalian epithelial cell culture via the NF-κB pathway. Moreover, purified SspH2 and NOD1 directly interact, where NOD1 potentiates SspH2 E3 ubiquitin ligase activity. Mass spectrometry and mutational analyses identified four key lysine residues in NOD1 that are required for its enhanced activation by SspH2, but not its basal activity. These critical lysine residues are positioned in the same region of NOD1 and define a surface on the receptor that appears to be targeted by SspH2. Overall, this work provides evidence for post-translational modification of NOD1 by ubiquitin and uncovers a unique mechanism of spatially selective ubiquitination to enhance the activation of an archetypal NLR.

GBSR: Graph-based suspiciousness refinement for improving fault localization

Fault Localization (FL) is an important and time-consuming phase of software debugging. The essence of FL lies in the process of calculating the suspiciousness of different program entities (e.g., statements) and generating a ranking list to guide developers in their code inspection. Nonetheless, a prevalent challenge within existing FL methodologies is the propensity for program entities with analogous execution information to receive a similar suspiciousness. This phenomenon can lead to confusion among developers, thereby reducing the effectiveness of debugging significantly. To alleviate this issue, we introduce fine-grained contextual information (such as partial code structural, coverage, and features from mutation analysis) to enrich the characteristics of program entities. Graphical structures are proposed to organize such information, where the passed and failed tests are constructed separately with the consideration of their differential impacts. In order to support the analysis of multidimensional features and the representation of large-scale programs, the PageRank algorithm is adopted to compute each program entity’s weight. Rather than altering the fundamental FL process, we leverage these computed weights to refine the suspiciousness produced by various FL techniques, thereby providing developers with a more precise and actionable ranking of potential fault locations. The proposed strategy Graph-Based Suspiciousness Refinement (GBSR) is evaluated on 243 real-world faulty programs from the Defects4J. The results demonstrate that GBSR can improve the accuracy of various FL techniques. Specifically, for the refinement with traditional SBFL and MBFL techniques, the number of faults localized by the first position of the ranking list (Top-1) is increased by 189% and 68%, respectively. Furthermore, GBSR can also boost the state-of-the-art learning-based FL technique Grace by achieving a 2.8% performance improvement in Top-1.

The Phenotype-Genotype Correlations of FMF Patients: A Single Center Study

Purpose: Familial Mediterranean Fever (FMF) is an autosomal recessive, auto-inflammatory disease that is characterized by recurrent fever and polyserositis episodes like peritonitis, arthritis, and pleuritis. The disease occurs most commonly in populations of Jewish, Turkish, Armenian, and Arabian origin. We aimed to show the genotype-phenotype relationship in FMF patients in our centre. Materials and Methods: We conducted a retrospective analysis of the medical registries of 200 FMF pediatric patients from Sisli Hamidiye Etfal Education and Research Hospital, Department of Pediatrics, Pediatric Nephrology Policlinic between 2011 and 2017. The diagnosis was based on the Tel-Hashomer clinical diagnostic criteria. We used the severity score recommended by Pras in 1998. Clinical data and baseline investigations were collected. Mutation analysis was performed by the amplification-refractory mutation system (ARMS)-PCR method. Findings: Females represented 54% and ages ranged from 40-210 months. The most frequent symptoms were abdominal pain, fever, and arthralgia.Among the 200 patients, 41 (20,5%) were homozygous, 63 (31,5%) were compound heterozygous, and 96 (48%) were heterozygous for MEFV mutations. The first 3 most common mutations were heterozygotus R202Q, M694V/R202Q and heterozygotus E148Q respectively., which was statistically significant. M694V mutation was positive in at least one allele in 72 patients studied. The symptom of fever and arthralgia were found to be significantly higher in patients with M694V mutation. Also, the number of episodes in one year before treatment, PRAS score and the values of fibrinogen during episodes were higher in M694V patients as a result, the age at diagnosis was found to be earlier in patients with homozygotus M694V mutation. The ratio of female gender and myalgia was significantly higher in patinets with the mutation of R202Q homozygotus. The number of episodes in one year before treatment was lower in R202Q patients when compared to the M694V carriers, therefore the initiation age to colchicine was later in the R202Q group which was statistically significant. İmplications to Theory, Practice and Policy: Our results support the notion that the genotype influences the phenotype as regards clinical manifestations, disease severity, and colchicine response.

Mutational analysis of the mammalian mitochondrial Ca2+/H+ exchanger TMBIM5/MICS1

Mutational analysis of the mammalian mitochondrial Ca2+/H+ exchanger TMBIM5/MICS1

The Genetic Determinants and Genomic Consequences of Non-Leukemogenic Somatic Point Mutations.

Clonal hematopoiesis (CH) is defined by the expansion of a lineage of genetically identical cells in blood. Genetic lesions that confer a fitness advantage, such as point mutations or mosaic chromosomal alterations (mCAs) in genes associated with hematologic malignancy, are frequent mediators of CH. However, recent analyses of both single cell-derived colonies of hematopoietic cells and population sequencing cohorts have revealed CH frequently occurs in the absence of known driver genetic lesions. To characterize CH without known driver genetic lesions, we used 51,399 deeply sequenced whole genomes from the NHLBI TOPMed sequencing initiative to perform simultaneous germline and somatic mutation analyses among individuals without leukemogenic point mutations (LPM), which we term CH-LPMneg. We quantified CH by estimating the total mutation burden. Because estimating somatic mutation burden without a paired-tissue sample is challenging, we developed a novel statistical method, the Genomic and Epigenomic informed Mutation (GEM) rate, that uses external genomic and epigenomic data sources to distinguish artifactual signals from true somatic mutations. We performed a genome-wide association study of GEM to discover the germline determinants of CH-LPMneg. After fine-mapping and variant-to-gene analyses, we identified seven genes associated with CH-LPMneg ( TCL1A, TERT, SMC4, NRIP1, PRDM16 , MSRA , SCARB1 ), and one locus associated with a sex-associated mutation pathway ( SRGAP2C) . We performed a secondary analysis excluding individuals with mCAs, finding that the genetic architecture was largely unaffected by their inclusion. Functional analyses of SMC4 and NRIP1 implicated altered HSC self-renewal and proliferation as the primary mediator of mutation burden in blood. We then performed comprehensive multi-tissue transcriptomic analyses, finding that the expression levels of 404 genes are associated with GEM. Finally, we performed phenotypic association meta-analyses across four cohorts, finding that GEM is associated with increased white blood cell count and increased risk for incident peripheral artery disease, but is not significantly associated with incident stroke or coronary disease events. Overall, we develop GEM for quantifying mutation burden from WGS without a paired-tissue sample and use GEM to discover the genetic, genomic, and phenotypic correlates of CH-LPMneg.

PIK3CA mutation analysis in circulating tumor cells of patients with hormone receptor positive metastatic breast cancer

In metastatic breast cancer (MBC), blood is a source of circulating tumor cells (CTCs). CTCs may serve as a ‘‘real-time liquid biopsy” as they represent metastatic tumor genetics better than primary tumor. PIK3CA is one of the most important oncogenes in treatment-unresponsive breast cancers. The aim of this study was to detect PIK3CA mutations and hereditary cancer variants in CTCs from MBC patients. Forty-seven blood samples were obtained from 20 MBC patients from at least 1/3 consecutive time points. CTCs were quantified using the CellSearch system and isolated from 11/20 patients with ≥5/7.5 ml CTCs (14/47 blood samples) using the DEPArray system. DNA was extracted and amplified to perform Sanger sequencing on PIK3CA gene. Sequencing revealed a pathogenic PIK3CA mutation in 2/11 (18 %) cases. Subsequently, we evaluated a 26-target hereditary gene panel by Next Generation Sequencing and identified a concomitant pathogenic mutation in the TP53 gene in a patient with a PIK3CA mutation. No pathogenic germline variants were found. Our data support the conclusion that CTCs analysis may be used to identify mutations in patients to identify those more likely to metastasize.

DNA mismatch repair controls the mutagenicity of Polymerase ζ-dependent translesion synthesis at methylated guanines

By replicating damaged nucleotides, error-prone DNA translesion synthesis (TLS) enables the completion of replication, albeit at the expense of fidelity. TLS of helix-distorting DNA lesions, that usually have reduced capacity of basepairing, comprises insertion opposite the lesion followed by extension, the latter in particular by polymerase ζ (Pol ζ). However, little is known about involvement of Pol ζ in TLS of non- or poorly-distorting, but miscoding, lesions such as O6-methyldeoxyguanosine (O6-medG). Using purified Pol ζ we describe that the enzyme can misincorporate thymidine opposite O6-medG and efficiently extend from terminal mismatches, suggesting its involvement in the mutagenicity of O6-medG. Surprisingly, O6-medG lesions induced by the methylating agent N-methyl-N’-nitro-N-nitrosoguanidine (MNNG) appeared more, rather than less, mutagenic in Pol ζ-deficient mouse embryonic fibroblasts (MEFs) than in wild type MEFs. This suggested that in vivo Pol ζ participates in non-mutagenic TLS of O6-medG. However, we found that the Pol ζ-dependent misinsertions at O6-medG lesions are efficiently corrected by DNA mismatch repair (MMR), which masks the error-proneness of Pol ζ. We also found that the MNNG-induced mutational signature is determined by the adduct spectrum, and modulated by MMR. The signature mimicked single base substitution signature 11 in the catalogue of somatic mutations in cancer, associated with treatment with the methylating drug temozolomide. Our results unravel the individual roles of the major contributors to methylating drug-induced mutagenesis. Moreover, these results warrant caution as to the classification of TLS as mutagenic or error-free based on in vitro data or on the analysis of mutations induced in MMR-proficient cells.

Mutational Signatures in Colorectal Cancer: Translational Insights, Clinical Applications, and Limitations.

A multitude of exogenous and endogenous processes have the potential to result in DNA damage. While the repair mechanisms are typically capable of correcting this damage, errors in the repair process can result in mutations. The findings of research conducted in 2012 indicate that mutations do not occur randomly but rather follow specific patterns that can be attributed to known or inferred mutational processes. The process of mutational signature analysis allows for the inference of the predominant mutational process for a given cancer sample, with significant potential for clinical applications. A deeper comprehension of these mutational signatures in CRC could facilitate enhanced prevention strategies, facilitate the comprehension of genotoxic drug activity, predict responses to personalized treatments, and, in the future, inform the development of targeted therapies in the context of precision oncology. The efforts of numerous researchers have led to the identification of several mutational signatures, which can be categorized into different mutational signature references. In CRC, distinct mutational signatures are identified as correlating with mismatch repair deficiency, polymerase mutations, and chemotherapy treatment. In this context, a mutational signature analysis offers considerable potential for enhancing minimal residual disease (MRD) tests in stage II (high-risk) and stage III CRC post-surgery, stratifying CRC based on the impacts of genetic and epigenetic alterations for precision oncology, identifying potential therapeutic vulnerabilities, and evaluating drug efficacy and guiding therapy, as illustrated in a proof-of-concept clinical trial.

Deep mutational scanning of EccD3 reveals the molecular basis of its essentiality in the mycobacterium ESX secretion system.

Tuberculosis remains the deadliest infectious disease in the world and requires novel therapeutic targets. The ESX-3 secretion system, which is essential for iron and zinc homeostasis and thus M. tuberculosis survival, is a promising target. In this study, we perform a deep mutational scan on the ESX-3 core protein EccD3 in the model organism M. smegmatis. We systematically investigated the functional roles of 145 residues across the soluble ubiquitin-like domain, the conformationally distinct flexible linker, and selected transmembrane helices of EccD3. Our data combined with structural comparisons to ESX-5 complexes support a model where EccD3 stabilizes the complex, with the hinge motif within the linker being particularly sensitive to disruption. Our study is the first deep mutational scan in mycobacteria, which could help guide drug development toward novel treatment of tuberculosis. This study underscores the importance of context-specific mutational analyses for discovering essential protein interactions within mycobacterial systems.

Comprehensive analysis of somatic mutations and structural variations in domestic pig.

Understanding somatic mutations and structural variations in domestic pigs (Sus scrofa domestica) is critical due to their increasing importance as model organisms in biomedical research. In this study, we conducted a comprehensive analysis through whole-genome sequencing of skin, organs, and blood samples. By examining two pig pedigrees, we investigated the inheritance and sharedness of structural variants among fathers, mothers, and offsprings. Utilizing single-cell clonal expansion techniques, we observed significant variations in the number of somatic mutations across different tissues. An in-house developed pipeline enabled precise filtering and analysis of these mutations, resulting in the construction of individual phylogenetic trees for two pigs. These trees explored the developmental relationships between different tissues, revealing insights into clonal expansions from various anatomical locations. This study enhances the understanding of pig genomes, affirming their increasing value in clinical and genomic research, and provides a foundation for future studies in other animals, paralleling previous studies in mice and humans. This approach not only deepens our understanding of mammalian genomic variations but also strengthens the role of pigs as a crucial model in human health and disease research.

Comprehensive analysis of Novel mutations in CCM1/KRIT1 and CCM2/MGC4607 and their clinical implications in Cerebral Cavernous malformations

BackgroundCerebral Cavernous Malformations (CCM) is a genetic disease characterized by vascular abnormalities in the brain and spinal cord, affecting 0.4-0.5 % of the population. We identified two novel pathogenic mutations, CCM1/KRIT1 c.811delT (p.Trp271GlyfsTer5) and CCM2/MGC4607 c.613_614insGG p.Glu205GlyfsTer31), which disrupt crucial protein domains and potentially alter disease progression. ObjectiveThe study aims to comprehensively analyze a Brazilian cohort of CCM patients, integrating genetic, clinical, and structural aspects. Specifically, we sought to identify novel mutations within the CCM complex, and explore their potential impact on disease progression. MethodsWe conducted a detailed examination of neuroradiological and clinical features in both symptomatic and asymptomatic CCM patients, performing genetic analyses through sequencing of the CCM1/KRIT1, CCM2/MGC4607, and CCM3/PDCD10 genes In silico structural predictions were carried out using PolyPhen-2, SIFT, and Human Genomics Community tools. Protein-protein interactions and docking analyses were explored using the STRING database. ResultsGenetic analysis identifies 6 pathogenic mutations, 4 likely pathogenic, 1 variants of uncertain significance, and 7 unclassified mutations, including the novel mutations in CCM1 c.811delT and CCM2 c.613_614insGG. In silico structural analysis revealed significant alterations in protein structure, supporting their pathogenicity. Protein-protein interaction analysis indicated nuanced impacts on cellular processes. Clinically, we observed a broad spectrum of symptoms, including seizures and focal neurological deficits. However, no statistically significant differences were found in lesion burden, age of first symptom onset, or sex between the identified CCM1/KRIT1 and CCM2/MGC4607 mutations among all patients studied. ConclusionThis study enhances the understanding of CCM by linking clinical variability, genetic mutations, and structural effects. The identification of these novel mutations opens new avenues for research and potential therapeutic strategies.