DNA Deletions Research Articles

Arsenic-Induced Cardiovascular Diseases and their Correlation with Mitochondrial DNA Copy Number, Deletion, and Telomere Length in Bangladeshi Population.

Arsenic contamination is a global health concern, primarily through contaminated groundwater and its entry into the food chain. The association between arsenic exposure and cardiovascular diseases (CVDs) is particularly alarming due to CVDs being the leading cause of death worldwide. Arsenic exposure has also been linked to changes in telomere length, mitochondrial DNA copy number (mtDNAcn), and deletion, further increasing the risk of CVDs. We aimed to determine whether arsenic exposure alters telomere length and mtDNAcn and deletion in a total of 50 CVD patients who underwent open heart surgery hailed from known arsenic-affected and unaffected areas in Bangladesh. Amount of arsenic was determined from the collected nails and cardiac tissues. Relative telomere length and mtDNAcn and deletion were quantified by qRT-PCR. The patients from arsenic-contaminated areas had higher average arsenic deposits in their fingers and toenails (P < 0.05) and higher cardiac tissue injury scores (P < 0.05). Moreover, approximately 1.5-fold shorter telomere length (P < 0.05, r = - 0.775), 1.2-fold decreased mtDNAcn (P < 0.05, r =- 0.797), and an 81-fold higher amount of mitochondrial DNA deletion (P < 0.05, r = 0.784) were observed in the patients who had higher arsenic deposition in their nails. Higher levels of arsenic exposure were found to be linked to shorter telomere length, decreased mtDNAcn, and increased mitochondrial DNA deletion in the patients from As-affected areas. It can also be anticipated that the correlation of arsenic exposure with telomere length, mtDNAcn, and deletion can be used as biomarkers for early diagnosis of arsenic-induced cardiovascular diseases.

A large cohort of deletional high hemoglobin F determinants in Thailand: A molecular revisited and identification of a novel mutation

Background and aimsHigh hemoglobin F determinants can be classified into hereditary persistence of fetal hemoglobin (HPFH) and δβ-thalassemia with different phenotype. We report the molecular basis and hematological features in a large cohort of deletional high Hb F determinants in Thailand. Materials and methodsSubjects (n = 28,177) encountered during 2015–2022 were reviewed, and those with phenotypically suspected of having high Hb F determinants were selected. Combined PCR, multiplex ligation-dependent probe amplification, next-generation sequencing, and DNA sequencing were used to identify the mutations. ResultsAmong 28,177 subjects investigated, 300 (1.06 %) were found to carry deletional high Hb F determinants in a total of 302 alleles, including heterozygote, compound heterozygote with β-hemoglobinopathies, and homozygote. DNA analysis identified eight different DNA deletions, including δβ0-thalassemia (12.6 kb deletion) (73.8 %), HPFH-6 (14.9 %), Indian deletion-inversion Aγδβ0-thalassemia (3.6 %), Thai deletion-inversion-insertion Aγδβ0-thalassemia (3.0 %), SEA-HPFH (3.0 %), Chinese Aγδβ0-thalassemia (1.0 %), Thai δβ0-thalassemia (11.3 kb deletion) (0.3 %), and a novel δβ0-thalassemia (137.1 kb deletion) (0.3 %). In addition, three novel genetic interactions, including Chinese Aγδβ0-thalassemia/Hb E, δβ0-thalassemia/Indian deletion-inversion Aγδβ0-thalassemia, and homozygous δβ0-thalassemia were found. Hematological features and Hb analysis results of 20 different genotypes were recorded. Multiplex gap-PCR assays for detection of these genetic determinants were described. ConclusionsDeletional high Hb F determinants are common and heterogeneous in Thailand. Data on the prevalence, molecular spectrum, phenotypic expression, and complex interactions of these genetic determinants should prove useful in the study and a prevention and control program of hemoglobinopathies in the region.

Mitochondrial transfer and implications for muscle function in idiopathic inflammatory myopathies.

Impairment in cellular bioenergetics as either the cause, consequence, or major contributor of tissue damage has drawn increasing scientific curiosity across aging and chronic health conditions, with mitochondrial dysfunction emerging as a central mechanism in the pathogenesis of a variety of inflammatory and degenerative disorders. Beyond bioenergetics, mitochondria play critical regulatory roles in programmed cell death of dysfunctional/defective cells as well as in metabolite synthesis and metabolic signalling. Further, extra-cellular exposure to fragmentation of injured mitochondria is associated with incitement of systemic and organ-based inflammation. Thus, mitochondrial function has recently drawn intense, spectral scientific interest as an integral component across maladies.In muscle, mitochondrial dysfunction is clinically associated with atrophy and diminished endurance. Direct myo-histopathological evidence characterising loss of mitochondrial integrity as a hallmark of muscle compromise was first noticed in inclusion body myositis (IBM). This was followed by the discovery of multiple deletions in mitochondrial DNA in sarcopenia, IBM, and other inflammatory myopathies, like dermatomyositis. Though fraught with bioethical considerations, the transplant technology of mitochondrial transfer is swiftly gaining prominence in cellular biology and muscle physiology to remediate mitochondrial diminution and dysfunction. Assembling seminal works and recent developments, this review ventures into the rapidly evolving landscape of mitochondrial transfer, focusing on its implications on muscle function, and offers an integrated perspective on the potential roles of mitochondrial transfer and its implications for preserving and restoring muscle health. Presented here is a consolidated viewpoint on mitochondrial transfer in idiopathic inflammatory myopathies.

CHCHD10 mutations induce tissue-specific mitochondrial DNA deletions with a distinct signature.

Mutations affecting the mitochondrial intermembrane space protein CHCHD10 cause human disease, but it is not known why different amino acid substitutions cause markedly different clinical phenotypes, including amyotrophic lateral sclerosis-frontotemporal dementia, spinal muscular atrophy Jokela-type, isolated autosomal dominant mitochondrial myopathy and cardiomyopathy. CHCHD10 mutations have been associated with deletions of mitochondrial DNA (mtDNA deletions), raising the possibility that these explain the clinical variability. Here, we sequenced mtDNA obtained from hearts, skeletal muscle, livers and spinal cords of WT and Chchd10 G58R or S59L knockin mice to characterise the mtDNA deletion signatures of the two mutant lines. We found that the deletion levels were higher in G58R and S59L mice than in WT mice in some tissues depending on the Chchd10 genotype, and the deletion burden increased with age. Furthermore, we observed that the spinal cord was less prone to the development of mtDNA deletions than the other tissues examined. Finally, in addition to accelerating the rate of naturally occurring deletions, Chchd10 mutations also led to the accumulation of a novel set of deletions characterised by shorter direct repeats flanking the deletion breakpoints. Our results indicate that Chchd10 mutations in mice induce tissue-specific deletions which may also contribute to the clinical phenotype associated with these mutations in humans.

Adrenocortical function in patients with Single Large Scale Mitochondrial DNA Deletions: a retrospective single centre cohort study.

Single Large Scale Mitochondrial DNA Deletions (SLSMDs), Pearson Syndrome (PS) and Kearns-Sayre Syndrome (KSS), are systemic diseases with multiple endocrine abnormalities. The adrenocortical function has not been systematically investigated with a few anecdotal reports of overt adrenal insufficiency (AI). The study aimed to assess the adrenocortical function in a large cohort of SLSMDs. A retrospective monocentric longitudinal study involved a cohort of 18 SLSMDs patients. Adrenocortical function was evaluated by baseline adrenocorticotrophic hormone (ACTH) and cortisol measurements and by high- (HDT) and low-dose (LDT) ACTH stimulation tests and compared with 92 healthy controls (HC). Baseline adrenocortical function was impaired in 39% of patients and by the end of the study, 66% of PS and 25% of KSS showed an insufficient increase after ACTH stimulation, with cortisol deficiency due to primary AI in most PS and subclinical AI in KSS. Symptomatic AI was recorded in 44% of patients. Peak cortisol levels after ACTH stimulation tests were significantly lower in patients than in HC (P < .0001), with a more reduced response to LDT vs HDT (P < .05). Our study highlights that cortisol deficiency due to primary AI represents a relevant part of the clinical spectrum in SLSMDs, with more severe impairment in PS than in KSS. Basal and after-stimulus assessment of adrenocortical axis should be early and regularly investigated to identify any degree of adrenocortical dysfunction. The study allowed the elaboration of a diagnostic process designed for the diagnosis, treatment, and follow-up of adrenocortical abnormalities in SLSMDs.

Prime editing: Mechanism insight and recent applications in plants.

Prime editing (PE) technology utilizes an extended prime editing guide RNA (pegRNA) to direct a fusion peptide consisting of nCas9 (H840) and reverse transcriptase (RT) to a specific location in the genome. This enables the installation of base changes at the targeted site using the extended portion of the pegRNA through RT activity. The resulting product of the RT reaction forms a 3' flap, which can be incorporated into the genomic site through a series of biochemical steps involving DNA repair and synthesis pathways. PE has demonstrated its effectiveness in achieving almost all forms of precise gene editing, such as base conversions (all types), DNA sequence insertions and deletions, chromosomal translocation and inversion and long DNA sequence insertion at safe harbour sites within the genome. In plant science, PE could serve as a groundbreaking tool for precise gene editing, allowing the creation of desired alleles to improve crop varieties. Nevertheless, its application has encountered limitations due to efficiency constraints, particularly in dicotyledonous plants. In this review, we discuss the step-by-step mechanism of PE, shedding light on the critical aspects of each step while suggesting possible solutions to enhance its efficiency. Additionally, we present an overview of recent advancements and future perspectives in PE research specifically focused on plants, examining the key technical considerations of its applications.

Exploring the Potential of Cytomegalovirus-Based Vectors: A Review.

Viral vectors have emerged as powerful tools for delivering and expressing foreign genes, playing a pivotal role in gene therapy. Among these vectors, cytomegalovirus (CMV) stands out as a promising viral vector due to its distinctive attributes including large packaging capacity, ability to achieve superinfection, broad host range, capacity to induce CD8+ T cell responses, lack of integration into the host genome, and other qualities that make it an appealing vector candidate. Engineered attenuated CMV strains such as Towne and AD169 that have a ~15 kb genomic DNA deletion caused by virus passage guarantee human safety. CMV's large genome enables the efficient incorporation of substantial foreign genes as demonstrated by CMV vector-based therapies for SIV, tuberculosis, cancer, malaria, aging, COVID-19, and more. CMV is capable of reinfecting hosts regardless of prior infection or immunity, making it highly suitable for multiple vector administrations. In addition to its broad cellular tropism and sustained high-level gene expression, CMV triggers robust, virus-specific CD8+ T cell responses, offering a significant advantage as a vaccine vector. To date, successful development and testing of murine CMV (MCMV) and rhesus CMV (RhCMV) vectors in animal models have demonstrated the efficacy of CMV-based vectors. These investigations have explored the potential of CMV vectors for vaccines against HIV, cancer, tuberculosis, malaria, and other infectious pathogens, as well as for other gene therapy applications. Moreover, the generation of single-cycle replication CMV vectors, produced by deleting essential genes, ensures robust safety in an immunocompromised population. The results of these studies emphasize CMV's effectiveness as a gene delivery vehicle and shed light on the future applications of a CMV vector. While challenges such as production complexities and storage limitations need to be addressed, ongoing efforts to bridge the gap between animal models and human translation continue to fuel the optimism surrounding CMV-based vectors. This review will outline the properties of CMV vectors and discuss their future applications as well as possible limitations.

Mitochondrial dysfunction as a probable mechanism for triggering inflammatory joint diseases

The article concerns the contribution of mitochondrial dysfunction to the development of inflammatory joint diseases. Mitochondria are the main suppliers of adenosine triphosphate (ATP). Reactive oxygen species (ROS) are a by-product of this metabolic process. Mitochondria also have an effective antioxidant mechanism: there is a certain balance between the ROS formation and their inactivation. Accumulation with age of mutations (single nucleotide substitutions, e.g., transversions, transitions, and deletions) in mitochondrial DNA, may cause a disorder in selective destruction (utilization) of damaged and dysfunctional mitochondria (mitophagy) thus leading to imbalance between the ROS production and their neutralization. This process is triggered by both internal factors (ROS overproduction) and external factors, i.e., tissue damage / injury and infection. The failure of quality control mechanisms resulting from disruption of mitophagy leads to a significant increase in terminally damaged mitochondria, which become a threat to cell survival. High level of genetic mutations accumulating with age in mitochondrial genome causes an increased formation of ROS, which, in turn, are one of the leading activators of the cytosolic NLRP3 protein, the main component of inflammasome type of the same name. Increased inflammasome formation ultimately triggers caspase-1 dependent production of pro-inflammatory interleukins-1β(IL-1β) and 18 (IL-18). Inadequate removal of damaged mitochondria leads to hyperactivation of inflammatory signaling pathways and, subsequently, to chronic systemic inflammation and development of inflammatory diseases, including primary osteoarthritis (OA). To assess the level of mitochondrial dysfunction, we assessed the numbers of mitochondrial genome copies in post-mitotic muscle cells in 48 patients aged 45 to 95 years who were diagnosed with OA of the knee or hip joints. As a result of our study, we have discovered and confirmed some regularities of human mtDNA mutations corresponding to those in vertebrates, and, in particular, in mammals. Degenerate mutation spectra (without classification of mutations by mtDNA chains and the context of surrounding nucleotides) were constructed for mtDNA in general, and for each individual sample. It was demonstrated that, in one-third of muscle samples, the critical threshold of mtDNA heteroplasmy was exceeded, at which the aberrant biochemical phenotype, in terms of oxidative phosphorylation functioning, (OXPHOS) becomes dominant. Of note, the heteroplasmy rates are lower in older patients who have had significant physical activity during their lives (sports, moderate physical work, etc.). Moreover, the heteroplasmy showed an inverse correlation with high mtDNA copy number. The results obtained can be used to diagnose pathologies in elderly, and the process of healthy aging.

-Thalassemia: Report of two cases in a family.

Case 1 presented with severe anemia and received an intrauterine blood cell transfusion at 33weeks of gestation. The anemia spontaneously improved in early infancy. Case 2, the father of Case 1, had an uneventful birth with no evidence of anemia, though microcytic anemia was observed during childhood. The genetic analysis of the β-globin gene cluster identified a novel heterozygous deletion of DNA extending from the Gγ-globin gene downstream to the β-globin gene, confirming a diagnosis of (G γA γδβ)0 -thalassemia. In cases where thalassemia is suspected based on blood tests, a genetic diagnosis should be performed for the sake of the offspring.

Quantifying human genome parameters in aging.

Healthy human longevity is a global goal of the world health system. Determining the causes and processes influencing human longevity is the primary fundamental goal facing the scientific community. Currently, the main efforts of the scientific community are aimed at identifying the qualitative characteristics of the genome that determine the trait. At the same time, when evaluating qualitative characteristics, there are many challenges that make it difficult to establish associations. Quantitative traits are burdened with such problems to a lesser extent, but they are largely overlooked in current genomic studies of aging and longevity. Although there is a wide repertoire of quantitative trait analyses based on genomic data, most opportunities are ignored by authors, which, along with the inaccessibility of published data, leads to the loss of this important information. This review focuses on describing quantitative traits important for understanding aging and necessary for analysis in further genomic studies, and recommends the inclusion of the described traits in the analysis. The review considers the relationship between quantitative characteristics of the mitochondrial genome and aging, longevity, and age-related neurodegenerative diseases, such as the frequency of extensive mitochondrial DNA (mtDNA) deletions, mtDNA half-life, the frequency of A>G replacements in the mtDNA heavy chain, the number of mtDNA copies; special attention is paid to the mtDNA methylation sign. A separate section of this review is devoted to the correlation of telomere length parameters with age, as well as the association of telomere length with the amount of mitochondrial DNA. In addition, we consider such a quantitative feature as the rate of accumulation of somatic mutations with aging in relation to the lifespan of living organisms. In general, it may be noted that there are quite serious reasons to suppose that various quantitative characteristics of the genome may be directly or indirectly associated with certain aspects of aging and longevity. At the same time, the available data are clearly insufficient for definitive conclusions and the determination of causal relationships.

Myopathy and Ophthalmologic Abnormalities in Association With Multiple Skeletal Muscle Mitochondrial DNA Deletions.

Establishing a molecular diagnosis of mitochondrial diseases due to pathogenic mitochondrial DNA (mtDNA) variants can be difficult because of varying levels of tissue heteroplasmy, and identifying these variants is important for clinical management. Here, we present clinical and molecular findings in 8 adult patients with classical features of mitochondrial ophthalmologic and/or muscle disease and multiple mtDNA deletions isolated to muscle. The patients were identified via a retrospective review of patients seen in both a tertiary ophthalmology center and a genetics clinic with a clinical diagnosis of chronic progressive external ophthalmoplegia, optic nerve abnormalities, and/or mitochondrial myopathy. Age at onset of symptoms ranged from 18 to 61 years. Ocular manifestations included bilateral optic neuropathy in one patient, bilateral optic disc cupping without optic neuropathy in 2 patients, ptosis in 4 patients, and ocular motility deficits in 2 patients. Five patients had generalized weakness. Pathogenic variants in mtDNA were not found in the blood or buccal sample from any patient, but 7 of 8 patients had multiple mtDNA deletions identified in muscle tissue. One patient had a single mtDNA deletion identified in the muscle. Heteroplasmy was less than 15% for all of the identified deletions, with the exception of one deletion that had a heteroplasmy of 50%-60%. None of the patients were found to have a nuclear gene variant known to be associated with mitochondrial DNA maintenance. mtDNA deletions were identified in adult patients with ophthalmologic and/or musle abnormalities and may underlie their clinical presentations.

D-galactose-induced mitochondrial oxidative damage and apoptosis in the cochlear stria vascularis of mice

BackgroundAge-related hearing loss, known as presbycusis, is the result of auditory system degeneration. Numerous studies have suggested that reactive oxygen species (ROS) and mitochondrial oxidative damage play important roles in the occurrence and progression of aging. The D-galactose (D-gal)-induced aging model is well known and widely utilized in aging research. Our previous studies demonstrate that administration of D-gal causes mitochondrial oxidative damage and causes subsequent dysfunction in the cochlear ribbon synapses, which in turn leads to hearing changes and early stage presbycusis. Stria vascularis (SV) cells are vital for hearing function. However, it is unclear to what extent D-gal induces oxidative damage and apoptosis in the cochlear SV of mice. In addition, the source of the causative ROS in the cochlear SV has not been fully investigated.MethodsIn this study, we investigated ROS generation in the cochlear SV of mice treated with D-gal. Hearing function was measured using the auditory brainstem response (ABR). Immunofluorescence was used to examine apoptosis and oxidative damage. Transmission electron microscopy was also used to investigate the mitochondrial ultrastructure. DNA fragmentation was determined using the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) assay. Mitochondrial membrane potential (MMP) and ATP were also measured.ResultsWe found that D-gal-treated mice exhibited a significant shift in the mean amplitude and latency of the ABR; a remarkable increase in the levels of NADPH oxidase (NOX-2), Uncoupling protein 2 (UCP2) and cleaved caspase-3 (c-Cas3) was observed, as well as an increase in the number of TUNEL-positive cells were observed in the SV of mice. Both the expression of the DNA oxidative damage biomarker 8-hydroxy-2-deoxyguanosine (8-OHdG) and a commonly occurring mitochondrial DNA deletion were markedly elevated in the SV of mice that had been treated with D-gal to induce aging. Conversely, the ATP level and MMP were significantly reduced in D-gal-induced aging mice. We also found alterations in the mitochondrial ultrastructure in the SV of aging mice, which include swollen and distorted mitochondrial shape, shortened and thickened microvilli, and the accumulation of lysosomes in the SV.ConclusionOur findings suggest that the impairment of cochlear SV during presbycusis may be caused by mitochondrial oxidative damage and subsequent apoptosis.

The MetaGens algorithm formetagenomic database lossy compression andsubject alignment.

The advancement of genetic sequencing techniques led to the production of a large volume of data. The extraction of genetic material from a sample is one of the early steps of the metagenomic study. With the evolution of the processes, the analysis of the sequenced data allowed the discovery of etiological agents and, by corollary, the diagnosis of infections. One of the biggest challenges of the technique is the huge volume of data generated with each new technology developed. To introduce an algorithm that may reduce the data volume, allowing faster DNA matching with the reference databases. Using techniques like lossy compression and substitution matrix, it is possible to match nucleotide sequences without losing the subject. This lossy compression explores the nature of DNA mutations, insertions and deletions and the possibility that different sequences are the same subject. The algorithm can reduce the overall size of the database to 15% of the original size. Depending on parameters, it may reduce up to 5% of the original size. Although is the same as the other platforms, the match algorithm is more sensible because it ignores the transitions and transversions, resulting in a faster way to obtain the diagnostic results. The first experiment results in an increase in speed 10 times faster than Blast while maintaining high sensitivity. This performance gain can be extended by combining other techniques already used in other studies, such as hash tables. Database URL https://github.com/ghc4/metagens.

Causes of DNA Damage and Genomic Instability: A Review

Genomic instability defines all genetic alterations resulting from excessive or high frequency of mutation; base pairs sequence alterations known as microsatellite instability (MSI) and aneuploidy also called chromosome instability (CIN), chromosomal re- arrangements within the genome or the susceptibility of genome to alterations that occur during cell division cycle. The genome of most cancer cells is highly unstable, mostly due to damage to tumour suppressor genes example tp53 which encodes p53 or other genes coordinating cell division. Telomere is a nucleoprotein complex which extends the physical ends of eukaryotic chromosomes, protecting it from degradation, counteracting sequence loss, protecting genes closer to the chromosomes end and inhibiting cell cycle arrest. Studies have implicated the dysfunction of telomere to the formation of sub-tetraploid aneuploid cells that exhibit tumourigenic capacity and formation of anueploid cells that can elude programmed cell death (apoptosis) and form tumour cells, thus initiating genomic instability. In this review, we presented an insight to the causes of DNA damage and its associated genomic instability. We found that many factors such as fragile sites, end replication problem, DNA double strand breaks, DNA replication defects, deletion, insertion and translocation all are responsible to the fragile nature of the genome.

Establishment of Biotesting System to Study Features of Innovative Multifunctional Biotextile

Abstract An established biotesting system designed to discover specific features of innovative multifunctional biotextile, encompassing integrated silica dioxide and succinite (Baltic amber) particles, is founded on reproducible, reliable, and relatively fast methods. The main idea starting this study was to create a system of test methods devoted to identification of specific features of biotextile materials designed to preserve living organisms from adverse environmental factors like enhanced electromagnetic radiation of different frequencies, without use of vivarium animals. Cultures of the freshwater macrophyte duckweed (Lemna minor) line Sta2 and fruit flies (Drosophila melanogaster) were chosen as model systems suitable for the study of the influence of electromagnetic field (EMF) radiation. The experiments showed changes of phenotypic features and growth parameters of test objects, as well as induction of point mutations of DNA (for example, insertions or deletions in chloroplast DNA and nucleotide substitutions in nuclear genes). The responses of test organisms induced by EMF were studied using microscopy, flow cytometry, and DNA sequencing methods. On the cell level, a new fast flow cytometry method for biotextile testing was developed: immature gametic (pollen) cell cultures were used as a highly sensitive model system (plant gametic cell response is comparable to human neutrophil response) to study the influence of EMF radiation. The method was based on the measurement of differences of fluorescence intensity between group of cells experimentally affected by EMF radiation and non-affected cells.

Enhanced mitochondrial G-quadruplex formation impedes replication fork progression leading to mtDNA loss in human cells.

Mitochondrial DNA (mtDNA) replication stalling is considered an initial step in the formation of mtDNA deletions that associate with genetic inherited disorders and aging. However, the molecular details of how stalled replication forks lead to mtDNA deletions accumulation are still unclear. Mitochondrial DNA deletion breakpoints preferentially occur at sequence motifs predicted to form G-quadruplexes (G4s), four-stranded nucleic acid structures that can fold in guanine-rich regions. Whether mtDNA G4s form in vivo and their potential implication for mtDNA instability is still under debate. In here, we developed new tools to map G4s in the mtDNA of living cells. We engineered a G4-binding protein targeted to the mitochondrial matrix of a human cell line and established the mtG4-ChIP method, enabling the determination of mtDNA G4s under different cellular conditions. Our results are indicative of transient mtDNA G4 formation in human cells. We demonstrate that mtDNA-specific replication stalling increases formation of G4s, particularly in the major arc. Moreover, elevated levels of G4 block the progression of the mtDNA replication fork and cause mtDNA loss. We conclude that stalling of the mtDNA replisome enhances mtDNA G4 occurrence, and that G4s not resolved in a timely manner can have a negative impact on mtDNA integrity.

Oncomine Dx Express CE-IVD liquid biopsy assay for non-small cell lung cancer: Performance review and analytical validation.

e21218 Background: Clinical diagnostics assays for oncology are becoming more readily available due to the advancement and democratization of Next-Generation Sequencing (NGS). Additionally, liquid biopsy can be used in NGS to detect genetic variants in circulating tumor DNA and RNA. Liquid biopsy is a less invasive option compared to traditional biopsy methods for early detection and continuous monitoring of cancer treatment outcomes. Here, we discuss screening of over 3,500 non-small cell lung cancer (NSCLC) samples, and the analytical validation of the Oncomine Dx Express Test (ODxET) and Genexus Dx Integrated Sequencer for detection of clinically significant variants in liquid biopsy samples. Methods: Our team screened over 3,500 NSCLC liquid biopsy samples in search of high priority clinical variants. Variants selected for analytical validation studies included ERBB2 exon 20 insertion, EGFR exon 20 insertion, EGFR exon 19 deletion, EGFR T790M, KRAS G12C, BRAF V600E, and RNA fusion isoforms including ALK, NTRK1/2/3, RET, and ROS1 oncogenic drivers. Over 550 screened samples were found to be positive for these variants of interest. This screening was performed on the Genexus Dx Integrated Sequencer according to the user guide. Results: The Genexus Dx Integrated Sequencer automates library preparation, sequencing, analysis, and reporting QC metrics and variant calls. Sequencing run setup is quick and straightforward, taking less than 15 minutes to start a run and just over 24 hours to go from nucleic acid to report. Limit of detection (LoD) for DNA SNVs, insertions, and deletions at 5 ng DNA input level ranged from 0.65% to 1.82% allelic frequency (AF), depending on the variant. The higher DNA input of 30 ng resulted in a lower LoD range, from 0.31% to 0.42% AF. RNA fusion and splice variant LoD at 5 ng sample input ranged from 9.9 to 19.6 molecular counts. The higher 30 ng input resulted in a lower LoD range for RNA variants as well, ranging from 6.4 to 8.0 molecular counts. In the analytical accuracy study, the false positive rate was found to be 0.2% for SNVs, 0% for insertions/deletions, and 0% for fusion targets. In the analytical reproducibility study, the average with-in run repeatability call rate (No Calls excluded) was 99.64% for DNA variants and 98.75% for RNA variants. Conclusions: Here, we demonstrated that the ODxET workflow on Genexus Dx Integrated Sequencer is a fast and efficient tool for testing clinical NSCLC liquid biopsy samples with high sensitivity and specificity.

Elucidating the Therapeutic Potential of Bis(Maltolato)OxoVanadium(IV): The Protective Role of Copper in Cellular Metabolism.

Vanadium (V) is a trace mineral whose biological activity, role as a micronutrient, and pharmacotherapeutic applications remain unknown. Over the last years, interest in V has increased due to its potential use as an antidiabetic agent mediated by its ability to improve glycemic metabolism. However, some toxicological aspects limit its potential therapeutic application. The present study aims to evaluate the effect of the co-treatment with copper (Cu) and bis(maltolato)oxovanadium(IV) (BMOV) as a possible strategy to reduce the toxicity of BMOV. Treating hepatic cells with BMOV reduced cell viability under the present conditions, but cell viability was corrected when cells were co-incubated with BMOV and Cu. Additionally, the effect of these two minerals on nuclear and mitochondrial DNA was evaluated. Co-treatment with both metals reduced the nuclear damage caused by BMOV. Moreover, treatment with these two metals simultaneously tended to reduce the ND1/ND4 deletion of the mitochondrial DNA produced with the treatment using BMOV alone. In conclusion, these results showed that combining Cu and V could effectively reduce the toxicity associated with V and enhance its potential therapeutic applications.

P53/TP53 Status Assessment in Gastroesophageal Adenocarcinoma.

Chromosomal instability (CIN) is very frequent in gastroesophageal adenocarcinoma (GEA) and it is characterized by TP53 deletions/mutations resulting in p53 nuclear accumulation, as revealed by immunohistochemistry (IHC), which considers the cases with "high" staining levels to be positive. Aiming to improve aberrant TP53 detection, droplet digital PCR (ddPCR) was used to evaluate TP53 deletion in formalin-fixed, paraffin-embedded DNA (FFPE-DNA) and cell-free DNA (cfDNA). To further investigate the mutational TP53 profile, next-generation sequencing (NGS) was performed in a subset of FFPE samples. After combining "low" and "high" IHC staining level groups, the proportion of deletion events was significantly higher compared to the "intermediate" group (72.9% vs. 47.5%, p-value = 0.002). The ddPCR TP53 deletion assay was feasible for cfDNA but only had good agreement (72.7%, Cohen's kappa = 0.48) with the assay performed with FFPE-DNA of the "low-level" group. NGS analysis confirmed that, in the "low-level" group, a high percentage (66.7%) of cases were aberrant, with disruptive mutations that probably led to p53 loss. Data suggested that p53 IHC alone underestimates the CIN phenotype in GEA and that molecular analysis in both solid and liquid biopsies could be integrated with it; in particular, in cases of completely negative staining.

Deletions of DNA in cancer and their possible uses for therapy.

Despite advances in treatments over the last decades, a uniformly reliable and free of side effects therapy of human cancers remains to be achieved. During chromosome replication, a premature halt of two converging DNA replication forks would cause incomplete replication and a cytotoxic chromosome nondisjunction during mitosis. In contrast to normal cells, most cancer cells bear numerous DNA deletions. A homozygous deletion permanently marks a cell and its descendants. Here, we propose an approach to cancer therapy in which a pair of sequence-specific roadblocks is placed solely at two cancer-confined deletion sites that are located ahead of two converging replication forks. We describe this method, termed "replication blocks specific for deletions" (RBSD), and another deletions-based approach as well. RBSD can be expanded by placing pairs of replication roadblocks on several different chromosomes. The resulting simultaneous nondisjunctions of these chromosomes in cancer cells would further increase the cancer-specific toxicity of RBSD.