Mutant Allele Research Articles

Molecular mechanisms of PI3K isoform dependence in embryonic growth.

The phosphoinositide 3-kinase (PI3K) pathway is an important signaling mechanism for cell proliferation and metabolism. Mutations that activate PIK3CA may make cells p110α dependent, but when phosphatase tensin homolog (PTEN) is lost, the p110β isoform of PI3Ks becomes more important. However, the exact mechanism underlying the prevalence of p110s remains unclear. In this study, our aim was to elucidate the processes behind PI3K isoform dependency in a cellular model of embryonic development. In order to understand PI3K isoform prevalence, mouse embryonic fibroblasts (MEFs) were used and p110β, PTEN and Rac1 activity was modulated using retroviral plasmids. Expression levels and cellular growth were assessed by performing immunoblots and crystal violet assays. The levels of PTEN had only a partial effect on the prevalence of PI3K isoforms in MEFs. The dependency on p110α diminished when PTEN was depleted. Of note, when PTEN expression was repressed, there was no full transition in dependency from one PI3K isoform to the other. Interestingly, the viability of PTEN-depleted MEFs became less dependent on p110α and more dependent on p110β when p110β was overexpressed. Nevertheless, the overexpression of p110β in conjunction with PTEN knock-downs did not result in a complete shift of isoforms in PI3Ks. Finally, we investigated Rac1 activation with a mutant allele and determined a more potent increase in p110β prominence in MEFs. These findings suggest that multiple cellular parameters, including PTEN status, PI3K isoform levels, and Rac1 activity, combine to influence PI3K isoform prevalence, rather than a single determinant.

Synergistic effects of novel penicillin-binding protein 1A amino acid substitutions contribute to high-level amoxicillin resistance of Helicobacter pylori.

The growing resistance to amoxicillin (AMX)-one of the main antibiotics used in Helicobacter pylori eradication therapy-is an increasing health concern. Several mutations of penicillin-binding protein 1A (PBP1A) are suspected of causing AMX resistance; however, only a limited set of these mutations have been experimentally explored. This study aimed to investigate four PBP1A mutations (i.e., T558S, N562H, T593A, and G595S) carried by strain KIN76, a high-level AMX-resistant clinical H. pylori isolate with an AMX minimal inhibition concentration (MIC) of 2 µg/mL. We transformed a recipient strain 26695 with the DNA containing one to four mutation allele combinations of the pbp1 gene from strain KIN76. Transformants were subjected to genomic exploration and antimicrobial susceptibility testing. The resistance was transformable, and the presence of two to four PBP1A mutations (T558S and N562H, or T593A and G595S), rather than separate single mutations, was necessary to synergistically increase the AMX MIC up to 16-fold compared with the wild-type (WT) strain 26695. An AMX binding assay of PBP1A was performed using these strains, and binding was visualized by chasing Bocillin, a fluorescent penicillin analog. This revealed that all four-mutation allele-transformed strains exhibited decreased affinity to AMX on PBP1A than the WT. Protein structure modeling indicated that functional modifications occur as a result of these amino acid substitutions. This study highlights a new synergistic AMX resistance mechanism and establishes new markers of AMX resistance in H. pylori.IMPORTANCEThe development of resistance to antibiotics, including amoxicillin, is hampering the eradication of Helicobacter pylori infection. The identification of mechanisms driving this resistance is crucial for the development of new therapeutic strategies. We have demonstrated in vitro the synergistic role of novel mutations in the pbp1 gene of H. pylori that is suspected to drive amoxicillin resistance. Also deepening our understanding of amoxicillin resistance mechanisms, this study establishes new molecular markers of amoxicillin resistance that may be useful in molecular-based antibiotic susceptibility testing approaches for clinical practice or epidemiologic investigations.

Association of Interleukin-6 gene (rs1800795) variant with waist-hip-ratio and Insulin resistance: A cross-sectional study in adult women

Obesity is a major contributing factor to metabolic disorders and public health problems worldwide. The goal of the current study was to determine if the Interleukin-6 gene rs1800795 variant is linked to waist-hip-ratio (WHR) and insulin resistance (IR) in North Indian adult women. The WHO guidelines were followed by 37 women who had a WHR >0.85 and 35 women who had a WHR <0.85, out of the 72 women who took part in the study. Every adult woman underwent anthropometric measures. Fasting blood glucose, serum Insulin, lipid levels, and IL-6 levels were measured, and IR was computed. The RFLP technique was used to polymorphise the IL-6 gene (174 G>C) variant. The study group's Insulin, HOMA-IR, BMI, lipid profile, TC/HDL ratio, HDL/LDL ratio, and serum IL-6 level were shown to differ significantly from the control. The percentage frequency of the IL-6 mutant genotype GC+CC (89.19%) and mutant allele C (75.68%) was higher in the study group (WHR >0.85). According to the findings, WHR, TC, TG, and HOMA-IR were substantially correlated (all p <0.05) with the mutant genotype GC+CC of the IL-6 174 G>C gene. This study indicates that the IL-6 rs1800795 gene (174 G>C) variant was substantially linked with metabolic risk variables, such as WHR and HOMA-IR.

Zebrafish Suppressor of Cytokine Signaling 4b (Socs4b) Is Dispensable for Development but May Regulate Epidermal Growth Factor Receptor Signaling.

The suppressor of cytokine signaling (SOCS) family of proteins were named after their defining role as negative feedback regulators of signaling initiated by numerous cytokine receptors. However, multiple members of the SOCS family likely function outside of this paradigm, including SOCS4. Zebrafish possess two SOCS4 paralogues, with socs4a previously shown to participate in central nervous system development and function. This study examined the role of the other paralogue, socs4b, through expression analysis and functional investigations in vivo and in vitro. This revealed maternal deposition of socs4b mRNA, specific zygotic expression during late embryogenesis, including in the brain, eye and intestine, and broad adult expression that was highest in the brain. A mutant allele, socs4bΔ18, was generated by genome editing, in which the start codon was deleted. Fish homozygous for this likely hypomorphic allele showed no overt developmental phenotypes. However, in vitro studies suggested the Socs4b protein may be able to regulate EGFR signaling.

De Novo Deep Intron ELANE Mutation Resulting in Severe Congenital Neutropenia.

Severe congenital neutropenia (SCN) comprises a diverse range of rare hematological disorders characterized by recurrent, often life-threatening infections that manifest within the first months of life. Mutations in the ELANE gene are the most prevalent cause of SCN. While over 230 mutations in ELANE have been documented, including substitutions, frameshifts, nonsense mutations, and splice site alterations, the occurrence of deep intronic mutations has not been previously reported. Herein, we present the case of a young girl who exhibited recurrent fever, respiratory infections, skin abscesses, and gingivitis shortly after birth. Laboratory analysis revealed markedly diminished neutrophil levels alongside elevated monocyte and eosinophil counts. Bone marrow examination disclosed a halt in myelopoiesis maturation. ELANE gene full-length sequencing identified a novel de novo deep intron mutation in ELANE (c.598 + 79G > T), subsequently confirmed by Sanger sequencing. cDNA sequencing of the patient demonstrated aberrant gene splicing. Utilizing a mini-gene splicing assay for ELANE intronic variants, we identified a mutant ELANE allele (c.597 + 1_597 + 83ins) leading to the creation of a premature termination codon (p.Gly200ValfsTer40). Confocal microscopy revealed heightened expression of myeloperoxidase and neutrophil elastase in the patient, suggesting a potential role for the unfolded protein response in the pathogenesis of the deep intron ELANE mutation. In summary, our findings illustrate the first reported instance of de novo deep intron ELANE mutations associated with SCN, underscoring the importance of exploring deep intronic regions in SCN patients lacking identifiable disease-causing gene mutations.

Clinical features and CPS1 variants in Chinese patients with carbamoyl phosphate synthetase 1 deficiency

BackgroundCarbamoyl phosphate synthetase 1 (CPS1) deficiency (OMIM 237300), an autosomal recessive rare and severe urea cycle disorder, is associated with hyperammonemia and high mortality.MethodsHerein we present 12 genetic variants identified in seven clinically well-characterized Chinese patients with CPS1 deficiency who were admitted to the Children’s Medical Center of Peking University First Hospital from September 2014 to August 2023.ResultsSeven patients (two male and five female patients including two sisters) experienced symptoms onset between 2 days and 13 years of age, and they were diagnosed with CPS1 deficiency between 2 months and 20 years. Peak blood ammonia levels ranged from 160 to 1,000 µmol/L. Three patients showed early-onset CPS1 deficiency, with only one surviving after treatment with sodium phenylbutyrate, N-carbamoyl-L-glutamate, and liver transplantation at 4 months, showing a favorable outcome. The remaining four patients had late-onset CPS1 deficiency, presenting with mental retardation, psychiatric symptoms, and self-selected low-protein diets. Among the 12 CPS1 variants identified in these patients, 10 were novel, with all patients exhibiting compound heterozygosity for CPS1 mutant alleles. Seven variants (c.149T > C, c.616 A > T, c.1145 C > T, c.1294G > A, c.3029 C > T, c.3503 A > T, and c.3793 C > T) resulted in single amino acid substitutions. Three frameshift variations (c.2493del, c.3067dup, and c.3241del) were identified, leading to enzyme truncation. One mutation (c.3506_3508del) caused an in-frame single amino acid deletion, while another (c.2895 + 2T > C) resulted in aberrant splicing.ConclusionsExcept for two known variants, all other variants were identified as novel. No hotspot variants were observed among the patients. Our data contribute to expanding the mutation spectrum of CPS1.

Mutations in nuclear genes encoding mitochondrial ribosome proteins restore pollen fertility in S male-sterile maize.

The interaction of plant mitochondrial and nuclear genetic systems is exemplified by mitochondria-encoded cytoplasmic male sterility (CMS) under the control of nuclear restorer-of-fertility genes. The S type of CMS in maize is characterized by a pollen collapse phenotype and a unique paradigm for fertility restoration in which numerous nuclear restorer-of-fertility lethal mutations rescue pollen function but condition homozygous-lethal seed phenotypes. Two nonallelic restorer mutations recovered from Mutator transposon-active lines were investigated to determine the mechanisms of pollen fertility restoration and seed lethality. Mu Illumina sequencing of transposon-flanking regions identified insertion alleles of nuclear genes encoding mitochondrial ribosomal proteins RPL6 and RPL14 as candidate restorer-of-fertility lethal mutations. Both candidates were associated with lowered abundance of mitochondria-encoded proteins in developing maize pollen, and the rpl14 mutant candidate was confirmed by independent insertion alleles. While the restored pollen functioned despite reduced accumulation of mitochondrial respiratory proteins, normal-cytoplasm plants heterozygous for the mutant alleles showed a significant pollen transmission bias in favor of the nonmutant Rpl6 and Rpl14 alleles. CMS-S fertility restoration affords a unique forward genetic approach to investigate the mitochondrial requirements for, and contributions to, pollen and seed development.

Resistance to ACCase and ALS-inhibiting herbicides detected in targeted sampling of Lolium rigidum (rigid ryegrass) populations from cereal crops in Morocco, Algeria, and Tunisia

Lolium rigidum Gaud. (rigid ryegrass) is one of the most widespread weeds in cereal crops in Morocco, Algeria and Tunisia. This weed has evolved resistance to various herbicide modes of action in this region. ACCase and ALS inhibiting herbicides are mainly used in the major-cereal growing regions to control rigid ryegrass. Through a questionnaire, regions where farmers reported less control of herbicide treatments were registered in the three Maghreb countries. Registered fields were visited for collection and 75 field populations were screened with two ACCase and two ALS herbicides. Target site resistance (TSR) was diagnosed using Illumina Next Generation Sequencing (NGS) technology. The sensitivity bioassay results revealed over 60% of sampled populations to be resistant to pinoxaden and/or clodinafop and about 40% to be resistant to iodosulfuron + mesosulfuron and/or pyroxsulam. In addition, 53% of populations displayed resistance (R) to the two herbicide modes of action tested among the regions. In total, 16 ACCase and 11 ALS mutant alleles were identified, carrying out an amino-acid substitution and conferring herbicide resistance in 3700 of the analyzed plants. Most ACCase and ALS mutations were detected at codons Ile1781 and Pro197, respectively. Not only does this study demonstrate the presence of both cross and multiple resistance, it also highlights the non-ACCase and non-ALS -based resistance mechanisms that could confer resistance to herbicides with different modes of action which complicates the resistance management strategies. In the three Maghreb countries, this challenge is even more prominent due to few modes of action being available for rigid ryegrass control due to low-cost market and the prevalence of generic herbicides.

BLADE-ON-PETIOLE interacts with CYCLOIDEA to fine-tune CYCLOIDEA-mediated flower symmetry in monkeyflowers (Mimulus).

Morphological novelties, or key innovations, are instrumental to the diversification of the organisms. In plants, one such innovation is the evolution of zygomorphic flowers, which is thought to promote outcrossing and increase flower morphological diversity. We isolated three allelic mutants from two Mimulus species displaying altered floral symmetry and identified the causal gene as the ortholog of Arabidopsis BLADE-ON-PETIOLE. We found that MlBOP and MlCYC2A physically interact and this BOP-CYC interaction module is highly conserved across the angiosperms. Furthermore, MlBOP self-ubiquitinates and suppresses MlCYC2A self-activation. MlCYC2A, in turn, impedes MlBOP ubiquitination. Thus, this molecular tug-of-war between MlBOP and MlCYC2A fine-tunes the expression of MlCYC2A, contributing to the formation of bilateral symmetry in flowers, a key trait in angiosperm evolution.

Outward askew endodermal cell divisions reveal INFLORESCENCE AND ROOT APICES RECEPTOR KINASE functions in division orientation.

Oriented cell divisions establish plant tissue and organ patterning and produce different cell types; this is particularly true of the highly organized Arabidopsis (Arabidopsis thaliana) root meristem. Mutant alleles of INFLORESCENCE AND ROOT APICES RECEPTOR KINASE (IRK) exhibit excess cell divisions in the root endodermis. IRK is a transmembrane receptor kinase that localizes to the outer polar domain of these cells, suggesting that directional signal perception is necessary to repress endodermal cell division. Here, a detailed examination revealed many of the excess endodermal divisions in irk have division planes that specifically skew towards the outer lateral side. Therefore, we termed them 'outward askew' divisions. Expression of an IRK truncation lacking the kinase domain retains polar localization and prevents outward askew divisions in irk; however, the roots exhibit excess periclinal endodermal divisions. Using cell identity markers, we show that the daughters of outward askew divisions transition from endodermal to cortical identity similar to those of periclinal divisions. These results extend the requirement for IRK beyond repression of cell division activity to include cell division plane positioning. Based on its polarity, we propose that IRK at the outer lateral endodermal cell face participates in division plane positioning to ensure normal root ground tissue patterning.

Therapeutic targeting of RNA for neurological and neuromuscular disease.

Neurological and neuromuscular diseases resulting from familial, sporadic, or de novo mutations have devasting personal, familial, and societal impacts. As the initial product of DNA transcription, RNA transcripts and their associated ribonucleoprotein complexes provide attractive targets for modulation by increasing wild-type or blocking mutant allele expression, thus relieving downstream pathological consequences. Therefore, it is unsurprising that many existing and under-development therapeutics have focused on targeting disease-associated RNA transcripts as a frontline drug strategy for these genetic disorders. This review focuses on the current range of RNA targeting modalities using examples of both dominant and recessive neurological and neuromuscular diseases.

An optimised CRISPR Cas9 and Cas12a mutagenesis toolkit for Barley and Wheat

BackgroundCRISPR Cas9 and Cas12a are the two most frequently used programmable nucleases reported in plant systems. There is now a wide range of component parts for both which likely have varying degrees of effectiveness and potentially applicability to different species. Our aim was to develop and optimise Cas9 and Cas12a based systems for highly efficient genome editing in the monocotyledons barley and wheat and produce a user-friendly toolbox facilitating simplex and multiplex editing in the cereal community.ResultsWe identified a Zea mays codon optimised Cas9 with 13 introns in conjunction with arrayed guides driven by U6 and U3 promoters as the best performer in barley where 100% of T0 plants were simultaneously edited in all three target genes. When this system was used in wheat > 90% of T0 plants were edited in all three subgenome targets. For Cas12a, an Arabidopsis codon optimised sequence with 8 introns gave the best editing efficiency in barley when combined with a tRNA based multiguide array, resulting in 90% mutant alleles in three simultaneously targeted genes. When we applied this Cas12a system in wheat 86% & 93% of T0 plants were mutated in two genes simultaneously targeted. We show that not all introns contribute equally to enhanced mutagenesis when inserted into a Cas12a coding sequence and that there is rationale for including multiple introns. We also show that the combined effect of two features which boost Cas12a mutagenesis efficiency (D156R mutation and introns) is more than the sum of the features applied separately.ConclusionBased on the results of our testing, we describe and provide a GoldenGate modular cloning system for Cas9 and Cas12a use in barley and wheat. Proven Cas nuclease and guide expression cassette options found in the toolkit will facilitate highly efficient simplex and multiplex mutagenesis in both species. We incorporate GRF-GIF transformation boosting cassettes in wheat options to maximise workflow efficiency.

Efficacy assessment of a novel pan-RAS inhibitor in KRAS-mutant and wild type colorectal 3D bioprinted organoid tumor tissue.

91 Background: Colorectal cancer (CRC) is the third leading type of cancer worldwide, with ~150,000 new cases in the US annually and a grim 14% 5-year survival for patients diagnosed at a late stage. A lack of treatment options leads to persistently poor prognosis for patients with advanced stage disease. KRAS mutations are well known drivers of CRC and other GI cancers. Multiple KRAS mutations occur in CRC, including G12D (34%), G12V (21%), G13D (20%), G12C (8%), and others (18%). Existing KRAS-targeted therapies have limited use in CRC, underscoring the need for pan-RAS inhibitors in treating CRC and other RAS driven cancers. Objective: Assess activity of ADT-007, our pan-RAS inhibitor, on wild-type (WT) and KRAS-mutant 3D bioprinted organoid tumor (BOT) tissue using our high-throughput ex vivo platform. Methods: Using previously established bioprinting protocols, WT and mutant BOTs were printed with HT29 and HCT116 cells, respectively. HT29 is an established human WT CRC cell line with known sensitivity to proteosome and survivin inhibitors. HCT116 is a KRASG13D mutant human CRC cell line. 3 sets of BOTs were generated and acclimated for 24h. One set was treated for 72h with proteosome inhibitor Bortezomib, another with survivin inhibitor YM155, and the third with our novel pan-RAS inhibitor ADT-007. Dose response curves were generated from both conventional ATP luminescence readouts and high-content imaging. Results: BOT tissue microarchitecture was validated and >200 µm diffusion in BOTs was confirmed using high-content imaging. Differential response was quantified using Cell TiterGlo endpoint assay as well as advanced image processing of high-content live/dead nuclear stained images captured at multiple z-plains. ADT-007 IC50 was found to be substantially lower for mutant HCT116 compared to that for WT HT29 cell line BOTs, which was consistent with separately conducted in vitro and in vivo studies. Conclusions: A pan-RAS inhibitor, such as ADT-007 with high selectivity for cancer cells with activated RAS that is not limited to a specific KRAS mutant allele or RAS isozyme, could have broader use for CRC and other RAS-driven cancers. Further, due to their potential to replicate biophysical characteristics of a tumor and its microenvironment, BOT based precision and personalized medicine platforms can provide more accurate drug efficacy readout compared to in vitro cancer models.

Less is more: CRISPR/Cas9-based mutations in DND1 gene enhance tomato resistance to powdery mildew with low fitness costs

Powdery mildew (PM), triggered by Oidium neolycopersici, represents a significant threat and a major concern for the productivity of tomato plants (Solanum lycopersicum L.). The presence of susceptibility (S) genes in plants facilitates pathogen proliferation and their dysfunction can lead to a recessively inherited broad-spectrum and durable type of resistance. Past studies have demonstrated that disrupting the function of DND1 (Defense No Death 1) increases plant resilience against various pathogens, such as powdery mildew (PM), but this comes at the cost of negatively affecting the overall health and vigor of the plant. To investigate the possibility of minimizing the adverse effects of the dnd1 mutation while boosting disease resistance, a CRISPR-Cas9 construct with four single guide RNAs targeting three exons of SlDND1 (Solyc02g088560.4.1) was designed and introduced into the tomato variety Moneymaker (MM) through Agrobacterium tumefaciens-mediated transformation. Three T1 lines (named E1, E3 and E4) were crossed with MM and then selfed to produce TF2 families. All the TF2 plants in homozygous state dnd1/dnd1, showed reduced PM symptoms compared to the heterozygous (DND1/dnd1) and wild type (DND1/DND1) ones. Two full knock-out (KO) mutant events (E1 and E4) encoding truncated DND1 proteins, exhibited clear dwarfness and auto-necrosis phenotypes, while mutant event E3 harbouring deletions of 3 amino acids, showed normal growth in height with less auto-necrotic spots. Analysis of the 3D structures of both the reference and the mutant proteins revealed significant conformational alterations in the protein derived from E3, potentially impacting its function. A dnd1/dnd1 TF2 line (TV181848-9, E3) underwent whole-genome sequencing using Illumina technology, which confirmed the absence of off-target mutations in selected genomic areas. Additionally, no traces of the Cas9 gene were detected, indicating its elimination through segregation. Our findings confirm the role of DND1 as an S-gene in tomato because impairment of this gene leads to a notable reduction in susceptibility to O. neolycopersici. Moreover, we provide, for the first time, a dnd1 mutant allele (E3) that exhibits fitness advantages in comparison with previously reported dnd1 mutant alleles, indicating a possible way to breed with dnd1 mutants.

A phase I/II study of nanoliposomal irinotecan and carboplatin in patients with advanced or metastatic, poorly differentiated gastroenteropancreatic neuroendocrine carcinoma characterized by tissue and liquid next-generation sequencing.

52 Background: For locally advanced or metastatic gastroenteropancreatic neuroendocrine carcinomas (GEP-NECs), standard chemotherapy typically incorporates etoposide or irinotecan plus platinum agents. Herein, we initiate a phase I/II trial to apply Nal-IRI in combination with carboplatin as the 1st line treatment in patients with GEP-NECs to define the dose-limiting toxicity (DLT) and recommended phase II dose (RP2D). Methods: In the phase I part, traditional 3+3 design was applied. Enrolled patients would receive escalating dose of 60 (level -1), 80 (starting dose, level 0) or 100 (level 1) mg/m2 salt-form nal-IRI plus carboplatin AUC=4 with maximum total dose of 600 mg on D1, every 21 days as a cycle. DLTs were evaluated during the first one cycle of treatment with tumor assessment every 6 weeks. Prophylaxis G-CSF was not allowed in the first cycle Paired testing of tissue (TBx) and liquid biopsy (LBx) was done by Illumina TSO500 platform before treatment. The second LBx would be repeated for confirmed partial responders when progression. Results: Totally 6 GEP-NEC patients with primary sites of colon in three, and each one in esophagus, ampulla of Vater and pancreas were enrolled to level 0. One patient had DLT of grade 4 neutropenia lasting for 3 days even under salvage G-CSF. Treatment-related adverse events showed grade 3/4 neutropenia (33.4%), grade 2 anemia (16.7%), grade 3 fatigue (16.7%), and grade 2 skin rash (16.7%). Four patients achieved confirmed partial response and 2 patients got stable disease, with the objective response rate of 66.7% (95% confidence interval: 22%-96%). After the data and safety monitoring board meeting, level 0 was decided as RP2D but not escalation to level 1 because of acceptable toxicity profiles and promising efficacies. Comprehensive genomic profiling showed that driver mutations were identified in three cases including two harboring BRAFV600E mutation and one with KRASG12D mutation. The other three cases lacking driver mutation showed frequent copy number alteration including 1 with MDM2 amplification (copy number 27) and a wild-type TP53. Homologous recombination deficiency (HRD), as determined by GIS score, was identified in a colon GEP-NEC who remained to be HRD-positive at progression but a higher TMB (from 4.7 to 10.2 Muts/Mb) and a higher BRAFV600E mutation allele frequency (from 24.1% to 43.6%) in tumor tissue, compared to the baseline profiling. Conclusions: Based on current data, the RP2D is 80 mg/m2 of nal-IRI (salt-form) and carboplatin of AUC=4, every 3 weeks. The extension phase II study in GEP-NEC is ongoing. Clinical trial information: NCT05385861 .

Recent Technologies towards Diagnostic and Therapeutic Applications of Circulating Nucleic Acids in Colorectal Cancers.

Liquid biopsy has emerged as a promising noninvasive approach for colorectal cancer (CRC) management. This review focuses on technologies detecting circulating nucleic acids, specifically circulating tumor DNA (ctDNA) and circulating RNA (cfRNA), as CRC biomarkers. Recent advancements in molecular technologies have enabled sensitive and specific detection of tumor-derived genetic material in bodily fluids. These include quantitative real-time PCR, digital PCR, next-generation sequencing (NGS), and emerging nanotechnology-based methods. For ctDNA analysis, techniques such as BEAMing and droplet digital PCR offer high sensitivity in detecting rare mutant alleles, while NGS approaches provide comprehensive genomic profiling. cfRNA detection primarily utilizes qRT-PCR arrays, microarray platforms, and RNA sequencing for profiling circulating microRNAs and discovering novel RNA biomarkers. These technologies show potential in early CRC detection, treatment response monitoring, minimal residual disease assessment, and tumor evolution tracking. However, challenges remain in standardizing procedures, optimizing detection limits, and establishing clinical utility across disease stages. This review summarizes current circulating nucleic acid detection technologies, their CRC applications, and discusses future directions for clinical implementation.

Loss of cped1 does not affect bone and lean tissue in zebrafish.

Human genetic studies have nominated Cadherin-like and PC-esterase Domain-containing 1 (CPED1) as a candidate target gene mediating bone mineral density (BMD) and fracture risk heritability. Recent efforts to define the role of CPED1 in bone in mouse and human models have revealed complex alternative splicing and inconsistent results arising from gene targeting, making its function in bone difficult to interpret. To better understand the role of CPED1 in adult bone mass and morphology, we turned to zebrafish, an emerging model for orthopaedic research. We analyzed two different cped1 mutant lines and performed deep phenotyping to characterize more than 200 measures of adult vertebral, craniofacial, and lean tissue morphology. We also examined alternative splicing of zebrafish cped1 and gene expression in various cell/tissue types. Our studies fail to support an essential role of cped1 in adult zebrafish bone. Specifically, homozygous mutants for both cped1 mutant alleles, which are expected to result in loss-of-function and impact all cped1 isoforms, exhibited no significant differences in the measures examined when compared to their respective wildtype controls, suggesting that cped1 does not significantly contribute to these traits. We identified sequence differences in critical residues of the catalytic triad between the zebrafish and mouse orthologs of CPED1, suggesting that differences in key residues, as well as distinct alternative splicing, could underlie different functions of CPED1 orthologs in the two species. Our studies demonstrate that cped1 is not required for normal adult zebrafish bone mass, lean mass, or bone and lean tissue morphology, adding to evidence that variants at 7q31.31 can act independently of CPED1 to influence BMD and fracture risk.

Crossing a CRISPR/Cas9 transgenic tomato plant with a wild-type plant yields diverse mutations in the F1 progeny.

Generating CRISPR/Cas9-mediated mutants in tomato (Solanum lycopersicum L.) involves screening shoots regenerated from cultured cells transformed with a T-DNA harboring sequences encoding Cas9 and single guide RNAs (sgRNAs). Production of transformants can be inconsistent and obtaining transformants in large numbers may be difficult, resulting in a limited variety of mutations. Here, I report a method for generating various types of mutations from one transgenic plant harboring the CRISPR/Cas9 system. In this method, a wild-type plant was crossed with a T0 biallelic mutant expressing two sgRNAs targeting the RIPENING INHIBITOR (RIN) gene, and the resulting F1 seedlings were classified using a kanamycin resistance marker on the T-DNA. Genotyping of the RIN locus revealed that kanamycin-sensitive F1 seedlings, which carried no T-DNA, always harbored the wild-type allele and a mutant allele from the transgenic parent. Kanamycin-resistant F1 seedlings, which do carry the T-DNA, harbored a variety of novel mutant alleles, but not the wild-type allele, suggesting that it was mutated during crossing. The novel mutations included one-base insertions or short deletions at each target site, or large deletions across the two target sites. This method was also successfully applied to produce mutations in Geranylgeranyl pyrophosphate synthase 2 (GGPS2). Because this method involves crossing rather than transformation, it can be readily scaled up to produce numerous novel mutations, even in plant species or cultivars for which transformation is inefficient. Therefore, when initial transgene experiments fail to induce the desired mutation, this method provides additional opportunities for generating mutants.

Patient-centric thresholding of Cobas® EGFR mutation Test v2 for surveillance of EGFR-mutated metastatic non-small cell lung cancer

Cobas EGFR mutation Test v2 was FDA-approved as qualitative liquid biopsy for actionable EGFR variants in non-small cell lung cancer (NSCLC). It generates semiquantitative index (SQI) values that correlate with mutant allele levels, but decision thresholds for clinical use in NSCLC surveillance are lacking. We conducted long-term ctDNA monitoring in 20 subjects with EGFR-mutated NSCLC; resulting in a 155 on-treatment samples. We defined optimal SQI intervals to predict/rule-out progression within 12 weeks from sampling and performed orthogonal calibration versus deep-sequencing and digital PCR. SQI showed significant diagnostic power (AUC 0.848, 95% CI 0.782–0.901). SQI below 5 (63% of samples) had 93% (95% CI 87–96%) NPV, while SQI above 10 (25% of samples) had 69% (95% CI 56–80%) PPV. Cobas EGFR showed perfect agreement with sequencing (Kappa 0.860; 95% CI 0.674–1.00) and digital PCR. SQI values strongly (r: 0.910, 95% 0.821–0.956) correlated to mutant allele concentrations with SQI of 5 and 10 corresponding to 6–9 (0.2–0.3%) and 64–105 (1.1–1.6%) mutant allele copies/mL (VAF) respectively. Our dual-threshold classifier of SQI 0/5/10 yielded informative results in 88% of blood draws with high NPV and good overall clinical utility for patient-centric surveillance of metastatic NSCLC.

Predicting recurrent glioblastoma clinical outcome to immune checkpoint inhibition and low-dose bevacizumab with tumor in situ fluid circulating tumor DNA analysis

ObjectiveMost recurrent glioblastoma (rGBM) patients do not benefit from immune checkpoint inhibition, emphasizing the necessity for response biomarkers. This study evaluates whether tumor in situ fluid (TISF) circulating tumor DNA (ctDNA) could serve as a biomarker for response to low-dose bevacizumab (Bev) plus anti-PD-1 therapy in rGBM patients, aiming to enhance systemic responses to immunotherapy.MethodsIn this phase II trial, 32 GBM patients with first recurrence after standard therapy were enrolled and then received tislelizumab plus low-dose Bev each cycle. TISF samples were analyzed for ctDNA using a 551-gene panel before each treatment.ResultsThe median progression-free survival (mPFS) and overall survival (mOS) were 8.2 months (95% CI, 5.2–11.1) and 14.3 months (95% CI, 6.5–22.1), respectively. The 12-month OS was 43.8%, and the objective response rate was 56.3%. Patients with more than 20% reduction in the mutant allele fraction and tumor mutational burden after treatment were significantly associated with better prognosis compared to baseline TISF-ctDNA. Among detectable gene mutations, patients with MUC16 mutation, EGFR mutation & amplification, SRSF2 amplification, and H3F3B amplification were significantly associated with worse prognosis.ConclusionsLow-dose Bev plus anti-PD-1 therapy significantly improves OS in rGBM patients, offering guiding significance for future individualized treatment strategies. TISF-ctDNA can monitor rGBM patients' response to combination therapy and guide treatment.Clinical trial registrationThis trial is registered with ClinicalTrials.gov, NCT05540275.